WO1998039480A1 - Procedes et compