WO2003066804A2 - Thermus scotoductus nucleic acid polymerases - Google Patents

Thermus scotoductus nucleic acid polymerases Download PDF

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WO2003066804A2
WO2003066804A2 PCT/US2002/029102 US0229102W WO03066804A2 WO 2003066804 A2 WO2003066804 A2 WO 2003066804A2 US 0229102 W US0229102 W US 0229102W WO 03066804 A2 WO03066804 A2 WO 03066804A2
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nucleic acid
polymerase
seq
amino acid
dna
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PCT/US2002/029102
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French (fr)
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WO2003066804A3 (en
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Elena V. Bolchakova
James E. Rozzelle
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Applera Corporation
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Priority to JP2003566155A priority Critical patent/JP2005516611A/en
Priority to EP02805994A priority patent/EP1466013A4/en
Priority to CA002459718A priority patent/CA2459718A1/en
Priority to AU2002366438A priority patent/AU2002366438A1/en
Publication of WO2003066804A2 publication Critical patent/WO2003066804A2/en
Priority to US10/799,369 priority patent/US20050042629A1/en
Publication of WO2003066804A3 publication Critical patent/WO2003066804A3/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
    • C12N9/1252—DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase

Definitions

  • the invention relates to nucleic acids and polypeptides for nucleic acid polymerases from thermophilic strains of Thermus scotoductus.
  • DNA polymerases are naturally-occurring intracellular enzymes used by a cell for replicating DNA by reading one nucleic acid strand and manufacturing its complement. Enzymes having DNA polymerase activity catalyze the formation of a bond between the 3' hydroxyl group at the growing end of a nucleic acid primer and the 5' phosphate group of a newly added nucleotide triphosphate.
  • Nucleotide triphosphates used for DNA synthesis are usually deoxyadenosine triphosphate (A), deoxythymidine triphosphate (T), deoxycytosine triphosphate (C) and deoxyguanosine triphosphate (G), but modified or altered versions of these nucleotides can also be used.
  • DNA polymerase I is the most abundant polymerase and is generally responsible for certain types of DNA repair, including a repairlike reaction that permits the joining of Okazaki fragments during DNA replication.
  • Polymerase I is essential for the repair of DNA damage induced by UV irradiation and radiomimetic drugs.
  • DNA Polymerase II is thought to play a role in repairing DNA damage that induces the SOS response. In mutants that lack both polymerase I and III, polymerase II repairs UV-induced lesions.
  • Polymerase I and II are monomeric polymerases while polymerase III is a multisubunit complex. Enzymes having DNA polymerase activity are often used in vitro for a variety of biochemical applications including cDNA synthesis and DNA sequencing reactions. See Sambrook e al., Molecular Cloning: A Laboratory Manual (3rd ed. Cold Spring Harbor Laboratory Press, 2001, hereby incorporated by reference. DNA polymerases are also used for amplification of nucleic acids by methods such as the polymerase chain reaction (PCR) (Mullis et al., U.S. Pat. Nos.
  • PCR polymerase chain reaction
  • RNA transcription-mediated amplification methods e.g., Kacian et al., PCT Publication No. WO91/01384, incorporated by reference.
  • DNA amplification utilizes cycles of primer extension through the use of a DNA polymerase activity, followed by thermal denaturation of the resulting double-stranded nucleic acid in order to provide a new template for another round of primer annealing and extension. Because the high temperatures necessary for strand denaturation result in the irreversible inactivations of many DNA polymerases, the discovery and use of DNA polymerases able to remain active at temperatures above about 37 °C provides an advantage in cost and labor efficiency. Thermostable DNA polymerases have been discovered in a number of thermophilic organisms including Thermus aquaticus, Thermus thermophilus, and species within the genera the Bacillus, Thermococcus, Sulfobus, and Pyrococcus.
  • thermostable DNA polymerase derived from Thermus aquaticus has been described by Lawyer, et al., J. Biol. Chem. 264:6427- 6437 (1989) and Gelfand et al, U.S. Pat. No. 5,079,352.
  • the cloning and expression of truncated versions of that DNA polymerase are further described in Lawyer et al., in PCR Methods and Applications, 2:275-287 (1993), and Barnes, PCT Publication No. WO92/06188 (1992).
  • Sullivan reports the cloning of a mutated version of the Taq DNA polymerase in EPO Publication No. 0482714A1 (1992).
  • a DNA polymerase from Thermus thermophilus has also been cloned and expressed. Asakura et al., J. Ferment. Bioeng. (Japan), 74:265- 269 (1993).
  • the properties of the various polymerases vary. Accordingly, new polymerases are needed that have improved sequence discrimination, better salt tolerance, combined reverse transcription and DNA polymerase activities, varying degrees of thermostability, improved tolerance for labeled or dideoxy nucleotides and other valuable properties.
  • the invention provides nucleic acid polymerase enzymes isolated from a thermophilic organism, Thermus scotoductus.
  • the invention provides nucleic acid polymerases from several Thermus scotoductus strains including strain X-l (ATCC Deposit No. 27978), strain SM3 and strain Vi7a.
  • the invention provides an isolated nucleic acid encoding a Thermus scotoductus nucleic acid polymerase.
  • Such a nucleic acid can have a polynucleotide sequence comprising any one of SEQ ID NO:l-12. Nucleic acids complementary to any one of SEQ ID NO: 1-12 are also included within the invention.
  • the invention provides an isolated nucleic acid encoding a polypeptide having at least 93% identity to an amino acid sequence comprising any one of SEQ ID NO: 13-28.
  • the invention also provides vectors comprising these isolated nucleic acids, including expression vectors comprising a promoter operably linked to any of the isolated nucleic acids of the invention.
  • Host cells comprising such isolated nucleic acids and vectors are also provided by the invention, particularly host cells capable of expressing a thermostable polypeptide, where the polypeptide has nucleic acid polymerase or DNA polymerase activity.
  • the invention provides an isolated nucleic acid encoding a derivative nucleic acid polymerase comprising any one of amino acid sequences SEQ ID NO: 13- 16 having a mutation that decreases 5-3' exonuclease activity. Such a derivative nucleic acid polymerase has decreased 5-3' exonuclease activity relative to a nucleic acid polymerase comprising any one of amino acid sequences SEQ ID NO: 13-16.
  • the invention provides an isolated nucleic acid encoding a derivative nucleic acid polymerase comprising any one of amino acid sequences SEQ ID NO:13-16 having a mutation that reduces discrimination against dideoxynucleotide triphosphates. Such a derivative nucleic acid polymerase has reduced discrimination against dideoxynucleotide triphosphates relative to a nucleic acid polymerase comprising any one of amino acid sequences SEQ ID NO:13-16.
  • the invention also provides isolated polypeptides that can include an amino acid sequence with at least 93% identity to any one of SEQ ID NO: 13-28.
  • the isolated polypeptides provided by the invention preferably have an amino acid sequence with at least 95% sequence identity to any one of SEQ ID NO: 13- 28.
  • Such polypeptides can also have nucleic acid polymerase or DNA polymerase activity.
  • DNA polymerase activity can, for example, be about 50,000 U/mg protein to about 500,000 U/mg protein.
  • the invention further provides a method of synthesizing DNA that includes contacting a polypeptide comprising any one of SEQ ID NO:13-28 with a DNA under conditions sufficient to permit polymerization of DNA.
  • the invention also provides a method of synthesizing DNA from an RNA template that includes contacting a polypeptide comprising any one of SEQ ID NO: 13-28 with an RNA template under conditions sufficient to permit synthesis of DNA (e.g. reverse transcription).
  • the invention further provides a method for thermocyclic amplification of nucleic acid that comprises contacting a nucleic acid with a thermostable polypeptide having any one of SEQ ID NO: 13-28 under conditions suitable for amplification of the nucleic acid, and amplifying the nucleic acid.
  • Such amplification can be, for example, by Strand Displacement Amplification or Polymerase Chain Reaction.
  • the invention also provides a method of primer extending DNA comprising contacting a polypeptide comprising of SEQ ID NO: 13-28 with a DNA under conditions sufficient to permit polymerization of DNA. Such primer extension can be performed, for example, to sequence DNA or to amplify DNA.
  • the invention further provides a method of making a nucleic acid polymerase comprising any one of SEQ ID NO: 13-28, the method comprising incubating a host cell comprising a nucleic acid that encodes a polypeptide comprising any one of SEQ ID NO: 13-28, operably linked to a promoter under conditions sufficient for RNA transcription and translation. In one embodiment, the method uses a nucleic acid that comprises any one of SEQ ID NO: 1-12.
  • the invention is also directed to a nucleic acid polymerase or DNA polymerase made by this method.
  • the invention also provides a kit that includes a container containing a nucleic acid polymerase comprising an amino acid sequence with at least 93% identity to any one of SEQ ID NO: 13-28.
  • the kit can also contain an unlabeled nucleotide, a labeled nucleotide, a balanced mixture of nucleotides, a chain terminating nucleotide, a nucleotide analog, a buffer solution, a solution containing magnesium, a cloning vector, a restriction endonuclease, a sequencing primer, a solution containing reverse transcriptase, or a DNA or RNA amplification primer.
  • kits can, for example, be adapted for performing DNA sequencing, DNA amplification, RNA amplification, reverse transcription or primer extension reactions.
  • Figure 1 provides a comparison of amino acid sequences for polymerases from Thermus aquaticus (Taq; SEQ ID NO:48), Thermus thermophilus (Tth; SEQ ID NO:49), Thermus filiformis (Tfi; SEQ ID NO:50) and strain X-l Thermus scotoductus strain X-l (Tsc; SEQ ED NO: 13).
  • Figure 2 provides a comparison of amino acid sequences for three strains of Thermus scotoductus polymerases: strain X-l (SEQ ID NO:13), strain SM3 (SEQ ID NO: 15), and strain Vi7a (SEQ ID NO: 16).
  • the present invention relates to nucleic acid and amino acid sequences encoding nucleic acid polymerases from thermophilic organisms.
  • the present invention provides a nucleic acid polymerase from Thermus scotoductus.
  • the nucleic acid polymerases of the invention can be used in a variety of procedures, including DNA synthesis, reverse transcription, DNA primer extension, DNA sequencing and DNA amplification procedures.
  • amino acid sequence refers to the positional arrangement and identity of amino acids in a peptide, polypeptide or protein molecule. Use of the term “amino acid sequence” is not meant to limit the amino acid sequence to the complete, native amino acid sequence of a peptide, polypeptide or protein.
  • nucleic acid such as a vector or a gene
  • nucleic acid segment is comprised of more than one nucleic acid segment and that at least two nucleic acid segments are of distinct origin. Such nucleic acid segments are fused together by recombinant techniques resulting in a nucleic acid sequence, which does not occur naturally.
  • coding region refers to the nucleotide sequence that codes for a protein of interest.
  • the coding region of a protein is bounded on the 5' side by the nucleotide triplet "ATG” that encodes the initiator methionine and on the 3' side by one of the three triplets that specify stop codons (i.e., TAA, TAG, TGA).
  • ATG nucleotide triplet
  • Constant expression refers to expression using a constitutive promoter.
  • Constant promoter refers to a promoter that is able to express the gene that it controls in all, or nearly all, phases of the life cycle of the cell.
  • “Complementary” or “complementarity” are used to define the degree of base-pairing or hybridization between nucleic acids.
  • adenine (A) can form hydrogen bonds or base pair with thymine (T) and guanine (G) can form hydrogen bonds or base pair with cytosine (C).
  • T thymine
  • G guanine
  • C cytosine
  • A is complementary to T
  • G is complementary to C.
  • Complementarity may be complete when all bases in a double-stranded nucleic acid are base paired.
  • complementarity may be "partial,” in which only some of the bases in a nucleic acid are matched according to the base pairing rules.
  • the degree of complementarity between nucleic acid strands has an effect on the efficiency and strength of hybridization between nucleic acid strands.
  • the "derivative" of a reference nucleic acid, protein, polypeptide or peptide is a nucleic acid, protein, polypeptide or peptide, respectively, with a related but different sequence or chemical structure than the respective reference nucleic acid, protein, polypeptide or peptide.
  • a derivative nucleic acid, protein, polypeptide or peptide is generally made purposefully to enhance or incorporate some chemical, physical or functional property that is absent or only weakly present in the reference nucleic acid, protein, polypeptide or peptide.
  • a derivative nucleic acid generally can differ in nucleotide sequence from a reference nucleic acid whereas a derivative protein, polypeptide or peptide can differ in amino acid sequence from the reference protein, polypeptide or peptide, respectively.
  • sequence differences can be one or more substitutions, insertions, additions, deletions, fusions and truncations, which can be present in any combination. Differences can be minor (e.g., a difference of one nucleotide or amino acid) or more substantial.
  • sequence of the derivative is not so different from the reference that one of skill in the art would not recognize that the derivative and reference are related in structure and/or function. Generally, differences are limited so that the reference and the derivative are closely similar overall and, in many regions, identical.
  • a “variant” differs from a “derivative" nucleic acid, protein, polypeptide or peptide in that the variant can have silent structural differences that do not significantly change the chemical, physical or functional properties of the reference nucleic acid, protein, polypeptide or peptide.
  • the differences between the reference and derivative nucleic acid, protein, polypeptide or peptide are intentional changes made to improve one or more chemical, physical or functional properties of the reference nucleic acid, protein, polypeptide or peptide.
  • DNA polymerase activity refers to the ability of an enzyme to synthesize new DNA strands by the incorporation of deoxynucleoside triphosphates.
  • a protein that can direct the synthesis of new DNA strands by the incorporation of deoxynucleoside triphosphates in a template-dependent manner is said to be “capable of DNA synthetic activity.”
  • the term “5' exonuclease activity” refers to the presence of an activity in a protein that is capable of removing nucleotides from the 5' end of a nucleic acid.
  • 3' exonuclease activity refers to the presence of an activity in a protein that is capable of removing nucleotides from the 3' end of a nucleic acid.
  • “Expression” refers to the transcription and/or translation of an endogenous or exogeneous gene in an organism. Expression generally refers to the transcription and stable accumulation of mRNA. Expression may also refer to the production of protein.
  • “Expression cassette” means a nucleic acid sequence capable of directing expression of a particular nucleotide sequence.
  • Expression cassettes generally comprise a promoter operably linked to the nucleotide sequence to be expressed (e.g., a coding region) that is operably linked to termination signals.
  • Expression cassettes also typically comprise sequences required for proper translation of the nucleotide sequence.
  • the expression cassette comprising the nucleotide sequence of interest may be chimeric, meaning that at least one of its components is heterologous with respect to at least one of its other components.
  • the expression of the nucleotide sequence in the expression cassette may be under the control of a constitutive promoter or of an inducible promoter that initiates transcription only when the host cell is exposed to some particular external stimulus. In the case of a multicellular organism, the promoter can also be specific to a particular tissue or organ or stage of development.
  • the term “gene” is used broadly to refer to any segment of nucleic acid associated with a biological function.
  • the term “gene” encompasses the coding region of a protein, polypeptide, peptide or structural RNA.
  • the term “gene” also includes sequences up to a distance of about 2 kb on either end of a coding region. These sequences are referred to as “flanking" sequences or regions (these flanking sequences are located 5' or 3' to the non- translated sequences present on the mRNA transcript).
  • the 5' flanking region may contain regulatory sequences such as promoters and enhancers or other recognition or binding sequences for proteins that control or influence the transcription of the gene.
  • the 3' flanking region may contain sequences that direct the termination of transcription, post-transcriptional cleavage and polyadenylation as well as recognition sequences for other proteins.
  • a protein or polypeptide encoded in a gene can be full length or any portion thereof, so that all activities or functional properties are retained, or so that only selected activities (e.g., enzymatic activity, ligand binding, or signal transduction) of the full-length protein or polypeptide are retained.
  • the protein or polypeptide can include any sequences necessary for the production of a proprotein or precursor polypeptide.
  • the term "native gene" refers to gene that is naturally present in the genome of an untransformed cell.
  • Gene refers to the complete genetic material that is naturally present in an organism and is transmitted from one generation to the next.
  • heterologous nucleic acid or “exogenous nucleic acid” refer to a nucleic acid that originates from a source foreign to the particular host cell or, if from the same source, is modified from its original form.
  • a heterologous gene in a host cell includes a gene that is endogenous to the particular host cell but has been modified through, for example, the use of DNA shuffling.
  • the terms also include non-naturally occurring multiple copies of a naturally occurring nucleic acid.
  • the terms refer to a nucleic acid segment that is foreign or heterologous to the cell, or normally found within the cell but in a position within the cell or genome where it is not ordinarily found.
  • the term "homology” refers to a degree of similarity between a nucleic acid and a reference nucleic acid or between a polypeptide and a reference polypeptide. Homology may be partial or complete. Complete homology indicates that the nucleic acid or amino acid sequences are identical. A partially homologous nucleic acid or amino acid sequence is one that is not identical to the reference nucleic acid or amino acid sequence. Hence, a partially homologous nucleic acid has one or more nucleotide differences in its sequence relative to the nucleic acid to which it is being compared. The degree of homology can be determined by sequence comparison.
  • DNA-DNA or DNA-RNA hybridization under various hybridization conditions, can provide an estimate of the degree of homology between nucleic acids, (see, e.g., Haines and Higgins (eds.), Nucleic Acid Hybridization, IRL Press, Oxford, U.K.).
  • Hybridization refers to the process of annealing complementary nucleic acid strands by forming hydrogen bonds between nucleotide bases on the complementary nucleic acid strands. Hybridization, and the strength of the association between the nucleic acids, is impacted by such factors as the degree of complementary between the hybridizing nucleic acids, the stringency of the conditions involved, the T m of the formed hybrid, and the G:C ratio within the nucleic acids.
  • “Inducible promoter” refers to a regulated promoter that can be turned on in one or more cell types by an external stimulus, such as a chemical, light, hormone, stress, temperature or a pathogen.
  • An "initiation site” is region surrounding the position of the first nucleotide that is part of the transcribed sequence, which is defined as position +1. All nucleotide positions of the gene are numbered by reference to the first nucleotide of the transcribed sequence, which resides within the initiation site. Downstream sequences (i.e., sequences in the 3' direction) are denominated positive, while upstream sequences (i.e., sequences in the 5' direction) are denominated negative.
  • nucleic acid or an “isolated” or “purified” polypeptide is a nucleic acid or polypeptide that, by the hand of man, exists apart from its native environment and is therefore not a product of nature.
  • An isolated nucleic acid or polypeptide may exist in a purified form or may exist in a non- native environment such as, for example, a transgenic host cell.
  • invader oligonucleotide refers to an oligonucleotide that contains sequences at its 3' end that are substantially the same as sequences located at the 5' end of a probe oligonucleotide. These regions will compete for hybridization to the same segment along a complementary target nucleic acid.
  • label refers to any atom or molecule that can be used to provide a detectable (preferably quantifiable) signal, and that can be attached to a nucleic acid or protein. Labels may provide signals detectable by fluorescence, radioactivity, colorimetry, gravimetry, X-ray diffraction or absorption, magnetism, enzymatic activity, and the like.
  • nucleic acid refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form, composed of monomers (nucleotides) containing a sugar, phosphate and a base that is either a purine or pyrimidine. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences as well as the reference sequence explicitly indicated.
  • oligonucleotide as used herein is defined as a molecule comprised of two or more deoxyribonucleotides or ribonucleotides, preferably more than three, and usually more than ten. There is no precise upper limit on the size of an oligonucleotide. However, in general, an oligonucleotide is shorter than about 250 nucleotides, preferably shorter than about 200 nucleotides and more preferably shorter than about 100 nucleotides. The exact size will depend on many factors, which in turn depends on the ultimate function or use of the oligonucleotide.
  • the oligonucleotide may be generated in any manner, including chemical synthesis, DNA replication, reverse transcription, or a combination thereof.
  • the terms "open reading frame” and “ORF” refer to the amino acid sequence encoded between translation initiation and termination codons of a coding sequence.
  • the terms "initiation codon” and “termination codon” refer to a unit of three adjacent nucleotides ('codon') in a coding sequence that specifies initiation and chain termination, respectively, of protein synthesis (mRNA translation).
  • operably linked means joined as part of the same nucleic acid molecule, so that the function of one is affected by the other.
  • “operably linked” also means that two or more nucleic acids are suitably positioned and oriented so that they can function together. Nucleic acids are often operably linked to permit transcription of a coding region to be initiated from the promoter.
  • a regulatory sequence is said to be “operably linked to” or “associated with” a nucleic acid sequence that codes for an RNA or a polypeptide if the two sequences are situated such that the regulatory sequence affects expression of the coding region (i.e., that the coding sequence or functional RNA is under the transcriptional control of the promoter). Coding regions can be operably-linked to regulatory sequences in sense or antisense orientation.
  • probe oligonucleotide refers to an oligonucleotide that interacts with a target nucleic acid to form a cleavage structure in the presence or absence of an invader oligonucleotide.
  • the probe oligonucleotide and target form a cleavage structure and cleavage occurs within the probe oligonucleotide.
  • the presence of an invader oligonucleotide upstream of the probe oligonucleotide can shift the site of cleavage within the probe oligonucleotide (relative to the site of cleavage in the absence of the invader).
  • Promoter refers to a nucleotide sequence, usually upstream (5') to a coding region, which controls the expression of the coding region by providing the recognition site for RNA polymerase and other factors required for proper transcription.
  • Promoter includes but is not limited a minimal promoter that is a short DNA sequence comprised of a TATA- box.
  • a promoter includes other sequences that serve to specify the site of transcription initiation and control or regulate expression, for example, enhancers.
  • an “enhancer” is a segment of DNA that can stimulate promoter activity and may be an innate element of the promoter or a heterologous element inserted to enhance the level or tissue specificity of a promoter.
  • Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even be comprised of synthetic DNA segments.
  • a promoter may also contain DNA segments that are involved in the binding of protein factors that control the effectiveness of transcription initiation in response to physiological or developmental conditions.
  • protein protein
  • peptide and “polypeptide” are used interchangeably herein.
  • regulatory sequences and “regulatory elements” refer to nucleotide sequences that control some aspect of the expression of nucleic acid sequences. Such sequences or elements can be located upstream (5' non-coding sequences), within, or downstream (3' non-coding sequences) of a coding sequence. "Regulatory sequences” and “regulatory elements” influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences include enhancers, introns, promoters, polyadenylation signal sequences, splicing signals, termination signals, and translation leader sequences. They include natural and synthetic sequences.
  • selectable marker refers to a gene that encodes an observable or selectable trait that is expressed and can be detected in an organism having that gene. Selectable markers are often linked to a nucleic acid of interest that may not encode an observable trait, in order to trace or select the presence of the nucleic acid of interest. Any selectable marker known to one of skill in the art can be used with the nucleic acids of the invention. Some selectable markers allow the host to survive under circumstances where, without the marker, the host would otherwise die. Examples of selectable markers include antibiotic resistance, for example, tetracycline or ampicillin resistance.
  • stringency is used to define the conditions of temperature, ionic strength, and the presence of other compounds such as organic solvents, under which nucleic acid hybridizations are conducted. With “high stringency” conditions, nucleic acid base pairing will occur only between nucleic acids that have a high frequency of complementary base sequences. With “weak” or “low” stringency conditions nucleic acids the frequency of complementary sequences is usually less, so that nucleic acids with differing sequences can be detected and/or isolated.
  • substantially similar and substantially homologous refer to nucleotide and amino acid sequences that represent functional equivalents of the instant inventive sequences. For example, altered nucleotide sequences that simply reflect the degeneracy of the genetic code but nonetheless encode amino acid sequences that are identical to the inventive amino acid sequences are substantially similar to the inventive sequences. In addition, amino acid sequences that are substantially similar to the instant sequences are those wherein overall amino acid identity is sufficient to provide an active, thermally stable nucleic acid polymerase.
  • amino acid sequences that are substantially similar to the sequences of the invention are those wherein the overall amino acid identity is 80% or greater, preferably 90% or greater, such as 91%, 92%, 93%, or 94%, and more preferably 95% or greater, such as 96%, 97%o, 98%, or 99% relative to the amino acid sequences of the invention.
  • a “terminating agent,” “terminating nucleotide” or “terminator” in relation to DNA synthesis or sequencing refers to compounds capable of specifically terminating a DNA sequencing reaction at a specific base, such compounds include but are not limited to, dideoxynucleosides having a 2', 3' dideoxy structure (e.g., ddATP, ddCTP, ddGTP and ddTTP).
  • Thermostable means that a nucleic acid polymerase remains active at a temperature greater than about 37 °C.
  • the nucleic acid polymerases of the invention remain active at a temperature greater than about 42 °C. More preferably, the nucleic acid polymerases of the invention remain active at a temperature greater than about 50 °C. Even more preferably, the nucleic acid polymerases of the invention remain active after exposure to a temperature greater than about 60 °C. Most preferably, the nucleic acid polymerases of the invention remain active despite exposure to a temperature greater than about 70 °C.
  • a "transgene” refers to a gene that has been introduced into the genome by transformation and is stably maintained.
  • Transgenes may include, for example, genes that are either heterologous or homologous to the genes of a particular organism to be transformed. Additionally, transgenes may comprise native genes inserted into a non-native organism, or chimeric genes.
  • endogenous gene refers to a native gene in its natural location in the genome of an organism.
  • a “foreign” or “exogenous” gene refers to a gene not normally found in the host organism but one that is introduced by gene transfer.
  • transformation refers to the transfer of a nucleic acid fragment into the genome of a host cell, resulting in genetically stable inheritance.
  • transgenic cells containing the transformed nucleic acid fragments are referred to as "transgenic” cells, and organisms comprising transgenic cells are referred to as “transgenic organisms.” Transformation may be accomplished by a variety of means known to the art including calcium DNA co-precipitation, electroporation, viral infection, and the like.
  • variant of a reference nucleic acid, protein, polypeptide or peptide is a nucleic acid, protein, polypeptide or peptide, respectively, with a related but different sequence than the respective reference nucleic acid, protein, polypeptide or peptide.
  • the differences between variant and reference nucleic acids, proteins, polypeptides or peptides are silent or conservative differences.
  • a variant nucleic acid differs in nucleotide sequence from a reference nucleic acid whereas a variant nucleic acid, protein, polypeptide or peptide differs in amino acid sequence from the reference protein, polypeptide or peptide, respectively.
  • a variant and reference nucleic acid, protein, polypeptide or peptide may differ in sequence by one or more substitutions, insertions, additions, deletions, fusions and truncations, which may be present in any combination. Differences can be minor (e.g., a difference of one nucleotide or amino acid) or more substantial.
  • the structure and function of the variant is not so different from the reference that one of skill in the art would not recognize that the variant and reference are related in structure and/or function. Generally, differences are limited so that the reference and the variant are closely similar overall and, in many regions, identical.
  • the term "vector" is used to refer to a nucleic acid that can transfer another nucleic acid segment(s) into a cell.
  • a “vector” includes, inter alia, any plasmid, cosmid, phage or nucleic acid in double- or single-stranded, linear or circular form that may or may not be self transmissible or mobilizable. It can transform prokaryotic or eukaryotic host cells either by integration into the cellular genome or by existing extrachromosomally (e.g., autonomous replicating plasmid with an origin of replication). Vectors used in bacterial systems often contain an origin of replication that allows the vector to replicate independently of the bacterial chromosome.
  • expression vector refers to a vector containing an expression cassette.
  • wild-type refers to a gene or gene product that has the characteristics of that gene or gene product when isolated from a naturally occurring source.
  • a wild-type gene is the gene form most frequently observed in a population and thus arbitrarily is designed the "normal” or “wild-type” form of the gene.
  • variant or “derivative” refers to a gene or gene product that displays modifications in sequence and or functional properties (i.e., altered characteristics) when compared to the wild-type gene or gene product.
  • Naturally-occurring derivatives can be isolated. They are identified by the fact that they have altered characteristics when compared to the wild-type gene or gene product.
  • the invention provides isolated nucleic acids encoding Thermus scotoductus nucleic acid polymerases as well as derivatives fragments and variant nucleic acids thereof that encode an active, thermally stable nucleic acid polymerase.
  • one aspect of the invention includes the nucleic acid polymerases encoded by the polynucleotide sequences contained in Thermus scotoductus strain X-l (ATCC Deposit No. 27978).
  • Another aspect of the invention provides the nucleic acid polymerases of Thermus scotoductus strains SM3 and Vi7a. Any nucleic acid encoding any one of amino acid sequences SEQ ID NO: 13-28, which are amino acid sequences for wild type and several derivative Thermus scotoductus nucleic acid polymerases, are also contemplated by the present invention.
  • the invention provides a nucleic acid of SEQ ID NO:l, a wild type Thermus scotoductus, strain X-l, nucleic acid encoding a nucleic acid polymerase.
  • the invention provides nucleic acids encoding a wild type nucleic acid polymerase from Thermus scotoductus, strain SM3, having, for example, SEQ ID NO:2.
  • the invention provides nucleic acids encoding a wild type nucleic acid polymerase from Thermus scotoductus, strain Vi7a, having, for example, SEQ ID NO:3.
  • CTACGGCGTC CTCTACGGCA TGTCCGCCCA CCGGCTTTCG 2040
  • CATGGCCTTC AACATGCCGG TCCAGGGGAC CGCCGCGGAT 2280 TTGATGAAAC TGGCCATGGT GAAGCTCTTT CCCAGGCTTC 2320
  • the invention provides a nucleic acid of SEQ ID NO:4, a derivative nucleic acid related to Thermus scotoductus, strain X-l, having GAC (encoding Asp) in place of GGG (encoding Gly) at positions 136- 138.
  • SEQ ID NO :4 is provided below.
  • GCCCAGGAGG CCAAGCGGAC CATGGAGGAG GTGTGGCCCC 2440 TGAAGGTGCC CTTGGAGGTG GAAGTGGGCA TCGGGGAGGA 2480
  • the invention provides a nucleic acid of SEQ ID NO:5, a derivative nucleic acid related to Thermus scotoductus, strain SM3, having GAC (encoding Asp) in place of GGG (encoding Gly) at positions 136- 138.
  • SEQ ID NO:5 is provided below.
  • the invention provides a nucleic acid of SEQ ID NO: 1
  • SEQ ID NO: 6 a derivative nucleic acid related to Thermus scotoductus, strain Vi7a, having GAC (encoding Asp) in place of GGG (encoding Gly) at positions 136- 138.
  • GAC encoding Asp
  • GGG encoding Gly
  • CTACGGCGTC CTCTACGGCA TGTCCGCCCA CCGGCTTTCG 2040 GGAGAGCTGG CCATCCCCTA CGAGGAAGCG GTGGCCTTCA 2080
  • GCCCAGGAGG CCAAGCGGAC CATGGAGGAG GTGTGGCCCC 2440 TGAAGGTGCC CTTGGAGGTG GAGGTGGGTA TCGGGGAGGA 2480
  • the invention provides a nucleic acid of SEQ ID NO: 7, a derivative nucleic acid related to Thermus scotoductus, strain X-l, having TAC (encoding Tyr) in place of TTC (encoding Phe) at positions 2002- 04.
  • SEQ ID NO:7 is provided below:
  • the invention provides a nucleic acid of SEQ ID NO:8, a derivative nucleic acid related to Thermus scotoductus, strain SM3, having TAC (encoding Tyr) in place of TTC (encoding Phe) at positions 2002- 04.
  • SEQ ED NO:8 is provided below:
  • the invention provides a nucleic acid of SEQ ID NO: 9, a derivative nucleic acid related to Thermus scotoductus, strain Vi7a, having TAC (encoding Tyr) in place of TTC (encoding Phe) at positions 2101- 03.
  • SEQ ED NO:9 is provided below:
  • the invention provides a nucleic acid of SEQ ED
  • SEQ ED NO: 10 a derivative nucleic acid related to Thermus scotoductus, strain X-l, having GAC (encoding Asp) in place of GGG (encoding Gly) at positions 136- 138 and having TAC (encoding Tyr) in place of TTC (encoding Phe) at positions 2002-04.
  • GAC encode Asp
  • TAC encoding Tyr
  • the invention provides a nucleic acid of SEQ D NO: 11 , a derivative nucleic acid related to Thermus scotoductus, strain SM3, having GAC (encoding Asp) in place of GGG (encoding Gly) at positions 136- 138 and having TAC (encoding Tyr) in place of TTC (encoding Phe) at positions 2002-04.
  • SEQ D NO:8 is provided below:
  • the invention provides a nucleic acid of SEQ ED
  • TAC encoding Tyr
  • TTC encoding Phe
  • GAC encoding Asp
  • nucleic acids of the invention have homology to portions of the nucleic acids encoding the thermostable DNA polymerases of Thermus aquaticus and Thermus thermophilus (see Figure 1). However, significant portions of the nucleic acid sequences of the present invention are distinct.
  • the invention also encompasses fragment and variant nucleic acids of SEQ ED NO: 1-12.
  • Nucleic acid "fragments" encompassed by the invention are of two general types. First, fragment nucleic acids that do not encode a full- length nucleic acid polymerase but do encode a thermally stable polypeptide with nucleic acid polymerase activity are encompassed within the invention. Second, fragment nucleic acids useful as hybridization probes but that generally do not encode polymerases retaining biological activity are also encompassed within the invention.
  • fragments of nucleotide sequences such as SEQ TD NO:l - 12 may be as small as about 9 nucleotides, about 12 nucleotides, about 15 nucleotides, about 17 nucleotides, about 18 nucleotides, about 20 nucleotides, about 50 nucleotides, about 100 nucleotides or more.
  • a fragment nucleic acid of the invention can have any upper size limit so long as it is related in sequence to the nucleic acids of the invention but is not full length.
  • variants are substantially similar or substantially homologous sequences.
  • variants include those sequences that, because of the degeneracy of the genetic code, encode the identical amino acid sequence of the native nucleic acid polymerase protein.
  • variant nucleic acids also include those that encode polypeptides that do not have amino acid sequences identical to that of a native nucleic acid polymerase protein, but that encode an active, thermally stable nucleic acid polymerase with conservative changes in the amino acid sequence.
  • the genetic code is "degenerate,” meaning that several trinucleotide codons can encode the same amino acid. This degeneracy is apparent from Table 1.
  • nucleic acid sequence of the variant may be silent and may not alter the amino acid sequence encoded by the nucleic acid.
  • nucleic acid sequence alterations are silent, a variant nucleic acid will encode a polypeptide with the same amino acid sequence as the reference nucleic acid. Therefore, a particular nucleic acid sequence of the invention also encompasses variants with degenerate codon substitutions, and complementary sequences thereof, as well as the sequence explicitly specified by a SEQ ED NO.
  • degenerate codon substitutions may be achieved by generating sequences in which the reference codon is replaced by any of the codons for the amino acid specified by the reference codon.
  • the third position of one or more selected codons can be substituted with mixed-base and/or deoxyinosine residues as disclosed by Batzer et al, Nucleic Acid Res., 19, 5081 (1991) and/or Ohtsuka et al., J. Biol. Chem., 260, 2605 (1985); Rossolini et al., Mol. Cell. Probes, 8, 91 (1994).
  • variant and reference nucleic acids of the invention may differ in the encoded amino acid sequence by one or more substitutions, additions, insertions, deletions, fusions and truncations, which may be present in any combination, so long as an active, thermally stable nucleic acid polymerase is encoded by the variant nucleic acid.
  • variant nucleic acids will not encode exactly the same amino acid sequence as the reference nucleic acid, but have conservative sequence changes.
  • variant nucleic acids with silent and conservative changes can be defined and characterized by the degree of homology to the reference nucleic acid.
  • Preferred variant nucleic acids are "substantially homologous" to the reference nucleic acids of the invention. As recognized by one of skill in the art, such substantially similar nucleic acids can hybridize under stringent conditions with the reference nucleic acids identified by SEQ ED NOs herein. These types of substantially homologous nucleic acids are encompassed by this invention.
  • nucleic acid derivatives and variants of the invention will have at least 90%, 91%, 92%, 93% or 94% sequence identity to the reference nucleotide sequence defined herein.
  • nucleic acids of the invention will have at least at least 95%, 96%, 97%, 98%, or 99% sequence identity to the reference nucleotide sequence defined herein.
  • Variant nucleic acids can be detected and isolated by standard hybridization procedures.
  • Hybridization to detect or isolate such sequences is generally carried out under stringent conditions.
  • Stringent hybridization conditions and “stringent hybridization wash conditions” in the context of nucleic acid hybridization experiments such as Southern and Northern hybridization are sequence dependent, and are different under different environmental parameters. Longer sequences hybridize specifically at higher temperatures.
  • An extensive guide to the hybridization of nucleic acids is found in Tijssen, Laboratory Techniques in Biochemistry and Molecular biology-Hybridization with Nucleic Acid Probes, page 1, chapter 2 "Overview of principles of hybridization and the strategy of nucleic acid probe assays" Elsevier, New York (1993). See also, J.
  • the invention also provides methods for detection and isolation of derivative or variant nucleic acids encoding nucleic acid polymerase activity.
  • the methods involve hybridizing at least a portion of a nucleic acid comprising any one of SEQ ED NO: 1-12 to a sample nucleic acid, thereby forming a hybridization complex; and detecting the hybridization complex.
  • the presence of the complex correlates with the presence of a derivative or variant nucleic acid encoding at least a segment of nucleic acid polymerase.
  • the portion of a nucleic acid comprising any one of SEQ ED NO: 1-12 used for hybridization is at least fifteen nucleotides, and hybridization is under hybridization conditions that are sufficiently stringent to permit detection and isolation of substantially homologous nucleic acids.
  • a nucleic acid sample is amplified by the polymerase chain reaction using primer oligonucleotides selected from any one of SEQ ID NO:l- 12.
  • highly stringent hybridization and wash conditions are selected to be about 5 °C lower than the thermal melting point (T m ) for the specific double-stranded sequence at a defined ionic strength and pH.
  • T m thermal melting point
  • highly stringent hybridization and wash conditions are selected to be about 5 °C lower than the thermal melting point (T m ) for the specific double-stranded sequence at a defined ionic strength and pH.
  • T m thermal melting point
  • stringency conditions can be adjusted to allow some mismatching in sequences so that lower degrees of similarity are detected (heterologous probing).
  • stringent conditions will be those in which the salt concentration is less than about 1.5 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C for short probes (e.g., 10 to 50 nucleotides) and at least about 60°C for long probes (e.g., greater than 50 nucleotides).
  • Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide.
  • Exemplary moderate stringency conditions include hybridization in 40 to 45% formamide, 1.0 M NaCl, 1% SDS at 37°C, and a wash in 0.5X to IX SSC at 55 to 60°C.
  • Exemplary high stringency conditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37°C, and a wash in 0. IX SSC at 60 to 65°C.
  • the degree of complementarity or homology of hybrids obtained during hybridization is typically a function of post-hybridization washes, the critical factors being the ionic strength and temperature of the final wash solution.
  • the type and length of hybridizing nucleic acids also affects whether hybridization will occur and whether any hybrids formed will be stable under a given set of hybridization and wash conditions.
  • the T m can be approximated from the equation of Meinkoth and Wahl Anal. Biochem.
  • T m 81.5°C + 16.6 (log M) +0.41 (%GC) - 0.61 (% form) - 500/L; where M is the molarity of monovalent cations, %GC is the percentage of guanosine and cytosine nucleotides in the DNA, % form is the percentage of formamide in the hybridization solution, and L is the length of the hybrid in base pairs.
  • the T m is the temperature (under defined ionic strength and pH) at which 50% of a complementary target sequence hybridizes to a perfectly matched probe. Very stringent conditions are selected to be equal to the T m for a particular probe.
  • An example of stringent hybridization conditions for hybridization of complementary nucleic acids that have more than 100 complementary residues on a filter in a Southern or Northern blot is 50% formamide with 1 mg of heparin at 42 °C, with the hybridization being carried out overnight.
  • An example of highly stringent conditions is 0.1 5 M NaCl at 72 °C for about 15 minutes.
  • An example of stringent wash conditions is a 0.2x SSC wash at 65 °C for 15 minutes (see also, Sambrook, infra). Often, a high stringency wash is preceded by a low stringency wash to remove background probe signal.
  • stringent conditions typically involve salt concentrations of less than about l.OM Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3, and the temperature is typically at least about 30°C.
  • Stringent conditions can also be achieved with the addition of destabilizing agents such as formamide.
  • destabilizing agents such as formamide.
  • a signal to noise ratio of 2x (or higher) than that observed for an unrelated probe in the particular hybridization assay indicates detection of a specific hybridization.
  • Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the proteins that they encode are substantially identical. This occurs, e.g., when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code.
  • a reference nucleotide sequence preferably hybridizes to the reference nucleotide sequence in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO 4 , 1 mM EDTA at 50°C with washing in 2X SSC, 0.1% SDS at 50°C, more desirably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO 4 , 1 mM EDTA at 50°C with washing in IX SSC, 0.1% SDS at 50°C, more desirably still in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO 4 , 1 mM EDTA at 50°C with washing in 0.5X SSC, 0.1% SDS at 50°C, preferably in 7% sodium dodecyl sulfate
  • T m is reduced by about 1°C for each 1% of mismatching.
  • T m , hybridization, and or wash conditions can be adjusted to hybridize to sequences of the desired sequence identity. For example, if sequences with >90% identity are sought, the T m can be decreased 10°C.
  • stringent conditions are selected to be about 5°C lower than the thermal melting point
  • T m for the specific sequence and its complement at a defined ionic strength and pH.
  • severely stringent conditions can utilize a hybridization and/or wash at 1, 2, 3, or 4°C lower than the thermal melting point (T m ); moderately stringent conditions can utilize a hybridization and/or wash at 6, 7, 8, 9, or 10°C lower than the thermal melting point (T m ); low stringency conditions can utilize a hybridization and/or wash at 11, 12, 13, 14, 15, or 20°C lower than the thermal melting point (T m ).
  • Computer analyses can also be utilized for comparison of sequences to determine sequence identity. Such analyses include, but are not limited to: CLUSTAL in the PC/Gene program (available from Intelligenetics, Mountain View, California); the ALIGN program (Version 2.0) and GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Version 8 (available from Genetics Computer Group (GCG), 575 Science Drive, Madison, Wisconsin, USA). Alignments using these programs can be performed using the default parameters.
  • the CLUSTAL program is well described by Higgins et al. Gene 73:237 244 (1988); Higgins et al. CABIOS 5:151-153 (1989); Corpet et al. Nucleic Acids Res.
  • PSI-BLAST in BLAST 2.0
  • PSI-BLAST can be used to perform an iterated search that detects distant relationships between molecules. See Altschul et al., supra.
  • the default parameters of the respective programs e.g. BLASTN for nucleotide sequences, BLASTX for proteins
  • the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA, 89, 10915 (1989)). See http://www.ncbi.nlm.nih.gov. Alignment may also be performed manually by inspection.
  • comparison of nucleotide sequences for determination of percent sequence identity to the nucleic acid polymerase sequences disclosed herein is preferably made using the BlastN program (version 1.4.7 or later) with its default parameters or any equivalent program.
  • equivalent program is intended any sequence comparison program that, for any two sequences in question, generates an alignment having identical nucleotide or amino acid residue matches and an identical percent sequence identity when compared to the corresponding alignment generated by the preferred program.
  • Nucleic acids of the invention may be used for the recombinant expression of the nucleic acid polymerase polypeptides of the invention.
  • recombinant expression of a nucleic acid polymerase polypeptide of the invention is effected by introducing a nucleic acid encoding that polypeptide into an expression vector adapted for use in particular type of host cell.
  • the nucleic acids of the invention can be introduced and expressed in any host organism, for example, in both prokaryotic or eukaryotic host cells. Examples of host cells include bacterial cells, yeast cells, cultured insect cell lines, and cultured mammalian cells lines.
  • the recombinant host cell system is selected that processes and post-translationally modifies nascent polypeptides in a manner similar to that of the organism from which the nucleic acid polymerase was derived.
  • prokaryotic organisms are preferred, for example, Escherichia coli. Accordingly, the invention provides host cells comprising the expression vectors of the invention.
  • the nucleic acids to be introduced can be conveniently placed in expression cassettes for expression in an organism of interest.
  • Such expression cassettes will comprise a transcriptional initiation region linked to a nucleic acid of the invention.
  • Expression cassettes preferably also have a plurality of restriction sites for insertion of the nucleic acid to be under the transcriptional regulation of various control elements.
  • the expression cassette additionally may contain selectable marker genes. Suitable control elements such as enhancers/promoters, splice junctions, polyadenylation signals, etc. may be placed in close proximity to the coding region of the gene if needed to permit proper initiation of transcription and/or correct processing of the primary RNA transcript.
  • the coding region utilized in the expression vectors of the present invention may contain endogenous enhancers/promoters, splice junctions, intervening sequences, polyadenylation signals, etc., or a combination of both endogenous and exogenous control elements.
  • the nucleic acid in the vector is under the control of, and operably linked to, an appropriate promoter or other regulatory elements for transcription in a host cell.
  • the vector may be a bi-functional expression vector that functions in multiple hosts.
  • the transcriptional cassette generally includes in the 5 '-3' direction of transcription, a promoter, a transcriptional and translational initiation region, a DNA sequence of interest, and a transcriptional and translational termination region functional in the organism.
  • the termination region may be native with the transcriptional initiation region, may be native with the DNA sequence of interest, or may be derived from another source. Efficient expression of recombinant nucleic acids in prokaryotic and eukaryotic cells generally requires regulatory control elements directing the efficient termination and polyadenylation of the resulting transcript. Transcription termination signals are generally found downstream of the polyadenylation signal and are a few hundred nucleotides in length.
  • the term "poly A site” or "poly A sequence” as used herein denotes a nucleic acid sequence that directs both the termination and polyadenylation of the nascent RNA transcript. Efficient polyadenylation of the recombinant transcript is desirable as transcripts lacking a poly A tail are unstable and are rapidly degraded.
  • Nucleic acids encoding nucleic acid polymerase may be introduced into bacterial host cells by a method known to one of skill in the art.
  • nucleic acids encoding a thermophilic nucleic acid polymerase can be introduced into bacterial cells by commonly used transformation procedures such as by treatment with calcium chloride or by electroporation.
  • nucleic acids encoding the thermophilic nucleic acid polymerase may be introduced into eukaryotic host cells by a number of means including calcium phosphate co- precipitation, spheroplast fusion, electroporation and the like.
  • transformation may be affected by treatment of the host cells with lithium acetate or by electroporation.
  • one aspect of the invention is to provide expression vectors and host cells comprising a nucleic acid encoding a nucleic acid polymerase polypeptide of the invention.
  • a wide range of expression vectors are well known in the art. Description of various expression vectors and how to use them can be found among other places in U.S. Pat. Nos. 5,604,118; 5,583,023; 5,432,082; 5,266,490; 5,063,158; 4,966,841; 4,806,472; 4,801,537; and Goedel et al., Gene Expression Technology, Methods of Enzymology, Vol. 185, Academic Press, San Diego (1989). The expression of nucleic acid polymerases in recombinant cell systems is a well-established technique.
  • nucleic acid polymerase examples include U.S. Pat. Nos. 5,602,756; 5,545,552; 5,541,311; 5,500,363; 5,489,523; 5,455,170; 5,352,778; 5,322,785; and 4,935,361.
  • Recombinant DNA and molecular cloning techniques that can be used to help make and use aspects of the invention are described by Sambrook et al., Molecular Cloning: A Laboratory Manual Vol.1-3, Cold Spring Harbor laboratory, Cold Spring Harbor, N.Y. (2001); Ausubel (ed.), Current Protocols in Molecular Biology, John Wiley and Sons, Enc. (1994); T. Maniatis, E. F.
  • Nucleic Acid Polymerase Enzymes The invention provides Thermus scotoductus nucleic acid polymerase polypeptides, as well as fragments thereof and variant nucleic acid Polymerase polypeptides that are active and thermally stable. Any polypeptide containing amino acid sequence having any one of SEQ ED NO: 13-28, which are the amino acid sequences for wild type and derivative Thermus scotoductus nucleic acid polymerases, are contemplated by the present invention.
  • the polypeptides of the invention are isolated or substantially purified polypeptides. En particular, the isolated polypeptides of the invention are substantially free of proteins normally present in Thermus scotoductus bacteria.
  • the invention provides a polypeptide of SEQ ED NO: 13, a wild type Thermus scotoductus nucleic acid polymerase polypeptide from strain X-l with three additional amino acids at the N-terminus:
  • VPD ASRVKS IREAAERMAF NMPVQGTAAD 760 LMKLAMVK F PR QE GARM LLQVHDELVL EAPKEQAEEV 800
  • the invention provides SEQ ED NO: 14, a wild type Thermus scotoductus nucleic acid polymerase enzyme, from strain X-l that does not have the three additional amino acids at the N-terminus that are present in SEQ ED NO: 13.
  • SEQ ID NO: 14 is provided below.
  • VPDLASRVKS IREAAERMAF NMPVQGTAAD 760 LMKLAMVKLF PRLQELGARM LLQVHDELVL EAPKEQAEEV 800
  • the invention provides SEQ ED NO: 15, a wild type Thermus scotoductus nucleic acid polymerase enzyme from strain SM3.
  • SEQ ED NO : 15 is provided below.
  • the invention provides SEQ ED NO: 16, a wild type Thermus scotoductus nucleic acid polymerase enzyme from strain Vi7a.
  • SEQ ED NO: 16 is provided below.
  • VPDLASRVKS IREAAERMAF NMPVQGTAAD 760
  • the sequences of wild type Thermus scotoductus nucleic acid polymerases are distinct from the amino acid sequence of Thermus aquaticus DNA Polymerase. There are about 51 conservative amino acid differences and about 62 nonconservative amino acid differences. For example, one region of dissimilarity is between approximate amino acid positions 51 and 65, where the sequence of the Thermus scotoductus polymerase has about four amino acid differences (in bold): LLKALREDG DVNEVVFDAK APSFRHQTYE (SEQ ED ⁇ O:39).
  • Another region of dissimilarity is between approximate amino acid positions 201 and 236, where the sequence of the Thermus scotoductus polymerase has about seven amino acid differences (in bold): GEKTAAKLIREWGSLENLLKHLEQV KPASV REKILS (SEQ ED NO:40).
  • Another region of dissimilarity is between about positions 311 and 350, where the sequence of the Thermus scotoductus polymerase has about seven amino acid changes (in bold): VGYVLSRPEPMWAELN
  • ALAAAWEGRVYRAEDPLEALRGLG SEQ ID NO:41.
  • Another region of dissimilarity is between about positions 415 and 435, where the sequence of the Thermus scotoductus polymerase has about five amino acid changes (in bold): RLYAALLERLKGEERLLWLYE (SEQ ED NO:42).
  • Another region of dissimilarity is between about positions 531 and 562, where the sequence of the Thermus scotoductus polymerase has about six amino acid changes (in bold): PIVDR1LQYRELSKLK GTYED PLPALVHPKTN (SEQ ED NO:43).
  • Another region of dissimilarity is between about positions 801 and 836, where the sequence of the Thermus scotoductus polymerase has about eight amino acid changes (in bold): EEVAQEAKRT MEEVWPLKNPLENEVGIGEDWLSAKA (SEQ ED ⁇ O:44).
  • EEVAQEAKRT MEEVWPLKNPLENEVGIGEDWLSAKA SEQ ED ⁇ O:44.
  • many regions of the Thermus scotoductus polymerase differ from the Thermus aquaticus and Thermus thermophilus DNA polymerases.
  • DNA polymerases possess activities in addition to a DNA polymerase activity. Such activities include, for example, a 5'-3' exonuclease activity and/or a 3'-5' exonuclease activity.
  • the 3'-5' exonuclease activity improves the accuracy of the newly synthesized strand by removing incorrect bases that may have been incorporated.
  • DNA polymerases in which such activity is low or absent are prone to errors in the incorporation of nucleotide residues into the primer extension strand.
  • Taq DNA polymerase has been reported to have low 3'-5' exonuclease activity. See Lawyer et al., J. Biol Chem. 264:6427-6437.
  • thermostable DNA polymerase used for such purposes.
  • thermostable nucleic acid polymerase with an attenuated 5 '-3' exonuclease activity, or in which such activity is absent is a desired characteristic of an enzyme for biochemical applications.
  • DNA polymerase enzymes have been described where a modification has been introduced in a DNA polymerase that accomplishes this object. For example, the Klenow fragment of E.
  • coli DNA polymerase I can be produced as a proteolytic fragment of the holoenzyme in which the domain of the protein controlling the 5'-3' exonuclease activity has been removed. The Klenow fragment still retains the polymerase activity and the 3'-5' exonuclease activity.
  • Barnes, PCT Publication No. WO92/06188 (1992) and Gelfand et al., U.S. Pat. No. 5,079,352 have produced 5'-3' exonuclease-deficient recombinant Thermus aquaticus DNA polymerases.
  • Ishino et al., EPO Publication No. 0517418A2 have produced a 5'-3' exonuclease-deficient DNA polymerase derived from Bacillus caldotenax.
  • the invention provides a polypeptide that is a derivative Thermus scotoductus polypeptide with reduced or eliminated 5 '-3' exonuclease activity.
  • a polypeptide that is a derivative Thermus scotoductus polypeptide with reduced or eliminated 5 '-3' exonuclease activity.
  • the invention contemplates any polypeptide derived from the Thermus scotoductus polypeptides of the invention that has reduced or eliminated such 5 '-3' exonuclease activity.
  • Xu et al. Biochemical and mutational studies of the 5'-3' exonuclease of DNA polymerase I of Escherichia coli. J. Mol. Biol. 1997 May 2; 268(2):284-302.
  • the invention provides a Thermus scotoductus nucleic acid polymerase polypeptide from strain X-l in which Asp is used in place of Gly at position 46.
  • This polypeptide has SEQ ED NO: 17 and reduced 5'- 3' exonuclease activity.
  • SEQ ED NO: 17 is provided below.
  • VPDLASRVKS IREAAERMAF NMPVQGTAAD 760
  • the invention provides a Thermus scotoductus nucleic acid polymerase polypeptide from strain X-l in which Asp is used in place of Gly at position 46.
  • This polypeptide has SEQ ED NO: 18 and reduced 5' 3' exonuclease activity.
  • SEQ ED NO: 18 is provided below.
  • VPDLASRVKS IREAAERMAF NMPVQGTAAD 760
  • the invention provides a Thermus scotoductus nucleic acid polymerase polypeptide from strain SM3 in which Asp is used in place of Gly at position 46.
  • This polypeptide has SEQ ED NO: 19 and reduced 5' 3' exonuclease activity.
  • SEQ ED NO: 19 is provided below.
  • VPDLASRVKS IREAAERMAF NMPVQGTAAD 760 LMKLAMVKLF PRLQELGARM LLQVHDELVL EAPKEQAEEV 800
  • the invention provides a Thermus scotoductus nucleic acid polymerase polypeptide from strain Vi7a in which Asp is used in place of Gly at position 46.
  • This polypeptide has SEQ ID NO:20 and reduced 5' 3' exonuclease activity.
  • SEQ ED NO:20 is provided below.
  • VPDLASRVKS IREAAERMAF NMPVQGTAAD 760
  • the invention provides a polypeptide of SEQ ID NO:21, a derivative Thermus scotoductus polypeptide from strain X-l with reduced bias against ddNTP incorporation.
  • SEQ ID NO:21 has Tyr in place of Phe at position 668.
  • the sequence of SEQ ED NO:21 is below.
  • VPDLASRVKS IREAAERMAF NMPVQGTAAD 760 LMKLAMVKLF PRLQELGARM LLQVHDELVL EAPKEQAEEV 800
  • the invention provides a polypeptide of SEQ ID NO:22, a derivative Thermus scotoductus polypeptide from strain X-l with reduced bias against ddNTP incorporation.
  • SEQ ID NO:22 has Tyr in place of Phe at position 668. The sequence of SEQ ID NO:22 is below.
  • VPDLASRVKS IREAAERMAF NMPVQGTAAD 760 LMKLAMVKLF PRLQELGARM LLQVHDELVL EAPKEQAEEV 800
  • the invention provides a polypeptide of SEQ ED NO: 23, a derivative Thermus scotoductus polypeptide from strain SM3 with reduced bias against ddNTP incorporation.
  • SEQ ED NO:23 has Tyr in place of Phe at position 668.
  • the sequence of SEQ ID NO:23 is below.
  • VPDLASRVKS IREAAERMAF NMPVQGTAAD 760
  • the invention provides a polypeptide of SEQ ED NO:24, a derivative Thermus scotoductus polypeptide from strain Vi7a with reduced bias against ddNTP incorporation.
  • SEQ ED NO:24 has Tyr in place of Phe at position 668.
  • the invention provides a polypeptide of SEQ ED NO:25, a derivative Thermus scotoductus polypeptide from strain X-l with reduced 5 '-3' exonuclease activity and reduced bias against ddNTP incorporation.
  • SEQ ID NO:25 has Asp in place of Gly at position 46 and Tyr in place of Phe at position 668. The sequence of SEQ ID NO:25 is below.
  • VPDLASRVKS IREAAERMAF NMPVQGTAAD 760
  • the invention provides a polypeptide of SEQ ED
  • SEQ ED NO:26 a derivative Thermus scotoductus polypeptide from strain X-l with reduced 5'-3' exonuclease activity and reduced bias against ddNTP incorporation.
  • SEQ ED NO:26 has Asp in place of Gly at position 46 and Tyr in place of Phe at position 668. The sequence of SEQ ED NO: 26 is below.
  • VPDLASRVKS IREAAERMAF NMPVQGTAAD 760 LMKLAMVKLF PRLQELGARM LLQVHDELVL EAPKEQAEEV 800
  • the invention provides a polypeptide of SEQ ED NO:27 a derivative Thermus scotoductus polypeptide from strain SM3 with reduced 5 '-3' exonuclease activity and reduced bias against ddNTP inco ⁇ oration.
  • SEQ ED NO:27 has Asp in place of Gly at position 46 and Tyr in place of Phe at position 668. The sequence of SEQ ED NO:27 is below.
  • the invention provides a polypeptide of SEQ ED NO:28 a derivative Thermus scotoductus polypeptide from strain Ni7a with reduced 5 '-3' exonuclease activity and reduced bias against dd ⁇ TP inco ⁇ oration.
  • SEQ ED ⁇ O:28 has Asp in place of Gly at position 46 and Tyr in place of Phe at position 46 and 668.
  • the sequence of SEQ ID NO:28 is below.
  • VPDLASRVKS IREAAERMAF NMPVQGTAAD 760
  • nucleic acid polymerase polypeptides of the invention have homology to portions of the amino acid sequences of the thermostable DNA polymerases of Thermus aquaticus and Thermus thermophilus (see Figure 1). However, significant portions of the amino acid sequences of the present invention are distinct, including SEQ ED NO: 39-44.
  • derivative and variant polypeptides of the invention are derived from the wild type nucleic acid polymerase by deletion or addition of one or more amino acids to the N-terminal and/or C-terminal end of the wild type polypeptide; deletion or addition of one or more amino acids at one or more sites within the wild type polypeptide; or substitution of one or more amino acids at one or more sites within the wild type polypeptide.
  • the polypeptides of the invention may be altered in various ways including amino acid substitutions, deletions, truncations, and insertions.
  • variant and derivative polypeptides may result, for example, from genetic polymo ⁇ hism or from human manipulation. Methods for such manipulations are generally known in the art.
  • amino acid sequence variants of the polypeptides can be prepared by mutations in the DNA. Methods for mutagenesis and nucleotide sequence alterations are well known in the art. See, for example, Kunkel, Proc. Natl. Acad. Sci. USA, 82, 488 (1985); Kunkel et al., Methods in Enzymol., 154, 367 (1987); U. S. Patent No. 4,873,192; Walker and Gaastra, eds., Techniques in Molecular Biology, MacMillan Publishing Company, New York (1983) and the references cited therein.
  • the derivatives and variants of the isolated polypeptides of the invention have identity with at least about 92% of the amino acid positions of any one of SEQ ED NO: 13-28 and have nucleic acid polymerase activity and/or are thermally stable.
  • polypeptide derivatives and variants have identity with at least about 95% of the amino acid positions of any one of SEQ ED NO: 13-28 and have nucleic acid polymerase activity and/or are thermally stable.
  • polypeptide derivatives and variants have identity with at least about 98% of the amino acid positions of any one of SEQ ED NO: 13-28 and have nucleic acid polymerase activity and/or are thermally stable.
  • Amino acid residues of the isolated polypeptides and polypeptide derivatives and variants can be genetically encoded L-amino acids, naturally occurring non-genetically encoded L-amino acids, synthetic L-amino acids or D- enantiomers of any of the above.
  • the amino acid notations used herein for the twenty genetically encoded L-amino acids and common non-encoded amino acids are conventional and are as shown in Table 2.
  • Polypeptide variants that are encompassed within the scope of the invention can have one or more amino acids substituted with an amino acid of similar chemical and/or physical properties, so long as these variant polypeptides retain polymerase activity and/or remain thermally stable.
  • Derivative polypeptides can have one or more amino acids substituted with amino acids having different chemical and/or physical properties, so long as these variant polypeptides retain polymerase activity and/or remain thermally stable.
  • amino acids that are substitutable for each other in the present variant polypeptides generally reside within similar classes or subclasses. As known to one of skill in the art, amino acids can be placed into three main classes: hydrophilic amino acids, hydrophobic amino acids and cysteine-like amino acids, depending primarily on the characteristics of the amino acid side chain. These main classes may be further divided into subclasses. Hydrophilic amino acids include amino acids having acidic, basic or polar side chains and hydrophobic amino acids include amino acids having aromatic or apolar side chains. Apolar amino acids may be further subdivided to include, among others, aliphatic amino acids.
  • Hydrophobic Amino Acid refers to an amino acid having a side chain that is uncharged at physiological pH and that is repelled by aqueous solution.
  • genetically encoded hydrophobic amino acids include lie, Leu and Val.
  • non-genetically encoded hydrophobic amino acids include t- BuA.
  • Amino Acid refers to a hydrophobic amino acid having a side chain containing at least one ring having a conjugated ⁇ -electron system (aromatic group).
  • the aromatic group may be further substituted with substituent groups such as alkyl, alkenyl, alkynyl, hydroxyl, sulfonyl, nitro and amino groups, as well as others.
  • substituent groups such as alkyl, alkenyl, alkynyl, hydroxyl, sulfonyl, nitro and amino groups, as well as others.
  • genetically encoded aromatic amino acids include phenylalanine, tyrosine and tryptophan. Commonly encountered non- genetically encoded aromatic amino acids include phenylglycine, 2- naphthylalanine, ⁇ -2-thienylalanine, 1 ,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, 4-chlorophenylalanine, 2-fluorophenylalanine, 3-fluorophenylalanine and 4-fIuorophenylalanine.
  • Apolar Amino Acid refers to a hydrophobic amino acid having a side chain that is generally uncharged at physiological pH and that is not polar.
  • Examples of genetically encoded apolar amino acids include glycine, proline and methionine.
  • Examples of non-encoded apolar amino acids include Cha.
  • “Aliphatic Amino Acid” refers to an apolar amino acid having a saturated or unsaturated straight chain, branched or cyclic hydrocarbon side chain.
  • genetically encoded aliphatic amino acids include Ala, Leu, Val and He.
  • non-encoded aliphatic amino acids include Nle.
  • Hydrophilic Amino Acid refers to an amino acid having a side chain that is attracted by aqueous solution.
  • examples of genetically encoded hydrophilic amino acids include Ser and Lys.
  • examples of non-encoded hydrophilic amino acids include Cit and hCys.
  • Acidic Amino Acid refers to a hydrophilic amino acid having a side chain pK value of less than 7. Acidic amino acids typically have negatively charged side chains at physiological pH due to loss of a hydrogen ion. Examples of genetically encoded acidic amino acids include aspartic acid (aspartate) and glutamic acid (glutamate).
  • Basic Amino Acid refers to a hydrophilic amino acid having a side chain pK value of greater than 7.
  • Basic amino acids typically have positively charged side chains at physiological pH due to association with hydronium ion.
  • genetically encoded basic amino acids include arginine, lysine and histidine.
  • non-genetically encoded basic amino acids include the non-cyclic amino acids ornithine, 2,3-diaminopropionic acid, 2,4-diaminobutyric acid and homoarginine.
  • Poly Amino Acid refers to a hydrophilic amino acid having a side chain that is uncharged at physiological pH, but which has a bond in which the pair of electrons shared in common by two atoms is held more closely by one of the atoms.
  • genetically encoded polar amino acids include asparagine and glutamine.
  • non-genetically encoded polar amino acids include citrulline, N-acetyl lysine and methionine sulfoxide.
  • cyste-Like Amino Acid refers to an amino acid having a side chain capable of forming a covalent linkage with a side chain of another amino acid residue, such as a disulfide linkage.
  • cysteine-like amino acids generally have a side chain containing at least one thiol (SH) group.
  • examples of genetically encoded cysteine-like amino acids include cysteine.
  • examples of non-genetically encoded cysteine-like amino acids include homocysteine and penicillamine.
  • the above classifications are not absolute. Several amino acids exhibit more than one characteristic property, and can therefore be included in more than one category.
  • tyrosine has both an aromatic ring and a polar hydroxyl group.
  • tyrosine has dual properties and can be included in both the aromatic and polar categories.
  • cysteine in addition to being able to form disulfide linkages, cysteine also has apolar character.
  • cysteine can be used to confer hydrophobicity to a polypeptide.
  • Certain commonly encountered amino acids that are not genetically encoded and that can be present, or substituted for an amino acid, in the variant polypeptides of the invention include, but are not limited to, ⁇ -alanine (b-Ala) and other omega-amino acids such as 3-aminopropionic acid (Dap), 2,3- diaminopropionic acid (Dpr), 4-aminobutyric acid and so forth; ⁇ - aminoisobutyric acid (Aib); e-aminohexanoic acid (Aha); ⁇ -aminovaleric acid (Ava); N-methylglycine (MeGly); ornithine (Orn); citrulline (Cit); t-butylalanine (t-BuA); t-butylglycine (t-BuG); N-methylisoleucine (Melle); phenylglycine (Phg); cyclohexylalanine (Cha); norleucine (Nle); 2-na
  • -2- thienylalanine (Thi); methionine sulfoxide (MSO); homoarginine (hArg); N- acetyl lysine (AcLys); 2,3-diaminobutyric acid (Dab); 2,3-diaminobutyric acid (Dbu); p-aminophenylalanine (Phe(pNH 2 )); N-methyl valine (Me Val); homocysteine (hCys) and homoserine (hSer).
  • These amino acids also fall into the categories defined above.
  • Table 3 is for illustrative pu ⁇ oses only and does not pu ⁇ ort to be an exhaustive list of amino acid residues that may comprise the variant and derivative polypeptides described herein.
  • Other amino acid residues that are useful for making the variant and derivative polypeptides described herein can be found, e.g., in Fasman, 1989, CRC Practical Handbook of Biochemistry and Molecular Biology, CRC Press, Inc., and the references cited therein.
  • Amino acids not specifically mentioned herein can be conveniently classified into the above-described categories on the basis of known behavior and/or their characteristic chemical and/or physical properties as compared with amino acids specifically identified.
  • Polypeptides of the invention can have any amino acid substituted by any similarly classified amino acid to create a variant peptide, so long as the peptide variant is thermally stable and/or retains DNA Polymerase activity.
  • thermostable polymerases may be used to provide thermostable polymerases containing novel properties. For example, placement of codons 289-422 from the Thermus scotoductus DNA polymerase coding sequence after codons 1-288 of the
  • Thermus aquaticus DNA polymerase would yield a novel thermostable nucleic acid polymerase containing the 5' - 3' exonuclease domain of Thermus aquaticus
  • DNA polymerase (1-289), the 3' - 5' exonuclease domain of Thermus scotoductus nucleic acid polymerase (289-422), and the DNA polymerase domain of Thermus aquaticus DNA polymerase (423-832).
  • the 5' - exonuclease domain of Thermus scotoductus nucleic acid polymerase (289-422)
  • the DNA polymerase domain of Thermus aquaticus DNA polymerase (423-832).
  • 3' exonuclease domain and the 3' - 5' exonuclease domain of Thermus scotoductus nucleic acid polymerase may be fused to the DNA polymerase
  • Thermus aquaticus DNA polymerase (dNTP binding and primer/template binding domains) portions of Thermus aquaticus DNA polymerase (about codons 423-832).
  • the donors and recipients need not be limited to Thermus aquaticus and Thermus scotoductus polymerases.
  • Thermus thermophilus DNA polymerase 3' - 5' exonuclease, 5' - 3' exonuclease and DNA polymerase domains can similarly be exchanged for those in the Thermus scotoductus polymerases of the invention.
  • thermostability or polymerase activity (Erlich et al., (1991) Science 252: 1643-1651, Barnes, W.M., (1992) Gene 112:29-35., Lawyer et al., (1989) JBC 264:6427-6437).
  • Other N-terminal deletions similarly have been shown to maintain thermostability and activity (Vainshtein et al., (1996) Protein Science 5:1785-1792 and references therein.) Therefore this invention also includes similarly truncated forms of any of the wild type or variant polymerases provided herein.
  • the invention is also directed to an active truncated variant of any of the polymerases provided by the invention in which the first 330 amino acids are removed.
  • the invention provides SEQ ED NO:45, a truncated form of a polymerase in which the N-terminal 289 amino acids have been removed from the wild type Thermus scotoductus polymerase from strain X-l.
  • the invention provides SEQ ID NO:46 a truncated form of a polymerase in which the N-terminal 289 amino acids have been removed from the wild type Thermus scotoductus polymerase from strain SM3.
  • the invention provides SEQ ED NO:47 a truncated form of a polymerase in which the N-terminal 289 amino acids have been removed from the wild type Thermus scotoductus polymerase from strain Vi7a.
  • polypeptides of the invention encompass both naturally occurring proteins as well as variations, truncations and modified forms thereof. Such variants will continue to possess the desired activity.
  • the deletions, insertions, and substitutions of the polypeptide sequence encompassed herein are not expected to produce radical changes in the characteristics of the polypeptide.
  • One skilled in the art can readily evaluate the thermal stability and polymerase activity of the polypeptides and variant polypeptides of the invention by routine screening assays.
  • Kits and compositions containing the present polypeptides are substantially free of cellular material. Such preparations and compositions have less than about 30%, 20%, 10%, 5%, (by dry weight) of contaminating bacterial cellular protein.
  • nucleic acid polymerase polypeptides and variant polypeptides can be assessed by any procedure known to one of skill in the art.
  • DNA synthetic activity of the variant and non- variant polymerase polypeptides of the invention can be tested in standard DNA sequencing or DNA primer extension reaction.
  • One such assay can be performed in a 100 ⁇ l (final volume) reaction mixture, containing, for example, 0.1 mM dCTP, dTTP, dGTP, ⁇ - 32 P-dATP, 0.3 mg/ml activated calf thymus DNA and 0.5 mg/ml BSA in a buffer containing: 50 mM KCl, 1 mM DTT, 10 mM MgCl and 50 mM of a buffering compound such as PIPES, Tris or Triethylamine.
  • a buffering compound such as PIPES, Tris or Triethylamine.
  • reaction mixture A dilution to 0.1 units/ ⁇ l of each polymerase enzyme is prepared, and 5 ⁇ l of such a dilution is added to the reaction mixture, followed by incubation at 60 °C for 10 minutes. Reaction products can be detected by determining the amount of 32 P inco ⁇ orated into DNA or by observing the products after separation on a polyacrylamide gel.
  • the thermostable enzyme of this invention may be used for any pu ⁇ ose in which DNA Polymerase or reverse transcriptase activity is necessary or desired.
  • the present nucleic acid polymerase polypeptides can be used in one or more of the following procedures: DNA sequencing, DNA amplification, RNA amplification, reverse transcription, DNA synthesis and/or primer extension.
  • the nucleic acid polymerase polypeptides of the invention can be used to amplify DNA by polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • the nucleic acid polymerase polypeptides of the invention can be used to sequence DNA by Sanger sequencing procedures.
  • the nucleic acid polymerase polypeptides of the invention can also be used in primer extension reactions.
  • the nucleic acid polymerase polypeptides of the invention can also be used for reverse transcription.
  • the nucleic acid polymerase polypeptides of the invention can be used test for single nucleotide polymo ⁇ hisms (SNPs) by single nucleotide primer extension using terminator nucleotides. Any such procedures and related procedures, for example, polynucleotide or primer labeling, minisequencing and the like are contemplated for use with the present nucleic acid polymerase polypeptides.
  • Methods of the invention comprise the step of extending a primed polynucleotide template with at least one labeled nucleotide, wherein the extension is catalyzed by a nucleic acid polymerase of the invention.
  • Nucleic acid polymerases used for Sanger sequencing can produce fluorescently labeled products that are analyzed on an automated fluorescence-based sequencing apparatus such as an Applied Biosystems 310 or 377 (Applied Biosystems,
  • the nucleic acid polymerase polypeptides of the invention are used for DNA amplification. Any procedure that employs a DNA polymerase can be used, for example, in polymerase chain reaction (PCR) assays, strand displacement amplification and other amplification procedures. Strand displacement amplification can be used as described in Walker et al (1992) Nucl. Acids Res. 20, 1691-1696.
  • PCR polymerase chain reaction
  • the PCR process for amplifying a target sequence consists of introducing a large excess of two oligonucleotide primers to the DNA mixture containing the desired target sequence, followed by a precise sequence of thermal cycling in the presence of a nucleic acid polymerase.
  • the two primers are complementary to their respective strands of the double stranded target sequence.
  • the mixture is denatured and the primers annealed to their complementary sequences within the target molecule.
  • the primers are extended with a polymerase so as to form a new pair of complementary strands.
  • the steps of denaturation, primer annealing and polymerase extension can be repeated many times. Each round of denaturation, annealing and extension constitutes one "cycle.” There can be numerous cycles, and the amount of amplified DNA produced increases with the number of cycles. Hence, to obtain a high concentration of an amplified target nucleic acid, many cycles are performed.
  • PCR nucleic acid amplification method The steps involve in PCR nucleic acid amplification method are described in more detail below.
  • the nucleic acid to be amplified is described as being double-stranded.
  • the process is equally useful for amplifying a single-stranded nucleic acid, such as an mRNA, although the ultimate product is generally double- stranded DNA.
  • the first step involves the synthesis of a complementary strand (one of the two amplification primers can be used for this pu ⁇ ose), and the succeeding steps proceed as follows: (a) contacting each nucleic acid strand with four different nucleoside triphosphates and one oligonucleotide primer for each strand of the specific sequence being amplified, wherein each primer is selected to be substantially complementary to the different strands of the specific sequence, such that the extension product synthesized from one primer, when it is separated from its complement, can serve as a template for synthesis of the extension product of the other primer, such contacting being at a temperature that allows hybridization of each primer to a complementary nucleic acid strand;
  • step (b) contacting each nucleic acid strand; at the same time as or after step (a), with a nucleic acid polymerase of the invention that enables combination of the nucleoside triphosphates to form primer extension products complementary to each strand of the specific nucleic acid sequence; (c) maintaining the mixture from step (b) at an effective temperature for an effective time to promote the activity of the enzyme and to synthesize, for each different sequence being amplified, an extension product of each primer that is complementary to each nucleic acid strand template, but not so high as to separate each extension product from the complementary strand template;
  • step (d) heating the mixture from step (c) for an effective time and at an effective temperature to separate the primer extension products from the templates on which they were synthesized to produce single-stranded molecules but not so high as to denature irreversibly the enzyme; (e) cooling the mixture from step (d) for an effective time and to an effective temperature to promote hybridization of a primer to each of the single- stranded molecules produced in step (d); and
  • step (f) maintaining the mixture from step (e) at an effective temperature for an effective time to promote the activity of the enzyme and to synthesize, for each different sequence being amplified, an extension product of each primer that is complementary to each nucleic acid template produced in step (d) but not so high as to separate each extension product from the complementary strand template.
  • the effective times and temperatures in steps (e) and (f) may coincide, so that steps (e) and (f) can be carried out simultaneously. Steps (d)-(f) are repeated until the desired level of amplification is obtained.
  • the amplification method is useful not only for producing large amounts of a specific nucleic acid sequence of known sequence but also for producing nucleic acid sequences that are known to exist but are not completely specified.
  • the greater the knowledge about the bases at both ends of the sequence the greater can be the specificity of the primers for the target nucleic acid sequence.
  • Thermally stable nucleic acid polymerases are therefore generally used for PCR because they can function at the high temperatures used for melting double stranded target DNA and annealing the primers during each cycle of the PCR reaction. High temperature results in thermodynamic conditions that favor primer hybridization with the target sequences and not hybridization with non- target sequences (H. A. Erlich (ed.), PCR Technology, Stockton Press [1989]).
  • the thermostable nucleic acid polymerases of the present invention satisfy the requirements for effective use in amplification reactions such as PCR.
  • the present polymerases do not become irreversibly denatured (inactivated) when subjected to the required elevated temperatures for the time necessary to melt double-stranded nucleic acids during the amplification process.
  • Irreversible denaturation for pu ⁇ oses herein refers to permanent and complete loss of enzymatic activity.
  • the heating conditions necessary for nucleic acid denaturation will depend, e.g., on the buffer salt concentration and the composition and length of the nucleic acids being denatured, but typically denaturation can be done at temperatures ranging from about 90 °C to about 105°C
  • the time required for denaturation depends mainly on the temperature and the length of the duplex nucleic acid. Typically the time needed for denaturation ranges from a few seconds up to four minutes. Higher temperatures may be required as the salt concentration of the buffer, or the length and/or GC composition of the nucleic acid is increased.
  • the nucleic acid polymerases of the invention do not become irreversibly denatured for relatively short exposures to temperatures of about 90°C to 100°C.
  • thermostable polymerases of the invention have an optimum temperature at which they function that is higher than about 45 °C. Temperatures below 45 °C facilitate hybridization of primer to template, but depending on salt composition and concentration and primer composition and length, hybridization of primer to template can occur at higher temperatures (e.g., 45 °C to 70 °C), which may promote specificity of the primer hybridization reaction.
  • the polymerases of the invention exhibit activity over a broad temperature range from about 37°C to about 90°C
  • the present polymerases have particular utility for PCR not only because of their thermal stability but also because of their ability to synthesize DNA using an RNA template and because of their fidelity in replicating the template nucleic acid.
  • reverse transcriptase In most PCR reactions that start with an RNA template, reverse transcriptase must be added. However, use of reverse transcriptase has certain drawbacks. First, it is not stable at higher temperatures. Hence, once the initial complementary DNA (cDNA) has been made by reverse transcriptase and the thermal cycles of PCR are started, the original RNA template is not used as a template in the amplification reaction. Second, reverse transcriptase does not produce a cDNA copy with particularly good sequence fidelity.
  • the amplified product can include a pool of nucleic acids with diverse sequences.
  • the nucleic acid polymerases of the invention that can accurately reverse transcribe RNA and replicate the sequence of the template RNA or DNA with high fidelity is highly desirable.
  • any nucleic acid can act as a "target nucleic acid” for the PCR methods of the invention.
  • target when used in reference to the polymerase chain reaction, refers to the region of nucleic acid bounded by the primers used for polymerase chain reaction.
  • any cDNA, RNA, oligonucleotide or polynucleotide can be amplified with the appropriate set of primer molecules.
  • the amplified segments created by the PCR process itself are, themselves, efficient templates for subsequent PCR amplifications.
  • the length of the amplified segment of the desired target sequence is determined by the relative positions of the primers with respect to each other, and therefore, this length is readily controlled.
  • the amplified target nucleic acid can be detected by any method known to one of skill in the art.
  • target nucleic acids are often amplified to such an extent that they form a band visible on a size separation gel.
  • Target nucleic acids can also be detected by hybridization with a labeled probe; by inco ⁇ oration of biotinylated primers during PCR followed by avidin-enzyme conjugate detection; by inco ⁇ oration of 32 P-labeled deoxynucleotide triphosphates during PCR, and the like.
  • the amount of amplification can also be monitored, for example, by use of a reporter-quencher oligonucleotide as described in U.S. Patent 5,723,591, and a nucleic acid polymerase of the invention that has 5' - 3' nuclease activity.
  • the reporter-quencher oligonucleotide has an attached reporter molecule and an attached quencher molecule that is capable of quenching the fluorescence of the reporter molecule when the two are in proximity. Quenching occurs when the reporter-quencher oligonucleotide is not hybridized to a complementary nucleic acid because the reporter molecule and the quencher molecule tend to be in proximity or at an optimal distance for quenching.
  • the reporter-quencher oligonucleotide When hybridized, the reporter-quencher oligonucleotide emits more fluorescence than when unhybridized because the reporter molecule and the quencher molecule tend to be further apart.
  • the reporter-quencher oligonucleotide is designed to hybridize 3' to an amplification primer. During amplification, the 5' - 3' nuclease activity of the polymerase digests the reporter oligonucleotide probe, thereby separating the reporter molecule from the quencher molecule. As the amplification is conducted, the fluorescence of the reporter molecule increases. Accordingly, the amount of amplification performed can be quantified based on the increase of fluorescence observed.
  • Oligonucleotides used for PCR primers are usually about 9 to about 75 nucleotides, preferably about 17 to about 50 nucleotides in length.
  • an oligonucleotide for use in PCR reactions is about 40 or fewer nucleotides in length (e.g., 9, 12, 15, 18, 20, 21, 24, 27, 30, 35, 40, or any number between 9 and 40).
  • specific primers are at least about 14 nucleotides in length. For optimum specificity and cost effectiveness, primers of 16-24 nucleotides in length are generally preferred.
  • primers for use processes such as PCR.
  • potential primers for nucleic acid amplification can be used as probes to determine whether the primer is selective for a single target and what conditions permit hybridization of a primer to a target within a sample or complex mixture of nucleic acids.
  • the present invention also contemplates use of the present nucleic acid polymerases in combination with other procedures or enzymes.
  • the polymerases can be used in combination with additional reverse transcriptase or another DNA polymerase. See U.S. Pat. No. 5,322,770, inco ⁇ orated by reference herein.
  • nucleic acid polymerases of the invention with 5' - 3' exonuclease activity are used to detect target nucleic acids in an invader- directed cleavage assay.
  • This type of assay is described, for example, in U.S. Patent 5,994,069.
  • the 5' - 3' exonuclease of DNA polymerases is not really an exonuclease that progressively cleaves nucleotides from the 5' end of a nucleic acid, but rather a nuclease that can cleave certain types of nucleic acid structures to produce oligonucleotide cleavage products. Such cleavage is sometimes called structure-specific cleavage.
  • the invader-directed cleavage assay employs at least one pair of oligonucleotides that interact with a target nucleic acid to form a cleavage structure for the 5' - 3' nuclease activity of the nucleic acid polymerase. Distinctive cleavage products are released when the cleavage structure is cleaved by the 5' - 3' nuclease activity of the polymerase. Formation of such a target- dependent cleavage structure and the resulting cleavage products is indicative of the presence of specific target nucleic acid sequences in the test sample.
  • the 5' - 3' nuclease activity of the present polymerases is needed as well at least one pair of oligonucleotides that interact with a target nucleic acid to form a cleavage structure for the 5' - 3' nuclease.
  • the first oligonucleotide sometimes termed the "probe”
  • the invader oligonucleotide can hybridize adjacent and upstream of the probe oligonucleotide.
  • the target sites to which the probe and invader oligonucleotides hybridize overlap such that the 3' segment of the invader oligonucleotide overlaps with the 5' segment of the probe oligonucleotide.
  • the 5' - 3' nuclease of the present polymerases can cleave the probe oligonucleotide at an internal site to produce distinctive fragments that are diagnostic of the presence of the target nucleic acid in a sample. Further details and methods for adapting the invader-directed cleavage assay to particular situations can be found in U.S. Patent 5,994,069.
  • nucleotide analogs can also be used with the present methods, kits and with the nucleic acid polymerases.
  • Such nucleotide analogs can be modified or non-naturally occurring nucleotides such as 7-deaza purines (i.e., 7-deaza-dATP and 7-deaza-dGTP).
  • Nucleotide analogs include base analogs and comprise modified forms of deoxyribonucleotides as well as ribonucleotides.
  • nucleotide analog when used in reference to targets present in a PCR mixture refers to the use of nucleotides other than dATP, dGTP, dCTP and dTTP; thus, the use of dUTP (a naturally occurring dNTP) in a PCR would comprise the use of a nucleotide analog in the PCR.
  • a PCR product generated using dUTP, 7-deaza-dATP, 7-deaza-dGTP or any other nucleotide analog in the reaction mixture is said to contain nucleotide analogs.
  • kits that contain at least one of the nucleic acid polymerases of the invention.
  • Individual kits may be adapted for performing one or more of the following procedures: DNA sequencing, DNA amplification, RNA Amplification and/or primer extension.
  • Kits of the invention comprise a DNA polymerase polypeptide of the invention and at least one nucleotide.
  • a nucleotide provided in the kits of the invention can be labeled or unlabeled.
  • Kits preferably can also contain instructions on how to perform the procedures for which the kits are adapted.
  • the subject kit may further comprise at least one other reagent required for performing the method the kit is adapted to perform.
  • additional reagents include: another unlabeled nucleotide, another labeled nucleotide, a balance mixture of nucleotides, one or more chain terminating nucleotides, one or more nucleotide analogs, buffer solution(s), magnesium solution(s), cloning vectors, restriction endonucleases, sequencing primers, reverse transcriptase, and DNA or RNA amplification primers.
  • the reagents included in the kits of the invention may be supplied in premeasured units so as to provide for greater precision and accuracy. Typically, kits reagents and other components are placed and contained in separate vessels.
  • a reaction vessel, test tube, microwell tray, microtiter dish or other container can also be included in the kit.
  • Different labels can be used on different reagents so that each reagent can be distinguished from another.
  • EXAMPLE 1 Cloning of Thermus scotoductus, Strain X-l Polymerase
  • Thermus scotoductus (Tsc) strain X-l was obtained from ATCC (ATCC Deposit No. 27978). The lyophilized bacteria were revived in ATCC Culture Medium 461 (Castenholz TYE medium) and grown overnight to stationary phase. Thermus scotoductus genomic DNA was prepared using a Quiagen genomic DNA preparation protocol and kit (Quiagen).
  • the first forward and reverse primers were designed by analysis of 5' and 3' terminal homologous conserved regions of the DNA sequences of Thermus aquaticus (Taq), Thermus thermophilus (Tth), Thermus filiformis (Tfi), Thermus caldophilus (that was determined to actually be Tth strain GK24), and Thermus flavus (believed to be Thermus igniterrae).
  • a fragment of a Thermus scotoductus polymerase gene was amplified using N-terminal primer 5'- ggc cac cac ctg gcc tac -3' (SEQ ED NO:29) and C-terminal primer 5'- ccc ace tec ace tec ag -3' (SEQ ED NO:30).
  • the following PCR reaction mixture contained 2.5 ⁇ l of lOx Amplitaq buffer (ABi), 2mM MgCl, 60 ng DNA template, 2.5mM (each) dNTP, 20pmol of each primer, and 1.25 units of Amplitaq DNA polymerase in a 25 ⁇ l total reaction volume.
  • the reaction mixture was heated to 80°C and then the primers were added. This was followed by a predenaturation step (96°C for 30 s); PCR cycling for 30 cycles (97°C for 3 s, 56°C for 30 s, 72°C for 3 min) with a finishing step (72°C for 7 min). This produced an approximate 1.5 kb DNA fragment that was cloned and sequenced. This cloned fragment showed some homology to the Tth Polymerase I gene (Genebank accession number 466573) between nucleotide numbers 644 and 1973.
  • the primer used was 5'- ctg gcc atg ctg aag etc ttt -3' (SEQ ED NO:31) and a 2-step thermocycling protocol. A predenaturation step (95°C for 5 min) was followed by 80 cycles (97°C for 5sec, 60°C for 4min). Reaction mixture consisted of 16 ⁇ l Big Dye VI Ready Reaction mix, 2.8 ug DNA, 15 pmol primer in a 40 ⁇ l total reaction volume.
  • the sequencing of the Thermus scotoductus gene from genomic DNA revealed that the 5' terminal sequence of the wild-type Thermus scotoductus gene is 5'- ata agg gcg atg ctg ccc etc ttt gag-5' (SEQ ID NO: 32) that would indicate that the ATG is the start codon of the wild-type gene.
  • the N-terminus of Taq, Tth and Tfi enzymes have two methionine amino acid residues at their N terminal end separated by two amino acids.
  • the ATA codon was changed to ATG.
  • Thermus scotoductus nucleic acid polymerase coding region was carried out using the 5' forward primer 5' - cat atg agg gcg atg ctg ccc etc -3' (SEQ ED NO:33). Another consideration when designing this primer was to introduce a recognition site for the restriction enzyme Nde I (catatg, SEQ ID NO:34). This sequence was introduced to facilitate subcloning of the coding region into other plasmid vectors.
  • the first cloned portion of the Thermus scotoductus, strain X-l polymerase gene was only 1.2 kb. This represented approximately half of the full-length gene.
  • a PCR reaction was carried out using the 5' forward primer (SEQ ED NO:33) described in the previous paragraph and a new primer designed near the same homologous 3' region of the known Thermus polymerase genes.
  • the sequence of this primer was 5'- etc cac etc cag ggg cac -3' (SEQ ID NO:35).
  • the PCR reaction was the same mixture as above. The cycling conditions were altered slightly in order to promote greater specificity.
  • the reaction mixture was heated to 80°C and then the primers were added. This was followed by a predenaturation step (96°C for 2 min); PCR cycling for 10 cycles (97°C for 10 s, 70°C for 3 min), 25 cycles (97°C for 10 s, 60°C for 3 min), with a finishing step (72°C for 7 min). This produced a 2.4kb fragment that was cloned and sequenced. This left to be sequenced a short 3' terminal region of the Thermus scotoductus, strain X-l polymerase gene.
  • a new primer was designed to obtain the remaining unknown 3' sequence: 5'- ctg gcc atg gtg aag etc ttt -3' (SEQ ED NO:36).
  • the genomic sequencing protocol was the same as described for the previous genomic DNA sequencing reaction for the 5' terminus.
  • a primer was designed to be used with the 5' terminal primer described above to amplify the full length Thermus scotoductus polymerase gene. This primer is complementary to the 3' terminal sequence. It also has a Sal I recognition site (gtcgac, SEQ ID NO:37) overlapping with the stop codon.
  • the sequence of the primer is 5'-gtc gac tag gcc ttg gcg aaa gcc a -3' (SEQ ID NO:38).
  • Thermus scotoductus polymerase genes Three different cloned Thermus scotoductus polymerase genes were sequenced independently in order to rule out PCR errors.
  • the resulting consensus sequence is the natural Thermus scotoductus polymerase gene sequence of this invention (SEQ ID NO: 14).
  • the amino acid numbering used in this description of the invention is based on a recombinant form of the Thermus scotoductus polymerase protein that has an additional three amino acids at its N- terminus (SEQ ED NO: 13).
  • SEQ ED NO: 14 is the sequence for the wild type Thermus scotoductus polymerase from strain X-l.
  • the amino acid sequence of the strain X-l Thermus scotoductus polymerase has several differences when compared with the amino acid sequence of Thermus aquaticus DNA Polymerase, including about 51 conservative amino acid changes and about 62 nonconservative amino acid changes. For example, one region of dissimilarity is between amino acid positions at approximately 51 and about 65, where the sequence of the Thermus scotoductus polymerase has about four amino acid changes (in bold):
  • LLKALREDG DVVIVVFDAK APSFRHQTYE SEQ ED NO:39. Another region of dissimilarity is between amino acid positions at approximately 201 and about 236, where the sequence of the Thermus scotoductus polymerase has about seven amino acid changes (in bold): GEKTAAKLIREWGSLENLLKHLEQV KPASV REKILS (SEQ ID NO:40). Another region of dissimilarity is between amino acid positions at approximately 311 and about 350, where the sequence of the Thermus scotoductus polymerase has about seven amino acid changes (in bold): VGYVLSRPEPMWAELN ALAAAWEGRNYRAEDPLEALRGLG (SEQ ED ⁇ O:41).
  • Another region of dissimilarity is between amino acid positions at approximately 415 and about 435, where the sequence of the Thermus scotoductus polymerase has about five amino acid changes (in bold): RLYAALLERLKGEERLLWLYE (SEQ ID NO:42).
  • Another region of dissimilarity is between amino acid positions at approximately 531 and about 562, where the sequence of the Thermus scotoductus polymerase has about six amino acid changes (in bold): PEVDRILQYRELSKLK GTYED PLPALVHPKTN (SEQ ID NO:43).
  • Another region of dissimilarity is between amino acid positions at approximately 801 and about 836, where the sequence of the Thermus scotoductus polymerase has about eight amino acid changes (in bold): EEVAQEAKRT MEEVWPLKVPLEVEVGIGEDWLSAKA (SEQ ID NO:44).
  • EEVAQEAKRT MEEVWPLKVPLEVEVGIGEDWLSAKA SEQ ID NO:44.
  • many regions of the Thermus scotoductus polymerase differ from the Thermus aquaticus and Thermus thermophilus DNA Polymerases.
  • Thermus scotoductus polymerase in a form suitable for dye-terminator DNA sequencing, two amino acid substitutions were made. These are the FS (Tabor and Richardson, 1995 PNAS 92: 6339-6343) and exo- minus (G46D mutation) mutations. To reduce the exonuclease activity to very low levels, the mutation G46D was introduced. To reduce the discrimination between ddNTP's and dNTP's, the mutation F666Y was introduced.
  • Mutagenesis was carried out using the modified QuickChangeTM (Stratagene) PCR mutagenesis protocol described in Sawano & Miyawaki (2000), Nucleic Acids Research Vol. 28. The mutated gene was resequenced completely to confirm the introduction of the mutations and to ensure that no PCR errors were introduced.
  • QuickChangeTM QuickChangeTM
  • Thermus scotoductus, strain X-l, polymerase gene (FS, exo- ) was removed from the cloning vector by restriction digest with Ndel and Sail.
  • the 2.4kb gene was ligated into the pT7 expression vector (Brookhaven National Laboratories, Long Island, NY). This resulting vector containing the Thermus scotoductus polymerase (fs, exo-) gene was used to transform BL21 E. coli cells (Invitrogen).
  • Thermus scotoductus To isolate the Thermus scotoductus, strain X-l polymerase, the cells were thawed and resuspended in 2.5 volumes (wet weight) of 50mM Tris (pH 7.2), 400mM NaCl, ImM EDTA. The cell walls were disrupted by sonication and the resulting E. coli cell debris were removed by centrifugation. The resulting lysate was pasteurized in a water bath (75°C for 45 min), denaturing and precipitating the majority of the non-thermostable E. coli proteins and leaving the thermostable Thermus scotoductus, strain X-l polymerase in solution. E.
  • coli genomic DNA was removed by coprecipitation with 0.3% Polyethyleneimine (PEI).
  • PEI Polyethyleneimine
  • the cleared lysate is then applied to two columns in series: (1) a Biorex 70 cation exchange resin that chelates excess PEI and (2) a heparin-agarose column (dimensions to be provided) that retains the polymerase.
  • This column is washed with 5 column volumes of 20mM Tris (pH 8.5), 5%glycerol, lOOmM NaCl, O.lmM EDTA, 0.05% Triton X-100 and 0.05% Tween-20 (KTA).
  • KTA Triton X-100
  • the eluted Tsc Polymerase was concentrated and the buffer exchanged using a Millipore concentration filter (30kD M.W. cutoff).
  • the concentrated protein was stored at in KTA (no salt) plus 50% glycerol at - 20°C.
  • the activity of the polymerase was measured using the standard salmon sperm DNA radiometric activity assay and sequencing was tested using the Big Dye Version 3.
  • the enzyme is active in 40-80mM Tris, 1.0-2.0mM MgCl at a dNTP mix consisting of 0.2mM dATP, 0.2mM dCTP, 0.2mM dUTP, and 0.3mM dlTP, at pH 8.0-10.0, with optimal activity between pH 9.0 and 9.58.
  • the enzyme is also active in KCl concentrations from 0 to lOOmM, indicating that the T. scotoductus, strain X-l polymerase is more salt-tolerant than either Tfil or Taq, but not quite as salt-tolerant as Tth.
  • EXAMPLE 3 Thermus scotoductus Strains SM3 and Vi7a The same primers used to amplify the full-length gene encoding the polymerase from Thermus scotoductus (Tsc) strain X-l were used to amplify the polymerase genes from two additional strains of Thermus scotoductus: strain SM3 and strain Vi7a.
  • the PCR reaction mixture used to amplify nucleic acids encoding the Thermus scotoductus polymerase from strains SM3 and Vi7a contained 2.5 ⁇ l of lOx Amplitaq reaction buffer (Applied Biosystems), 2 mM MgCl 2 , 70 to 100 ng genomic DNA template, 0.2 mM (each) dNTPs, 20 pmol of each primer, and 1.25 units of Amplitaq in a 25 ⁇ l total reaction volume.
  • the reaction was started by adding a premix containing enzyme, MgCl , dNTPs, buffer and water to another premix containing primer and template preheated at 80°C
  • the entire reaction mixture was then denatured (30 sec at 96°C) followed by 30 PCR cycles (97°C for 3 sec, 62°C for 30 sec, 72°C for 3 min) with a finishing step (72°C for 7 min).
  • PCR reactions each produced approximate 2.5 kb DNA fragments.
  • the amplified fragments were purified from the PCR reaction mixes using a Quiagen PCR cleanup kit (Quiagen).
  • the Thermus scotoductus fragments were ligated into the inducible expression vector pCR®4-TOPO® (Invitrogen, Carlsbad, CA).
  • Three different cloned Thermus scotoductus polymerase genes from each strain were sequenced independently in order to rule out PCR errors. The resulting consensus sequences for the wild-type genes are reported in Figures 1 and 3 below.
  • Thermus scotoductus, strain SM3 polymerase has five positions that have different amino acids compared to strain X-l.
  • the Thermus scotoductus strain Vi7a polymerase has four differences when compared to the amino acid sequence of the polymerase from strain X-l. These are indicated with boldface in Figure 2.
  • the "FS, exo-minus form of both Thermus scotoductus polymerase genes were subcloned into the pet expression vector using the Ndel and Sal I restriction sites.
  • BL21 cells (Invitrogen) were transformed with this expression construct.
  • the cells were grown in one liter of Terrific Broth (Maniatis) to an optical density of 1.2OD and the proteins were ove ⁇ roduced by four-hour induction with 1.0 mM EPTG.
  • the cells were harvested by centrifugation, washed in 50 mM Tris (pH 7.5), 5mM EDTA, 5% glycerol, lOmM EDTA to remove growth media, and the cell pellet frozen at -80°C
  • Thermus scotoductus To isolate the Thermus scotoductus, strain SM3 and Vi7a polymerases, the cells were thawed and resuspended in 2.5 volumes (wet weight) of 50mM Tris (pH 7.2), 400mM NaCl, ImM EDTA. The cell walls were disrupted by sonication and the resulting E. coli cell debris was removed by centrifugation. The resulting lysate was pasteurized in a water bath (75°C for 45 min), denaturing and precipitating the majority of the non-thermostable E. coli proteins and leaving the thermostable Thermus scotoductus polymerase in solution. E. coli genomic DNA was removed by coprecipitation with 0.3% Polyethyleneimine (PEI).
  • PEI Polyethyleneimine
  • the cleared lysate was then applied to two columns in series: (1) a Biorex 70 cation exchange resin that chelates excess PEI and (2) a heparin-agarose column that retains the polymerase. This column was washed with 5 column volumes of 20mM Tris (pH 8.5), 5%glycerol, 1 OOmM NaCl,
  • the proteins were then eluted with a 0.1 to 1.0M NaCl linear gradient.
  • the polymerases eluted at 0.8M NaCl.
  • the eluted Thermus scotoductus polymerases were concentrated and the buffer exchanged using a Millipore concentration filter (30kD M.W. cutoff).
  • the concentrated proteins were stored at in KTA (no salt) plus 50% glycerol at - 20°C.
  • the activity of the polymerases were measured using a nicked salmon sperm DNA radiometric activity assay. Both enzymes are being tested for use in sequencing using the Big DyeTM V 3.0.
  • the enzymes are active in 40-80mM Tris, 1.0-2.0mM MgCl at a dNTP mix consisting of 0.2mM dATP, 0.2mM dCTP, 0.2mM dUTP, and 0.3mM dlTP, at pH 8.0-10.0, with optimal activity between pH 9.0 and 9.58.

Abstract

The invention provides nucleic acids and polypeptides for a nucleic acid polymerase from a thermophilic organism, Thermus scotoductus. The invention also provides methods for using these nucleic acids and polypeptides.

Description

THERMUS SCOTODUCTUS NUCLEIC ACID POLYMERASES
This application relates to U.S. Provisional Application No. 60/322,218, filed September 14, 2001 and U.S. Provisional Application No. 60/334489, filed November 30, 2001.
FIELD OF THE INVENTION The invention relates to nucleic acids and polypeptides for nucleic acid polymerases from thermophilic strains of Thermus scotoductus.
BACKGROUND OF THE INVENTION
DNA polymerases are naturally-occurring intracellular enzymes used by a cell for replicating DNA by reading one nucleic acid strand and manufacturing its complement. Enzymes having DNA polymerase activity catalyze the formation of a bond between the 3' hydroxyl group at the growing end of a nucleic acid primer and the 5' phosphate group of a newly added nucleotide triphosphate. Nucleotide triphosphates used for DNA synthesis are usually deoxyadenosine triphosphate (A), deoxythymidine triphosphate (T), deoxycytosine triphosphate (C) and deoxyguanosine triphosphate (G), but modified or altered versions of these nucleotides can also be used. The order in which the nucleotides are added is dictated by hydrogen-bond formation between A and T nucleotide bases and between G and C nucleotide bases. Bacterial cells contain three types of DNA polymerases, termed polymerase I, II and III. DNA polymerase I is the most abundant polymerase and is generally responsible for certain types of DNA repair, including a repairlike reaction that permits the joining of Okazaki fragments during DNA replication. Polymerase I is essential for the repair of DNA damage induced by UV irradiation and radiomimetic drugs. DNA Polymerase II is thought to play a role in repairing DNA damage that induces the SOS response. In mutants that lack both polymerase I and III, polymerase II repairs UV-induced lesions. Polymerase I and II are monomeric polymerases while polymerase III is a multisubunit complex. Enzymes having DNA polymerase activity are often used in vitro for a variety of biochemical applications including cDNA synthesis and DNA sequencing reactions. See Sambrook e al., Molecular Cloning: A Laboratory Manual (3rd ed. Cold Spring Harbor Laboratory Press, 2001, hereby incorporated by reference. DNA polymerases are also used for amplification of nucleic acids by methods such as the polymerase chain reaction (PCR) (Mullis et al., U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,800,159, incorporated by reference) and RNA transcription-mediated amplification methods (e.g., Kacian et al., PCT Publication No. WO91/01384, incorporated by reference).
DNA amplification utilizes cycles of primer extension through the use of a DNA polymerase activity, followed by thermal denaturation of the resulting double-stranded nucleic acid in order to provide a new template for another round of primer annealing and extension. Because the high temperatures necessary for strand denaturation result in the irreversible inactivations of many DNA polymerases, the discovery and use of DNA polymerases able to remain active at temperatures above about 37 °C provides an advantage in cost and labor efficiency. Thermostable DNA polymerases have been discovered in a number of thermophilic organisms including Thermus aquaticus, Thermus thermophilus, and species within the genera the Bacillus, Thermococcus, Sulfobus, and Pyrococcus. A full length thermostable DNA polymerase derived from Thermus aquaticus (Taq) has been described by Lawyer, et al., J. Biol. Chem. 264:6427- 6437 (1989) and Gelfand et al, U.S. Pat. No. 5,079,352. The cloning and expression of truncated versions of that DNA polymerase are further described in Lawyer et al., in PCR Methods and Applications, 2:275-287 (1993), and Barnes, PCT Publication No. WO92/06188 (1992). Sullivan reports the cloning of a mutated version of the Taq DNA polymerase in EPO Publication No. 0482714A1 (1992). A DNA polymerase from Thermus thermophilus has also been cloned and expressed. Asakura et al., J. Ferment. Bioeng. (Japan), 74:265- 269 (1993). However, the properties of the various polymerases vary. Accordingly, new polymerases are needed that have improved sequence discrimination, better salt tolerance, combined reverse transcription and DNA polymerase activities, varying degrees of thermostability, improved tolerance for labeled or dideoxy nucleotides and other valuable properties.
SUMMARY OF THE INVENTION
The invention provides nucleic acid polymerase enzymes isolated from a thermophilic organism, Thermus scotoductus. The invention provides nucleic acid polymerases from several Thermus scotoductus strains including strain X-l (ATCC Deposit No. 27978), strain SM3 and strain Vi7a. In one embodiment, the invention provides an isolated nucleic acid encoding a Thermus scotoductus nucleic acid polymerase. Such a nucleic acid can have a polynucleotide sequence comprising any one of SEQ ID NO:l-12. Nucleic acids complementary to any one of SEQ ID NO: 1-12 are also included within the invention. In another embodiment, the invention provides an isolated nucleic acid encoding a polypeptide having at least 93% identity to an amino acid sequence comprising any one of SEQ ID NO: 13-28. The invention also provides vectors comprising these isolated nucleic acids, including expression vectors comprising a promoter operably linked to any of the isolated nucleic acids of the invention. Host cells comprising such isolated nucleic acids and vectors are also provided by the invention, particularly host cells capable of expressing a thermostable polypeptide, where the polypeptide has nucleic acid polymerase or DNA polymerase activity.
In another embodiment, the invention provides an isolated nucleic acid encoding a derivative nucleic acid polymerase comprising any one of amino acid sequences SEQ ID NO: 13- 16 having a mutation that decreases 5-3' exonuclease activity. Such a derivative nucleic acid polymerase has decreased 5-3' exonuclease activity relative to a nucleic acid polymerase comprising any one of amino acid sequences SEQ ID NO: 13-16. In another embodiment, the invention provides an isolated nucleic acid encoding a derivative nucleic acid polymerase comprising any one of amino acid sequences SEQ ID NO:13-16 having a mutation that reduces discrimination against dideoxynucleotide triphosphates. Such a derivative nucleic acid polymerase has reduced discrimination against dideoxynucleotide triphosphates relative to a nucleic acid polymerase comprising any one of amino acid sequences SEQ ID NO:13-16.
The invention also provides isolated polypeptides that can include an amino acid sequence with at least 93% identity to any one of SEQ ID NO: 13-28. The isolated polypeptides provided by the invention preferably have an amino acid sequence with at least 95% sequence identity to any one of SEQ ID NO: 13- 28. Such polypeptides can also have nucleic acid polymerase or DNA polymerase activity. Such DNA polymerase activity can, for example, be about 50,000 U/mg protein to about 500,000 U/mg protein.
The invention further provides a method of synthesizing DNA that includes contacting a polypeptide comprising any one of SEQ ID NO:13-28 with a DNA under conditions sufficient to permit polymerization of DNA. The invention also provides a method of synthesizing DNA from an RNA template that includes contacting a polypeptide comprising any one of SEQ ID NO: 13-28 with an RNA template under conditions sufficient to permit synthesis of DNA (e.g. reverse transcription). The invention further provides a method for thermocyclic amplification of nucleic acid that comprises contacting a nucleic acid with a thermostable polypeptide having any one of SEQ ID NO: 13-28 under conditions suitable for amplification of the nucleic acid, and amplifying the nucleic acid. Such amplification can be, for example, by Strand Displacement Amplification or Polymerase Chain Reaction. The invention also provides a method of primer extending DNA comprising contacting a polypeptide comprising of SEQ ID NO: 13-28 with a DNA under conditions sufficient to permit polymerization of DNA. Such primer extension can be performed, for example, to sequence DNA or to amplify DNA. The invention further provides a method of making a nucleic acid polymerase comprising any one of SEQ ID NO: 13-28, the method comprising incubating a host cell comprising a nucleic acid that encodes a polypeptide comprising any one of SEQ ID NO: 13-28, operably linked to a promoter under conditions sufficient for RNA transcription and translation. In one embodiment, the method uses a nucleic acid that comprises any one of SEQ ID NO: 1-12. The invention is also directed to a nucleic acid polymerase or DNA polymerase made by this method.
The invention also provides a kit that includes a container containing a nucleic acid polymerase comprising an amino acid sequence with at least 93% identity to any one of SEQ ID NO: 13-28. The kit can also contain an unlabeled nucleotide, a labeled nucleotide, a balanced mixture of nucleotides, a chain terminating nucleotide, a nucleotide analog, a buffer solution, a solution containing magnesium, a cloning vector, a restriction endonuclease, a sequencing primer, a solution containing reverse transcriptase, or a DNA or RNA amplification primer. Such kits can, for example, be adapted for performing DNA sequencing, DNA amplification, RNA amplification, reverse transcription or primer extension reactions.
DESCRIPTION OF THE FIGURES
Figure 1 provides a comparison of amino acid sequences for polymerases from Thermus aquaticus (Taq; SEQ ID NO:48), Thermus thermophilus (Tth; SEQ ID NO:49), Thermus filiformis (Tfi; SEQ ID NO:50) and strain X-l Thermus scotoductus strain X-l (Tsc; SEQ ED NO: 13). Figure 2 provides a comparison of amino acid sequences for three strains of Thermus scotoductus polymerases: strain X-l (SEQ ID NO:13), strain SM3 (SEQ ID NO: 15), and strain Vi7a (SEQ ID NO: 16).
DETAILED DESCRIPTION OF THE INVENTION The present invention relates to nucleic acid and amino acid sequences encoding nucleic acid polymerases from thermophilic organisms. In particular, the present invention provides a nucleic acid polymerase from Thermus scotoductus. The nucleic acid polymerases of the invention can be used in a variety of procedures, including DNA synthesis, reverse transcription, DNA primer extension, DNA sequencing and DNA amplification procedures.
Definitions
The term "amino acid sequence" refers to the positional arrangement and identity of amino acids in a peptide, polypeptide or protein molecule. Use of the term "amino acid sequence" is not meant to limit the amino acid sequence to the complete, native amino acid sequence of a peptide, polypeptide or protein.
"Chimeric" is used to indicate that a nucleic acid, such as a vector or a gene, is comprised of more than one nucleic acid segment and that at least two nucleic acid segments are of distinct origin. Such nucleic acid segments are fused together by recombinant techniques resulting in a nucleic acid sequence, which does not occur naturally.
The term "coding region" refers to the nucleotide sequence that codes for a protein of interest. The coding region of a protein is bounded on the 5' side by the nucleotide triplet "ATG" that encodes the initiator methionine and on the 3' side by one of the three triplets that specify stop codons (i.e., TAA, TAG, TGA). "Constitutive expression" refers to expression using a constitutive promoter.
"Constitutive promoter" refers to a promoter that is able to express the gene that it controls in all, or nearly all, phases of the life cycle of the cell. "Complementary" or "complementarity" are used to define the degree of base-pairing or hybridization between nucleic acids. For example, as is known to one of skill in the art, adenine (A) can form hydrogen bonds or base pair with thymine (T) and guanine (G) can form hydrogen bonds or base pair with cytosine (C). Hence, A is complementary to T and G is complementary to C. Complementarity may be complete when all bases in a double-stranded nucleic acid are base paired. Alternatively, complementarity may be "partial," in which only some of the bases in a nucleic acid are matched according to the base pairing rules. The degree of complementarity between nucleic acid strands has an effect on the efficiency and strength of hybridization between nucleic acid strands.
The "derivative" of a reference nucleic acid, protein, polypeptide or peptide, is a nucleic acid, protein, polypeptide or peptide, respectively, with a related but different sequence or chemical structure than the respective reference nucleic acid, protein, polypeptide or peptide. A derivative nucleic acid, protein, polypeptide or peptide is generally made purposefully to enhance or incorporate some chemical, physical or functional property that is absent or only weakly present in the reference nucleic acid, protein, polypeptide or peptide. A derivative nucleic acid generally can differ in nucleotide sequence from a reference nucleic acid whereas a derivative protein, polypeptide or peptide can differ in amino acid sequence from the reference protein, polypeptide or peptide, respectively. Such sequence differences can be one or more substitutions, insertions, additions, deletions, fusions and truncations, which can be present in any combination. Differences can be minor (e.g., a difference of one nucleotide or amino acid) or more substantial. However, the sequence of the derivative is not so different from the reference that one of skill in the art would not recognize that the derivative and reference are related in structure and/or function. Generally, differences are limited so that the reference and the derivative are closely similar overall and, in many regions, identical. A "variant" differs from a "derivative" nucleic acid, protein, polypeptide or peptide in that the variant can have silent structural differences that do not significantly change the chemical, physical or functional properties of the reference nucleic acid, protein, polypeptide or peptide. In contrast, the differences between the reference and derivative nucleic acid, protein, polypeptide or peptide are intentional changes made to improve one or more chemical, physical or functional properties of the reference nucleic acid, protein, polypeptide or peptide.
The terms "DNA polymerase activity," "synthetic activity" and "polymerase activity" are used interchangeably and refer to the ability of an enzyme to synthesize new DNA strands by the incorporation of deoxynucleoside triphosphates. A protein that can direct the synthesis of new DNA strands by the incorporation of deoxynucleoside triphosphates in a template-dependent manner is said to be "capable of DNA synthetic activity." The term "5' exonuclease activity" refers to the presence of an activity in a protein that is capable of removing nucleotides from the 5' end of a nucleic acid. The term "3' exonuclease activity" refers to the presence of an activity in a protein that is capable of removing nucleotides from the 3' end of a nucleic acid.
"Expression" refers to the transcription and/or translation of an endogenous or exogeneous gene in an organism. Expression generally refers to the transcription and stable accumulation of mRNA. Expression may also refer to the production of protein.
"Expression cassette" means a nucleic acid sequence capable of directing expression of a particular nucleotide sequence. Expression cassettes generally comprise a promoter operably linked to the nucleotide sequence to be expressed (e.g., a coding region) that is operably linked to termination signals. Expression cassettes also typically comprise sequences required for proper translation of the nucleotide sequence. The expression cassette comprising the nucleotide sequence of interest may be chimeric, meaning that at least one of its components is heterologous with respect to at least one of its other components. The expression of the nucleotide sequence in the expression cassette may be under the control of a constitutive promoter or of an inducible promoter that initiates transcription only when the host cell is exposed to some particular external stimulus. In the case of a multicellular organism, the promoter can also be specific to a particular tissue or organ or stage of development.
The term "gene" is used broadly to refer to any segment of nucleic acid associated with a biological function. The term "gene" encompasses the coding region of a protein, polypeptide, peptide or structural RNA. The term "gene"also includes sequences up to a distance of about 2 kb on either end of a coding region. These sequences are referred to as "flanking" sequences or regions (these flanking sequences are located 5' or 3' to the non- translated sequences present on the mRNA transcript). The 5' flanking region may contain regulatory sequences such as promoters and enhancers or other recognition or binding sequences for proteins that control or influence the transcription of the gene. The 3' flanking region may contain sequences that direct the termination of transcription, post-transcriptional cleavage and polyadenylation as well as recognition sequences for other proteins. A protein or polypeptide encoded in a gene can be full length or any portion thereof, so that all activities or functional properties are retained, or so that only selected activities (e.g., enzymatic activity, ligand binding, or signal transduction) of the full-length protein or polypeptide are retained. The protein or polypeptide can include any sequences necessary for the production of a proprotein or precursor polypeptide. The term "native gene" refers to gene that is naturally present in the genome of an untransformed cell.
"Genome" refers to the complete genetic material that is naturally present in an organism and is transmitted from one generation to the next. The terms "heterologous nucleic acid," or "exogenous nucleic acid" refer to a nucleic acid that originates from a source foreign to the particular host cell or, if from the same source, is modified from its original form. Thus, a heterologous gene in a host cell includes a gene that is endogenous to the particular host cell but has been modified through, for example, the use of DNA shuffling. The terms also include non-naturally occurring multiple copies of a naturally occurring nucleic acid. Thus, the terms refer to a nucleic acid segment that is foreign or heterologous to the cell, or normally found within the cell but in a position within the cell or genome where it is not ordinarily found.
The term "homology" refers to a degree of similarity between a nucleic acid and a reference nucleic acid or between a polypeptide and a reference polypeptide. Homology may be partial or complete. Complete homology indicates that the nucleic acid or amino acid sequences are identical. A partially homologous nucleic acid or amino acid sequence is one that is not identical to the reference nucleic acid or amino acid sequence. Hence, a partially homologous nucleic acid has one or more nucleotide differences in its sequence relative to the nucleic acid to which it is being compared. The degree of homology can be determined by sequence comparison. Alternatively, as is understood by those skilled in the art, DNA-DNA or DNA-RNA hybridization, under various hybridization conditions, can provide an estimate of the degree of homology between nucleic acids, (see, e.g., Haines and Higgins (eds.), Nucleic Acid Hybridization, IRL Press, Oxford, U.K.).
"Hybridization" refers to the process of annealing complementary nucleic acid strands by forming hydrogen bonds between nucleotide bases on the complementary nucleic acid strands. Hybridization, and the strength of the association between the nucleic acids, is impacted by such factors as the degree of complementary between the hybridizing nucleic acids, the stringency of the conditions involved, the Tm of the formed hybrid, and the G:C ratio within the nucleic acids.
"Inducible promoter" refers to a regulated promoter that can be turned on in one or more cell types by an external stimulus, such as a chemical, light, hormone, stress, temperature or a pathogen. An "initiation site" is region surrounding the position of the first nucleotide that is part of the transcribed sequence, which is defined as position +1. All nucleotide positions of the gene are numbered by reference to the first nucleotide of the transcribed sequence, which resides within the initiation site. Downstream sequences (i.e., sequences in the 3' direction) are denominated positive, while upstream sequences (i.e., sequences in the 5' direction) are denominated negative.
An "isolated" or "purified" nucleic acid or an "isolated" or "purified" polypeptide is a nucleic acid or polypeptide that, by the hand of man, exists apart from its native environment and is therefore not a product of nature. An isolated nucleic acid or polypeptide may exist in a purified form or may exist in a non- native environment such as, for example, a transgenic host cell.
The term "invader oligonucleotide" refers to an oligonucleotide that contains sequences at its 3' end that are substantially the same as sequences located at the 5' end of a probe oligonucleotide. These regions will compete for hybridization to the same segment along a complementary target nucleic acid.
The term "label" refers to any atom or molecule that can be used to provide a detectable (preferably quantifiable) signal, and that can be attached to a nucleic acid or protein. Labels may provide signals detectable by fluorescence, radioactivity, colorimetry, gravimetry, X-ray diffraction or absorption, magnetism, enzymatic activity, and the like.
The term "nucleic acid" refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form, composed of monomers (nucleotides) containing a sugar, phosphate and a base that is either a purine or pyrimidine. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences as well as the reference sequence explicitly indicated.
The term "oligonucleotide" as used herein is defined as a molecule comprised of two or more deoxyribonucleotides or ribonucleotides, preferably more than three, and usually more than ten. There is no precise upper limit on the size of an oligonucleotide. However, in general, an oligonucleotide is shorter than about 250 nucleotides, preferably shorter than about 200 nucleotides and more preferably shorter than about 100 nucleotides. The exact size will depend on many factors, which in turn depends on the ultimate function or use of the oligonucleotide. The oligonucleotide may be generated in any manner, including chemical synthesis, DNA replication, reverse transcription, or a combination thereof. The terms "open reading frame" and "ORF" refer to the amino acid sequence encoded between translation initiation and termination codons of a coding sequence. The terms "initiation codon" and "termination codon" refer to a unit of three adjacent nucleotides ('codon') in a coding sequence that specifies initiation and chain termination, respectively, of protein synthesis (mRNA translation).
"Operably linked" means joined as part of the same nucleic acid molecule, so that the function of one is affected by the other. In general, "operably linked" also means that two or more nucleic acids are suitably positioned and oriented so that they can function together. Nucleic acids are often operably linked to permit transcription of a coding region to be initiated from the promoter. For example, a regulatory sequence is said to be "operably linked to" or "associated with" a nucleic acid sequence that codes for an RNA or a polypeptide if the two sequences are situated such that the regulatory sequence affects expression of the coding region (i.e., that the coding sequence or functional RNA is under the transcriptional control of the promoter). Coding regions can be operably-linked to regulatory sequences in sense or antisense orientation.
The term "probe oligonucleotide" refers to an oligonucleotide that interacts with a target nucleic acid to form a cleavage structure in the presence or absence of an invader oligonucleotide. When annealed to the target nucleic acid, the probe oligonucleotide and target form a cleavage structure and cleavage occurs within the probe oligonucleotide. The presence of an invader oligonucleotide upstream of the probe oligonucleotide can shift the site of cleavage within the probe oligonucleotide (relative to the site of cleavage in the absence of the invader).
"Promoter" refers to a nucleotide sequence, usually upstream (5') to a coding region, which controls the expression of the coding region by providing the recognition site for RNA polymerase and other factors required for proper transcription. "Promoter" includes but is not limited a minimal promoter that is a short DNA sequence comprised of a TATA- box. Hence, a promoter includes other sequences that serve to specify the site of transcription initiation and control or regulate expression, for example, enhancers. Accordingly, an "enhancer" is a segment of DNA that can stimulate promoter activity and may be an innate element of the promoter or a heterologous element inserted to enhance the level or tissue specificity of a promoter. It is capable of operating in both orientations (normal or flipped), and is capable of functioning even when moved either upstream or downstream from the promoter. Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even be comprised of synthetic DNA segments. A promoter may also contain DNA segments that are involved in the binding of protein factors that control the effectiveness of transcription initiation in response to physiological or developmental conditions. The terms "protein," "peptide" and "polypeptide" are used interchangeably herein.
"Regulatory sequences" and "regulatory elements" refer to nucleotide sequences that control some aspect of the expression of nucleic acid sequences. Such sequences or elements can be located upstream (5' non-coding sequences), within, or downstream (3' non-coding sequences) of a coding sequence. "Regulatory sequences" and "regulatory elements" influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences include enhancers, introns, promoters, polyadenylation signal sequences, splicing signals, termination signals, and translation leader sequences. They include natural and synthetic sequences.
As used herein, the term "selectable marker" refers to a gene that encodes an observable or selectable trait that is expressed and can be detected in an organism having that gene. Selectable markers are often linked to a nucleic acid of interest that may not encode an observable trait, in order to trace or select the presence of the nucleic acid of interest. Any selectable marker known to one of skill in the art can be used with the nucleic acids of the invention. Some selectable markers allow the host to survive under circumstances where, without the marker, the host would otherwise die. Examples of selectable markers include antibiotic resistance, for example, tetracycline or ampicillin resistance. As used herein the term "stringency" is used to define the conditions of temperature, ionic strength, and the presence of other compounds such as organic solvents, under which nucleic acid hybridizations are conducted. With "high stringency" conditions, nucleic acid base pairing will occur only between nucleic acids that have a high frequency of complementary base sequences. With "weak" or "low" stringency conditions nucleic acids the frequency of complementary sequences is usually less, so that nucleic acids with differing sequences can be detected and/or isolated.
The terms "substantially similar" and "substantially homologous" refer to nucleotide and amino acid sequences that represent functional equivalents of the instant inventive sequences. For example, altered nucleotide sequences that simply reflect the degeneracy of the genetic code but nonetheless encode amino acid sequences that are identical to the inventive amino acid sequences are substantially similar to the inventive sequences. In addition, amino acid sequences that are substantially similar to the instant sequences are those wherein overall amino acid identity is sufficient to provide an active, thermally stable nucleic acid polymerase. For example, amino acid sequences that are substantially similar to the sequences of the invention are those wherein the overall amino acid identity is 80% or greater, preferably 90% or greater, such as 91%, 92%, 93%, or 94%, and more preferably 95% or greater, such as 96%, 97%o, 98%, or 99% relative to the amino acid sequences of the invention. A "terminating agent," "terminating nucleotide" or "terminator" in relation to DNA synthesis or sequencing refers to compounds capable of specifically terminating a DNA sequencing reaction at a specific base, such compounds include but are not limited to, dideoxynucleosides having a 2', 3' dideoxy structure (e.g., ddATP, ddCTP, ddGTP and ddTTP).
"Thermostable" means that a nucleic acid polymerase remains active at a temperature greater than about 37 °C. Preferably, the nucleic acid polymerases of the invention remain active at a temperature greater than about 42 °C. More preferably, the nucleic acid polymerases of the invention remain active at a temperature greater than about 50 °C. Even more preferably, the nucleic acid polymerases of the invention remain active after exposure to a temperature greater than about 60 °C. Most preferably, the nucleic acid polymerases of the invention remain active despite exposure to a temperature greater than about 70 °C. A "transgene" refers to a gene that has been introduced into the genome by transformation and is stably maintained. Transgenes may include, for example, genes that are either heterologous or homologous to the genes of a particular organism to be transformed. Additionally, transgenes may comprise native genes inserted into a non-native organism, or chimeric genes. The term "endogenous gene" refers to a native gene in its natural location in the genome of an organism. A "foreign" or "exogenous" gene refers to a gene not normally found in the host organism but one that is introduced by gene transfer. The term "transformation" refers to the transfer of a nucleic acid fragment into the genome of a host cell, resulting in genetically stable inheritance. Host cells containing the transformed nucleic acid fragments are referred to as "transgenic" cells, and organisms comprising transgenic cells are referred to as "transgenic organisms." Transformation may be accomplished by a variety of means known to the art including calcium DNA co-precipitation, electroporation, viral infection, and the like.
The "variant" of a reference nucleic acid, protein, polypeptide or peptide, is a nucleic acid, protein, polypeptide or peptide, respectively, with a related but different sequence than the respective reference nucleic acid, protein, polypeptide or peptide. The differences between variant and reference nucleic acids, proteins, polypeptides or peptides are silent or conservative differences. A variant nucleic acid differs in nucleotide sequence from a reference nucleic acid whereas a variant nucleic acid, protein, polypeptide or peptide differs in amino acid sequence from the reference protein, polypeptide or peptide, respectively. A variant and reference nucleic acid, protein, polypeptide or peptide may differ in sequence by one or more substitutions, insertions, additions, deletions, fusions and truncations, which may be present in any combination. Differences can be minor (e.g., a difference of one nucleotide or amino acid) or more substantial. However, the structure and function of the variant is not so different from the reference that one of skill in the art would not recognize that the variant and reference are related in structure and/or function. Generally, differences are limited so that the reference and the variant are closely similar overall and, in many regions, identical. The term "vector" is used to refer to a nucleic acid that can transfer another nucleic acid segment(s) into a cell. A "vector" includes, inter alia, any plasmid, cosmid, phage or nucleic acid in double- or single-stranded, linear or circular form that may or may not be self transmissible or mobilizable. It can transform prokaryotic or eukaryotic host cells either by integration into the cellular genome or by existing extrachromosomally (e.g., autonomous replicating plasmid with an origin of replication). Vectors used in bacterial systems often contain an origin of replication that allows the vector to replicate independently of the bacterial chromosome. The term "expression vector" refers to a vector containing an expression cassette. The term "wild-type" refers to a gene or gene product that has the characteristics of that gene or gene product when isolated from a naturally occurring source. A wild-type gene is the gene form most frequently observed in a population and thus arbitrarily is designed the "normal" or "wild-type" form of the gene. In contrast, the term "variant" or "derivative" refers to a gene or gene product that displays modifications in sequence and or functional properties (i.e., altered characteristics) when compared to the wild-type gene or gene product. Naturally-occurring derivatives can be isolated. They are identified by the fact that they have altered characteristics when compared to the wild-type gene or gene product.
Polymerase Nucleic Acids The invention provides isolated nucleic acids encoding Thermus scotoductus nucleic acid polymerases as well as derivatives fragments and variant nucleic acids thereof that encode an active, thermally stable nucleic acid polymerase. Thus, one aspect of the invention includes the nucleic acid polymerases encoded by the polynucleotide sequences contained in Thermus scotoductus strain X-l (ATCC Deposit No. 27978). Another aspect of the invention provides the nucleic acid polymerases of Thermus scotoductus strains SM3 and Vi7a. Any nucleic acid encoding any one of amino acid sequences SEQ ID NO: 13-28, which are amino acid sequences for wild type and several derivative Thermus scotoductus nucleic acid polymerases, are also contemplated by the present invention.
In one embodiment, the invention provides a nucleic acid of SEQ ID NO:l, a wild type Thermus scotoductus, strain X-l, nucleic acid encoding a nucleic acid polymerase.
ATGAGGGCGA TGCTGCCCCT CTTTGAGCCC AAGGGCCGGG 40
TGCTTCTGGT GGACGGCCAC CACCTGGCCT ACCGTACCTT 80
TTTTGCCCTG AAGGGCCTCA CCACCAGCCG CGGGGAGCCG 120
GTCCAGGCGG TGTACGGGTT TGCCAAGAGC CTTTTGAAGG 160
CGCTAAGGGA AGACGGGGAT GTGGTGATCG TGGTGTTTGA 200 CGCCAAGGCC CCCTCCTTCC GCCACCAGAC CTACGAGGCC 240
TACAAGGCGG GGCGGGCTCC CACCCCCGAG GACTTTCCCC 280
GGCAGCTTGC CCTTATCAAG GAGATGGTGG ACCTTTTGGG 320
CCTGGAGCGC CTCGAGGTGC CGGGCTTTGA GGCGGATGAC 360
GTCCTGGCTA CCCTGGCCAA GAAGGCGGAA AAGGAAGGCT 400 ACGAGGTGCG CATCCTCACC GCGGACCGGG ACCTTTACCA 440
GCTTCTTTCG GAGCGAATCT CCATCCTTCA CCCGGAGGGT 480
TACCTGATCA CCCCGGAGTG GCTTTGGGAG AAGTATGGGC 520 TTAAGCCTTC CCAGTGGGTG GACTACCGGG CCTTGGCCGG 560 GGACCCTTCC GACAACATCC CCGGCGTGAA GGGCATCGGG 600 GAGAAGACGG CGGCCAAGCT GATCCGGGAG TGGGGAAGCC 640 TGGAAAACCT TCTTAAGCAC CTGGAACAGG TGAAACCTGC 680 CTCCGTGCGG GAGAAGATCC TTAGCCACAT GGAGGACCTC 720 AAGCTATCCC TGGAGCTATC CCGGGTGCGC ACGGACTTGC 760 CCCTTCAGGT GGACTTCGCC CGGCGCCGGG AGCCGGACCG 800 GGAGGGGCTT AAGGCCTTTT TGGAGAGGCT GGAGTTCGGA 840 AGCCTCCTCC ACGAGTTCGG CCTGTTGGAA AGCCCGGTGG 880 CGGCGGAGGA AGCTCCCTGG CCGCCCCCCG AGGGAGCCTT 920 CGTGGGGTAC GTTCTTTCCC GCCCCGAGCC CATGTGGGCG 960 GAGCTTAACG CCTTGGCCGC CGCCTGGGAG GGAAGGGTTT 1000 ACCGGGCGGA GGATCCCTTG GAGGCCTTGC GGGGGCTTGG 1040 GGAGGTGAGG GGGCTTTTGG CCAAGGACCT GGCGGTGCTG 1080 GCCCTGAGGG AAGGGATTGC CCTGGCACCG GGCGACGACC 1120 CCATGCTCCT CGCCTACCTC CTGGATCCTT CCAACACCGC 1160 CCCCGAAGGG GTAGCCCGGC GCTACGGGGG GGAGTGGACC 1200 GAGGAGGCGG GGGAAAGGGC GTTGCTTTCC GAAAGGCTTT 1240 ACGCCGCCCT CCTGGAGCGG CTTAAGGGGG AGGAGAGGCT 1280 TCTTTGGCTT TACGAGGAGG TGGAAAAGCC CCTTTCGCGG 1320 GTCCTGGCCC ACATGGAGGC CACGGGGGTA CGGTTGGATG 1360 TGGCCTACTT AAAGGCCCTT TCCCTGGAGG TGGAGGCGGA 1400 GCTCAGGCGC CTCGAGGAGG AGGTCCACCG CCTGGCCGGG 1440 CATCCTTTCA ACCTGAACTC CCGGGACCAG CTGGAAAGGG 1480 TCCTCTTTGA CGAGCTTGGG CTTCCCGCCA TCGGCAAGAC 1520 GGAGAAGACG GGCAAGCGCT CCACCAGCGC CGCCGTTTTG 1560 GAGGCCTTGC GGGAGGCTCA TCCCATCGTG GACCGCATCC 1600 TTCAGTACCG GGAGCTTTCC AAGCTCAAGG GAACCTACAT 1640 CGATCCCTTG CCTGCCCTGG TCCACCCCAA GACGAACCGC 1680 CTCCACACCC GTTTCAACCA GACGGCCACC GCCACGGGGA 1720 GGCTTAGCAG CTCGGATCCC AACCTGCAAA ATATCCCCGT 1760 GCGCACCCCT TTGGGCCAGC GGATCCGCCG GGCCTTCGTG 1800 GCCGAGGAGG GGTGGAGGCT GGTGGTTTTG GACTACAGCC 1840 AGATTGAGCT CAGGGTCCTG GCGCACCTTT CCGGGGACGA 1880 GAACCTAATC CGGGTCTTCC AGGAGGGCCA GGACATCCAC 1920 ACCCAGACGG CCAGCTGGAT GTTCGGCGTG CCCCCAGAGG 1960 CCGTGGATTC CCTGATGCGT CGGGCGGCCA AGACCATCAA 2000 CTTCGGCGTC CTCTACGGCA TGTCCGCCCA CCGGCTTTCG 2040 GGAGAGCTGG CCATCCCCTA CGAGGAGGCG GTGGCCTTCA 2080 TCGAGCGGTA TTTCCAGAGC TACCCCAAGG TGCGGGCCTG 2120 GATTGAGAAA ACCCTGGCGG AAGGACGGGA ACGGGGCTAT 2160 GTGGAAACCC TCTTTGGCCG CCGGCGCTAC GTGCCCGACT 2200 TGGCTTCCCG GGTGAAGAGC ATCCGGGAGG CAGCGGAGCG 2240 CATGGCCTTC AACATGCCGG TCCAGGGGAC CGCCGCGGAT 2280 TTGATGAAAC TGGCCATGGT GAAGCTCTTT CCCAGGCTTC 2320 AGGAGCTGGG GGCCAGGATG CTTTTGCAGG TGCACGACGA 2360 ACTGGTCCTC GAGGCTCCCA AGGAGCAAGC GGAGGAAGTC 2400 GCCCAGGAGG CCAAGCGGAC CATGGAGGAG GTGTGGCCCC 2440 TGAAGGTGCC CTTGGAGGTG GAAGTGGGCA TCGGGGAGGA 2480 CTGGCTTTCC GCCAAGGCCT AG 2502
In another embodiment, the invention provides nucleic acids encoding a wild type nucleic acid polymerase from Thermus scotoductus, strain SM3, having, for example, SEQ ID NO:2.
ATGAGGGCGA TGCTGCCCCT CTTTGAGCCC AAGGGCCGGG 40 TGCTTCTGGT GGACGGCCAC CACCTGGCCT ACCGTACCTT 80
TTTTGCCCTG AAGGGCCTCA CCACCAGCCG CGGGGAGCCG 120
GTCCAGGCGG TGTACGGGTT TGCCAAGAGC CTTTTGAAGG 160
CGCTAAGGGA AGACGGGGAT GTGGTGATCG TGGTGTTTGA 200
CGCCAAGGCC CCCTCCTTCC GCCACCAGAC CTACGAGGCC 240 TACAAGGCGG GGCGGGCTCC CACCCCCGAG GACTTTCCCC 280
GGCAGCTTGC CCTTATCAAG GAGATGGTGG ACCTTTTGGG 320
CCTGGAGCGC CTCGAAGTGC CGGGTTTTGA GGCGGATGAC 360
GTCCTGGCCA CCCTGGCCAA GAAGGCGGAA AAGGAAGGCT 400
ACGAGGTGCG CATCCTCACC GCGGACCGGG ACCTTTACCA 440 GCTTCTTTCG GACCGAATCT CCATCCTTCA CCCGGAGGGT 480
TACCTGATCA CCCCGGAGTG GCTTTGGGAG AAGTATGGGC 520
TTAAGCCTTC CCAGTGGGTG GACTACCGGG CCTTGGCCGG 560
GGACCCTTCC GACAACATCC CCGGCGTGAA GGGCATCGGG 600
GAGAAGACGG CGGCCAAGCT GATCCGGGAG TGGGGAAGCC 640 TGGAAAACCT TCTTAAGCAC CTGGAACAGG TGAAACCTGC 680 CTCCGTGCGG GAGAAGATCC TTAGCCACAT GGAGGACCTC 720 AAGCTATCCC TGGAGCTTTC CCGGGTGCAC ACGGAGTTGC 760 CCCTTCAGGT GGACTTCGCC CGGCGCCGGG AGCCGGACCG 800 GGAAGGGCTT AAGGCCTTTT TGGAGAGGCT GGAGTTCGGA 840 AGCCTCCTCC ACGAGTTCGG CCTGTTGGAA AGCCCGGTGG 880 CGGCGGAGGA AGCTCCCTGG CCGCCCCCCG AGGGAGCCTT 920 CGTGGGGTAC GTTCTTTCCC GCCCCGAGCC CATGTGGGCG 960 GAGCTTAACG CCTTGGCCGC CGCCTGGGAG GGAAGGGTTT 1000 ACCGGGCGGA GGATCCCTTG GAGGCCTTGC GGGGGCTTGG 1040 GGAGGTGAGG GGGCTTTTGG CCAAGGACCT GGCGGTGCTG 1080 GCCCTGAGGG AAGGGATTGC CCTGGCACAG GGCGACGACC 1120 CCATGCTCCT CGCCTACCTC CTGGATCCTT CCAACACCGC 1160 CCCCGAAGGG GTAGCCCGGC GCTACGGGGG GGAGTGGACC 1200 GAGGAGGCGG GGGAAAGGGC GCTGCTTTCC GAAAGGCTTT 1240 ACGCCGCCCT CCTGGAGCGG CTTAAGGGGG AGGAGAGGCT 1280 TCTTTGGCTT TACGAGGAGG TGGAAAAGCC CCTTTCGCGG 1320 GTCCTGGCCC ACATGGAGGC CACGGGGGTA TGGTTGGATG 1360 TGGCCTACTT GAAGGCCCTT TCCCTGGAGG TGGAGGCGGA 1400 GCTCAGGCGC CTCGAGGAGG AGGTCCACCG ACTGGCCGGG 1440 CATCCTTTCA ACCTGAACTC CCGGGACCAG CTGGAAAGGG 1480 TCCTCTTTGA CGAGCTTGGG CTTCCCGCCA TCGGCAAGAC 1520 GGAGAAGACG GGTAAGCGTT CCACCAGCGC CGCCGTTTTG 1560 GAGGCTTTGA GGGAGGCTCA TCCCATAGTG GACCGCATCC 1600 TCCAGTACCG GGAGCTTTCC AAGCTCAAGG GAACGTACAT 1640 CGATCCCTTG CCCGCCCTGG TCCACCCCAA GACGAACCGC 1680 CTCCACACCC GTTTCAACCA GACGGCCACC GCCACGGGGA 1720 GGCTTAGCAG CTCGGATCCC AACCTGCAAA ATATCCCCGT 1760 GCGCACCCCT TTAGGCCAGC GGATCCGCCG GGCCTTCGTG 1800 GCCGAGGAGG GGTGGAGGCT GGTGGTTTTG GACTACAGCC 1840 AGATTGAGCT CAGGGTCCTG GCGCACCTTT CCGGGGACGA 1880 GAACCTGATC CGGGTCTTCC AAGAGGGCCA GGACATCCAC 1920 ACCCAGACGG CCAGCTGGAT GTTCGGCGTG CCCCCAGAGG 1960 CCGTGGATTC CCTGATGCGC CGGGCGGCCA AGACCATCAA 2000 CTTCGGCGTC CTCTACGGCA TGTCCGCCCA CCGGCTTTCG 2040 GGAGAGCTGG CCATCCCCTA CGAGGAAGCG GTGGCCTTCA 2080 TCGAGCGGTA TTTCCAGAGC TACCCCAAGG TACGGGCCTG 2120 GATTGAGAAA ACCCTGGCGG AAGGACGGGA GCGGGGCTAT 2160 GTGGAAACCC TCTTTGGCCG CCGGCGCTAT GTGCCCGACT 2200 TGGCTTCCCG GGTGAAGAGC ATCCGGGAGG CAGCGGAGCG 2240 CATGGCCTTC AACATGCCGG TCCAGGGGAC CGCCGCGGAT 2280 TTGATGAAAC TGGCCATGGT GAAGCTCTTT CCCAGGCTTC 2320 AGGAGCTGGG GGCCAGGATG CTTTTGCAGG TGCACGACGA 2360 ACTGGTCCTC GAGGCTCCCA AGGAGCAAGC GGAGGAAGTC 2400 GCCCAGGAGG CCAAGCGGAC CATGGAGGAG GTGTGGCCCC 2440 TGAAGGTGCC CTTGGAGGTG GAGGTGGGTA TCGGGGAGGA 2480 CTGGCTTTCC GCCAAGGCCT AGTCGAC 2507
In another embodiment, the invention provides nucleic acids encoding a wild type nucleic acid polymerase from Thermus scotoductus, strain Vi7a, having, for example, SEQ ID NO:3.
ATGAGGGCGA TGCTGCCCCT CTTTGAGCCC AAGGGCCGGG 40 TGCTTCTGGT GGACGGCCAC CACCTGGCCT ACCGTACCTT 80
TTTTGCCCTG AAGGGCCTCA CCACCAGCCG CGGGGAGCCG 120
GTCCAGGCGG TGTACGGGTT TGCCAAGAGC CTTTTGAAGG 160
CGCTAAGGGA AGACGGGGAT GTGGTGATCG TGGTGTTTGA 200
CGCCAAGGCC CCCTCCTTCC GCCACCAGAC CTACGAGGCC 240 TACAAGGCGG GGCGGGCTCC CACCCCCGAG GACTTTCCCC 280
GGCAGCTTGC CCTTATCAAG GAGATGGTGG ACCTTTTGGG 320
CCTGGAGCGC CTCGAAGTGC CGGGTTTTGA GGCGGATGAC 360
GTCCTGGCCA CCCTGGCCAA GAAGGCGGAA AAGGAAGGCT 400
ACGAGGTGCG CATCCTCACC GCGGACCGGG ACCTTTACCA 440 GCTTCTTTCG GACCGAATCT CCATCCTTCA CCCGGAGGGT 480
TACCTGATTA CCCCGGAGTG GCTTTGGGAG AAGTATGGGC 520
TTAAGCCTTC CCAGTGGGTG GACTACCGGG CCTTGGCCGG 560
GGACCCTTCC GACAACATCC CCGGCGTGAA GGGCATCGGG 600 GAGAAGACGG CGGCCAAGCT GATCCGGGAG TGGGGAAGCC 640 TGGAAAACCT TCTTAAGCAC CTGGAACAGG TGAAACCTGC 680 CTCCGTGCGG GAGAAGATCC TTAGCCACAT GGAGGACCTC 720 AAGCTATCCC TGGAGCTTTC CCGGGTGCAC ACGGAGTTGC 760 CCCTTCAGGT GGACTTCGCC CGGCGCCGGG AGCCGGACCG 800 GGAAGGGCTT AAGGCCTTTT TGGAGAGGCT GGAGTTCGGA 840 AGCCTCCTCC ACGAGTTCGG CCTGTTGGAA AGCCCGGTGG 880 CGGCGGAGGA AGCTCCCTGG CCGCCCCCCG AGGGAGCCTT 920 CGTGGGGTAC GTTCTTTCCC GCCCCGAGCC CATGTGGGCG 960 GAGCTTAACG CCTTGGCCGC CGCCTGGGAG GGAAGGGTTT 1000 ACCGGGCGGA GGATCCCTTG GAGGCCTTGC GGGGGCTTGG 1040 GGAGGTGAGG GGGCTTTTGG CCAAGGACCT GGCGGTGCTG 1080 GCCCTGAGGG AAGGGATTGC CCTGGCACCG GGCGACGACC 1120 CCATGCTCCT CGCCTACCTC CTGGATCCTT CCAACACCGC 1160 CCCCGAAGGG GTAGCCCGGC GCTACGGGGG GGAGTGGACC 1200 GAGGAGGCGG GGGAAAGGGC GCTGCTTTCC GAAAGGCTTT 1240 ACGCCGCCCT CCTGGAGCGG CTTAAGGGGG AGGAGAGGCT 1280 TCTTTGGCTT TACGAGGAGG TGGAAAAGCC CCTTTCGCGG 1320 GTCCTGGCCC ACATGGAGGC CACGGGGGTA TGGTTGGATG 1360 TGGCCTACTT GAAGGCCCTT TCCCTGGAGG TGGAGGCGGA 1400 GCTCAGGCGC CTCGAGGAGG AGGTCCACCG ACTGGCCGGG 1440 CATCCTTTCA ACCTGAACTC CCGGGACCAG CTGGAAAGGG 1480 TCCTCTTTGA CGAGCTTGGG CTTCCCGCCA TCGGCAAGAC 1520 GGAGAAGACG GGTAAGCGTT CCACCAGCGC CGCCGTTTTG 1560 GAGGCTTTGA GGGAGGCTCA TCCCATAGTG GACCGCATCC 1600 TCCAGTACCG GGAGCTTTCC AAGCTCAAGG GAACGTACAT 1640 CGATCCCTTG CCCGCCCTGG TCCACCCCAA GACGAACCGC 1680 CTCCACACCC GTTTCAACCA GACGGCCACC GCCACGGGGA 1720 GGCTTAGCAG CTCGGATCCC AACCTGCAAA ATATCCCCGT 1760 GCGCACCCCT TTAGGCCAGC GGATCCGCCG GGCCTTCGTG 1800 GCCGAGGAGG GGTGGAGGCT GGTGGTTTTG GACTACAGCC 1840 AGATTGAGCT CAGGGTCCTG GCGCACCTTT CCGGGGACGA 1880 GAACCTGATC CGGGTCTTCC AAGAGGGCCA GGACATCCAC 1920 ACCCAGACGG CCAGCTGGAT GTTCGGCGTG CCCCCAGAGG 1960
CCGTGGATTC CCTGATGCGC CGGGCGGCCA AGACCATCAA 2000
CTACGGCGTC CTCTACGGCA TGTCCGCCCA CCGGCTTTCG 2040
GGAGAGCTGG CCATCCCCTA CGAGGAAGCG GTGGCCTTCA 2080 TCGAGCGGTA TTTCCAGAGC TTCCCCAAGG TACGGGCCTG 2120
GATTGAGAAA ACCCTGGCGG AAGGACGGGA GCGGGGCTAT 2160
GTGGAAACCC TCTTTGGCCG CCGGCGCTAT GTGCCCGACT 2200
TGGCTTCCCG GGTGAAGAGC ATCCGGGAGG CAGCGGAGCG 2240
CATGGCCTTC AACATGCCGG TCCAGGGGAC CGCCGCGGAT 2280 TTGATGAAAC TGGCCATGGT GAAGCTCTTT CCCAGGCTTC 2320
AGGAGCTGGG GGCCAGGATG CTTTTGCAGG TGCACGACGA 2360
ACTGGTCCTC GAGGCTCCCA AGGAGCAAGC GGAGGAAGTC 2400
GCCCAGGAGG CCAAGCGGAC CATGGAGGAG GTGTGGCCCC 2440
TGAAGGTGCC CTTGGAGGTG GAGGTGGGTA TCGGGGAGGA 2480 CTGGCTTTCC GCCAAGGCCT AGTCGAC 2507
In another embodiment, the invention provides a nucleic acid of SEQ ID NO:4, a derivative nucleic acid related to Thermus scotoductus, strain X-l, having GAC (encoding Asp) in place of GGG (encoding Gly) at positions 136- 138. SEQ ID NO :4 is provided below.
ATGAGGGCGA TGCTGCCCCT CTTTGAGCCC AAGGGCCGGG 40 TGCTTCTGGT GGACGGCCAC CACCTGGCCT ACCGTACCTT 80
TTTTGCCCTG AAGGGCCTCA CCACCAGCCG CGGGGAGCCG 120
GTCCAGGCGG TGTACGACTT TGCCAAGAGC CTTTTGAAGG 160 CGCTAAGGGA AGACGGGGAT GTGGTGATCG TGGTGTTTGA 200
CGCCAAGGCC CCCTCCTTCC GCCACCAGAC CTACGAGGCC 240
TACAAGGCGG GGCGGGCTCC CACCCCCGAG GACTTTCCCC 280
GGCAGCTTGC CCTTATCAAG GAGATGGTGG ACCTTTTGGG 320
CCTGGAGCGC CTCGAGGTGC CGGGCTTTGA GGCGGATGAC 360 GTCCTGGCTA CCCTGGCCAA GAAGGCGGAA AAGGAAGGCT 400
ACGAGGTGCG CATCCTCACC GCGGACCGGG ACCTTTACCA 440
GCTTCTTTCG GAGCGAATCT CCATCCTTCA CCCGGAGGGT 480
TACCTGATCA CCCCGGAGTG GCTTTGGGAG AAGTATGGGC 520
TTAAGCCTTC CCAGTGGGTG GACTACCGGG CCTTGGCCGG 560 GGACCCTTCC GACAACATCC CCGGCGTGAA GGGCATCGGG 600 GAGAAGACGG CGGCCAAGCT GATCCGGGAG TGGGGAAGCC 640 TGGAAAACCT TCTTAAGCAC CTGGAACAGG TGAAACCTGC 680 CTCCGTGCGG GAGAAGATCC TTAGCCACAT GGAGGACCTC 720 AAGCTATCCC TGGAGCTATC CCGGGTGCGC ACGGACTTGC 760 CCCTTCAGGT GGACTTCGCC CGGCGCCGGG AGCCGGACCG 800 GGAGGGGCTT AAGGCCTTTT TGGAGAGGCT GGAGTTCGGA 840 AGCCTCCTCC ACGAGTTCGG CCTGTTGGAA AGCCCGGTGG 880 CGGCGGAGGA AGCTCCCTGG CCGCCCCCCG AGGGAGCCTT 920 CGTGGGGTAC GTTCTTTCCC GCCCCGAGCC CATGTGGGCG 960 GAGCTTAACG CCTTGGCCGC CGCCTGGGAG GGAAGGGTTT 1000 ACCGGGCGGA GGATCCCTTG GAGGCCTTGC GGGGGCTTGG 1040 GGAGGTGAGG GGGCTTTTGG CCAAGGACCT GGCGGTGCTG 1080 GCCCTGAGGG AAGGGATTGC CCTGGCACCG GGCGACGACC 1120 CCATGCTCCT CGCCTACCTC CTGGATCCTT CCAACACCGC 1160 CCCCGAAGGG GTAGCCCGGC GCTACGGGGG GGAGTGGACC 1200 GAGGAGGCGG GGGAAAGGGC GTTGCTTTCC GAAAGGCTTT 1240 ACGCCGCCCT CCTGGAGCGG CTTAAGGGGG AGGAGAGGCT 1280 TCTTTGGCTT TACGAGGAGG TGGAAAAGCC CCTTTCGCGG 1320 GTCCTGGCCC ACATGGAGGC CACGGGGGTA CGGTTGGATG 1360 TGGCCTACTT AAAGGCCCTT TCCCTGGAGG TGGAGGCGGA 1400 GCTCAGGCGC CTCGAGGAGG AGGTCCACCG CCTGGCCGGG 1440 CATCCTTTCA ACCTGAACTC CCGGGACCAG CTGGAAAGGG 1480 TCCTCTTTGA CGAGCTTGGG CTTCCCGCCA TCGGCAAGAC 1520 GGAGAAGACG GGCAAGCGCT CCACCAGCGC CGCCGTTTTG 1560 GAGGCCTTGC GGGAGGCTCA TCCCATCGTG GACCGCATCC 1600 TTCAGTACCG GGAGCTTTCC AAGCTCAAGG GAACCTACAT 1640 CGATCCCTTG CCTGCCCTGG TCCACCCCAA GACGAACCGC 1680 CTCCACACCC GTTTCAACCA GACGGCCACC GCCACGGGGA 1720 GGCTTAGCAG CTCGGATCCC AACCTGCAAA ATATCCCCGT 1760 GCGCACCCCT TTGGGCCAGC GGATCCGCCG GGCCTTCGTG 1800 GCCGAGGAGG GGTGGAGGCT GGTGGTTTTG GACTACAGCC 1840 AGATTGAGCT CAGGGTCCTG GCGCACCTTT CCGGGGACGA 1880 GAACCTAATC CGGGTCTTCC AGGAGGGCCA GGACATCCAC 1920 ACCCAGACGG CCAGCTGGAT GTTCGGCGTG CCCCCAGAGG 1960 CCGTGGATTC CCTGATGCGT CGGGCGGCCA AGACCATCAA 2000 CTTCGGCGTC CTCTACGGCA TGTCCGCCCA CCGGCTTTCG 2040 GGAGAGCTGG CCATCCCCTA CGAGGAGGCG GTGGCCTTCA 2080 TCGAGCGGTA TTTCCAGAGC TACCCCAAGG TGCGGGCCTG 2120
GATTGAGAAA ACCCTGGCGG AAGGACGGGA ACGGGGCTAT 2160
GTGGAAACCC TCTTTGGCCG CCGGCGCTAC GTGCCCGACT 2200
TGGCTTCCCG GGTGAAGAGC ATCCGGGAGG CAGCGGAGCG 2240 CATGGCCTTC AACATGCCGG TCCAGGGGAC CGCCGCGGAT 2280
TTGATGAAAC TGGCCATGGT GAAGCTCTTT CCCAGGCTTC 2320
AGGAGCTGGG GGCCAGGATG CTTTTGCAGG TGCACGACGA 2360
ACTGGTCCTC GAGGCTCCCA AGGAGCAAGC GGAGGAAGTC 2400
GCCCAGGAGG CCAAGCGGAC CATGGAGGAG GTGTGGCCCC 2440 TGAAGGTGCC CTTGGAGGTG GAAGTGGGCA TCGGGGAGGA 2480
CTGGCTTTCC GCCAAGGCCT AG 2502
In another embodiment, the invention provides a nucleic acid of SEQ ID NO:5, a derivative nucleic acid related to Thermus scotoductus, strain SM3, having GAC (encoding Asp) in place of GGG (encoding Gly) at positions 136- 138. SEQ ID NO:5 is provided below.
ATGAGGGCGA TGCTGCCCCT CTTTGAGCCC AAGGGCCGGG 40 TGCTTCTGGT GGACGGCCAC CACCTGGCCT ACCGTACCTT 80 TTTTGCCCTG AAGGGCCTCA CCACCAGCCG CGGGGAGCCG 120 GTCCAGGCGG TGTACGACTT TGCCAAGAGC CTTTTGAAGG 160
CGCTAAGGGA AGACGGGGAT GTGGTGATCG TGGTGTTTGA 200
CGCCAAGGCC CCCTCCTTCC GCCACCAGAC CTACGAGGCC 240 TACAAGGCGG GGCGGGCTCC CACCCCCGAG GACTTTCCCC 280
GGCAGCTTGC CCTTATCAAG GAGATGGTGG ACCTTTTGGG 320
CCTGGAGCGC CTCGAAGTGC CGGGTTTTGA GGCGGATGAC 360
GTCCTGGCCA CCCTGGCCAA GAAGGCGGAA AAGGAAGGCT 400
ACGAGGTGCG CATCCTCACC GCGGACCGGG ACCTTTACCA 440 GCTTCTTTCG GACCGAATCT CCATCCTTCA CCCGGAGGGT 480
TACCTGATCA CCCCGGAGTG GCTTTGGGAG AAGTATGGGC 520
TTAAGCCTTC CCAGTGGGTG GACTACCGGG CCTTGGCCGG 560
GGACCCTTCC GACAACATCC CCGGCGTGAA GGGCATCGGG 600
GAGAAGACGG CGGCCAAGCT GATCCGGGAG TGGGGAAGCC 640 TGGAAAACCT TCTTAAGCAC CTGGAACAGG TGAAACCTGC 680 CTCCGTGCGG GAGAAGATCC TTAGCCACAT GGAGGACCTC 720 AAGCTATCCC TGGAGCTTTC CCGGGTGCAC ACGGAGTTGC 760 CCCTTCAGGT GGACTTCGCC CGGCGCCGGG AGCCGGACCG 800 GGAAGGGCTT AAGGCCTTTT TGGAGAGGCT GGAGTTCGGA 840 AGCCTCCTCC ACGAGTTCGG CCTGTTGGAA AGCCCGGTGG 880 CGGCGGAGGA AGCTCCCTGG CCGCCCCCCG AGGGAGCCTT 920 CGTGGGGTAC GTTCTTTCCC GCCCCGAGCC CATGTGGGCG 960 GAGCTTAACG CCTTGGCCGC CGCCTGGGAG GGAAGGGTTT 1000 ACCGGGCGGA GGATCCCTTG GAGGCCTTGC GGGGGCTTGG 1040 GGAGGTGAGG GGGCTTTTGG CCAAGGACCT GGCGGTGCTG 1080 GCCCTGAGGG AAGGGATTGC CCTGGCACAG GGCGACGACC 1120 CCATGCTCCT CGCCTACCTC CTGGATCCTT CCAACACCGC 1160 CCCCGAAGGG GTAGCCCGGC GCTACGGGGG GGAGTGGACC 1200 GAGGAGGCGG GGGAAAGGGC GCTGCTTTCC GAAAGGCTTT 1240 ACGCCGCCCT CCTGGAGCGG CTTAAGGGGG AGGAGAGGCT 1280 TCTTTGGCTT TACGAGGAGG TGGAAAAGCC CCTTTCGCGG 1320 GTCCTGGCCC ACATGGAGGC CACGGGGGTA TGGTTGGATG 1360 TGGCCTACTT GAAGGCCCTT TCCCTGGAGG TGGAGGCGGA 1400 GCTCAGGCGC CTCGAGGAGG AGGTCCACCG ACTGGCCGGG 1440 CATCCTTTCA ACCTGAACTC CCGGGACCAG CTGGAAAGGG 1480 TCCTCTTTGA CGAGCTTGGG CTTCCCGCCA TCGGCAAGAC 1520 GGAGAAGACG GGTAAGCGTT CCACCAGCGC CGCCGTTTTG 1560 GAGGCTTTGA GGGAGGCTCA TCCCATAGTG GACCGCATCC 1600 TCCAGTACCG GGAGCTTTCC AAGCTCAAGG GAACGTACAT 1640 CGATCCCTTG CCCGCCCTGG TCCACCCCAA GACGAACCGC 1680 CTCCACACCC GTTTCAACCA GACGGCCACC GCCACGGGGA 1720 GGCTTAGCAG CTCGGATCCC AACCTGCAAA ATATCCCCGT 1760 GCGCACCCCT TTAGGCCAGC GGATCCGCCG GGCCTTCGTG 1800 GCCGAGGAGG GGTGGAGGCT GGTGGTTTTG GACTACAGCC 1840 AGATTGAGCT CAGGGTCCTG GCGCACCTTT CCGGGGACGA 1880 GAACCTGATC CGGGTCTTCC AAGAGGGCCA GGACATCCAC 1920 ACCCAGACGG CCAGCTGGAT GTTCGGCGTG CCCCCAGAGG 1960 CCGTGGATTC CCTGATGCGC CGGGCGGCCA AGACCATCAA 2000 CTTCGGCGTC CTCTACGGCA TGTCCGCCCA CCGGCTTTCG 2040 GGAGAGCTGG CCATCCCCTA CGAGGAAGCG GTGGCCTTCA 2080 TCGAGCGGTA TTTCCAGAGC TACCCCAAGG TACGGGCCTG 2120 GATTGAGAAA ACCCTGGCGG AAGGACGGGA GCGGGGCTAT 2160 GTGGAAACCC TCTTTGGCCG CCGGCGCTAT GTGCCCGACT 2200 TGGCTTCCCG GGTGAAGAGC ATCCGGGAGG CAGCGGAGCG 2240 CATGGCCTTC AACATGCCGG TCCAGGGGAC CGCCGCGGAT 2280 TTGATGAAAC TGGCCATGGT GAAGCTCTTT CCCAGGCTTC 2320 AGGAGCTGGG GGCCAGGATG CTTTTGCAGG TGCACGACGA 2360 ACTGGTCCTC GAGGCTCCCA AGGAGCAAGC GGAGGAAGTC 2400 GCCCAGGAGG CCAAGCGGAC CATGGAGGAG GTGTGGCCCC 2440 TGAAGGTGCC CTTGGAGGTG GAGGTGGGTA TCGGGGAGGA 2480 CTGGCTTTCC GCCAAGGCCT AGTCGAC 2507
In another embodiment, the invention provides a nucleic acid of SEQ ID
NO: 6, a derivative nucleic acid related to Thermus scotoductus, strain Vi7a, having GAC (encoding Asp) in place of GGG (encoding Gly) at positions 136- 138. SEQ ID NO:6 is provided below.
ATGAGGGCGA TGCTGCCCCT CTTTGAGCCC AAGGGCCGGG 40
TGCTTCTGGT GGACGGCCAC CACCTGGCCT ACCGTACCTT 80
TTTTGCCCTG AAGGGCCTCA CCACCAGCCG CGGGGAGCCG 120
GTCCAGGCGG TGTACGACTT TGCCAAGAGC CTTTTGAAGG 160
CGCTAAGGGA AGACGGGGAT GTGGTGATCG TGGTGTTTGA 200 CGCCAAGGCC CCCTCCTTCC GCCACCAGAC CTACGAGGCC 240
TACAAGGCGG GGCGGGCTCC CACCCCCGAG GACTTTCCCC 280
GGCAGCTTGC CCTTATCAAG GAGATGGTGG ACCTTTTGGG 320
CCTGGAGCGC CTCGAAGTGC CGGGTTTTGA GGCGGATGAC 360
GTCCTGGCCA CCCTGGCCAA GAAGGCGGAA AAGGAAGGCT 400 ACGAGGTGCG CATCCTCACC GCGGACCGGG ACCTTTACCA 440
GCTTCTTTCG GACCGAATCT CCATCCTTCA CCCGGAGGGT 480
TACCTGATTA CCCCGGAGTG GCTTTGGGAG AAGTATGGGC 520
TTAAGCCTTC CCAGTGGGTG GACTACCGGG CCTTGGCCGG 560 GGACCCTTCC GACAACATCC CCGGCGTGAA GGGCATCGGG 600 GAGAAGACGG CGGCCAAGCT GATCCGGGAG TGGGGAAGCC 640 TGGAAAACCT TCTTAAGCAC CTGGAACAGG TGAAACCTGC 680 CTCCGTGCGG GAGAAGATCC TTAGCCACAT GGAGGACCTC 720 AAGCTATCCC TGGAGCTTTC CCGGGTGCAC ACGGAGTTGC 760 CCCTTCAGGT GGACTTCGCC CGGCGCCGGG AGCCGGACCG 800 GGAAGGGCTT AAGGCCTTTT TGGAGAGGCT GGAGTTCGGA 840 AGCCTCCTCC ACGAGTTCGG CCTGTTGGAA AGCCCGGTGG 880 CGGCGGAGGA AGCTCCCTGG CCGCCCCCCG AGGGAGCCTT 920 CGTGGGGTAC GTTCTTTCCC GCCCCGAGCC CATGTGGGCG 960 GAGCTTAACG CCTTGGCCGC CGCCTGGGAG GGAAGGGTTT 1000 ACCGGGCGGA GGATCCCTTG GAGGCCTTGC GGGGGCTTGG 1040 GGAGGTGAGG GGGCTTTTGG CCAAGGACCT GGCGGTGCTG 1080 GCCCTGAGGG AAGGGATTGC CCTGGCACCG GGCGACGACC 1120 CCATGCTCCT CGCCTACCTC CTGGATCCTT CCAACACCGC 1160 CCCCGAAGGG GTAGCCCGGC GCTACGGGGG GGAGTGGACC 1200 GAGGAGGCGG GGGAAAGGGC GCTGCTTTCC GAAAGGCTTT 1240 ACGCCGCCCT CCTGGAGCGG CTTAAGGGGG AGGAGAGGCT 1280 TCTTTGGCTT TACGAGGAGG TGGAAAAGCC CCTTTCGCGG 1320 GTCCTGGCCC ACATGGAGGC CACGGGGGTA TGGTTGGATG 1360 TGGCCTACTT GAAGGCCCTT TCCCTGGAGG TGGAGGCGGA 1400 GCTCAGGCGC CTCGAGGAGG AGGTCCACCG ACTGGCCGGG 1440 CATCCTTTCA ACCTGAACTC CCGGGACCAG CTGGAAAGGG 1480 TCCTCTTTGA CGAGCTTGGG CTTCCCGCCA TCGGCAAGAC 1520 GGAGAAGACG GGTAAGCGTT CCACCAGCGC CGCCGTTTTG 1560 GAGGCTTTGA GGGAGGCTCA TCCCATAGTG GACCGCATCC 1600 TCCAGTACCG GGAGCTTTCC AAGCTCAAGG GAACGTACAT 1640 CGATCCCTTG CCCGCCCTGG TCCACCCCAA GACGAACCGC 1680 CTCCACACCC GTTTCAACCA GACGGCCACC GCCACGGGGA 1720 GGCTTAGCAG CTCGGATCCC AACCTGCAAA ATATCCCCGT 1760 GCGCACCCCT TTAGGCCAGC GGATCCGCCG GGCCTTCGTG 1800 GCCGAGGAGG GGTGGAGGCT GGTGGTTTTG GACTACAGCC 1840 AGATTGAGCT CAGGGTCCTG GCGCACCTTT CCGGGGACGA 1880 GAACCTGATC CGGGTCTTCC AAGAGGGCCA GGACATCCAC 1920
ACCCAGACGG CCAGCTGGAT GTTCGGCGTG CCCCCAGAGG 1960 CCGTGGATTC CCTGATGCGC CGGGCGGCCA AGACCATCAA 2000
CTACGGCGTC CTCTACGGCA TGTCCGCCCA CCGGCTTTCG 2040 GGAGAGCTGG CCATCCCCTA CGAGGAAGCG GTGGCCTTCA 2080
TCGAGCGGTA TTTCCAGAGC TTCCCCAAGG TACGGGCCTG 2120
GATTGAGAAA ACCCTGGCGG AAGGACGGGA GCGGGGCTAT 2160
GTGGAAACCC TCTTTGGCCG CCGGCGCTAT GTGCCCGACT 2200
TGGCTTCCCG GGTGAAGAGC ATCCGGGAGG CAGCGGAGCG 2240 CATGGCCTTC AACATGCCGG TCCAGGGGAC CGCCGCGGAT 2280
TTGATGAAAC TGGCCATGGT GAAGCTCTTT CCCAGGCTTC 2320
AGGAGCTGGG GGCCAGGATG CTTTTGCAGG TGCACGACGA 2360
ACTGGTCCTC GAGGCTCCCA AGGAGCAAGC GGAGGAAGTC 2400
GCCCAGGAGG CCAAGCGGAC CATGGAGGAG GTGTGGCCCC 2440 TGAAGGTGCC CTTGGAGGTG GAGGTGGGTA TCGGGGAGGA 2480
CTGGCTTTCC GCCAAGGCCT AGTCGAC 2507
In another embodiment, the invention provides a nucleic acid of SEQ ID NO: 7, a derivative nucleic acid related to Thermus scotoductus, strain X-l, having TAC (encoding Tyr) in place of TTC (encoding Phe) at positions 2002- 04. SEQ ID NO:7 is provided below:
ATGAGGGCGA TGCTGCCCCT CTTTGAGCCC AAGGGCCGGG 40
TGCTTCTGGT GGACGGCCAC CACCTGGCCT ACCGTACCTT 80
TTTTGCCCTG AAGGGCCTCA CCACCAGCCG CGGGGAGCCG 120 GTCCAGGCGG TGTACGGGTT TGCCAAGAGC CTTTTGAAGG 160
CGCTAAGGGA AGACGGGGAT GTGGTGATCG TGGTGTTTGA 200
CGCCAAGGCC CCCTCCTTCC GCCACCAGAC CTACGAGGCC 240
TACAAGGCGG GGCGGGCTCC CACCCCCGAG GACTTTCCCC 280
GGCAGCTTGC CCTTATCAAG GAGATGGTGG ACCTTTTGGG 320 CCTGGAGCGC CTCGAGGTGC CGGGCTTTGA GGCGGATGAC 360
GTCCTGGCTA CCCTGGCCAA GAAGGCGGAA AAGGAAGGCT 400
ACGAGGTGCG CATCCTCACC GCGGACCGGG ACCTTTACCA 440
GCTTCTTTCG GAGCGAATCT CCATCCTTCA CCCGGAGGGT 480
TACCTGATCA CCCCGGAGTG GCTTTGGGAG AAGTATGGGC 520 TTAAGCCTTC CCAGTGGGTG GACTACCGGG CCTTGGCCGG 560 GGACCCTTCC GACAACATCC CCGGCGTGAA GGGCATCGGG 600 GAGAAGACGG CGGCCAAGCT GATCCGGGAG TGGGGAAGCC 640 TGGAAAACCT TCTTAAGCAC CTGGAACAGG TGAAACCTGC 680 CTCCGTGCGG GAGAAGATCC TTAGCCACAT GGAGGACCTC 720 AAGCTATCCC TGGAGCTATC CCGGGTGCGC ACGGACTTGC 760 CCCTTCAGGT GGACTTCGCC CGGCGCCGGG AGCCGGACCG 800 GGAGGGGCTT AAGGCCTTTT TGGAGAGGCT GGAGTTCGGA 840 AGCCTCCTCC ACGAGTTCGG CCTGTTGGAA AGCCCGGTGG 880 CGGCGGAGGA AGCTCCCTGG CCGCCCCCCG AGGGAGCCTT 920 CGTGGGGTAC GTTCTTTCCC GCCCCGAGCC CATGTGGGCG 960 GAGCTTAACG CCTTGGCCGC CGCCTGGGAG GGAAGGGTTT 1000 ACCGGGCGGA GGATCCCTTG GAGGCCTTGC GGGGGCTTGG 1040 GGAGGTGAGG GGGCTTTTGG CCAAGGACCT GGCGGTGCTG 1080 GCCCTGAGGG AAGGGATTGC CCTGGCACCG GGCGACGACC 1120 CCATGCTCCT CGCCTACCTC CTGGATCCTT CCAACACCGC 1160 CCCCGAAGGG GTAGCCCGGC GCTACGGGGG GGAGTGGACC 1200 GAGGAGGCGG GGGAAAGGGC GTTGCTTTCC GAAAGGCTTT Ϊ240 ACGCCGCCCT CCTGGAGCGG CTTAAGGGGG AGGAGAGGCT 1280 TCTTTGGCTT TACGAGGAGG TGGAAAAGCC CCTTTCGCGG 1320 GTCCTGGCCC ACATGGAGGC CACGGGGGTA CGGTTGGATG 1360 TGGCCTACTT AAAGGCCCTT TCCCTGGAGG TGGAGGCGGA 1400 GCTCAGGCGC CTCGAGGAGG AGGTCCACCG CCTGGCCGGG 1440 CATCCTTTCA ACCTGAACTC CCGGGACCAG CTGGAAAGGG 1480 TCCTCTTTGA CGAGCTTGGG CTTCCCGCCA TCGGCAAGAC 1520 GGAGAAGACG GGCAAGCGCT CCACCAGCGC CGCCGTTTTG 1560 GAGGCCTTGC GGGAGGCTCA TCCCATCGTG GACCGCATCC 1600 TTCAGTACCG GGAGCTTTCC AAGCTCAAGG GAACCTACAT 1640 CGATCCCTTG CCTGCCCTGG TCCACCCCAA GACGAACCGC 1680 CTCCACACCC GTTTCAACCA GACGGCCACC GCCACGGGGA 1720 GGCTTAGCAG CTCGGATCCC AACCTGCAAA ATATCCCCGT 1760 GCGCACCCCT TTGGGCCAGC GGATCCGCCG GGCCTTCGTG 1800 GCCGAGGAGG GGTGGAGGCT GGTGGTTTTG GACTACAGCC 1840 AGATTGAGCT CAGGGTCCTG GCGCACCTTT CCGGGGACGA 1880 GAACCTAATC CGGGTCTTCC AGGAGGGCCA GGACATCCAC 1920 ACCCAGACGG CCAGCTGGAT GTTCGGCGTG CCCCCAGAGG 1960 CCGTGGATTC CCTGATGCGT CGGGCGGCCA AGACCATCAA 2000 CTACGGCGTC CTCTACGGCA TGTCCGCCCA CCGGCTTTCG 2040 GGAGAGCTGG CCATCCCCTA CGAGGAGGCG GTGGCCTTCA 2080 TCGAGCGGTA TTTCCAGAGC TACCCCAAGG TGCGGGCCTG 2120 GATTGAGAAA ACCCTGGCGG AAGGACGGGA ACGGGGCTAT 2160 GTGGAAACCC TCTTTGGCCG CCGGCGCTAC GTGCCCGACT 2200 TGGCTTCCCG GGTGAAGAGC ATCCGGGAGG CAGCGGAGCG 2240 CATGGCCTTC AACATGCCGG TCCAGGGGAC CGCCGCGGAT 2280 TTGATGAAAC TGGCCATGGT GAAGCTCTTT CCCAGGCTTC 2320 AGGAGCTGGG GGCCAGGATG CTTTTGCAGG TGCACGACGA 2360 ACTGGTCCTC GAGGCTCCCA AGGAGCAAGC GGAGGAAGTC 2400 GCCCAGGAGG CCAAGCGGAC CATGGAGGAG GTGTGGCCCC 2440 TGAAGGTGCC CTTGGAGGTG GAAGTGGGCA TCGGGGAGGA 2480 CTGGCTTTCC GCCAAGGCCT AG 2502
In another embodiment, the invention provides a nucleic acid of SEQ ID NO:8, a derivative nucleic acid related to Thermus scotoductus, strain SM3, having TAC (encoding Tyr) in place of TTC (encoding Phe) at positions 2002- 04. SEQ ED NO:8 is provided below:
ATGAGGGCGA TGCTGCCCCT CTTTGAGCCC AAGGGCCGGG 40
TGCTTCTGGT GGACGGCCAC CACCTGGCCT ACCGTACCTT 80
TTTTGCCCTG AAGGGCCTCA CCACCAGCCG CGGGGAGCCG 120
GTCCAGGCGG TGTACGGGTT TGCCAAGAGC CTTTTGAAGG 160
CGCTAAGGGA AGACGGGGAT GTGGTGATCG TGGTGTTTGA 200 CGCCAAGGCC CCCTCCTTCC GCCACCAGAC CTACGAGGCC 240
TACAAGGCGG GGCGGGCTCC CACCCCCGAG GACTTTCCCC 280
GGCAGCTTGC CCTTATCAAG GAGATGGTGG ACCTTTTGGG 320
CCTGGAGCGC CTCGAAGTGC CGGGTTTTGA GGCGGATGAC 360
GTCCTGGCCA CCCTGGCCAA GAAGGCGGAA AAGGAAGGCT 400 ACGAGGTGCG CATCCTCACC GCGGACCGGG ACCTTTACCA 440
GCTTCTTTCG GACCGAATCT CCATCCTTCA CCCGGAGGGT 480
TACCTGATCA CCCCGGAGTG GCTTTGGGAG AAGTATGGGC 520
TTAAGCCTTC CCAGTGGGTG GACTACCGGG CCTTGGCCGG 560
GGACCCTTCC GACAACATCC CCGGCGTGAA GGGCATCGGG 600 GAGAAGACGG CGGCCAAGCT GATCCGGGAG TGGGGAAGCC 640 TGGAAAACCT TCTTAAGCAC CTGGAACAGG TGAAACCTGC 680 CTCCGTGCGG GAGAAGATCC TTAGCCACAT GGAGGACCTC 720 AAGCTATCCC TGGAGCTTTC CCGGGTGCAC ACGGAGTTGC 760 CCCTTCAGGT GGACTTCGCC CGGCGCCGGG AGCCGGACCG 800 GGAAGGGCTT AAGGCCTTTT TGGAGAGGCT GGAGTTCGGA 840 AGCCTCCTCC ACGAGTTCGG CCTGTTGGAA AGCCCGGTGG 880 CGGCGGAGGA AGCTCCCTGG CCGCCCCCCG AGGGAGCCTT 920 CGTGGGGTAC GTTCTTTCCC GCCCCGAGCC CATGTGGGCG 960 GAGCTTAACG CCTTGGCCGC CGCCTGGGAG GGAAGGGTTT 1000 ACCGGGCGGA GGATCCCTTG GAGGCCTTGC GGGGGCTTGG 1040 GGAGGTGAGG GGGCTTTTGG CCAAGGACCT GGCGGTGCTG 1080 GCCCTGAGGG AAGGGATTGC CCTGGCACAG GGCGACGACC 1120 CCATGCTCCT CGCCTACCTC CTGGATCCTT CCAACACCGC 1160 CCCCGAAGGG GTAGCCCGGC GCTACGGGGG GGAGTGGACC 1200 GAGGAGGCGG GGGAAAGGGC GCTGCTTTCC GAAAGGCTTT 1240 ACGCCGCCCT CCTGGAGCGG CTTAAGGGGG AGGAGAGGCT 1280 TCTTTGGCTT TACGAGGAGG TGGAAAAGCC CCTTTCGCGG 1320 GTCCTGGCCC ACATGGAGGC CACGGGGGTA TGGTTGGATG 1360 TGGCCTACTT GAAGGCCCTT TCCCTGGAGG TGGAGGCGGA 1400 GCTCAGGCGC CTCGAGGAGG AGGTCCACCG ACTGGCCGGG 1440 CATCCTTTCA ACCTGAACTC CCGGGACCAG CTGGAAAGGG 1480 TCCTCTTTGA CGAGCTTGGG CTTCCCGCCA TCGGCAAGAC 1520 GGAGAAGACG GGTAAGCGTT CCACCAGCGC CGCCGTTTTG 1560 GAGGCTTTGA GGGAGGCTCA TCCCATAGTG GACCGCATCC 1600 TCCAGTACCG GGAGCTTTCC AAGCTCAAGG GAACGTACAT 1640 CGATCCCTTG CCCGCCCTGG TCCACCCCAA GACGAACCGC 1680 CTCCACACCC GTTTCAACCA GACGGCCACC GCCACGGGGA 1720 GGCTTAGCAG CTCGGATCCC AACCTGCAAA ATATCCCCGT 1760 GCGCACCCCT TTAGGCCAGC GGATCCGCCG GGCCTTCGTG 1800 GCCGAGGAGG GGTGGAGGCT GGTGGTTTTG GACTACAGCC 1840 AGATTGAGCT CAGGGTCCTG GCGCACCTTT CCGGGGACGA 1880 GAACCTGATC CGGGTCTTCC AAGAGGGCCA GGACATCCAC 1920 ACCCAGACGG CCAGCTGGAT GTTCGGCGTG CCCCCAGAGG 1960 CCGTGGATTC CCTGATGCGC CGGGCGGCCA AGACCATCAA 2000 CTACGGCGTC CTCTACGGCA TGTCCGCCCA CCGGCTTTCG 2040 GGAGAGCTGG CCATCCCCTA CGAGGAAGCG GTGGCCTTCA 2080 TCGAGCGGTA TTTCCAGAGC TACCCCAAGG TACGGGCCTG 2120 GATTGAGAAA ACCCTGGCGG AAGGACGGGA GCGGGGCTAT 2160 GTGGAAACCC TCTTTGGCCG CCGGCGCTAT GTGCCCGACT 2200 TGGCTTCCCG GGTGAAGAGC ATCCGGGAGG CAGCGGAGCG 2240 CATGGCCTTC AACATGCCGG TCCAGGGGAC CGCCGCGGAT 2280 TTGATGAAAC TGGCCATGGT GAAGCTCTTT CCCAGGCTTC 2320 AGGAGCTGGG GGCCAGGATG CTTTTGCAGG TGCACGACGA 2360 ACTGGTCCTC GAGGCTCCCA AGGAGCAAGC GGAGGAAGTC 2400 GCCCAGGAGG CCAAGCGGAC CATGGAGGAG GTGTGGCCCC 2440 TGAAGGTGCC CTTGGAGGTG GAGGTGGGTA TCGGGGAGGA 2480 CTGGCTTTCC GCCAAGGCCT AGTCGAC 2507
In another embodiment, the invention provides a nucleic acid of SEQ ID NO: 9, a derivative nucleic acid related to Thermus scotoductus, strain Vi7a, having TAC (encoding Tyr) in place of TTC (encoding Phe) at positions 2101- 03. SEQ ED NO:9 is provided below:
ATGAGGGCGA TGCTGCCCCT CTTTGAGCCC AAGGGCCGGG 40
TGCTTCTGGT GGACGGCCAC CACCTGGCCT ACCGTACCTT 80
TTTTGCCCTG AAGGGCCTCA CCACCAGCCG CGGGGAGCCG 120 GTCCAGGCGG TGTACGGGTT TGCCAAGAGC CTTTTGAAGG 160
CGCTAAGGGA AGACGGGGAT GTGGTGATCG TGGTGTTTGA 200
CGCCAAGGCC CCCTCCTTCC GCCACCAGAC CTACGAGGCC 240
TACAAGGCGG GGCGGGCTCC CACCCCCGAG GACTTTCCCC 280
GGCAGCTTGC CCTTATCAAG GAGATGGTGG ACCTTTTGGG 320 CCTGGAGCGC CTCGAAGTGC CGGGTTTTGA GGCGGATGAC 360
GTCCTGGCCA CCCTGGCCAA GAAGGCGGAA AAGGAAGGCT 400
ACGAGGTGCG CATCCTCACC GCGGACCGGG ACCTTTACCA 440
GCTTCTTTCG GACCGAATCT CCATCCTTCA CCCGGAGGGT 480 TACCTGATTA CCCCGGAGTG GCTTTGGGAG AAGTATGGGC 520 TTAAGCCTTC CCAGTGGGTG GACTACCGGG CCTTGGCCGG 560 GGACCCTTCC GACAACATCC CCGGCGTGAA GGGCATCGGG 600 GAGAAGACGG CGGCCAAGCT GATCCGGGAG TGGGGAAGCC 640 TGGAAAACCT TCTTAAGCAC CTGGAACAGG TGAAACCTGC 680 CTCCGTGCGG GAGAAGATCC TTAGCCACAT GGAGGACCTC 720 AAGCTATCCC TGGAGCTTTC CCGGGTGCAC ACGGAGTTGC 760 CCCTTCAGGT GGACTTCGCC CGGCGCCGGG AGCCGGACCG 800 GGAAGGGCTT AAGGCCTTTT TGGAGAGGCT GGAGTTCGGA 840 AGCCTCCTCC ACGAGTTCGG CCTGTTGGAA AGCCCGGTGG 880 CGGCGGAGGA AGCTCCCTGG CCGCCCCCCG AGGGAGCCTT 920 CGTGGGGTAC GTTCTTTCCC GCCCCGAGCC CATGTGGGCG 960 GAGCTTAACG CCTTGGCCGC CGCCTGGGAG GGAAGGGTTT 1000 ACCGGGCGGA GGATCCCTTG GAGGCCTTGC GGGGGCTTGG 1040 GGAGGTGAGG GGGCTTTTGG CCAAGGACCT GGCGGTGCTG 1080 GCCCTGAGGG AAGGGATTGC CCTGGCACCG GGCGACGACC 1120 CCATGCTCCT CGCCTACCTC CTGGATCCTT CCAACACCGC 1160 CCCCGAAGGG GTAGCCCGGC GCTACGGGGG GGAGTGGACC 1200 GAGGAGGCGG GGGAAAGGGC GCTGCTTTCC GAAAGGCTTT 1240 ACGCCGCCCT CCTGGAGCGG CTTAAGGGGG AGGAGAGGCT 1280 TCTTTGGCTT TACGAGGAGG TGGAAAAGCC CCTTTCGCGG 1320 GTCCTGGCCC ACATGGAGGC CACGGGGGTA TGGTTGGATG 1360 TGGCCTACTT GAAGGCCCTT TCCCTGGAGG TGGAGGCGGA 1400 GCTCAGGCGC CTCGAGGAGG AGGTCCACCG ACTGGCCGGG 1440 CATCCTTTCA ACCTGAACTC CCGGGACCAG CTGGAAAGGG 1480 TCCTCTTTGA CGAGCTTGGG CTTCCCGCCA TCGGCAAGAC 1520 GGAGAAGACG GGTAAGCGTT CCACCAGCGC CGCCGTTTTG 1560 GAGGCTTTGA GGGAGGCTCA TCCCATAGTG GACCGCATCC 1600 TCCAGTACCG GGAGCTTTCC AAGCTCAAGG GAACGTACAT 1640 CGATCCCTTG CCCGCCCTGG TCCACCCCAA GACGAACCGC 1680 CTCCACACCC GTTTCAACCA GACGGCCACC GCCACGGGGA 1720 GGCTTAGCAG CTCGGATCCC AACCTGCAAA ATATCCCCGT 1760 GCGCACCCCT TTAGGCCAGC GGATCCGCCG GGCCTTCGTG 1800 GCCGAGGAGG GGTGGAGGCT GGTGGTTTTG GACTACAGCC 1840 AGATTGAGCT CAGGGTCCTG GCGCACCTTT CCGGGGACGA 1880 GAACCTGATC CGGGTCTTCC AAGAGGGCCA GGACATCCAC 1920 ACCCAGACGG CCAGCTGGAT GTTCGGCGTG CCCCCAGAGG 1960 CCGTGGATTC CCTGATGCGC CGGGCGGCCA AGACCATCAA 2000 . CTACGGCGTC CTCTACGGCA TGTCCGCCCA CCGGCTTTCG 2040 GGAGAGCTGG CCATCCCCTA CGAGGAAGCG GTGGCCTTCA 2080 TCGAGCGGTA TTTCCAGAGC TACCCCAAGG TACGGGCCTG 2120 GATTGAGAAA ACCCTGGCGG AAGGACGGGA GCGGGGCTAT 2160 GTGGAAACCC TCTTTGGCCG CCGGCGCTAT GTGCCCGACT 2200 TGGCTTCCCG GGTGAAGAGC ATCCGGGAGG CAGCGGAGCG 2240 CATGGCCTTC AACATGCCGG TCCAGGGGAC CGCCGCGGAT 2280 TTGATGAAAC TGGCCATGGT GAAGCTCTTT CCCAGGCTTC 2320 AGGAGCTGGG GGCCAGGATG CTTTTGCAGG TGCACGACGA 2360 ACTGGTCCTC GAGGCTCCCA AGGAGCAAGC GGAGGAAGTC 2400 GCCCAGGAGG CCAAGCGGAC CATGGAGGAG GTGTGGCCCC 2440 TGAAGGTGCC CTTGGAGGTG GAGGTGGGTA TCGGGGAGGA 2480 CTGGCTTTCC GCCAAGGCCT AGTCGAC 2507
In another embodiment, the invention provides a nucleic acid of SEQ ED
NO: 10, a derivative nucleic acid related to Thermus scotoductus, strain X-l, having GAC (encoding Asp) in place of GGG (encoding Gly) at positions 136- 138 and having TAC (encoding Tyr) in place of TTC (encoding Phe) at positions 2002-04. SEQ ED NO: 10 is provided below:
ATGAGGGCGA TGCTGCCCCT CTTTGAGCCC AAGGGCCGGG 40
TGCTTCTGGT GGACGGCCAC CACCTGGCCT ACCGTACCTT 80
TTTTGCCCTG AAGGGCCTCA CCACCAGCCG CGGGGAGCCG 120 GTCCAGGCGG TGTACGACTT TGCCAAGAGC CTTTTGAAGG 160
CGCTAAGGGA AGACGGGGAT GTGGTGATCG TGGTGTTTGA 200
CGCCAAGGCC CCCTCCTTCC GCCACCAGAC CTACGAGGCC 240
TACAAGGCGG GGCGGGCTCC CACCCCCGAG GACTTTCCCC 280
GGCAGCTTGC CCTTATCAAG GAGATGGTGG ACCTTTTGGG 320 CCTGGAGCGC CTCGAGGTGC CGGGCTTTGA GGCGGATGAC 360 GTCCTGGCTA CCCTGGCCAA GAAGGCGGAA AAGGAAGGCT 400 ACGAGGTGCG CATCCTCACC GCGGACCGGG ACCTTTACCA 440 GCTTCTTTCG GAGCGAATCT CCATCCTTCA CCCGGAGGGT 480 TACCTGATCA CCCCGGAGTG GCTTTGGGAG AAGTATGGGC 520 TTAAGCCTTC CCAGTGGGTG GACTACCGGG CCTTGGCCGG 560 GGACCCTTCC GACAACATCC CCGGCGTGAA GGGCATCGGG 600 GAGAAGACGG CGGCCAAGCT GATCCGGGAG TGGGGAAGCC 640 TGGAAAACCT TCTTAAGCAC CTGGAACAGG TGAAACCTGC 680 CTCCGTGCGG GAGAAGATCC TTAGCCACAT GGAGGACCTC 720 AAGCTATCCC TGGAGCTATC CCGGGTGCGC ACGGACTTGC 760 CCCTTCAGGT GGACTTCGCC CGGCGCCGGG AGCCGGACCG 800 GGAGGGGCTT AAGGCCTTTT TGGAGAGGCT GGAGTTCGGA 840 AGCCTCCTCC ACGAGTTCGG CCTGTTGGAA AGCCCGGTGG 880 CGGCGGAGGA AGCTCCCTGG CCGCCCCCCG AGGGAGCCTT 920 CGTGGGGTAC GTTCTTTCCC GCCCCGAGCC CATGTGGGCG 960 GAGCTTAACG CCTTGGCCGC CGCCTGGGAG GGAAGGGTTT 1000 ACCGGGCGGA GGATCCCTTG GAGGCCTTGC GGGGGCTTGG 1040 GGAGGTGAGG GGGCTTTTGG CCAAGGACCT GGCGGTGCTG 1080 GCCCTGAGGG AAGGGATTGC CCTGGCACCG GGCGACGACC 1120 CCATGCTCCT CGCCTACCTC CTGGATCCTT CCAACACCGC 1160 CCCCGAAGGG GTAGCCCGGC GCTACGGGGG GGAGTGGACC 1200 GAGGAGGCGG GGGAAAGGGC GTTGCTTTCC GAAAGGCTTT 1240 ACGCCGCCCT CCTGGAGCGG CTTAAGGGGG AGGAGAGGCT 1280 TCTTTGGCTT TACGAGGAGG TGGAAAAGCC CCTTTCGCGG 1320 GTCCTGGCCC ACATGGAGGC CACGGGGGTA CGGTTGGATG 1360 TGGCCTACTT AAAGGCCCTT TCCCTGGAGG TGGAGGCGGA 1400 GCTCAGGCGC CTCGAGGAGG AGGTCCACCG CCTGGCCGGG 1440 CATCCTTTCA ACCTGAACTC CCGGGACCAG CTGGAAAGGG 1480 TCCTCTTTGA CGAGCTTGGG CTTCCCGCCA TCGGCAAGAC 1520 GGAGAAGACG GGCAAGCGCT CCACCAGCGC CGCCGTTTTG 1560 GAGGCCTTGC GGGAGGCTCA TCCCATCGTG GACCGCATCC 1600 TTCAGTACCG GGAGCTTTCC AAGCTCAAGG GAACCTACAT 1640 CGATCCCTTG CCTGCCCTGG TCCACCCCAA GACGAACCGC 1680 CTCCACACCC GTTTCAACCA GACGGCCACC GCCACGGGGA 1720 GGCTTAGCAG CTCGGATCCC AACCTGCAAA ATATCCCCGT 1760 GCGCACCCCT TTGGGCCAGC GGATCCGCCG GGCCTTCGTG 1800 GCCGAGGAGG GGTGGAGGCT GGTGGTTTTG GACTACAGCC 1840 AGATTGAGCT CAGGGTCCTG GCGCACCTTT CCGGGGACGA 1880 GAACCTAATC CGGGTCTTCC AGGAGGGCCA GGACATCCAC 1920 ACCCAGACGG CCAGCTGGAT GTTCGGCGTG CCCCCAGAGG 1960 CCGTGGATTC CCTGATGCGT CGGGCGGCCA AGACCATCAA 2000 CTACGGCGTC CTCTACGGCA TGTCCGCCCA CCGGCTTTCG 2040 GGAGAGCTGG CCATCCCCTA CGAGGAGGCG GTGGCCTTCA 2080 TCGAGCGGTA TTTCCAGAGC TACCCCAAGG TGCGGGCCTG 2120 GATTGAGAAA ACCCTGGCGG AAGGACGGGA ACGGGGCTAT 2160 GTGGAAACCC TCTTTGGCCG CCGGCGCTAC GTGCCCGACT 2200 TGGCTTCCCG GGTGAAGAGC ATCCGGGAGG CAGCGGAGCG 2240 CATGGCCTTC AACATGCCGG TCCAGGGGAC CGCCGCGGAT 2280 TTGATGAAAC TGGCCATGGT GAAGCTCTTT CCCAGGCTTC 2320 AGGAGCTGGG GGCCAGGATG CTTTTGCAGG TGCACGACGA 2360 ACTGGTCCTC GAGGCTCCCA AGGAGCAAGC GGAGGAAGTC 2400 GCCCAGGAGG CCAAGCGGAC CATGGAGGAG GTGTGGCCCC 2440 TGAAGGTGCC CTTGGAGGTG GAAGTGGGCA TCGGGGAGGA 2480 CTGGCTTTCC GCCAAGGCCT AG 2502
In another embodiment, the invention provides a nucleic acid of SEQ D NO: 11 , a derivative nucleic acid related to Thermus scotoductus, strain SM3, having GAC (encoding Asp) in place of GGG (encoding Gly) at positions 136- 138 and having TAC (encoding Tyr) in place of TTC (encoding Phe) at positions 2002-04. SEQ D NO:8 is provided below:
ATGAGGGCGA TGCTGCCCCT CTTTGAGCCC AAGGGCCGGG 40
TGCTTCTGGT GGACGGCCAC CACCTGGCCT ACCGTACCTT 80
TTTTGCCCTG AAGGGCCTCA CCACCAGCCG CGGGGAGCCG 120
GTCCAGGCGG TGTACGACTT TGCCAAGAGC CTTTTGAAGG 160
CGCTAAGGGA AGACGGGGAT GTGGTGATCG TGGTGTTTGA 200 CGCCAAGGCC CCCTCCTTCC GCCACCAGAC CTACGAGGCC 240
TACAAGGCGG GGCGGGCTCC CACCCCCGAG GACTTTCCCC 280
GGCAGCTTGC CCTTATCAAG GAGATGGTGG ACCTTTTGGG 320
CCTGGAGCGC CTCGAAGTGC CGGGTTTTGA GGCGGATGAC 360
GTCCTGGCCA CCCTGGCCAA GAAGGCGGAA AAGGAAGGCT 400 ACGAGGTGCG CATCCTCACC GCGGACCGGG ACCTTTACCA 440 GCTTCTTTCG GACCGAATCT CCATCCTTCA CCCGGAGGGT 480 TACCTGATCA CCCCGGAGTG GCTTTGGGAG AAGTATGGGC 520 TTAAGCCTTC CCAGTGGGTG GACTACCGGG CCTTGGCCGG 560 GGACCCTTCC GACAACATCC CCGGCGTGAA GGGCATCGGG 600 GAGAAGACGG CGGCCAAGCT GATCCGGGAG TGGGGAAGCC 640 TGGAAAACCT TCTTAAGCAC CTGGAACAGG TGAAACCTGC 680 CTCCGTGCGG GAGAAGATCC TTAGCCACAT GGAGGACCTC 720 AAGCTATCCC TGGAGCTTTC CCGGGTGCAC ACGGAGTTGC 760 CCCTTCAGGT GGACTTCGCC CGGCGCCGGG AGCCGGACCG 800 GGAAGGGCTT AAGGCCTTTT TGGAGAGGCT GGAGTTCGGA 840 AGCCTCCTCC ACGAGTTCGG CCTGTTGGAA AGCCCGGTGG 880 CGGCGGAGGA AGCTCCCTGG CCGCCCCCCG AGGGAGCCTT 920 CGTGGGGTAC GTTCTTTCCC GCCCCGAGCC CATGTGGGCG 960 GAGCTTAACG CCTTGGCCGC CGCCTGGGAG GGAAGGGTTT 1000 ACCGGGCGGA GGATCCCTTG GAGGCCTTGC GGGGGCTTGG 1040 GGAGGTGAGG GGGCTTTTGG CCAAGGACCT GGCGGTGCTG 1080 GCCCTGAGGG AAGGGATTGC CCTGGCACAG GGCGACGACC 1120 CCATGCTCCT CGCCTACCTC CTGGATCCTT CCAACACCGC 1160 CCCCGAAGGG GTAGCCCGGC GCTACGGGGG GGAGTGGACC 1200 GAGGAGGCGG GGGAAAGGGC GCTGCTTTCC GAAAGGCTTT 1240 ACGCCGCCCT CCTGGAGCGG CTTAAGGGGG AGGAGAGGCT 1280 TCTTTGGCTT TACGAGGAGG TGGAAAAGCC CCTTTCGCGG 1320 GTCCTGGCCC ACATGGAGGC CACGGGGGTA TGGTTGGATG 1360 TGGCCTACTT GAAGGCCCTT TCCCTGGAGG TGGAGGCGGA 1400 GCTCAGGCGC CTCGAGGAGG AGGTCCACCG ACTGGCCGGG 1440 CATCCTTTCA ACCTGAACTC CCGGGACCAG CTGGAAAGGG 1480 TCCTCTTTGA CGAGCTTGGG CTTCCCGCCA TCGGCAAGAC 1520 GGAGAAGACG GGTAAGCGTT CCACCAGCGC CGCCGTTTTG 1560 GAGGCTTTGA GGGAGGCTCA TCCCATAGTG GACCGCATCC 1600 TCCAGTACCG GGAGCTTTCC AAGCTCAAGG GAACGTACAT 1640 CGATCCCTTG CCCGCCCTGG TCCACCCCAA GACGAACCGC 1680 CTCCACACCC GTTTCAACCA GACGGCCACC GCCACGGGGA 1720 GGCTTAGCAG CTCGGATCCC AACCTGCAAA ATATCCCCGT 1760 GCGCACCCCT TTAGGCCAGC GGATCCGCCG GGCCTTCGTG 1800 GCCGAGGAGG GGTGGAGGCT GGTGGTTTTG GACTACAGCC 1840 AGATTGAGCT CAGGGTCCTG GCGCACCTTT CCGGGGACGA 1880 GAACCTGATC CGGGTCTTCC AAGAGGGCCA GGACATCCAC 1920 ACCCAGACGG CCAGCTGGAT GTTCGGCGTG CCCCCAGAGG 1960 CCGTGGATTC CCTGATGCGC CGGGCGGCCA AGACCATCAA 2000 CTACGGCGTC CTCTACGGCA TGTCCGCCCA CCGGCTTTCG 2040 GGAGAGCTGG CCATCCCCTA CGAGGAAGCG GTGGCCTTCA 2080 TCGAGCGGTA TTTCCAGAGC TACCCCAAGG TACGGGCCTG 2120 GATTGAGAAA ACCCTGGCGG AAGGACGGGA GCGGGGCTAT 2160 GTGGAAACCC TCTTTGGCCG CCGGCGCTAT GTGCCCGACT 2200 TGGCTTCCCG GGTGAAGAGC ATCCGGGAGG CAGCGGAGCG 2240 CATGGCCTTC AACATGCCGG TCCAGGGGAC CGCCGCGGAT 2280 TTGATGAAAC TGGCCATGGT GAAGCTCTTT CCCAGGCTTC 2320 AGGAGCTGGG GGCCAGGATG CTTTTGCAGG TGCACGACGA 2360
ACTGGTCCTC GAGGCTCCCA AGGAGCAAGC GGAGGAAGTC 2400
GCCCAGGAGG CCAAGCGGAC CATGGAGGAG GTGTGGCCCC 2440
TGAAGGTGCC CTTGGAGGTG GAGGTGGGTA TCGGGGAGGA 2480
CTGGCTTTCC GCCAAGGCCT AGTCGAC 2507
In another embodiment, the invention provides a nucleic acid of SEQ ED
NO: 12, a derivative nucleic acid related to Thermus scotoductus, strain Vi7a, having GAC (encoding Asp) in place of GGG (encoding Gly) at positions 136-
138 and having TAC (encoding Tyr) in place of TTC (encoding Phe) at positions 2101-03. SEQ ED NO:12 is provided below:
ATGAGGGCGA TGCTGCCCCT CTTTGAGCCC AAGGGCCGGG 40
TGCTTCTGGT GGACGGCCAC CACCTGGCCT ACCGTACCTT 80
TTTTGCCCTG AAGGGCCTCA CCACCAGCCG CGGGGAGCCG 120 GTCCAGGCGG TGTACGACTT TGCCAAGAGC CTTTTGAAGG 160
CGCTAAGGGA AGACGGGGAT GTGGTGATCG TGGTGTTTGA 200
CGCCAAGGCC CCCTCCTTCC GCCACCAGAC CTACGAGGCC 240
TACAAGGCGG GGCGGGCTCC CACCCCCGAG GACTTTCCCC 280 GGCAGCTTGC CCTTATCAAG GAGATGGTGG ACCTTTTGGG 320 CCTGGAGCGC CTCGAAGTGC CGGGTTTTGA GGCGGATGAC 360 GTCCTGGCCA CCCTGGCCAA GAAGGCGGAA AAGGAAGGCT 400 ACGAGGTGCG CATCCTCACC GCGGACCGGG ACCTTTACCA 440 GCTTCTTTCG GACCGAATCT CCATCCTTCA CCCGGAGGGT 480 TACCTGATTA CCCCGGAGTG GCTTTGGGAG AAGTATGGGC 520 TTAAGCCTTC CCAGTGGGTG GACTACCGGG CCTTGGCCGG 560 GGACCCTTCC GACAACATCC CCGGCGTGAA GGGCATCGGG 600 GAGAAGACGG CGGCCAAGCT GATCCGGGAG TGGGGAAGCC 640 TGGAAAACCT TCTTAAGCAC CTGGAACAGG TGAAACCTGC 680 CTCCGTGCGG GAGAAGATCC TTAGCCACAT GGAGGACCTC 720 AAGCTATCCC TGGAGCTTTC CCGGGTGCAC ACGGAGTTGC 760 CCCTTCAGGT GGACTTCGCC CGGCGCCGGG AGCCGGACCG 800 GGAAGGGCTT AAGGCCTTTT TGGAGAGGCT GGAGTTCGGA 840 AGCCTCCTCC ACGAGTTCGG CCTGTTGGAA AGCCCGGTGG 880 CGGCGGAGGA AGCTCCCTGG CCGCCCCCCG AGGGAGCCTT 920 CGTGGGGTAC GTTCTTTCCC GCCCCGAGCC CATGTGGGCG 960 GAGCTTAACG CCTTGGCCGC CGCCTGGGAG GGAAGGGTTT 1000 ACCGGGCGGA GGATCCCTTG GAGGCCTTGC GGGGGCTTGG 1040 GGAGGTGAGG GGGCTTTTGG CCAAGGACCT GGCGGTGCTG 1080 GCCCTGAGGG AAGGGATTGC CCTGGCACCG GGCGACGACC 1120 CCATGCTCCT CGCCTACCTC CTGGATCCTT CCAACACCGC 1160 CCCCGAAGGG GTAGCCCGGC GCTACGGGGG GGAGTGGACC 1200 GAGGAGGCGG GGGAAAGGGC GCTGCTTTCC GAAAGGCTTT 1240 ACGCCGCCCT CCTGGAGCGG CTTAAGGGGG AGGAGAGGCT 1280 TCTTTGGCTT TACGAGGAGG TGGAAAAGCC CCTTTCGCGG 1320 GTCCTGGCCC ACATGGAGGC CACGGGGGTA TGGTTGGATG 1360 TGGCCTACTT GAAGGCCCTT TCCCTGGAGG TGGAGGCGGA 1400 GCTCAGGCGC CTCGAGGAGG AGGTCCACCG ACTGGCCGGG 1440 CATCCTTTCA ACCTGAACTC CCGGGACCAG CTGGAAAGGG 1480 TCCTCTTTGA CGAGCTTGGG CTTCCCGCCA TCGGCAAGAC 1520 GGAGAAGACG GGTAAGCGTT CCACCAGCGC CGCCGTTTTG 1560 GAGGCTTTGA GGGAGGCTCA TCCCATAGTG GACCGCATCC 1600 TCCAGTACCG GGAGCTTTCC AAGCTCAAGG GAACGTACAT 1640 CGATCCCTTG CCCGCCCTGG TCCACCCCAA GACGAACCGC 1680 CTCCACACCC GTTTCAACCA GACGGCCACC GCCACGGGGA 1720 GGCTTAGCAG CTCGGATCCC AACCTGCAAA ATATCCCCGT 1760 GCGCACCCCT TTAGGCCAGC GGATCCGCCG GGCCTTCGTG 1800 GCCGAGGAGG GGTGGAGGCT GGTGGTTTTG GACTACAGCC 1840 AGATTGAGCT CAGGGTCCTG GCGCACCTTT CCGGGGACGA 1880 GAACCTGATC CGGGTCTTCC AAGAGGGCCA GGACATCCAC 1920 ACCCAGACGG CCAGCTGGAT GTTCGGCGTG CCCCCAGAGG 1960 CCGTGGATTC CCTGATGCGC CGGGCGGCCA AGACCATCAA 2000 CTACGGCGTC CTCTACGGCA TGTCCGCCCA CCGGCTTTCG 2040 GGAGAGCTGG CCATCCCCTA CGAGGAAGCG GTGGCCTTCA 2080 TCGAGCGGTA TTTCCAGAGC TACCCCAAGG TACGGGCCTG 2120 GATTGAGAAA ACCCTGGCGG AAGGACGGGA GCGGGGCTAT 2160 GTGGAAACCC TCTTTGGCCG CCGGCGCTAT GTGCCCGACT 2200 TGGCTTCCCG GGTGAAGAGC ATCCGGGAGG CAGCGGAGCG 2240 CATGGCCTTC AACATGCCGG TCCAGGGGAC CGCCGCGGAT 2280 TTGATGAAAC TGGCCATGGT GAAGCTCTTT CCCAGGCTTC 2320 AGGAGCTGGG GGCCAGGATG CTTTTGCAGG TGCACGACGA 2360 ACTGGTCCTC GAGGCTCCCA AGGAGCAAGC GGAGGAAGTC 2400 GCCCAGGAGG CCAAGCGGAC CATGGAGGAG GTGTGGCCCC 2440 TGAAGGTGCC CTTGGAGGTG GAGGTGGGTA TCGGGGAGGA 2480 CTGGCTTTCC GCCAAGGCCT AGTCGAC 2507
The substitution of TAC (encoding Tyr) for TTC (encoding Phe) at the indicated positions can reduce discrimination against ddNTP incorporation by DNA polymerase I. See, e.g., U.S. Patent 5,614,365 that is incorporated herein by reference. The substitution of GAC (encoding Asp) for GGG (encoding Gly) at the indicated positions removes the 5 '-3' exonuclease activity.
The nucleic acids of the invention have homology to portions of the nucleic acids encoding the thermostable DNA polymerases of Thermus aquaticus and Thermus thermophilus (see Figure 1). However, significant portions of the nucleic acid sequences of the present invention are distinct.
The invention also encompasses fragment and variant nucleic acids of SEQ ED NO: 1-12. Nucleic acid "fragments" encompassed by the invention are of two general types. First, fragment nucleic acids that do not encode a full- length nucleic acid polymerase but do encode a thermally stable polypeptide with nucleic acid polymerase activity are encompassed within the invention. Second, fragment nucleic acids useful as hybridization probes but that generally do not encode polymerases retaining biological activity are also encompassed within the invention. Thus, fragments of nucleotide sequences such as SEQ TD NO:l - 12 may be as small as about 9 nucleotides, about 12 nucleotides, about 15 nucleotides, about 17 nucleotides, about 18 nucleotides, about 20 nucleotides, about 50 nucleotides, about 100 nucleotides or more. In general, a fragment nucleic acid of the invention can have any upper size limit so long as it is related in sequence to the nucleic acids of the invention but is not full length.
As indicated above, "variants" are substantially similar or substantially homologous sequences. For nucleotide sequences, variants include those sequences that, because of the degeneracy of the genetic code, encode the identical amino acid sequence of the native nucleic acid polymerase protein. Variant nucleic acids also include those that encode polypeptides that do not have amino acid sequences identical to that of a native nucleic acid polymerase protein, but that encode an active, thermally stable nucleic acid polymerase with conservative changes in the amino acid sequence. As is known by one of skill in the art, the genetic code is "degenerate," meaning that several trinucleotide codons can encode the same amino acid. This degeneracy is apparent from Table 1.
Table 1
Figure imgf000040_0001
Figure imgf000041_0001
Hence, many changes in the nucleotide sequence of the variant may be silent and may not alter the amino acid sequence encoded by the nucleic acid. Where nucleic acid sequence alterations are silent, a variant nucleic acid will encode a polypeptide with the same amino acid sequence as the reference nucleic acid. Therefore, a particular nucleic acid sequence of the invention also encompasses variants with degenerate codon substitutions, and complementary sequences thereof, as well as the sequence explicitly specified by a SEQ ED NO. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the reference codon is replaced by any of the codons for the amino acid specified by the reference codon. In general, the third position of one or more selected codons can be substituted with mixed-base and/or deoxyinosine residues as disclosed by Batzer et al, Nucleic Acid Res., 19, 5081 (1991) and/or Ohtsuka et al., J. Biol. Chem., 260, 2605 (1985); Rossolini et al., Mol. Cell. Probes, 8, 91 (1994).
However, the invention is not limited to silent changes in the present nucleotide sequences but also includes variant nucleic acid sequences that conservatively alter the amino acid sequence of a polypeptide of the invention. According to the present invention, variant and reference nucleic acids of the invention may differ in the encoded amino acid sequence by one or more substitutions, additions, insertions, deletions, fusions and truncations, which may be present in any combination, so long as an active, thermally stable nucleic acid polymerase is encoded by the variant nucleic acid. Such variant nucleic acids will not encode exactly the same amino acid sequence as the reference nucleic acid, but have conservative sequence changes.
Variant nucleic acids with silent and conservative changes can be defined and characterized by the degree of homology to the reference nucleic acid. Preferred variant nucleic acids are "substantially homologous" to the reference nucleic acids of the invention. As recognized by one of skill in the art, such substantially similar nucleic acids can hybridize under stringent conditions with the reference nucleic acids identified by SEQ ED NOs herein. These types of substantially homologous nucleic acids are encompassed by this invention. Generally, nucleic acid derivatives and variants of the invention will have at least 90%, 91%, 92%, 93% or 94% sequence identity to the reference nucleotide sequence defined herein. Preferably, nucleic acids of the invention will have at least at least 95%, 96%, 97%, 98%, or 99% sequence identity to the reference nucleotide sequence defined herein. Variant nucleic acids can be detected and isolated by standard hybridization procedures.
Hybridization to detect or isolate such sequences is generally carried out under stringent conditions. "Stringent hybridization conditions" and "stringent hybridization wash conditions" in the context of nucleic acid hybridization experiments such as Southern and Northern hybridization are sequence dependent, and are different under different environmental parameters. Longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in Tijssen, Laboratory Techniques in Biochemistry and Molecular biology-Hybridization with Nucleic Acid Probes, page 1, chapter 2 "Overview of principles of hybridization and the strategy of nucleic acid probe assays" Elsevier, New York (1993). See also, J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, N.Y., pp 9.31-9.58 (1989); J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, N.Y. (3rd ed. 2001). The invention also provides methods for detection and isolation of derivative or variant nucleic acids encoding nucleic acid polymerase activity. The methods involve hybridizing at least a portion of a nucleic acid comprising any one of SEQ ED NO: 1-12 to a sample nucleic acid, thereby forming a hybridization complex; and detecting the hybridization complex. The presence of the complex correlates with the presence of a derivative or variant nucleic acid encoding at least a segment of nucleic acid polymerase. In general, the portion of a nucleic acid comprising any one of SEQ ED NO: 1-12 used for hybridization is at least fifteen nucleotides, and hybridization is under hybridization conditions that are sufficiently stringent to permit detection and isolation of substantially homologous nucleic acids. In an alternative embodiment, a nucleic acid sample is amplified by the polymerase chain reaction using primer oligonucleotides selected from any one of SEQ ID NO:l- 12.
Generally, highly stringent hybridization and wash conditions are selected to be about 5 °C lower than the thermal melting point (Tm) for the specific double-stranded sequence at a defined ionic strength and pH. For example, under "highly stringent conditions" or "highly stringent hybridization conditions" a nucleic acid will hybridize to its complement to a detectably greater degree than to other sequences (e.g., at least 2- fold over background). By controlling the stringency of the hybridization and/or washing conditions, nucleic acids that are 100% complementary can be identified.
' Alternatively, stringency conditions can be adjusted to allow some mismatching in sequences so that lower degrees of similarity are detected (heterologous probing). Typically, stringent conditions will be those in which the salt concentration is less than about 1.5 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C for short probes (e.g., 10 to 50 nucleotides) and at least about 60°C for long probes (e.g., greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. Exemplary low stringency conditions include hybridization with a buffer solution of 30 to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at 37°C, and a wash in IX to 2X SSC (20X SSC = 3.0 M NaCl and 0.3 M trisodium citrate) at 50 to 55 °C. Exemplary moderate stringency conditions include hybridization in 40 to 45% formamide, 1.0 M NaCl, 1% SDS at 37°C, and a wash in 0.5X to IX SSC at 55 to 60°C. Exemplary high stringency conditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37°C, and a wash in 0. IX SSC at 60 to 65°C.
The degree of complementarity or homology of hybrids obtained during hybridization is typically a function of post-hybridization washes, the critical factors being the ionic strength and temperature of the final wash solution. The type and length of hybridizing nucleic acids also affects whether hybridization will occur and whether any hybrids formed will be stable under a given set of hybridization and wash conditions. For DNA-DNA hybrids, the Tm can be approximated from the equation of Meinkoth and Wahl Anal. Biochem. 138:267-284 (1984); Tm 81.5°C + 16.6 (log M) +0.41 (%GC) - 0.61 (% form) - 500/L; where M is the molarity of monovalent cations, %GC is the percentage of guanosine and cytosine nucleotides in the DNA, % form is the percentage of formamide in the hybridization solution, and L is the length of the hybrid in base pairs. The Tm is the temperature (under defined ionic strength and pH) at which 50% of a complementary target sequence hybridizes to a perfectly matched probe. Very stringent conditions are selected to be equal to the Tm for a particular probe.
An example of stringent hybridization conditions for hybridization of complementary nucleic acids that have more than 100 complementary residues on a filter in a Southern or Northern blot is 50% formamide with 1 mg of heparin at 42 °C, with the hybridization being carried out overnight. An example of highly stringent conditions is 0.1 5 M NaCl at 72 °C for about 15 minutes. An example of stringent wash conditions is a 0.2x SSC wash at 65 °C for 15 minutes (see also, Sambrook, infra). Often, a high stringency wash is preceded by a low stringency wash to remove background probe signal. An example of medium stringency for a duplex of, e.g., more than 100 nucleotides, is lx SSC at 45 °C for 15 minutes. An example low stringency wash for a duplex of, e.g., more than 100 nucleotides, is 4-6x SSC at 40°C for 15 minutes. For short probes (e.g., about 10 to 50 nucleotides), stringent conditions typically involve salt concentrations of less than about l.OM Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3, and the temperature is typically at least about 30°C.
Stringent conditions can also be achieved with the addition of destabilizing agents such as formamide. En general, a signal to noise ratio of 2x (or higher) than that observed for an unrelated probe in the particular hybridization assay indicates detection of a specific hybridization. Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the proteins that they encode are substantially identical. This occurs, e.g., when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code. The following are examples of sets of hybridization/wash conditions that may be used to detect and isolate homologous nucleic acids that are substantially identical to reference nucleic acids of the present invention: a reference nucleotide sequence preferably hybridizes to the reference nucleotide sequence in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO4, 1 mM EDTA at 50°C with washing in 2X SSC, 0.1% SDS at 50°C, more desirably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO4, 1 mM EDTA at 50°C with washing in IX SSC, 0.1% SDS at 50°C, more desirably still in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO4, 1 mM EDTA at 50°C with washing in 0.5X SSC, 0.1% SDS at 50°C, preferably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO4, 1 mM EDTA at 50°C with washing in 0.1X SSC, 0.1% SDS at 50°C, more preferably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO4, 1 mM EDTA at 50°C with washing in 0.1X SSC, 0.1% SDS at 65 °C.
In general, Tm is reduced by about 1°C for each 1% of mismatching. Thus, Tm, hybridization, and or wash conditions can be adjusted to hybridize to sequences of the desired sequence identity. For example, if sequences with >90% identity are sought, the Tm can be decreased 10°C. Generally, stringent conditions are selected to be about 5°C lower than the thermal melting point
(Tm) for the specific sequence and its complement at a defined ionic strength and pH. However, severely stringent conditions can utilize a hybridization and/or wash at 1, 2, 3, or 4°C lower than the thermal melting point (Tm); moderately stringent conditions can utilize a hybridization and/or wash at 6, 7, 8, 9, or 10°C lower than the thermal melting point (Tm); low stringency conditions can utilize a hybridization and/or wash at 11, 12, 13, 14, 15, or 20°C lower than the thermal melting point (Tm).
If the desired degree of mismatching results in a Tm of less than 45 °C (aqueous solution) or 32°C (formamide solution), it is preferred to increase the SSC concentration so that a higher temperature can be used. An extensive guide to the hybridization of nucleic acids is found in Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Acid Probes, Part 1, Chapter 2 (Elsevier, New York); and Ausubel et al., eds. (1995) Current Protocols in Molecular Biology, Chapter 2 (Greene Publishing and Wiley - Enterscience, New York). See Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, New York). Using these references and the teachings herein on the relationship between Tm, mismatch, and hybridization and wash conditions, those of ordinary skill can generate variants of the present nucleic acid polymerase nucleic acids.
Computer analyses can also be utilized for comparison of sequences to determine sequence identity. Such analyses include, but are not limited to: CLUSTAL in the PC/Gene program (available from Intelligenetics, Mountain View, California); the ALIGN program (Version 2.0) and GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Version 8 (available from Genetics Computer Group (GCG), 575 Science Drive, Madison, Wisconsin, USA). Alignments using these programs can be performed using the default parameters. The CLUSTAL program is well described by Higgins et al. Gene 73:237 244 (1988); Higgins et al. CABIOS 5:151-153 (1989); Corpet et al. Nucleic Acids Res. 16:10881-90 (1988); Huang et al. CABIOS 8:155-65 (1992); and Pearson et al. Meth. Mol. Biol. 24:307-331 (1994). The ALIGN program is based on the algorithm of Myers and Miller, supra. The BLAST programs of Altschul et al., J. Mol. Biol. 215:403 (1990), are based on the algorithm of Karlin and Altschul supra. To obtain gapped alignments for comparison purposes, Gapped BLAST (in BLAST 2.0) can be utilized as described in Altschul et al. Nucleic Acids Res. 25:3389 (1997). Alternatively, PSI-BLAST (in BLAST 2.0) can be used to perform an iterated search that detects distant relationships between molecules. See Altschul et al., supra. When utilizing BLAST, Gapped BLAST, PSI-BLAST, the default parameters of the respective programs (e.g. BLASTN for nucleotide sequences, BLASTX for proteins) can be used. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, a cutoff of 100, M - 5, N = -4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA, 89, 10915 (1989)). See http://www.ncbi.nlm.nih.gov. Alignment may also be performed manually by inspection.
For purposes of the present invention, comparison of nucleotide sequences for determination of percent sequence identity to the nucleic acid polymerase sequences disclosed herein is preferably made using the BlastN program (version 1.4.7 or later) with its default parameters or any equivalent program. By "equivalent program" is intended any sequence comparison program that, for any two sequences in question, generates an alignment having identical nucleotide or amino acid residue matches and an identical percent sequence identity when compared to the corresponding alignment generated by the preferred program.
Expression of Nucleic Acids Encoding Polymerases
Nucleic acids of the invention may be used for the recombinant expression of the nucleic acid polymerase polypeptides of the invention. Generally, recombinant expression of a nucleic acid polymerase polypeptide of the invention is effected by introducing a nucleic acid encoding that polypeptide into an expression vector adapted for use in particular type of host cell. The nucleic acids of the invention can be introduced and expressed in any host organism, for example, in both prokaryotic or eukaryotic host cells. Examples of host cells include bacterial cells, yeast cells, cultured insect cell lines, and cultured mammalian cells lines. Preferably, the recombinant host cell system is selected that processes and post-translationally modifies nascent polypeptides in a manner similar to that of the organism from which the nucleic acid polymerase was derived. For purposes of expressing and isolating nucleic acid Polymerase polypeptides of the invention, prokaryotic organisms are preferred, for example, Escherichia coli. Accordingly, the invention provides host cells comprising the expression vectors of the invention.
The nucleic acids to be introduced can be conveniently placed in expression cassettes for expression in an organism of interest. Such expression cassettes will comprise a transcriptional initiation region linked to a nucleic acid of the invention. Expression cassettes preferably also have a plurality of restriction sites for insertion of the nucleic acid to be under the transcriptional regulation of various control elements. The expression cassette additionally may contain selectable marker genes. Suitable control elements such as enhancers/promoters, splice junctions, polyadenylation signals, etc. may be placed in close proximity to the coding region of the gene if needed to permit proper initiation of transcription and/or correct processing of the primary RNA transcript. Alternatively, the coding region utilized in the expression vectors of the present invention may contain endogenous enhancers/promoters, splice junctions, intervening sequences, polyadenylation signals, etc., or a combination of both endogenous and exogenous control elements. Preferably the nucleic acid in the vector is under the control of, and operably linked to, an appropriate promoter or other regulatory elements for transcription in a host cell. The vector may be a bi-functional expression vector that functions in multiple hosts. The transcriptional cassette generally includes in the 5 '-3' direction of transcription, a promoter, a transcriptional and translational initiation region, a DNA sequence of interest, and a transcriptional and translational termination region functional in the organism. The termination region may be native with the transcriptional initiation region, may be native with the DNA sequence of interest, or may be derived from another source. Efficient expression of recombinant nucleic acids in prokaryotic and eukaryotic cells generally requires regulatory control elements directing the efficient termination and polyadenylation of the resulting transcript. Transcription termination signals are generally found downstream of the polyadenylation signal and are a few hundred nucleotides in length. The term "poly A site" or "poly A sequence" as used herein denotes a nucleic acid sequence that directs both the termination and polyadenylation of the nascent RNA transcript. Efficient polyadenylation of the recombinant transcript is desirable as transcripts lacking a poly A tail are unstable and are rapidly degraded.
Nucleic acids encoding nucleic acid polymerase may be introduced into bacterial host cells by a method known to one of skill in the art. For example, nucleic acids encoding a thermophilic nucleic acid polymerase can be introduced into bacterial cells by commonly used transformation procedures such as by treatment with calcium chloride or by electroporation. If the thermophilic nucleic acid polymerase is to be expressed in eukaryotic host cells, nucleic acids encoding the thermophilic nucleic acid polymerase may be introduced into eukaryotic host cells by a number of means including calcium phosphate co- precipitation, spheroplast fusion, electroporation and the like. When the eukaryotic host cell is a yeast cell, transformation may be affected by treatment of the host cells with lithium acetate or by electroporation.
Thus, one aspect of the invention is to provide expression vectors and host cells comprising a nucleic acid encoding a nucleic acid polymerase polypeptide of the invention. A wide range of expression vectors are well known in the art. Description of various expression vectors and how to use them can be found among other places in U.S. Pat. Nos. 5,604,118; 5,583,023; 5,432,082; 5,266,490; 5,063,158; 4,966,841; 4,806,472; 4,801,537; and Goedel et al., Gene Expression Technology, Methods of Enzymology, Vol. 185, Academic Press, San Diego (1989). The expression of nucleic acid polymerases in recombinant cell systems is a well-established technique. Examples of the recombinant expression of nucleic acid polymerase can be found in U.S. Pat. Nos. 5,602,756; 5,545,552; 5,541,311; 5,500,363; 5,489,523; 5,455,170; 5,352,778; 5,322,785; and 4,935,361. Recombinant DNA and molecular cloning techniques that can be used to help make and use aspects of the invention are described by Sambrook et al., Molecular Cloning: A Laboratory Manual Vol.1-3, Cold Spring Harbor laboratory, Cold Spring Harbor, N.Y. (2001); Ausubel (ed.), Current Protocols in Molecular Biology, John Wiley and Sons, Enc. (1994); T. Maniatis, E. F. Fritsch and J. Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor laboratory, Cold Spring Harbor, N.Y. (1989); and by T. J. Silhavy, M.L. Berman, and L. W. Enquist, Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1984).
Nucleic Acid Polymerase Enzymes The invention provides Thermus scotoductus nucleic acid polymerase polypeptides, as well as fragments thereof and variant nucleic acid Polymerase polypeptides that are active and thermally stable. Any polypeptide containing amino acid sequence having any one of SEQ ED NO: 13-28, which are the amino acid sequences for wild type and derivative Thermus scotoductus nucleic acid polymerases, are contemplated by the present invention. The polypeptides of the invention are isolated or substantially purified polypeptides. En particular, the isolated polypeptides of the invention are substantially free of proteins normally present in Thermus scotoductus bacteria.
En one embodiment, the invention provides a polypeptide of SEQ ED NO: 13, a wild type Thermus scotoductus nucleic acid polymerase polypeptide from strain X-l with three additional amino acids at the N-terminus:
MRAMLPLFEP KGRVLLVDGH HLAYRTFFAL KG TTSRGEP 40 VQAVYGFAKS LLKALREDGD WIWFDAKA PSFRHQTYEA 80
YKAGRAPTPE DFPRQLALIK EMVDLLGLER LEVPGFEADD 120
VLATLAKKAE KEGYEVRILT ADRDLYQLLS ERISILHPEG 160 Y ITPE L E KYGLKPSQ V DYRALAGDPS DNIPGVKGIG 200
EKTAAKLIRE GSLEN LKH LEQVKPASVR EKI SHMEDL 240
K SLELSRVR TDLPLQVDFA RRREPDREGL KAF ERLEFG 280
SLLHEFG E SPVAAEEAP PPPEGAFVGY V SRPEPM A 320
ELNA AAA E GRVYRAEDPL EA RGLGEVR GLLAKDLAVL 360 ALREGIALAP GDDPMLLAYL DPSNTAPEG VARRYGGE T 400
EEAGERALLS ERLYAALLER LKGEER L L YEEVEKP SR 440
VLAHMEATGV R DVAYLKAL SLEVEAELRR LEEEVHRLAG 480
HPFNLNSRDQ ERVLFDELG LPAIGKTEKT GKRSTSAAVL 520
EALREAHPIV DRILQYRELS KLKGTYIDPL PALVHPKTNR 560 LHTRFNQTAT ATGRLSSSDP NLQNIPVRTP LGQRIRRAFV 600
AEEG RLWL DYSQIELRVL AH SGDENLI RVFQEGQDIH 640
TQTAS MFGV PPEAVDSLMR RAAKTINFGV LYGMSAHRLS 680
GELAIPYEEA VAFIERYFQS YPKVRAWIEK TLAEGRERGY 720
VETLFGRRRY VPD ASRVKS IREAAERMAF NMPVQGTAAD 760 LMKLAMVK F PR QE GARM LLQVHDELVL EAPKEQAEEV 800
AQEAKRTMEE V PLKVPLEV EVGIGED LS AKA 833
In another embodiment, the invention provides SEQ ED NO: 14, a wild type Thermus scotoductus nucleic acid polymerase enzyme, from strain X-l that does not have the three additional amino acids at the N-terminus that are present in SEQ ED NO: 13. SEQ ID NO: 14 is provided below.
MLPLFEP KGRVLLVDGH HLAYRTFFAL KGLTTSRGEP 40
VQAVYGFAKS LLKALREDGD WIWFDAKA PSFRHQTYEA 80 YKAGRAPTPE DFPRQLALIK EMVDLLGLER LEVPGFEADD 120
VLATLAKKAE KEGYEVRILT ADRDLYQLLS ERISILHPEG 160
YLITPEWLWE KYGLKPSQWV DYRALAGDPS DNIPGVKGIG 200
EKTAAKLIRE WGSLENLLKH LEQVKPASVR EKILSHMEDL 240
KLSLELSRVR TDLPLQVDFA RRREPDREGL KAFLERLEFG 280 SLLHEFGLLE SPVAAEEAPW PPPEGAFVGY VLSRPEPMWA 320
ELNALAAAWE GRVYRAEDPL EALRGLGEVR GLLAKDLAVL 360
ALREGIALAP GDDPMLLAYL LDPSNTAPEG VARRYGGEWT 400 EEAGERALLS ERLYAALLER LKGEERLLWL YEEVEKPLSR 440
VLAHMEATGV RLDVAYLKAL SLEVEAELRR LEEEVHRLAG 480
HPFNLNSRDQ LERVLFDELG LPAIGKTEKT GKRSTSAAVL 520
EALREAHPIV DRILQYRELS KLKGTYIDPL PALVHPKTNR 560 LHTRFNQTAT ATGRLSSSDP NLQNIPVRTP LGQRIRRAFV 600
AEEGWRLWL DYSQIELRVL AHLSGDENLI RVFQEGQDIH 640
TQTASWMFGV PPEAVDSLMR RAAKTINFGV LYGMSAHRLS 680
GELAIPYEEA VAFIERYFQS YPKVRAWIEK TLAEGRERGY 720
VETLFGRRRY VPDLASRVKS IREAAERMAF NMPVQGTAAD 760 LMKLAMVKLF PRLQELGARM LLQVHDELVL EAPKEQAEEV 800
AQEAKRTMEE VWPLKVPLEV EVGIGEDWLS AKA 833
En another embodiment, the invention provides SEQ ED NO: 15, a wild type Thermus scotoductus nucleic acid polymerase enzyme from strain SM3. SEQ ED NO : 15 is provided below.
MRAMLPLFEP KGRVLLVDGH HLAYRTFFAL KGLTTSRGEP 40
VQAVYGFAKS LLKALREDGD WIWFDAKA PSFRHQTYEA 80
YKAGRAPTPE DFPRQLALIK EMVDLLGLER LEVPGFEADD 120 VLATLAKKAE KEGYEVRILT ADRDLYQLLS DRISILHPEG 160
YLITPEWLWE KYGLKPSQWV DYRALAGDPS DNIPGVKGIG 200
EKTAAKLIRE WGSLENLLKH LEQVKPASVR EKILSHMEDL 240
KLSLELSRVH TELPLQVDFA RRREPDREGL KAFLERLEFG 280
SLLHEFGLLE SPVAAEEAPW PPPEGAFVGY VLSRPEPMWA 320 ELNALAAAWE GRVYRAEDPL EALRGLGEVR GLLAKDLAVL 360
ALREGIALAQ GDDPMLLAYL LDPSNTAPEG VARRYGGEWT 400
EEAGERALLS ERLYAALLER LKGEERLLWL YEEVEKPLSR 440
VLAHMEATGV WLDVAYLKAL SLEVEAELRR LEEEVHRLAG 480
HPFNLNSRDQ LERVLFDELG LPAIGKTEKT GKRSTSAAVL 520 EALREAHPIV DRILQYRELS KLKGTYIDPL PALVHPKTNR 560
LHTRFNQTAT ATGRLSSSDP NLQNIPVRTP LGQRIRRAFV 600
AEEGWRLWL DYSQIELRVL AHLSGDENLI RVFQEGQDIH 640
TQTASWMFGV PPEAVDSLMR RAAKTINFGV LYGMSAHRLS 680
GELAIPYEEA VAFIERYFQS YPKVRAWIEK TLAEGRERGY 720 VETLFGRRRY VPDLASRVKS IREAAERMAF NMPVQGTAAD 760
LMKLAMVKLF PRLQELGARM LLQVHDELVL EAPKEQAEEV 800
AQEAKRTMEE VWPLKVPLEV EVGIGEDWLS AKA 833
In another embodiment, the invention provides SEQ ED NO: 16, a wild type Thermus scotoductus nucleic acid polymerase enzyme from strain Vi7a. SEQ ED NO: 16 is provided below.
MRAMLPLFEP KGRVLLVDGH HLAYRTFFAL KGLTTSRGEP 40 VQAVYGFAKS LLKALREDGD WIWFDAKA PSFRHQTYEA 80
YKAGRAPTPE DFPRQLALIK EMVDLLGLER LEVPGFEADD 120
VLATLAKKAE KEGYEVRILT ADRDLYQLLS DRISILHPEG 160
YLITPEWLWE KYGLKPSQWV DYRALAGDPS DNIPGVKGIG 200 EKTAAKLIRE WGSLENLLKH LEQVKPASVR EKILSHMEDL 240
KLSLELSRVH TELPLQVDFA RRREPDREGL KAFLERLEFG 280
SLLHEFGLLE SPVAAEEAPW PPPEGAFVGY VLSRPEPMWA 320
ELNALAAAWE GRVYRAEDPL EALRGLGEVR GLLAKDLAVL 360
ALREGIALAP GDDPMLLAYL LDPSNTAPEG VARRYGGEWT 400 EEAGERALLS ERLYAALLER LKGEERLLWL YEEVEKPLSR 440
VLAHMEATGV WLDVAYLKAL SLEVEAELRR LEEEVHRLAG 480
HPFNLNSRDQ LERVLFDELG LPAIGKTEKT GKRSTSAAVL 520
EALREAHPIV DRILQYRELS KLKGTYIDPL PALVHPKTNR 560
LHTRFNQTAT ATGRLSSSDP NLQNIPVRTP LGQRIRRAFV 600 AEEGWRLWL DYSQIELRVL AHLSGDENLI RVFQEGQDIH 640
TQTASWMFGV PPEAVDSLMR RAAKTINFGV LYGMSAHRLS 680
GELAIPYEEA VAFIERYFQS YPKVRAWIEK TLAEGRERGY 720
VETLFGRRRY VPDLASRVKS IREAAERMAF NMPVQGTAAD 760
LMKLAMVKLF PRLQELGARM LLQVHDELVL EAPKEQAEEV 800 AQEAKRTMEE VWPLKVPLEV EVGIGEDWLS AKA 833
The sequences of wild type Thermus scotoductus nucleic acid polymerases are distinct from the amino acid sequence of Thermus aquaticus DNA Polymerase. There are about 51 conservative amino acid differences and about 62 nonconservative amino acid differences. For example, one region of dissimilarity is between approximate amino acid positions 51 and 65, where the sequence of the Thermus scotoductus polymerase has about four amino acid differences (in bold): LLKALREDG DVNEVVFDAK APSFRHQTYE (SEQ ED ΝO:39). Another region of dissimilarity is between approximate amino acid positions 201 and 236, where the sequence of the Thermus scotoductus polymerase has about seven amino acid differences (in bold): GEKTAAKLIREWGSLENLLKHLEQV KPASV REKILS (SEQ ED NO:40). Another region of dissimilarity is between about positions 311 and 350, where the sequence of the Thermus scotoductus polymerase has about seven amino acid changes (in bold): VGYVLSRPEPMWAELN
ALAAAWEGRVYRAEDPLEALRGLG (SEQ ID NO:41). Another region of dissimilarity is between about positions 415 and 435, where the sequence of the Thermus scotoductus polymerase has about five amino acid changes (in bold): RLYAALLERLKGEERLLWLYE (SEQ ED NO:42). Another region of dissimilarity is between about positions 531 and 562, where the sequence of the Thermus scotoductus polymerase has about six amino acid changes (in bold): PIVDR1LQYRELSKLK GTYED PLPALVHPKTN (SEQ ED NO:43). Another region of dissimilarity is between about positions 801 and 836, where the sequence of the Thermus scotoductus polymerase has about eight amino acid changes (in bold): EEVAQEAKRT MEEVWPLKNPLENEVGIGEDWLSAKA (SEQ ED ΝO:44). Hence, many regions of the Thermus scotoductus polymerase differ from the Thermus aquaticus and Thermus thermophilus DNA polymerases.
Many DNA polymerases possess activities in addition to a DNA polymerase activity. Such activities include, for example, a 5'-3' exonuclease activity and/or a 3'-5' exonuclease activity. The 3'-5' exonuclease activity improves the accuracy of the newly synthesized strand by removing incorrect bases that may have been incorporated. DNA polymerases in which such activity is low or absent are prone to errors in the incorporation of nucleotide residues into the primer extension strand. Taq DNA polymerase has been reported to have low 3'-5' exonuclease activity. See Lawyer et al., J. Biol Chem. 264:6427-6437. In applications such as nucleic acid amplification procedures in which the replication of DNA is often geometric in relation to the number of primer extension cycles, such errors can lead to serious artifactual problems such as sequence heterogeneity of the nucleic acid amplification product (amplicon). Thus, a 3'-5' exonuclease activity is a desired characteristic of a thermostable DNA polymerase used for such purposes.
By contrast, the 5'-3' exonuclease activity of DNA polymerase enzymes is often undesirable because this activity may digest nucleic acids, including primers, that have an unprotected 5' end. Thus, a thermostable nucleic acid polymerase with an attenuated 5 '-3' exonuclease activity, or in which such activity is absent, is a desired characteristic of an enzyme for biochemical applications. Various DNA polymerase enzymes have been described where a modification has been introduced in a DNA polymerase that accomplishes this object. For example, the Klenow fragment of E. coli DNA polymerase I can be produced as a proteolytic fragment of the holoenzyme in which the domain of the protein controlling the 5'-3' exonuclease activity has been removed. The Klenow fragment still retains the polymerase activity and the 3'-5' exonuclease activity. Barnes, PCT Publication No. WO92/06188 (1992) and Gelfand et al., U.S. Pat. No. 5,079,352 have produced 5'-3' exonuclease-deficient recombinant Thermus aquaticus DNA polymerases. Ishino et al., EPO Publication No. 0517418A2, have produced a 5'-3' exonuclease-deficient DNA polymerase derived from Bacillus caldotenax.
En another embodiment, the invention provides a polypeptide that is a derivative Thermus scotoductus polypeptide with reduced or eliminated 5 '-3' exonuclease activity. Several methods exist for reducing this activity, and the invention contemplates any polypeptide derived from the Thermus scotoductus polypeptides of the invention that has reduced or eliminated such 5 '-3' exonuclease activity. Xu et al., Biochemical and mutational studies of the 5'-3' exonuclease of DNA polymerase I of Escherichia coli. J. Mol. Biol. 1997 May 2; 268(2):284-302.
In one embodiment, the invention provides a Thermus scotoductus nucleic acid polymerase polypeptide from strain X-l in which Asp is used in place of Gly at position 46. This polypeptide has SEQ ED NO: 17 and reduced 5'- 3' exonuclease activity. SEQ ED NO: 17 is provided below.
MRAMLPLFEP KGRVLLVDGH HLAYRTFFAL KGLTTSRGEP 40
VQAVYDFAKS LLKALREDGD WIWFDAKA PSFRHQTYEA 80 YKAGRAPTPE DFPRQLALIK EMVDLLGLER LEVPGFEADD 120
VLATLAKKAE KEGYEVRILT ADRDLYQLLS ERISILHPEG 160
YLITPEWLWE KYGLKPSQWV DYRALAGDPS DNIPGVKGIG 200
EKTAAKLIRE WGSLENLLKH LEQVKPASVR EKILSHMEDL 240
KLSLELSRVR TDLPLQVDFA RRREPDREGL KAFLERLEFG 280 SLLHEFGLLE SPVAAEEAPW PPPEGAFVGY VLSRPEPMWA 320
ELNALAAAWE GRVYRAEDPL EALRGLGEVR GLLAKDLAVL 360
ALREGIALAP GDDPMLLAYL LDPSNTAPEG VARRYGGEWT 400
EEAGERALLS ERLYAALLER LKGEERLLWL YEEVEKPLSR 440
VLAHMEATGV RLDVAYLKAL SLEVEAELRR LEEEVHRLAG 480 HPFNLNSRDQ LERVLFDELG LPAIGKTEKT GKRSTSAAVL 520
EALREAHPIV DRILQYRELS KLKGTYIDPL PALVHPKTNR 560
LHTRFNQTAT ATGRLSSSDP NLQNIPVRTP LGQRIRRAFV 600
AEEGWRLWL DYSQIELRVL AHLSGDENLI RVFQEGQDIH 640
TQTASWMFGV PPEAVDSLMR RAAKTINFGV LYGMSAHRLS 680 GELAIPYEEA VAFIERYFQS YPKVRAWIEK TLAEGRERGY 720
VETLFGRRRY VPDLASRVKS IREAAERMAF NMPVQGTAAD 760
LMKLAMVKLF PRLQELGARM LLQVHDELVL EAPKEQAEEV 800
AQEAKRTMEE VWPLKVPLEV EVGIGEDWLS AKA 833
En another embodiment, the invention provides a Thermus scotoductus nucleic acid polymerase polypeptide from strain X-l in which Asp is used in place of Gly at position 46. This polypeptide has SEQ ED NO: 18 and reduced 5' 3' exonuclease activity. SEQ ED NO: 18 is provided below.
MLPLFEP KGRVLLVDGH HLAYRTFFAL KGLTTSRGEP 40 VQAVYDFAKS LLKALREDGD WIWFDAKA PSFRHQTYEA 80
YKAGRAPTPE DFPRQLALIK EMVDLLGLER LEVPGFEADD 120
VLATLAKKAE KEGYEVRILT ADRDLYQLLS ERISILHPEG 160
YLITPEWLWE KYGLKPSQWV DYRALAGDPS DNIPGVKGIG 200
EKTAAKLIRE WGSLENLLKH LEQVKPASVR EKILSHMEDL 240 KLSLELSRVR TDLPLQVDFA RRREPDREGL KAFLERLEFG 280
SLLHEFGLLE SPVAAEEAPW PPPEGAFVGY VLSRPEPMWA 320
ELNALAAAWE GRVYRAEDPL EALRGLGEVR GLLAKDLAVL 360
ALREGIALAP GDDPMLLAYL LDPSNTAPEG VARRYGGEWT 400
EEAGERALLS ERLYAALLER LKGEERLLWL YEEVEKPLSR 440 VLAHMEATGV RLDVAYLKAL SLEVEAELRR LEEEVHRLAG 480
HPFNLNSRDQ LERVLFDELG LPAIGKTEKT GKRSTSAAVL 520
EALREAHPIV DRILQYRELS KLKGTYIDPL PALVHPKTNR 560
LHTRFNQTAT ATGRLSSSDP NLQNIPVRTP LGQRIRRAFV 600
AEEGWRLWL DYSQIELRVL AHLSGDENLI RVFQEGQDIH 640 TQTASWMFGV PPEAVDSLMR RAAKTINFGV LYGMSAHRLS 680
GELAIPYEEA VAFIERYFQS YPKVRAWIEK TLAEGRERGY 720
VETLFGRRRY VPDLASRVKS IREAAERMAF NMPVQGTAAD 760
LMKLAMVKLF PRLQELGARM LLQVHDELVL EAPKEQAEEV 800
AQEAKRTMEE VWPLKVPLEV EVGIGEDWLS AKA 833
In another embodiment, the invention provides a Thermus scotoductus nucleic acid polymerase polypeptide from strain SM3 in which Asp is used in place of Gly at position 46. This polypeptide has SEQ ED NO: 19 and reduced 5' 3' exonuclease activity. SEQ ED NO: 19 is provided below.
MRAMLPLFEP KGRVLLVDGH HLAYRTFFAL KGLTTSRGEP 40
VQAVYDFAKS LLKALREDGD WIWFDAKA PSFRHQTYEA 80
YKAGRAPTPE DFPRQLALIK EMVDLLGLER LEVPGFEADD 120
VLATLAKKAE KEGYEVRILT ADRDLYQLLS DRISILHPEG 160 YLITPEWLWE KYGLKPSQWV DYRALAGDPS DNIPGVKGIG 200
EKTAAKLIRE WGSLENLLKH LEQVKPASVR EKILSHMEDL 240
KLSLELSRVH TELPLQVDFA RRREPDREGL KAFLERLEFG 280
SLLHEFGLLE SPVAAEEAPW PPPEGAFVGY VLSRPEPMWA 320
ELNALAAAWE GRVYRAEDPL EALRGLGEVR GLLAKDLAVL 360 ALREGIALAQ GDDPMLLAYL LDPSNTAPEG VARRYGGEWT 400 EEAGERALLS ERLYAALLER LKGEERLLWL YEEVEKPLSR 440
VLAHMEATGVnWLDVAYLKAL SLEVEAELRR LEEEVHRLAG 480
HPFNLNSRDQ LERVLFDELG LPAIGKTEKT GKRSTSAAVL 520
EALREAHPIV DRILQYRELS KLKGTYIDPL PALVHPKTNR 560 LHTRFNQTAT ATGRLSSSDP NLQNIPVRTP LGQRIRRAFV 600
AEEGWRLWL DYSQIELRVL AHLSGDENLI RVFQEGQDIH 640
TQTASWMFGV PPEAVDSLMR RAAKTINFGV LYGMSAHRLS 680
GELAIPYEEA VAFIERYFQS YPKVRAWIEK TLAEGRERGY 720
VETLFGRRRY VPDLASRVKS IREAAERMAF NMPVQGTAAD 760 LMKLAMVKLF PRLQELGARM LLQVHDELVL EAPKEQAEEV 800
AQEAKRTMEE VWPLKVPLEV EVGIGEDWLS AKA 833
In another embodiment, the invention provides a Thermus scotoductus nucleic acid polymerase polypeptide from strain Vi7a in which Asp is used in place of Gly at position 46. This polypeptide has SEQ ID NO:20 and reduced 5' 3' exonuclease activity. SEQ ED NO:20 is provided below.
MRAMLPLFEP KGRVLLVDGH HLAYRTFFAL KGLTTSRGEP 40 VQAVYDFAKS LLKALREDGD WIWFDAKA PSFRHQTYEA 80
YKAGRAPTPE DFPRQLALIK EMVDLLGLER LEVPGFEADD 120
VLATLAKKAE KEGYEVRILT ADRDLYQLLS DRISILHPEG 160
YLITPEWLWE KYGLKPSQWV DYRALAGDPS DNIPGVKGIG 200
EKTAAKLIRE WGSLENLLKH LEQVKPASVR EKILSHMEDL 240 KLSLELSRVH TELPLQVDFA RRREPDREGL KAFLERLEFG 280
SLLHEFGLLE SPVAAEEAPW PPPEGAFVGY VLSRPEPMWA 320
ELNALAAAWE GRVYRAEDPL EALRGLGEVR GLLAKDLAVL 360
ALREGIALAP GDDPMLLAYL LDPSNTAPEG VARRYGGEWT 400
EEAGERALLS ERLYAALLER LKGEERLLWL YEEVEKPLSR 440 VLAHMEATGV WLDVAYLKAL SLEVEAELRR LEEEVHRLAG 480
HPFNLNSRDQ LERVLFDELG LPAIGKTEKT GKRSTSAAVL 520
EALREAHPIV DRILQYRELS KLKGTYIDPL PALVHPKTNR 560
LHTRFNQTAT ATGRLSSSDP NLQNIPVRTP LGQRIRRAFV 600
AEEGWRLWL DYSQIELRVL AHLSGDENLI RVFQEGQDIH 640 TQTASWMFGV PPEAVDSLMR RAAKTINFGV LYGMSAHRLS 680
GELAIPYEEA VAFIERYFQS YPKVRAWIEK TLAEGRERGY 720
VETLFGRRRY VPDLASRVKS IREAAERMAF NMPVQGTAAD 760
LMKLAMVKLF PRLQELGARM LLQVHDELVL EAPKEQAEEV 800
AQEAKRTMEE VWPLKVPLEV EVGIGEDWLS AKA 833
In another embodiment, the invention provides a polypeptide of SEQ ID NO:21, a derivative Thermus scotoductus polypeptide from strain X-l with reduced bias against ddNTP incorporation. SEQ ID NO:21 has Tyr in place of Phe at position 668. The sequence of SEQ ED NO:21 is below. MRAMLPLFEP KGRVLLVDGH HLAYRTFFAL KGLTTSRGEP 40
VQAVYGFAKS LLKALREDGD WIWFDAKA PSFRHQTYEA 80
YKAGRAPTPE DFPRQLALIK EMVDLLGLER LEVPGFEADD 120
VLATLAKKAE KEGYEVRILT ADRDLYQLLS ERISILHPEG 160 YLITPEWLWE KYGLKPSQWV DYRALAGDPS DNIPGVKGIG 200
EKTAAKLIRE WGSLENLLKH LEQVKPASVR EKILSHMEDL 240
KLSLELSRVR TDLPLQVDFA RRREPDREGL KAFLERLEFG 280
SLLHEFGLLE SPVAAEEAPW PPPEGAFVGY VLSRPEPMWA 320
ELNALAAAWE GRVYRAEDPL EALRGLGEVR GLLAKDLAVL 360 ALREGIALAP GDDPMLLAYL LDPSNTAPEG VARRYGGEWT 400
EEAGERALLS ERLYAALLER LKGEERLLWL YEEVEKPLSR 440
VLAHMEATGV RLDVAYLKAL SLEVEAELRR LEEEVHRLAG 480
HPFNLNSRDQ LERVLFDELG LPAIGKTEKT GKRSTSAAVL 520
EALREAHPIV DRILQYRELS KLKGTYIDPL PALVHPKTNR 560 LHTRFNQTAT ATGRLSSSDP NLQNIPVRTP LGQRIRRAFV 600
AEEGWRLWL DYSQIELRVL AHLSGDENLI RVFQEGQDIH 640
TQTASWMFGV PPEAVDSLMR RAAKTINYGV LYGMSAHRLS 680
GELAIPYEEA VAFIERYFQS YPKVRAWIEK TLAEGRERGY 720
VETLFGRRRY VPDLASRVKS IREAAERMAF NMPVQGTAAD 760 LMKLAMVKLF PRLQELGARM LLQVHDELVL EAPKEQAEEV 800
AQEAKRTMEE VWPLKVPLEV EVGIGEDWLS AKA 833
In another embodiment, the invention provides a polypeptide of SEQ ID NO:22, a derivative Thermus scotoductus polypeptide from strain X-l with reduced bias against ddNTP incorporation. SEQ ID NO:22 has Tyr in place of Phe at position 668. The sequence of SEQ ID NO:22 is below.
MLPLFEP KGRVLLVDGH HLAYRTFFAL KGLTTSRGEP 40
VQAVYGFAKS LLKALREDGD WIWFDAKA PSFRHQTYEA 80 YKAGRAPTPE DFPRQLALIK EMVDLLGLER LEVPGFEADD 120
VLATLAKKAE KEGYEVRILT ADRDLYQLLS ERISILHPEG 160
YLITPEWLWE KYGLKPSQWV DYRALAGDPS DNIPGVKGIG 200
EKTAAKLIRE WGSLENLLKH LEQVKPASVR EKILSHMEDL 240
KLSLELSRVR TDLPLQVDFA RRREPDREGL KAFLERLEFG 280 SLLHEFGLLE SPVAAEEAPW PPPEGAFVGY VLSRPEPMWA 320
ELNALAAAWE GRVYRAEDPL EALRGLGEVR GLLAKDLAVL 360
ALREGIALAP GDDPMLLAYL LDPSNTAPEG VARRYGGEWT 400 EEAGERALLS ERLYAALLER LKGEERLLWL YEEVEKPLSR 440
VLAHMEATGV RLDVAYLKAL SLEVEAELRR LEEEVHRLAG 480
HPFNLNSRDQ LERVLFDELG LPAIGKTEKT GKRSTSAAVL 520
EALREAHPIV DRILQYRELS KLKGTYIDPL PALVHPKTNR 560 LHTRFNQTAT ATGRLSSSDP NLQNIPVRTP LGQRIRRAFV 600
AEEGWRLWL DYSQIELRVL AHLSGDENLI RVFQEGQDIH 640
TQTASWMFGV PPEAVDSLMR RAAKTINYGV LYGMSAHRLS 680
GELAIPYEEA VAFIERYFQS YPKVRAWIEK TLAEGRERGY 720
VETLFGRRRY VPDLASRVKS IREAAERMAF NMPVQGTAAD 760 LMKLAMVKLF PRLQELGARM LLQVHDELVL EAPKEQAEEV 800
AQEAKRTMEE VWPLKVPLEV EVGIGEDWLS AKA 833
In another embodiment, the invention provides a polypeptide of SEQ ED NO: 23, a derivative Thermus scotoductus polypeptide from strain SM3 with reduced bias against ddNTP incorporation. SEQ ED NO:23 has Tyr in place of Phe at position 668. The sequence of SEQ ID NO:23 is below.
MRAMLPLFEP KGRVLLVDGH HLAYRTFFAL KGLTTSRGEP 40 VQAVYGFAKS LLKALREDGD WIWFDAKA PSFRHQTYEA 80 YKAGRAPTPE DFPRQLALIK EMVDLLGLER LEVPGFEADD 120
VLATLAKKAE KEGYEVRILT ADRDLYQLLS DRISILHPEG 160
YLITPEWLWE KYGLKPSQWV DYRALAGDPS DNIPGVKGIG 200
EKTAAKLIRE WGSLENLLKH LEQVKPASVR EKILSHMEDL 240
KLSLELSRVH TELPLQVDFA RRREPDREGL KAFLERLEFG 280 SLLHEFGLLE SPVAAEEAPW PPPEGAFVGY VLSRPEPMWA 320
ELNALAAAWE GRVYRAEDPL EALRGLGEVR GLLAKDLAVL 360
ALREGIALAQ GDDPMLLAYL LDPSNTAPEG VARRYGGEWT 400
EEAGERALLS ERLYAALLER LKGEERLLWL YEEVEKPLSR 440
VLAHMEATGV WLDVAYLKAL SLEVEAELRR LEEEVHRLAG 480 HPFNLNSRDQ LERVLFDELG LPAIGKTEKT GKRSTSAAVL 520
EALREAHPIV DRILQYRELS KLKGTYIDPL PALVHPKTNR 560
LHTRFNQTAT ATGRLSSSDP NLQNIPVRTP LGQRIRRAFV 600
AEEGWRLWL DYSQIELRVL AHLSGDENLI RVFQEGQDIH 640
TQTASWMFGV PPEAVDSLMR RAAKTINYGV LYGMSAHRLS 680 GELAIPYEEA VAFIERYFQS YPKVRAWIEK TLAEGRERGY 720
VETLFGRRRY VPDLASRVKS IREAAERMAF NMPVQGTAAD 760
LMKLAMVKLF PRLQELGARM LLQVHDELVL EAPKEQAEEV 800
AQEAKRTMEE VWPLKVPLEV EVGIGEDWLS AKA 833
In another embodiment, the invention provides a polypeptide of SEQ ED NO:24, a derivative Thermus scotoductus polypeptide from strain Vi7a with reduced bias against ddNTP incorporation. SEQ ED NO:24 has Tyr in place of Phe at position 668. MRAMLPLFEP KGRVLLVDGH HLAYRTFFAL KGLTTSRGEP 40
VQAVYGFAKS LLKALREDGD WIWFDAKA PSFRHQTYEA 80
YKAGRAPTPE DFPRQLALIK EMVDLLGLER LEVPGFEADD 120 VLATLAKKAE KEGYEVRILT ADRDLYQLLS DRISILHPEG 160
YLITPEWLWE KYGLKPSQWV DYRALAGDPS DNIPGVKGIG 200
EKTAAKLIRE WGSLENLLKH LEQVKPASVR EKILSHMEDL 240
KLSLELSRVH TELPLQVDFA RRREPDREGL KAFLERLEFG 280
SLLHEFGLLE SPVAAEEAPW PPPEGAFVGY VLSRPEPMWA 320 ELNALAAAWE GRVYRAEDPL EALRGLGEVR GLLAKDLAVL 360
ALREGIALAP GDDPMLLAYL LDPSNTAPEG VARRYGGEWT 400
EEAGERALLS ERLYAALLER LKGEERLLWL YEEVEKPLSR 440
VLAHMEATGV WLDVAYLKAL SLEVEAELRR LEEEVHRLAG 480
HPFNLNSRDQ LERVLFDELG LPAIGKTEKT GKRSTSAAVL 520 EALREAHPIV DRILQYRELS KLKGTYIDPL PALVHPKTNR 560
LHTRFNQTAT ATGRLSSSDP NLQNIPVRTP LGQRIRRAFV 600
AEEGWRLWL DYSQIELRVL AHLSGDENLI RVFQEGQDIH 640
TQTASWMFGV PPEAVDSLMR RAAKTINYGV LYGMSAHRLS 680
GELAIPYEEA VAFIERYFQS YPKVRAWIEK TLAEGRERGY 720 VETLFGRRRY VPDLASRVKS IREAAERMAF NMPVQGTAAD 760
LMKLAMVKLF PRLQELGARM LLQVHDELVL EAPKEQAEEV 800
AQEAKRTMEE VWPLKVPLEV EVGIGEDWLS AKA 833
In another embodiment, the invention provides a polypeptide of SEQ ED NO:25, a derivative Thermus scotoductus polypeptide from strain X-l with reduced 5 '-3' exonuclease activity and reduced bias against ddNTP incorporation. SEQ ID NO:25 has Asp in place of Gly at position 46 and Tyr in place of Phe at position 668. The sequence of SEQ ID NO:25 is below.
MRAMLPLFEP KGRVLLVDGH HLAYRTFFAL KGLTTSRGEP 40
VQAVYDFAKS LLKALREDGD WIWFDAKA PSFRHQTYEA 80
YKAGRAPTPE DFPRQLALIK EMVDLLGLER LEVPGFEADD 120
VLATLAKKAE KEGYEVRILT ADRDLYQLLS ERISILHPEG 160
YLITPEWLWE KYGLKPSQWV DYRALAGDPS DNIPGVKGIG 200 EKTAAKLIRE WGSLENLLKH LEQVKPASVR EKILSHMEDL 240
KLSLELSRVR TDLPLQVDFA RRREPDREGL KAFLERLEFG 280
SLLHEFGLLE SPVAAEEAPW PPPEGAFVGY VLSRPEPMWA 320
ELNALAAAWE GRVYRAEDPL EALRGLGEVR GLLAKDLAVL 360
ALREGIALAP GDDPMLLAYL LDPSNTAPEG VARRYGGEWT 400 EEAGERALLS ERLYAALLER LKGEERLLWL YEEVEKPLSR 440
VLAHMEATGV RLDVAYLKAL SLEVEAELRR LEEEVHRLAG 480
HPFNLNSRDQ LERVLFDELG LPAIGKTEKT GKRSTSAAVL 520 EALREAHPIV DRILQYRELS KLKGTYIDPL PALVHPKTNR 560
LHTRFNQTAT ATGRLSSSDP NLQNIPVRTP LGQRIRRAFV 600
AEEGWRLWL DYSQIELRVL AHLSGDENLI RVFQEGQDIH 640
TQTASWMFGV PPEAVDSLMR RAAKTINYGV LYGMSAHRLS 680 GELAIPYEEA VAFIERYFQS YPKVRAWIEK TLAEGRERGY 720
VETLFGRRRY VPDLASRVKS IREAAERMAF NMPVQGTAAD 760
LMKLAMVKLF PRLQELGARM LLQVHDELVL EAPKEQAEEV 800
AQEAKRTMEE VWPLKVPLEV EVGIGEDWLS AKA 833
In another embodiment, the invention provides a polypeptide of SEQ ED
NO:26 a derivative Thermus scotoductus polypeptide from strain X-l with reduced 5'-3' exonuclease activity and reduced bias against ddNTP incorporation. SEQ ED NO:26 has Asp in place of Gly at position 46 and Tyr in place of Phe at position 668. The sequence of SEQ ED NO: 26 is below.
MLPLFEP KGRVLLVDGH HLAYRTFFAL KGLTTSRGEP 40 VQAVYDFAKS LLKALREDGD WIWFDAKA PSFRHQTYEA 80
YKAGRAPTPE DFPRQLALIK EMVDLLGLER LEVPGFEADD 120
VLATLAKKAE KEGYEVRILT ADRDLYQLLS ERISILHPEG 160 YLITPEWLWE KYGLKPSQWV DYRALAGDPS DNIPGVKGIG 200
EKTAAKLIRE WGSLENLLKH LEQVKPASVR EKILSHMEDL 240
KLSLELSRVR TDLPLQVDFA RRREPDREGL KAFLERLEFG 280
SLLHEFGLLE SPVAAEEAPW PPPEGAFVGY VLSRPEPMWA 320
ELNALAAAWE GRVYRAEDPL EALRGLGEVR GLLAKDLAVL 360 ALREGIALAP GDDPMLLAYL LDPSNTAPEG VARRYGGEWT 400
EEAGERALLS ERLYAALLER LKGEERLLWL YEEVEKPLSR 440
VLAHMEATGV RLDVAYLKAL SLEVEAELRR LEEEVHRLAG 480
HPFNLNSRDQ LERVLFDELG LPAIGKTEKT GKRSTSAAVL 520
EALREAHPIV DRILQYRELS KLKGTYIDPL PALVHPKTNR 560 LHTRFNQTAT ATGRLSSSDP NLQNIPVRTP LGQRIRRAFV 600
AEEGWRLWL DYSQIELRVL AHLSGDENLI RVFQEGQDIH 640
TQTASWMFGV PPEAVDSLMR RAAKTINYGV LYGMSAHRLS 680
GELAIPYEEA VAFIERYFQS YPKVRAWIEK TLAEGRERGY 720
VETLFGRRRY VPDLASRVKS IREAAERMAF NMPVQGTAAD 760 LMKLAMVKLF PRLQELGARM LLQVHDELVL EAPKEQAEEV 800
AQEAKRTMEE VWPLKVPLEV EVGIGEDWLS AKA 833 In another embodiment, the invention provides a polypeptide of SEQ ED NO:27 a derivative Thermus scotoductus polypeptide from strain SM3 with reduced 5 '-3' exonuclease activity and reduced bias against ddNTP incoφoration. SEQ ED NO:27 has Asp in place of Gly at position 46 and Tyr in place of Phe at position 668. The sequence of SEQ ED NO:27 is below.
MRAMLPLFEP KGRVLLVDGH HLAYRTFFAL KGLTTSRGEP 40
VQAVYDFAKS LLKALREDGD WIWFDAKA PSFRHQTYEA 80
YKAGRAPTPE DFPRQLALIK EMVDLLGLER LEVPGFEADD 120 VLATLAKKAE KEGYEVRILT ADRDLYQLLS DRISILHPEG 160
YLITPEWLWE KYGLKPSQWV DYRALAGDPS DNIPGVKGIG 200
EKTAAKLIRE WGSLENLLKH LEQVKPASVR EKILSHMEDL 240
KLSLELSRVH TELPLQVDFA RRREPDREGL KAFLERLEFG 280
SLLHEFGLLE SPVAAEEAPW PPPEGAFVGY VLSRPEPMWA 320 ELNALAAAWE GRVYRAEDPL EALRGLGEVR GLLAKDLAVL 360
ALREGIALAQ GDDPMLLAYL LDPSNTAPEG VARRYGGEWT 400
EEAGERALLS ERLYAALLER LKGEERLLWL YEEVEKPLSR 440
VLAHMEATGV WLDVAYLKAL SLEVEAELRR LEEEVHRLAG 480
HPFNLNSRDQ LERVLFDELG LPAIGKTEKT GKRSTSAAVL 520 EALREAHPIV DRILQYRELS KLKGTYIDPL PALVHPKTNR 560
LHTRFNQTAT ATGRLSSSDP NLQNIPVRTP LGQRIRRAFV 600
AEEGWRLWL DYSQIELRVL AHLSGDENLI RVFQEGQDIH 640
TQTASWMFGV PPEAVDSLMR RAAKTINYGV LYGMSAHRLS 680
GELAIPYEEA VAFIERYFQS YPKVRAWIEK TLAEGRERGY 720 VETLFGRRRY VPDLASRVKS IREAAERMAF NMPVQGTAAD 760
LMKLAMVKLF PRLQELGARM LLQVHDELVL EAPKEQAEEV 800
AQEAKRTMEE VWPLKVPLEV EVGIGEDWLS AKA 833
In another embodiment, the invention provides a polypeptide of SEQ ED NO:28 a derivative Thermus scotoductus polypeptide from strain Ni7a with reduced 5 '-3' exonuclease activity and reduced bias against ddΝTP incoφoration. SEQ ED ΝO:28 has Asp in place of Gly at position 46 and Tyr in place of Phe at position 46 and 668. The sequence of SEQ ID NO:28 is below. MRAMLPLFEP KGRVLLVDGH HLAYRTFFAL KGLTTSRGEP 40
VQAVYDFAKS LLKALREDGD WIWFDAKA PSFRHQTYEA 80
YKAGRAPTPE DFPRQLALIK EMVDLLGLER LEVPGFEADD 120
VLATLAKKAE KEGYEVRILT ADRDLYQLLS DRISILHPEG 160
YLITPEWLWE KYGLKPSQWV DYRALAGDPS DNIPGVKGIG 200 EKTAAKLIRE WGSLENLLKH LEQVKPASVR EKILSHMEDL 240
KLSLELSRVH TELPLQVDFA RRREPDREGL KAFLERLEFG 280
SLLHEFGLLE SPVAAEEAPW PPPEGAFVGY VLSRPEPMWA 320
ELNALAAAWE GRVYRAEDPL EALRGLGEVR GLLAKDLAVL 360
ALREGIALAP GDDPMLLAYL LDPSNTAPEG VARRYGGEWT 400 EEAGERALLS ERLYAALLER LKGEERLLWL YEEVEKPLSR 440
VLAHMEATGV WLDVAYLKAL SLEVEAELRR LEEEVHRLAG 480 HPFNLNSRDQ LERVLFDELG LPAIGKTEKT GKRSTSAAVL 520
EALREAHPIV DRILQYRELS KLKGTYIDPL PALVHPKTNR 560
LHTRFNQTAT ATGRLSSSDP NLQNIPVRTP LGQRIRRAFV 600
AEEGWRLWL DYSQIELRVL AHLSGDENLI RVFQEGQDIH 640
TQTASWMFGV PPEAVDSLMR RAAKTINYGV LYGMSAHRLS 680
GELAIPYEEA VAFIERYFQS YPKVRAWIEK TLAEGRERGY 720
VETLFGRRRY VPDLASRVKS IREAAERMAF NMPVQGTAAD 760
LMKLAMVKLF PRLQELGARM LLQVHDELVL EAPKEQAEEV 800
AQEAKRTMEE VWPLKVPLEV EVGIGEDWLS AKA 833
The nucleic acid polymerase polypeptides of the invention have homology to portions of the amino acid sequences of the thermostable DNA polymerases of Thermus aquaticus and Thermus thermophilus (see Figure 1). However, significant portions of the amino acid sequences of the present invention are distinct, including SEQ ED NO: 39-44.
As indicated above, derivative and variant polypeptides of the invention are derived from the wild type nucleic acid polymerase by deletion or addition of one or more amino acids to the N-terminal and/or C-terminal end of the wild type polypeptide; deletion or addition of one or more amino acids at one or more sites within the wild type polypeptide; or substitution of one or more amino acids at one or more sites within the wild type polypeptide. Thus, the polypeptides of the invention may be altered in various ways including amino acid substitutions, deletions, truncations, and insertions.
Such variant and derivative polypeptides may result, for example, from genetic polymoφhism or from human manipulation. Methods for such manipulations are generally known in the art. For example, amino acid sequence variants of the polypeptides can be prepared by mutations in the DNA. Methods for mutagenesis and nucleotide sequence alterations are well known in the art. See, for example, Kunkel, Proc. Natl. Acad. Sci. USA, 82, 488 (1985); Kunkel et al., Methods in Enzymol., 154, 367 (1987); U. S. Patent No. 4,873,192; Walker and Gaastra, eds., Techniques in Molecular Biology, MacMillan Publishing Company, New York (1983) and the references cited therein. Guidance as to appropriate amino acid substitutions that do not affect biological activity of the protein of interest may be found in the model of Dayhoff et al., Atlas of Protein Sequence and Structure, Natl. Biomed. Res. Found., Washington, CD. (1978), herein incoφorated by reference.
The derivatives and variants of the isolated polypeptides of the invention have identity with at least about 92% of the amino acid positions of any one of SEQ ED NO: 13-28 and have nucleic acid polymerase activity and/or are thermally stable. In a preferred embodiment, polypeptide derivatives and variants have identity with at least about 95% of the amino acid positions of any one of SEQ ED NO: 13-28 and have nucleic acid polymerase activity and/or are thermally stable. In a more preferred embodiment, polypeptide derivatives and variants have identity with at least about 98% of the amino acid positions of any one of SEQ ED NO: 13-28 and have nucleic acid polymerase activity and/or are thermally stable.
Amino acid residues of the isolated polypeptides and polypeptide derivatives and variants can be genetically encoded L-amino acids, naturally occurring non-genetically encoded L-amino acids, synthetic L-amino acids or D- enantiomers of any of the above. The amino acid notations used herein for the twenty genetically encoded L-amino acids and common non-encoded amino acids are conventional and are as shown in Table 2.
Table 2
Figure imgf000063_0001
Figure imgf000064_0001
Figure imgf000065_0001
Polypeptide variants that are encompassed within the scope of the invention can have one or more amino acids substituted with an amino acid of similar chemical and/or physical properties, so long as these variant polypeptides retain polymerase activity and/or remain thermally stable. Derivative polypeptides can have one or more amino acids substituted with amino acids having different chemical and/or physical properties, so long as these variant polypeptides retain polymerase activity and/or remain thermally stable.
Amino acids that are substitutable for each other in the present variant polypeptides generally reside within similar classes or subclasses. As known to one of skill in the art, amino acids can be placed into three main classes: hydrophilic amino acids, hydrophobic amino acids and cysteine-like amino acids, depending primarily on the characteristics of the amino acid side chain. These main classes may be further divided into subclasses. Hydrophilic amino acids include amino acids having acidic, basic or polar side chains and hydrophobic amino acids include amino acids having aromatic or apolar side chains. Apolar amino acids may be further subdivided to include, among others, aliphatic amino acids. The definitions of the classes of amino acids as used herein are as follows: "Hydrophobic Amino Acid" refers to an amino acid having a side chain that is uncharged at physiological pH and that is repelled by aqueous solution. Examples of genetically encoded hydrophobic amino acids include lie, Leu and Val. Examples of non-genetically encoded hydrophobic amino acids include t- BuA. "Aromatic Amino Acid" refers to a hydrophobic amino acid having a side chain containing at least one ring having a conjugated π-electron system (aromatic group). The aromatic group may be further substituted with substituent groups such as alkyl, alkenyl, alkynyl, hydroxyl, sulfonyl, nitro and amino groups, as well as others. Examples of genetically encoded aromatic amino acids include phenylalanine, tyrosine and tryptophan. Commonly encountered non- genetically encoded aromatic amino acids include phenylglycine, 2- naphthylalanine, β-2-thienylalanine, 1 ,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, 4-chlorophenylalanine, 2-fluorophenylalanine, 3-fluorophenylalanine and 4-fIuorophenylalanine. "Apolar Amino Acid" refers to a hydrophobic amino acid having a side chain that is generally uncharged at physiological pH and that is not polar. Examples of genetically encoded apolar amino acids include glycine, proline and methionine. Examples of non-encoded apolar amino acids include Cha. "Aliphatic Amino Acid" refers to an apolar amino acid having a saturated or unsaturated straight chain, branched or cyclic hydrocarbon side chain. Examples of genetically encoded aliphatic amino acids include Ala, Leu, Val and He. Examples of non-encoded aliphatic amino acids include Nle.
"Hydrophilic Amino Acid" refers to an amino acid having a side chain that is attracted by aqueous solution. Examples of genetically encoded hydrophilic amino acids include Ser and Lys. Examples of non-encoded hydrophilic amino acids include Cit and hCys.
"Acidic Amino Acid" refers to a hydrophilic amino acid having a side chain pK value of less than 7. Acidic amino acids typically have negatively charged side chains at physiological pH due to loss of a hydrogen ion. Examples of genetically encoded acidic amino acids include aspartic acid (aspartate) and glutamic acid (glutamate).
"Basic Amino Acid" refers to a hydrophilic amino acid having a side chain pK value of greater than 7. Basic amino acids typically have positively charged side chains at physiological pH due to association with hydronium ion. Examples of genetically encoded basic amino acids include arginine, lysine and histidine. Examples of non-genetically encoded basic amino acids include the non-cyclic amino acids ornithine, 2,3-diaminopropionic acid, 2,4-diaminobutyric acid and homoarginine. "Polar Amino Acid" refers to a hydrophilic amino acid having a side chain that is uncharged at physiological pH, but which has a bond in which the pair of electrons shared in common by two atoms is held more closely by one of the atoms. Examples of genetically encoded polar amino acids include asparagine and glutamine. Examples of non-genetically encoded polar amino acids include citrulline, N-acetyl lysine and methionine sulfoxide.
"Cysteine-Like Amino Acid" refers to an amino acid having a side chain capable of forming a covalent linkage with a side chain of another amino acid residue, such as a disulfide linkage. Typically, cysteine-like amino acids generally have a side chain containing at least one thiol (SH) group. Examples of genetically encoded cysteine-like amino acids include cysteine. Examples of non-genetically encoded cysteine-like amino acids include homocysteine and penicillamine. As will be appreciated by those having skill in the art, the above classifications are not absolute. Several amino acids exhibit more than one characteristic property, and can therefore be included in more than one category. For example, tyrosine has both an aromatic ring and a polar hydroxyl group. Thus, tyrosine has dual properties and can be included in both the aromatic and polar categories. Similarly, in addition to being able to form disulfide linkages, cysteine also has apolar character. Thus, while not strictly classified as a hydrophobic or apolar amino acid, in many instances cysteine can be used to confer hydrophobicity to a polypeptide. Certain commonly encountered amino acids that are not genetically encoded and that can be present, or substituted for an amino acid, in the variant polypeptides of the invention include, but are not limited to, β-alanine (b-Ala) and other omega-amino acids such as 3-aminopropionic acid (Dap), 2,3- diaminopropionic acid (Dpr), 4-aminobutyric acid and so forth; α- aminoisobutyric acid (Aib); e-aminohexanoic acid (Aha); δ-aminovaleric acid (Ava); N-methylglycine (MeGly); ornithine (Orn); citrulline (Cit); t-butylalanine (t-BuA); t-butylglycine (t-BuG); N-methylisoleucine (Melle); phenylglycine (Phg); cyclohexylalanine (Cha); norleucine (Nle); 2-naphthylalanine (2-Nal); 4- chlorophenylalanine (Phe(4-Cl)); 2-fluorophenylalanine (Phe(2-F)); 3- fluorophenylalanine (Phe(3-F)); 4-fluorophenylalanine (Phe(4-F)); penicillamine (Pen); 1, 2,3, 4-tetrahydroisoquinoline-3 -carboxylic acid (Tic); .beta. -2- thienylalanine (Thi); methionine sulfoxide (MSO); homoarginine (hArg); N- acetyl lysine (AcLys); 2,3-diaminobutyric acid (Dab); 2,3-diaminobutyric acid (Dbu); p-aminophenylalanine (Phe(pNH2)); N-methyl valine (Me Val); homocysteine (hCys) and homoserine (hSer). These amino acids also fall into the categories defined above.
The classifications of the above-described genetically encoded and non- encoded amino acids are summarized in Table 3, below. It is to be understood that Table 3 is for illustrative puφoses only and does not puφort to be an exhaustive list of amino acid residues that may comprise the variant and derivative polypeptides described herein. Other amino acid residues that are useful for making the variant and derivative polypeptides described herein can be found, e.g., in Fasman, 1989, CRC Practical Handbook of Biochemistry and Molecular Biology, CRC Press, Inc., and the references cited therein. Amino acids not specifically mentioned herein can be conveniently classified into the above-described categories on the basis of known behavior and/or their characteristic chemical and/or physical properties as compared with amino acids specifically identified.
TABLE 3
Figure imgf000068_0001
Polypeptides of the invention can have any amino acid substituted by any similarly classified amino acid to create a variant peptide, so long as the peptide variant is thermally stable and/or retains DNA Polymerase activity.
"Domain shuffling" or construction of "thermostable chimeric nucleic acid polymerases" may be used to provide thermostable polymerases containing novel properties. For example, placement of codons 289-422 from the Thermus scotoductus DNA polymerase coding sequence after codons 1-288 of the
Thermus aquaticus DNA polymerase would yield a novel thermostable nucleic acid polymerase containing the 5' - 3' exonuclease domain of Thermus aquaticus
DNA polymerase (1-289), the 3' - 5' exonuclease domain of Thermus scotoductus nucleic acid polymerase (289-422), and the DNA polymerase domain of Thermus aquaticus DNA polymerase (423-832). Alternatively, the 5' -
3' exonuclease domain and the 3' - 5' exonuclease domain of Thermus scotoductus nucleic acid polymerase may be fused to the DNA polymerase
(dNTP binding and primer/template binding domains) portions of Thermus aquaticus DNA polymerase (about codons 423-832). The donors and recipients need not be limited to Thermus aquaticus and Thermus scotoductus polymerases. Thermus thermophilus DNA polymerase 3' - 5' exonuclease, 5' - 3' exonuclease and DNA polymerase domains can similarly be exchanged for those in the Thermus scotoductus polymerases of the invention.
It has been demonstrated that the exonuclease domain of Thermus aquaticus Polymerase I can be removed from the amino terminus of the protein with out a significant loss of thermostability or polymerase activity (Erlich et al., (1991) Science 252: 1643-1651, Barnes, W.M., (1992) Gene 112:29-35., Lawyer et al., (1989) JBC 264:6427-6437). Other N-terminal deletions similarly have been shown to maintain thermostability and activity (Vainshtein et al., (1996) Protein Science 5:1785-1792 and references therein.) Therefore this invention also includes similarly truncated forms of any of the wild type or variant polymerases provided herein. For example, the invention is also directed to an active truncated variant of any of the polymerases provided by the invention in which the first 330 amino acids are removed.
Moreover, the invention provides SEQ ED NO:45, a truncated form of a polymerase in which the N-terminal 289 amino acids have been removed from the wild type Thermus scotoductus polymerase from strain X-l.
E SPVAAEEAPW 300
PPPEGAFVGY VLSRPEPMWA ELNALAAAWE GRVYRAEDPL 340
EALRGLGEVR GLLAKDLAVL ALREGIALAP GDDPMLLAYL 380 LDPSNTAPEG VARRYGGEWT EEAGERALLS ERLYAALLER 420
LKGEERLLWL YEEVEKPLSR VLAHMEATGV RLDVAYLKAL 460
SLEVEAELRR LEEEVHRLAG HPFNLNSRDQ LERVLFDELG 500
LPAIGKTEKT GKRSTSAAVL EALREAHPIV DRILQYRELS 540
KLKGTYIDPL PALVHPKTNR LHTRFNQTAT ATGRLSSSDP 580 NLQNIPVRTP LGQRIRRAFV AEEGWRLWL DYSQIELRVL 620
AHLSGDENLI RVFQEGQDIH TQTASWMFGV PPEAVDSLMR 660
RAAKTINFGV LYGMSAHRLS GELAIPYEEA VAFIERYFQS 700
YPKVRAWIEK TLAEGRERGY VETLFGRRRY VPDLASRVKS 740
IRE7AAERMAF NMPVQGTAAD LMKLAMVKLF PRLQELGARM 780 LLQVHDELVL EAPKEQAEEV AQEAKRTMEE VWPLKVPLEV 820
EVGIGEDWLS AKA 833
Moreover, the invention provides SEQ ID NO:46 a truncated form of a polymerase in which the N-terminal 289 amino acids have been removed from the wild type Thermus scotoductus polymerase from strain SM3.
E SPVAAEEAPW 300
PPPEGAFVGY VLSRPEPMWA ELNALAAAWE GRVYRAEDPL 340
EALRGLGEVR GLLAKDLAVL ALREGIALAQ GDDPMLLAYL 380
LDPSNTAPEG VARRYGGEWT EEAGERALLS ERLYAALLER 420 LKGEERLLWL YEEVEKPLSR VLAHMEATGV WLDVAYLKAL 460
SLEVEAELRR LEEEVHRLAG HPFNLNSRDQ LERVLFDELG 500
LPAIGKTEKT GKRSTSAAVL EALREAHPIV DRILQYRELS 540
KLKGTYIDPL PALVHPKTNR LHTRFNQTAT ATGRLSSSDP 580
NLQNIPVRTP LGQRIRRAFV AEEGWRLWL DYSQIELRVL 620 AHLSGDENLI RVFQEGQDIH TQTASWMFGV PPEAVDSLMR 660
RAAKTINFGV LYGMSAHRLS GELAIPYEEA VAFIERYFQS 700
YPKVRAWIEK TLAEGRERGY VETLFGRRRY VPDLASRVKS 740
IREAAERMAF NMPVQGTAAD LMKLAMVKLF PRLQELGARM 780
LLQVHDELVL EAPKEQAEEV AQEAKRTMEE VWPLKVPLEV 820 EVGIGEDWLS AKA 833
Moreover, the invention provides SEQ ED NO:47 a truncated form of a polymerase in which the N-terminal 289 amino acids have been removed from the wild type Thermus scotoductus polymerase from strain Vi7a. E SPVAAEEAPW 300
PPPEGAFVGY VLSRPEPMWA ELNALAAAWE GRVYRAEDPL 340 EALRGLGEVR GLLAKDLAVL ALREGIALAP GDDPMLLAYL 380
LDPSNTAPEG VARRYGGEWT EEAGERALLS ERLYAALLER 420
LKGEERLLWL YEEVEKPLSR VLAHMEATGV WLDVAYLKAL 460
SLEVEAELRR LEEEVHRLAG HPFNLNSRDQ LERVLFDELG 500 LPAIGKTEKT GKRSTSAAVL EALREAHPIV DRILQYRELS 540
KLKGTYIDPL PALVHPKTNR LHTRFNQTAT ATGRLSSSDP 580
NLQNIPVRTP LGQRIRRAFV AEEGWRLWL DYSQIELRVL 620
AHLSGDENLI RVFQEGQDIH TQTASWMFGV PPEAVDSLMR 660
RAAKTINFGV LYGMSAHRLS GELAIPYEEA VAFIERYFQS 700 YPKVRAWIEK TLAEGRERGY VETLFGRRRY VPDLASRVKS 740
IREAAERMAF NMPVQGTAAD LMKLAMVKLF PRLQELGARM 780
LLQVHDELVL EAPKEQAEEV AQEAKRTMEE VWPLKVPLEV 820
EVGIGEDWLS AKA 833
Thus, the polypeptides of the invention encompass both naturally occurring proteins as well as variations, truncations and modified forms thereof. Such variants will continue to possess the desired activity. The deletions, insertions, and substitutions of the polypeptide sequence encompassed herein are not expected to produce radical changes in the characteristics of the polypeptide. One skilled in the art can readily evaluate the thermal stability and polymerase activity of the polypeptides and variant polypeptides of the invention by routine screening assays.
Kits and compositions containing the present polypeptides are substantially free of cellular material. Such preparations and compositions have less than about 30%, 20%, 10%, 5%, (by dry weight) of contaminating bacterial cellular protein.
The activity of nucleic acid polymerase polypeptides and variant polypeptides can be assessed by any procedure known to one of skill in the art. For example, the DNA synthetic activity of the variant and non- variant polymerase polypeptides of the invention can be tested in standard DNA sequencing or DNA primer extension reaction. One such assay can be performed in a 100 μl (final volume) reaction mixture, containing, for example, 0.1 mM dCTP, dTTP, dGTP, α-32P-dATP, 0.3 mg/ml activated calf thymus DNA and 0.5 mg/ml BSA in a buffer containing: 50 mM KCl, 1 mM DTT, 10 mM MgCl and 50 mM of a buffering compound such as PIPES, Tris or Triethylamine. A dilution to 0.1 units/μl of each polymerase enzyme is prepared, and 5 μl of such a dilution is added to the reaction mixture, followed by incubation at 60 °C for 10 minutes. Reaction products can be detected by determining the amount of 32P incoφorated into DNA or by observing the products after separation on a polyacrylamide gel.
Uses for Nucleic Acid Polymerase Polypeptides
The thermostable enzyme of this invention may be used for any puφose in which DNA Polymerase or reverse transcriptase activity is necessary or desired. For example, the present nucleic acid polymerase polypeptides can be used in one or more of the following procedures: DNA sequencing, DNA amplification, RNA amplification, reverse transcription, DNA synthesis and/or primer extension. The nucleic acid polymerase polypeptides of the invention can be used to amplify DNA by polymerase chain reaction (PCR). The nucleic acid polymerase polypeptides of the invention can be used to sequence DNA by Sanger sequencing procedures. The nucleic acid polymerase polypeptides of the invention can also be used in primer extension reactions. The nucleic acid polymerase polypeptides of the invention can also be used for reverse transcription. The nucleic acid polymerase polypeptides of the invention can be used test for single nucleotide polymoφhisms (SNPs) by single nucleotide primer extension using terminator nucleotides. Any such procedures and related procedures, for example, polynucleotide or primer labeling, minisequencing and the like are contemplated for use with the present nucleic acid polymerase polypeptides. Methods of the invention comprise the step of extending a primed polynucleotide template with at least one labeled nucleotide, wherein the extension is catalyzed by a nucleic acid polymerase of the invention. Nucleic acid polymerases used for Sanger sequencing can produce fluorescently labeled products that are analyzed on an automated fluorescence-based sequencing apparatus such as an Applied Biosystems 310 or 377 (Applied Biosystems,
Foster City, Calif). Detailed protocols for Sanger sequencing are known to those skilled in the art and may be found, for example in Sambrook et al, Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989). En one embodiment, the nucleic acid polymerase polypeptides of the invention are used for DNA amplification. Any procedure that employs a DNA polymerase can be used, for example, in polymerase chain reaction (PCR) assays, strand displacement amplification and other amplification procedures. Strand displacement amplification can be used as described in Walker et al (1992) Nucl. Acids Res. 20, 1691-1696. The term "polymerase chain reaction" ("PCR") refers to the method of K. B. Mullis U.S. Pat. Nos. 4,683,195; 4,683,202; and 4,965,188, hereby incoφorated by reference, which describe a method for increasing the concentration of a segment of a target sequence in a mixture of genomic DNA or other DNA or RNA without cloning or purification. The PCR process for amplifying a target sequence consists of introducing a large excess of two oligonucleotide primers to the DNA mixture containing the desired target sequence, followed by a precise sequence of thermal cycling in the presence of a nucleic acid polymerase. The two primers are complementary to their respective strands of the double stranded target sequence. To effect amplification, the mixture is denatured and the primers annealed to their complementary sequences within the target molecule. Following annealing, the primers are extended with a polymerase so as to form a new pair of complementary strands. The steps of denaturation, primer annealing and polymerase extension can be repeated many times. Each round of denaturation, annealing and extension constitutes one "cycle." There can be numerous cycles, and the amount of amplified DNA produced increases with the number of cycles. Hence, to obtain a high concentration of an amplified target nucleic acid, many cycles are performed.
The steps involve in PCR nucleic acid amplification method are described in more detail below. For ease of discussion, the nucleic acid to be amplified is described as being double-stranded. However, the process is equally useful for amplifying a single-stranded nucleic acid, such as an mRNA, although the ultimate product is generally double- stranded DNA. In the amplification of a single- stranded nucleic acid, the first step involves the synthesis of a complementary strand (one of the two amplification primers can be used for this puφose), and the succeeding steps proceed as follows: (a) contacting each nucleic acid strand with four different nucleoside triphosphates and one oligonucleotide primer for each strand of the specific sequence being amplified, wherein each primer is selected to be substantially complementary to the different strands of the specific sequence, such that the extension product synthesized from one primer, when it is separated from its complement, can serve as a template for synthesis of the extension product of the other primer, such contacting being at a temperature that allows hybridization of each primer to a complementary nucleic acid strand;
(b) contacting each nucleic acid strand; at the same time as or after step (a), with a nucleic acid polymerase of the invention that enables combination of the nucleoside triphosphates to form primer extension products complementary to each strand of the specific nucleic acid sequence; (c) maintaining the mixture from step (b) at an effective temperature for an effective time to promote the activity of the enzyme and to synthesize, for each different sequence being amplified, an extension product of each primer that is complementary to each nucleic acid strand template, but not so high as to separate each extension product from the complementary strand template;
(d) heating the mixture from step (c) for an effective time and at an effective temperature to separate the primer extension products from the templates on which they were synthesized to produce single-stranded molecules but not so high as to denature irreversibly the enzyme; (e) cooling the mixture from step (d) for an effective time and to an effective temperature to promote hybridization of a primer to each of the single- stranded molecules produced in step (d); and
(f) maintaining the mixture from step (e) at an effective temperature for an effective time to promote the activity of the enzyme and to synthesize, for each different sequence being amplified, an extension product of each primer that is complementary to each nucleic acid template produced in step (d) but not so high as to separate each extension product from the complementary strand template. The effective times and temperatures in steps (e) and (f) may coincide, so that steps (e) and (f) can be carried out simultaneously. Steps (d)-(f) are repeated until the desired level of amplification is obtained.
The amplification method is useful not only for producing large amounts of a specific nucleic acid sequence of known sequence but also for producing nucleic acid sequences that are known to exist but are not completely specified. One need know only a sufficient number of bases at both ends of the sequence in sufficient detail so that two oligonucleotide primers can be prepared that will hybridize to different strands of the desired sequence at relative positions along the sequence such that an extension product synthesized from one primer, when separated from the template (complement), can serve as a template for extension of the other primer. The greater the knowledge about the bases at both ends of the sequence, the greater can be the specificity of the primers for the target nucleic acid sequence.
Thermally stable nucleic acid polymerases are therefore generally used for PCR because they can function at the high temperatures used for melting double stranded target DNA and annealing the primers during each cycle of the PCR reaction. High temperature results in thermodynamic conditions that favor primer hybridization with the target sequences and not hybridization with non- target sequences (H. A. Erlich (ed.), PCR Technology, Stockton Press [1989]). The thermostable nucleic acid polymerases of the present invention satisfy the requirements for effective use in amplification reactions such as PCR. The present polymerases do not become irreversibly denatured (inactivated) when subjected to the required elevated temperatures for the time necessary to melt double-stranded nucleic acids during the amplification process. Irreversible denaturation for puφoses herein refers to permanent and complete loss of enzymatic activity. The heating conditions necessary for nucleic acid denaturation will depend, e.g., on the buffer salt concentration and the composition and length of the nucleic acids being denatured, but typically denaturation can be done at temperatures ranging from about 90 °C to about 105°C The time required for denaturation depends mainly on the temperature and the length of the duplex nucleic acid. Typically the time needed for denaturation ranges from a few seconds up to four minutes. Higher temperatures may be required as the salt concentration of the buffer, or the length and/or GC composition of the nucleic acid is increased. The nucleic acid polymerases of the invention do not become irreversibly denatured for relatively short exposures to temperatures of about 90°C to 100°C.
The thermostable polymerases of the invention have an optimum temperature at which they function that is higher than about 45 °C. Temperatures below 45 °C facilitate hybridization of primer to template, but depending on salt composition and concentration and primer composition and length, hybridization of primer to template can occur at higher temperatures (e.g., 45 °C to 70 °C), which may promote specificity of the primer hybridization reaction. The polymerases of the invention exhibit activity over a broad temperature range from about 37°C to about 90°C
The present polymerases have particular utility for PCR not only because of their thermal stability but also because of their ability to synthesize DNA using an RNA template and because of their fidelity in replicating the template nucleic acid. In most PCR reactions that start with an RNA template, reverse transcriptase must be added. However, use of reverse transcriptase has certain drawbacks. First, it is not stable at higher temperatures. Hence, once the initial complementary DNA (cDNA) has been made by reverse transcriptase and the thermal cycles of PCR are started, the original RNA template is not used as a template in the amplification reaction. Second, reverse transcriptase does not produce a cDNA copy with particularly good sequence fidelity. With PCR, it is possible to amplify a single copy of a specific target or template nucleic acid to a level detectable by several different methodologies. However, if the sequence of the target nucleic acid is not replicated with fidelity, then the amplified product can include a pool of nucleic acids with diverse sequences. Hence, the nucleic acid polymerases of the invention that can accurately reverse transcribe RNA and replicate the sequence of the template RNA or DNA with high fidelity is highly desirable.
Any nucleic acid can act as a "target nucleic acid" for the PCR methods of the invention. The term "target," when used in reference to the polymerase chain reaction, refers to the region of nucleic acid bounded by the primers used for polymerase chain reaction. En addition to genomic DNA and mRNA, any cDNA, RNA, oligonucleotide or polynucleotide can be amplified with the appropriate set of primer molecules. In particular, the amplified segments created by the PCR process itself are, themselves, efficient templates for subsequent PCR amplifications. The length of the amplified segment of the desired target sequence is determined by the relative positions of the primers with respect to each other, and therefore, this length is readily controlled.
The amplified target nucleic acid can be detected by any method known to one of skill in the art. For example, target nucleic acids are often amplified to such an extent that they form a band visible on a size separation gel. Target nucleic acids can also be detected by hybridization with a labeled probe; by incoφoration of biotinylated primers during PCR followed by avidin-enzyme conjugate detection; by incoφoration of 32P-labeled deoxynucleotide triphosphates during PCR, and the like.
The amount of amplification can also be monitored, for example, by use of a reporter-quencher oligonucleotide as described in U.S. Patent 5,723,591, and a nucleic acid polymerase of the invention that has 5' - 3' nuclease activity. The reporter-quencher oligonucleotide has an attached reporter molecule and an attached quencher molecule that is capable of quenching the fluorescence of the reporter molecule when the two are in proximity. Quenching occurs when the reporter-quencher oligonucleotide is not hybridized to a complementary nucleic acid because the reporter molecule and the quencher molecule tend to be in proximity or at an optimal distance for quenching. When hybridized, the reporter-quencher oligonucleotide emits more fluorescence than when unhybridized because the reporter molecule and the quencher molecule tend to be further apart. To monitor amplification, the reporter-quencher oligonucleotide is designed to hybridize 3' to an amplification primer. During amplification, the 5' - 3' nuclease activity of the polymerase digests the reporter oligonucleotide probe, thereby separating the reporter molecule from the quencher molecule. As the amplification is conducted, the fluorescence of the reporter molecule increases. Accordingly, the amount of amplification performed can be quantified based on the increase of fluorescence observed. Oligonucleotides used for PCR primers are usually about 9 to about 75 nucleotides, preferably about 17 to about 50 nucleotides in length. Preferably, an oligonucleotide for use in PCR reactions is about 40 or fewer nucleotides in length (e.g., 9, 12, 15, 18, 20, 21, 24, 27, 30, 35, 40, or any number between 9 and 40). Generally specific primers are at least about 14 nucleotides in length. For optimum specificity and cost effectiveness, primers of 16-24 nucleotides in length are generally preferred.
Those skilled in the art can readily design primers for use processes such as PCR. For example, potential primers for nucleic acid amplification can be used as probes to determine whether the primer is selective for a single target and what conditions permit hybridization of a primer to a target within a sample or complex mixture of nucleic acids.
The present invention also contemplates use of the present nucleic acid polymerases in combination with other procedures or enzymes. For example, the polymerases can be used in combination with additional reverse transcriptase or another DNA polymerase. See U.S. Pat. No. 5,322,770, incoφorated by reference herein.
In another embodiment, nucleic acid polymerases of the invention with 5' - 3' exonuclease activity are used to detect target nucleic acids in an invader- directed cleavage assay. This type of assay is described, for example, in U.S. Patent 5,994,069. It is important to note that the 5' - 3' exonuclease of DNA polymerases is not really an exonuclease that progressively cleaves nucleotides from the 5' end of a nucleic acid, but rather a nuclease that can cleave certain types of nucleic acid structures to produce oligonucleotide cleavage products. Such cleavage is sometimes called structure-specific cleavage.
In general, the invader-directed cleavage assay employs at least one pair of oligonucleotides that interact with a target nucleic acid to form a cleavage structure for the 5' - 3' nuclease activity of the nucleic acid polymerase. Distinctive cleavage products are released when the cleavage structure is cleaved by the 5' - 3' nuclease activity of the polymerase. Formation of such a target- dependent cleavage structure and the resulting cleavage products is indicative of the presence of specific target nucleic acid sequences in the test sample.
Therefore, in the invader-directed cleavage procedure, the 5' - 3' nuclease activity of the present polymerases is needed as well at least one pair of oligonucleotides that interact with a target nucleic acid to form a cleavage structure for the 5' - 3' nuclease. The first oligonucleotide, sometimes termed the "probe," can hybridize within the target site but downstream of a second oligonucleotide, sometimes termed an "invader" oligonucleotide. The invader oligonucleotide can hybridize adjacent and upstream of the probe oligonucleotide. However, the target sites to which the probe and invader oligonucleotides hybridize overlap such that the 3' segment of the invader oligonucleotide overlaps with the 5' segment of the probe oligonucleotide. The 5' - 3' nuclease of the present polymerases can cleave the probe oligonucleotide at an internal site to produce distinctive fragments that are diagnostic of the presence of the target nucleic acid in a sample. Further details and methods for adapting the invader-directed cleavage assay to particular situations can be found in U.S. Patent 5,994,069.
One or more nucleotide analogs can also be used with the present methods, kits and with the nucleic acid polymerases. Such nucleotide analogs can be modified or non-naturally occurring nucleotides such as 7-deaza purines (i.e., 7-deaza-dATP and 7-deaza-dGTP). Nucleotide analogs include base analogs and comprise modified forms of deoxyribonucleotides as well as ribonucleotides. As used herein the term "nucleotide analog" when used in reference to targets present in a PCR mixture refers to the use of nucleotides other than dATP, dGTP, dCTP and dTTP; thus, the use of dUTP (a naturally occurring dNTP) in a PCR would comprise the use of a nucleotide analog in the PCR. A PCR product generated using dUTP, 7-deaza-dATP, 7-deaza-dGTP or any other nucleotide analog in the reaction mixture is said to contain nucleotide analogs.
The invention also provides kits that contain at least one of the nucleic acid polymerases of the invention. Individual kits may be adapted for performing one or more of the following procedures: DNA sequencing, DNA amplification, RNA Amplification and/or primer extension. Kits of the invention comprise a DNA polymerase polypeptide of the invention and at least one nucleotide. A nucleotide provided in the kits of the invention can be labeled or unlabeled. Kits preferably can also contain instructions on how to perform the procedures for which the kits are adapted.
Optionally, the subject kit may further comprise at least one other reagent required for performing the method the kit is adapted to perform. Examples of such additional reagents include: another unlabeled nucleotide, another labeled nucleotide, a balance mixture of nucleotides, one or more chain terminating nucleotides, one or more nucleotide analogs, buffer solution(s), magnesium solution(s), cloning vectors, restriction endonucleases, sequencing primers, reverse transcriptase, and DNA or RNA amplification primers. The reagents included in the kits of the invention may be supplied in premeasured units so as to provide for greater precision and accuracy. Typically, kits reagents and other components are placed and contained in separate vessels. A reaction vessel, test tube, microwell tray, microtiter dish or other container can also be included in the kit. Different labels can be used on different reagents so that each reagent can be distinguished from another. The following Examples further illustrate the invention and are not intended to limit the scope of the invention.
EXAMPLE 1: Cloning of Thermus scotoductus, Strain X-l Polymerase
Growth of bacteria and genomic DNA isolation
Thermus scotoductus (Tsc) strain X-l was obtained from ATCC (ATCC Deposit No. 27978). The lyophilized bacteria were revived in ATCC Culture Medium 461 (Castenholz TYE medium) and grown overnight to stationary phase. Thermus scotoductus genomic DNA was prepared using a Quiagen genomic DNA preparation protocol and kit (Quiagen).
Cloning methods
The first forward and reverse primers were designed by analysis of 5' and 3' terminal homologous conserved regions of the DNA sequences of Thermus aquaticus (Taq), Thermus thermophilus (Tth), Thermus filiformis (Tfi), Thermus caldophilus (that was determined to actually be Tth strain GK24), and Thermus flavus (believed to be Thermus igniterrae). A fragment of a Thermus scotoductus polymerase gene was amplified using N-terminal primer 5'- ggc cac cac ctg gcc tac -3' (SEQ ED NO:29) and C-terminal primer 5'- ccc ace tec ace tec ag -3' (SEQ ED NO:30). The following PCR reaction mixture contained 2.5μl of lOx Amplitaq buffer (ABi), 2mM MgCl, 60 ng DNA template, 2.5mM (each) dNTP, 20pmol of each primer, and 1.25 units of Amplitaq DNA polymerase in a 25 μl total reaction volume.
The reaction mixture was heated to 80°C and then the primers were added. This was followed by a predenaturation step (96°C for 30 s); PCR cycling for 30 cycles (97°C for 3 s, 56°C for 30 s, 72°C for 3 min) with a finishing step (72°C for 7 min). This produced an approximate 1.5 kb DNA fragment that was cloned and sequenced. This cloned fragment showed some homology to the Tth Polymerase I gene (Genebank accession number 466573) between nucleotide numbers 644 and 1973.
Direct sequencing of the genomic DNA was used to obtain the sequence of the 5' terminus of the Thermus scotoductus polymerase gene. The primer used was 5'- ctg gcc atg ctg aag etc ttt -3' (SEQ ED NO:31) and a 2-step thermocycling protocol. A predenaturation step (95°C for 5 min) was followed by 80 cycles (97°C for 5sec, 60°C for 4min). Reaction mixture consisted of 16μl Big Dye VI Ready Reaction mix, 2.8 ug DNA, 15 pmol primer in a 40μl total reaction volume. The sequencing of the Thermus scotoductus gene from genomic DNA revealed that the 5' terminal sequence of the wild-type Thermus scotoductus gene is 5'- ata agg gcg atg ctg ccc etc ttt gag-5' (SEQ ID NO: 32) that would indicate that the ATG is the start codon of the wild-type gene. However, the N-terminus of Taq, Tth and Tfi enzymes have two methionine amino acid residues at their N terminal end separated by two amino acids. In order to make the Thermus scotoductus N-terminus more similar to the other known Thermus DNA polymerases, and possibly to improve protein translation efficiency, the ATA codon was changed to ATG. This introduced an additional start for protein translation making the recombinant protein N terminus MRAM. The amplification of the full-length Thermus scotoductus nucleic acid polymerase coding region was carried out using the 5' forward primer 5' - cat atg agg gcg atg ctg ccc etc -3' (SEQ ED NO:33). Another consideration when designing this primer was to introduce a recognition site for the restriction enzyme Nde I (catatg, SEQ ID NO:34). This sequence was introduced to facilitate subcloning of the coding region into other plasmid vectors.
As described above, the first cloned portion of the Thermus scotoductus, strain X-l polymerase gene was only 1.2 kb. This represented approximately half of the full-length gene. In order to obtain a larger fragment of the Thermus scotoductus gene, a PCR reaction was carried out using the 5' forward primer (SEQ ED NO:33) described in the previous paragraph and a new primer designed near the same homologous 3' region of the known Thermus polymerase genes. The sequence of this primer was 5'- etc cac etc cag ggg cac -3' (SEQ ID NO:35). The PCR reaction was the same mixture as above. The cycling conditions were altered slightly in order to promote greater specificity. The reaction mixture was heated to 80°C and then the primers were added. This was followed by a predenaturation step (96°C for 2 min); PCR cycling for 10 cycles (97°C for 10 s, 70°C for 3 min), 25 cycles (97°C for 10 s, 60°C for 3 min), with a finishing step (72°C for 7 min). This produced a 2.4kb fragment that was cloned and sequenced. This left to be sequenced a short 3' terminal region of the Thermus scotoductus, strain X-l polymerase gene.
Based on the additional sequence of the larger fragment of the Thermus scotoductus polymerase gene, a new primer was designed to obtain the remaining unknown 3' sequence: 5'- ctg gcc atg gtg aag etc ttt -3' (SEQ ED NO:36). The genomic sequencing protocol was the same as described for the previous genomic DNA sequencing reaction for the 5' terminus. Once the sequence was obtained, a primer was designed to be used with the 5' terminal primer described above to amplify the full length Thermus scotoductus polymerase gene. This primer is complementary to the 3' terminal sequence. It also has a Sal I recognition site (gtcgac, SEQ ID NO:37) overlapping with the stop codon. This restriction site will facilitate subcloning into other plasmid DNA vectors. The sequence of the primer is 5'-gtc gac tag gcc ttg gcg aaa gcc a -3' (SEQ ID NO:38).
Three different cloned Thermus scotoductus polymerase genes were sequenced independently in order to rule out PCR errors. The resulting consensus sequence is the natural Thermus scotoductus polymerase gene sequence of this invention (SEQ ID NO: 14). The amino acid numbering used in this description of the invention is based on a recombinant form of the Thermus scotoductus polymerase protein that has an additional three amino acids at its N- terminus (SEQ ED NO: 13). However, SEQ ED NO: 14 is the sequence for the wild type Thermus scotoductus polymerase from strain X-l.
The amino acid sequence of the strain X-l Thermus scotoductus polymerase has several differences when compared with the amino acid sequence of Thermus aquaticus DNA Polymerase, including about 51 conservative amino acid changes and about 62 nonconservative amino acid changes. For example, one region of dissimilarity is between amino acid positions at approximately 51 and about 65, where the sequence of the Thermus scotoductus polymerase has about four amino acid changes (in bold):
LLKALREDG DVVIVVFDAK APSFRHQTYE (SEQ ED NO:39). Another region of dissimilarity is between amino acid positions at approximately 201 and about 236, where the sequence of the Thermus scotoductus polymerase has about seven amino acid changes (in bold): GEKTAAKLIREWGSLENLLKHLEQV KPASV REKILS (SEQ ID NO:40). Another region of dissimilarity is between amino acid positions at approximately 311 and about 350, where the sequence of the Thermus scotoductus polymerase has about seven amino acid changes (in bold): VGYVLSRPEPMWAELN ALAAAWEGRNYRAEDPLEALRGLG (SEQ ED ΝO:41). Another region of dissimilarity is between amino acid positions at approximately 415 and about 435, where the sequence of the Thermus scotoductus polymerase has about five amino acid changes (in bold): RLYAALLERLKGEERLLWLYE (SEQ ID NO:42). Another region of dissimilarity is between amino acid positions at approximately 531 and about 562, where the sequence of the Thermus scotoductus polymerase has about six amino acid changes (in bold): PEVDRILQYRELSKLK GTYED PLPALVHPKTN (SEQ ID NO:43). Another region of dissimilarity is between amino acid positions at approximately 801 and about 836, where the sequence of the Thermus scotoductus polymerase has about eight amino acid changes (in bold): EEVAQEAKRT MEEVWPLKVPLEVEVGIGEDWLSAKA (SEQ ID NO:44). Hence, many regions of the Thermus scotoductus polymerase differ from the Thermus aquaticus and Thermus thermophilus DNA Polymerases.
Modification of Strain X-l Polymerase Wild-Type Gene
In order to produce Thermus scotoductus polymerase in a form suitable for dye-terminator DNA sequencing, two amino acid substitutions were made. These are the FS (Tabor and Richardson, 1995 PNAS 92: 6339-6343) and exo- minus (G46D mutation) mutations. To reduce the exonuclease activity to very low levels, the mutation G46D was introduced. To reduce the discrimination between ddNTP's and dNTP's, the mutation F666Y was introduced.
Mutagenesis was carried out using the modified QuickChange™ (Stratagene) PCR mutagenesis protocol described in Sawano & Miyawaki (2000), Nucleic Acids Research Vol. 28. The mutated gene was resequenced completely to confirm the introduction of the mutations and to ensure that no PCR errors were introduced.
The Thermus scotoductus, strain X-l, polymerase gene (FS, exo- ) was removed from the cloning vector by restriction digest with Ndel and Sail. The 2.4kb gene was ligated into the pT7 expression vector (Brookhaven National Laboratories, Long Island, NY). This resulting vector containing the Thermus scotoductus polymerase (fs, exo-) gene was used to transform BL21 E. coli cells (Invitrogen).
EXAMPLE 2: Thermus scotoductus, Strain X-l Polymerase Expression and
Purification BL21 E. coli cells (Invitrogen) containing the pT7 expression vector with the Thermus scotoductus, strain X-l polymerase coding region were grown in one liter of Terrific Broth (Maniatis) to an optical density of 1.2OD and the polymerase protein was oveφroduced by four-hour induction with 1.0 mM EPTG. The cells were harvested by centrifugation, washed in 50 mM Tris (pH 7.5), 5mM EDTA, 5% glycerol, lOmM EDTA to remove growth media, and the cell pellet frozen at -80°C.
To isolate the Thermus scotoductus, strain X-l polymerase, the cells were thawed and resuspended in 2.5 volumes (wet weight) of 50mM Tris (pH 7.2), 400mM NaCl, ImM EDTA. The cell walls were disrupted by sonication and the resulting E. coli cell debris were removed by centrifugation. The resulting lysate was pasteurized in a water bath (75°C for 45 min), denaturing and precipitating the majority of the non-thermostable E. coli proteins and leaving the thermostable Thermus scotoductus, strain X-l polymerase in solution. E. coli genomic DNA was removed by coprecipitation with 0.3% Polyethyleneimine (PEI). The cleared lysate is then applied to two columns in series: (1) a Biorex 70 cation exchange resin that chelates excess PEI and (2) a heparin-agarose column (dimensions to be provided) that retains the polymerase. This column is washed with 5 column volumes of 20mM Tris (pH 8.5), 5%glycerol, lOOmM NaCl, O.lmM EDTA, 0.05% Triton X-100 and 0.05% Tween-20 (KTA). The protein was then eluted with a 0.1 to l.OM NaCl linear gradient. The polymerase eluted at 0.8M NaCl. The eluted Tsc Polymerase was concentrated and the buffer exchanged using a Millipore concentration filter (30kD M.W. cutoff). The concentrated protein was stored at in KTA (no salt) plus 50% glycerol at - 20°C.
The activity of the polymerase was measured using the standard salmon sperm DNA radiometric activity assay and sequencing was tested using the Big Dye Version 3. The enzyme is active in 40-80mM Tris, 1.0-2.0mM MgCl at a dNTP mix consisting of 0.2mM dATP, 0.2mM dCTP, 0.2mM dUTP, and 0.3mM dlTP, at pH 8.0-10.0, with optimal activity between pH 9.0 and 9.58. The enzyme is also active in KCl concentrations from 0 to lOOmM, indicating that the T. scotoductus, strain X-l polymerase is more salt-tolerant than either Tfil or Taq, but not quite as salt-tolerant as Tth.
EXAMPLE 3: Thermus scotoductus Strains SM3 and Vi7a The same primers used to amplify the full-length gene encoding the polymerase from Thermus scotoductus (Tsc) strain X-l were used to amplify the polymerase genes from two additional strains of Thermus scotoductus: strain SM3 and strain Vi7a. The PCR reaction mixture used to amplify nucleic acids encoding the Thermus scotoductus polymerase from strains SM3 and Vi7a contained 2.5μl of lOx Amplitaq reaction buffer (Applied Biosystems), 2 mM MgCl2, 70 to 100 ng genomic DNA template, 0.2 mM (each) dNTPs, 20 pmol of each primer, and 1.25 units of Amplitaq in a 25 μl total reaction volume. The reaction was started by adding a premix containing enzyme, MgCl , dNTPs, buffer and water to another premix containing primer and template preheated at 80°C The entire reaction mixture was then denatured (30 sec at 96°C) followed by 30 PCR cycles (97°C for 3 sec, 62°C for 30 sec, 72°C for 3 min) with a finishing step (72°C for 7 min).
These PCR reactions each produced approximate 2.5 kb DNA fragments. The amplified fragments were purified from the PCR reaction mixes using a Quiagen PCR cleanup kit (Quiagen). The Thermus scotoductus fragments were ligated into the inducible expression vector pCR®4-TOPO® (Invitrogen, Carlsbad, CA). Three different cloned Thermus scotoductus polymerase genes from each strain were sequenced independently in order to rule out PCR errors. The resulting consensus sequences for the wild-type genes are reported in Figures 1 and 3 below.
There are several silent changes at the DNA level among the three genes. Only the changes resulting in a different amino acid are noted in the alignment of amino acid sequences provided in Figure 2. The Thermus scotoductus, strain SM3 polymerase has five positions that have different amino acids compared to strain X-l. The Thermus scotoductus strain Vi7a polymerase has four differences when compared to the amino acid sequence of the polymerase from strain X-l. These are indicated with boldface in Figure 2.
Modification of Polymerases from Strains SM3 and Vi7a
In order to produce the polymerases from Thermus scotoductus strains SM3 and Vi7a in a form suitable for dye-terminator DNA sequencing, two amino acid substitutions were made in each gene. These are the FS mutation (U.S. Patent 5,614,365; Tabor and Richardson, 1995 PNAS 92: 6339-6343) and exo-minus mutation (G46D Patent, Joyce papers) that were described in the patent application. As described previously, mutagenesis was carried out using the modified QuickChange™ (Stratagene) PCR mutagenesis protocol described in Sawano & Miyawaki (2000), Nucleic Acids Research Vol. 28. The mutated genes was resequenced completely to confirm the introduction of the mutations and to ensure that no PCR errors were introduced.
Protein expression and purification
The "FS, exo-minus form of both Thermus scotoductus polymerase genes were subcloned into the pet expression vector using the Ndel and Sal I restriction sites. BL21 cells (Invitrogen) were transformed with this expression construct. The cells were grown in one liter of Terrific Broth (Maniatis) to an optical density of 1.2OD and the proteins were oveφroduced by four-hour induction with 1.0 mM EPTG. The cells were harvested by centrifugation, washed in 50 mM Tris (pH 7.5), 5mM EDTA, 5% glycerol, lOmM EDTA to remove growth media, and the cell pellet frozen at -80°C
To isolate the Thermus scotoductus, strain SM3 and Vi7a polymerases, the cells were thawed and resuspended in 2.5 volumes (wet weight) of 50mM Tris (pH 7.2), 400mM NaCl, ImM EDTA. The cell walls were disrupted by sonication and the resulting E. coli cell debris was removed by centrifugation. The resulting lysate was pasteurized in a water bath (75°C for 45 min), denaturing and precipitating the majority of the non-thermostable E. coli proteins and leaving the thermostable Thermus scotoductus polymerase in solution. E. coli genomic DNA was removed by coprecipitation with 0.3% Polyethyleneimine (PEI). The cleared lysate was then applied to two columns in series: (1) a Biorex 70 cation exchange resin that chelates excess PEI and (2) a heparin-agarose column that retains the polymerase. This column was washed with 5 column volumes of 20mM Tris (pH 8.5), 5%glycerol, 1 OOmM NaCl,
O.lmM EDTA, 0.05% Triton X-100 and 0.05% Tween-20 (KTA). The proteins were then eluted with a 0.1 to 1.0M NaCl linear gradient. The polymerases eluted at 0.8M NaCl. The eluted Thermus scotoductus polymerases were concentrated and the buffer exchanged using a Millipore concentration filter (30kD M.W. cutoff). The concentrated proteins were stored at in KTA (no salt) plus 50% glycerol at - 20°C.
The activity of the polymerases were measured using a nicked salmon sperm DNA radiometric activity assay. Both enzymes are being tested for use in sequencing using the Big Dye™ V 3.0. The enzymes are active in 40-80mM Tris, 1.0-2.0mM MgCl at a dNTP mix consisting of 0.2mM dATP, 0.2mM dCTP, 0.2mM dUTP, and 0.3mM dlTP, at pH 8.0-10.0, with optimal activity between pH 9.0 and 9.58. All publications and patents mentioned in the above specification are herein incoφorated by reference. Various modifications and variations of the described method and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in the relevant arts are intended to be within the scope of the following claims.

Claims

WHAT IS CLAIMED IS:
1. An isolated nucleic acid encoding a Thermus scotoductus strain X- 1 , ATCC Deposit No. 27978, nucleic acid polymerase.
2. The isolated nucleic acid of claim 1 wherein the nucleic acid polymerase is a DNA polymerase.
3. An isolated nucleic acid encoding a nucleic acid polymerase comprising any one of amino acid sequences SEQ ID NO: 13-28.
4. An isolated nucleic acid encoding a derivative nucleic acid polymerase comprising any one of amino acid sequences SEQ ED NO: 13-16 having a mutation that decreases 5-3' exonuclease activity.
5. The isolated nucleic acid of claim 4, wherein the derivative nucleic acid polymerase has decreased 5-3' exonuclease activity relative to a nucleic acid polymerase comprising any one of amino acid sequences SEQ ID NO: 13-16.
6. An isolated nucleic acid encoding a derivative nucleic acid polymerase comprising any one of amino acid sequences SEQ ED NO:13-16 having a mutation that reduces discrimination against dideoxynucleotide triphosphates.
7. The isolated nucleic acid of claim 6, wherein the derivative nucleic acid polymerase has reduced discrimination against dideoxynucleotide triphosphates relative to a nucleic acid polymerase comprising any one of amino acid sequences SEQ ID NO: 13- 16.
8. An isolated nucleic acid encoding a nucleic polymerase comprising any one of SEQ ED NO:l-12.
9. An isolated nucleic acid comprising a nucleotide sequence complementary to any one of SEQ ID NO : 1 - 12.
10. A vector comprising an isolated nucleic acid encoding a Thermus scotoductus strain X-l, ATCC Deposit No. 27978, nucleic acid polymerase.
11. The vector of claim 10 wherein the nucleic acid polymerase is a DNA polymerase.
12. A vector comprising an isolated nucleic acid encoding a nucleic acid polymerase comprising any one of amino acid sequences SEQ ID NO: 13-28.
13. A vector comprising an isolated nucleic acid encoding a derivative nucleic acid polymerase comprising any one of amino acid sequences SEQ ED NO: 13-16 having a mutation that decreases 5-3' exonuclease activity.
14. The vector of claim 13, wherein the derivative nucleic acid polymerase has decreased 5-3' exonuclease activity relative to a nucleic acid polymerase comprising any one of amino acid sequences SEQ ID NO:13-16.
15. A vector comprising an isolated nucleic acid encoding a derivative nucleic acid polymerase comprising any one of amino acid sequences SEQ ED NO: 13- 16 having a mutation that reduces discrimination against dideoxynucleotide triphosphates.
16. The vector of claim 15, wherein the derivative nucleic acid polymerase has reduced discrimination against dideoxynucleotide triphosphates relative to a nucleic acid polymerase comprising any one of amino acid sequences SEQ ED NO: 13-16.
17. A vector comprising an isolated nucleic acid encoding a nucleic polymerase comprising any one of SEQ ED NO: 1-12.
18. An expression vector comprising a promoter operably linked to an isolated nucleic acid encoding a Thermus scotoductus strain X-l, ATCC Deposit No. 27978, nucleic acid polymerase.
19. The vector of claim 19 wherein the nucleic acid polymerase is a DNA polymerase.
20. An expression vector comprising a promoter operably linked to an isolated nucleic acid encoding a nucleic acid polymerase comprising any one of amino acid sequences SEQ ID NO: 13-28.
21. An expression vector comprising a promoter operably linked to an isolated nucleic acid encoding a derivative nucleic acid polymerase comprising any one of amino acid sequences SEQ ID NO: 13-16 having a mutation that decreases 5-3' exonuclease activity.
22. The expression vector of claim 21, wherein the derivative nucleic acid polymerase has decreased 5-3' exonuclease activity relative to a nucleic acid polymerase comprising any one of amino acid sequences SEQ ID NO:13-16.
23. An expression vector comprising a promoter operably linked to an isolated nucleic acid encoding a derivative nucleic acid polymerase comprising any one of amino acid sequences SEQ ED NO: 13-16 having a mutation that reduces discrimination against dideoxynucleotide triphosphates.
24. The expression vector of claim 23, wherein the derivative nucleic acid polymerase has reduced discrimination against dideoxynucleotide triphosphates relative to a nucleic acid polymerase comprising any one of amino acid sequences SEQ ID NO:13-16.
25. An expression vector comprising a promoter operably linked to an isolated nucleic acid encoding a nucleic polymerase comprising any one of SEQ D NO:l-12.
26. A host cell comprising an isolated nucleic acid encoding a Thermus scotoductus strain X-l, ATCC Deposit No. 27978, nucleic acid polymerase.
27. The host cell of claim 26 wherein the nucleic acid polymerase is a DNA polymerase.
28. A host cell comprising an isolated nucleic acid encoding a nucleic acid polymerase comprising any one of amino acid sequences SEQ ID
NO: 13-28.
29. A host cell comprising an isolated nucleic acid encoding a derivative nucleic acid polymerase comprising any one of amino acid sequences SEQ ID NO:13-16 having a mutation that decreases 5-3' exonuclease activity.
30. The host cell of claim 29, wherein the derivative nucleic acid polymerase has decreased 5-3' exonuclease activity relative to a nucleic acid polymerase comprising any one of amino acid sequences SEQ ED
NO:13-16.
31. A host cell comprising an isolated nucleic acid encoding a derivative nucleic acid polymerase comprising any one of amino acid sequences SEQ ED NO:13-16 having a mutation that reduces discrimination against dideoxynucleotide triphosphates.
32. The host cell of claim 31, wherein the derivative nucleic acid polymerase has reduced discrimination against dideoxynucleotide triphosphates relative to a nucleic acid polymerase comprising any one of amino acid sequences SEQ ID NO: 13-16.
33. A host cell comprising an isolated nucleic acid encoding a nucleic polymerase comprising any one of SEQ ID NO: 1-12.
34. An isolated DNA polymerase polypeptide from Thermus scotoductus strain X-l, ATCC Deposit No. 27978.
35. The isolated nucleic acid polymerase of claim 34 wherein the nucleic acid polymerase is a DNA polymerase.
36. An isolated nucleic acid polymerase comprising any one of amino acid sequences SEQ ID NO: 13-28.
37. An isolated nucleic acid polymerase comprising any one of amino acid sequences SEQ ED NO: 13-16 having a mutation that decreases 5-3' exonuclease activity.
38. The isolated nucleic acid polymerase of claim 37, wherein the derivative nucleic acid polymerase has decreased 5-3' exonuclease activity relative to a nucleic acid polymerase comprising any one of amino acid sequences SEQ ED NO: 13-16.
39. An isolated nucleic acid polymerase comprising any one of amino acid sequences SEQ ED NO: 13- 16 having a mutation that reduces discrimination against dideoxynucleotide triphosphates.
40. The isolated nucleic acid polymerase of claim 39, wherein the derivative nucleic acid polymerase has reduced discrimination against dideoxynucleotide triphosphates relative to a nucleic acid polymerase comprising any one of amino acid sequences SEQ ED NO : 13 - 16.
41. A kit comprising a container containing a nucleic acid polymerase, wherein the nucleic acid polymerase comprises any one of amino acid sequences SEQ ID NO: 13-28.
42. The kit of claim 41 further comprising a container containing an unlabeled nucleotide, a labeled nucleotide, a balanced mixture of nucleotides, a chain terminating nucleotide, a nucleotide analog, a buffer solution, a solution containing magnesium, a cloning vector, a restriction endonuclease, a sequencing primer, a solution containing reverse transcriptase, or a DNA or RNA amplification primer.
43. The kit of claim 41 , adapted for performing DNA sequencing, DNA amplification, reverse transcription, RNA amplification or primer extension.
44. A method of making a nucleic acid polymerase comprising any one of SEQ ED NO: 13-28 that comprises, incubating under conditions sufficient for RNA transcription and translation a host cell comprising a nucleic acid encoding a polypeptide comprising any one of SEQ ID NO: 13-28 operably linked to a promoter.
45. The method of claim 44 wherein the nucleic acid comprises any one SEQ ED NO:l-12.
46. A nucleic acid polymerase made by the method of claim 44.
47. A method of synthesizing DNA comprising contacting a polypeptide comprising any one of SEQ ID NO: 13-28 with a DNA under conditions sufficient to permit polymerization of DNA.
48. A method for thermocyclic amplification of nucleic acid comprising: (a) contacting a nucleic acid with a thermostable polypeptide having any one of SEQ ID NO: 13-28 under conditions suitable for amplification of said nucleic acid; and (b) amplifying the nucleic acid.
49. The method of claim 48 wherein the thermocyclic amplification of the nucleic acid includes cycles of denaturation, primer annealing and primer extension.
50. The method of claim 48 wherein the thermocyclic amplification of the nucleic acid is performed by Strand Displacement Amplification.
51. The method of claim 48 wherein thermocyclic amplification of the nucleic acid is performed by Polymerase Chain Reaction.
52. A method of primer extension comprising contacting a polypeptide comprising any one of SEQ ID NO: 13-28 with a primer and a nucleic acid that is complementary to the primer under conditions sufficient to permit polymerization of DNA.
53. The method of claim 52 wherein the nucleic acid is DNA.
54. The method of claim 52 wherein the primer extension is done to sequence the nucleic acid.
55. The method of claim 52 wherein the primer extension is done to amplify the nucleic acid.
PCT/US2002/029102 2001-09-14 2002-09-13 Thermus scotoductus nucleic acid polymerases WO2003066804A2 (en)

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EP02805994A EP1466013A4 (en) 2001-09-14 2002-09-13 Thermus scotoductus nucleic acid polymerases
CA002459718A CA2459718A1 (en) 2001-09-14 2002-09-13 Thermus scotoductus nucleic acid polymerases
AU2002366438A AU2002366438A1 (en) 2001-09-14 2002-09-13 Thermus scotoductus nucleic acid polymerases
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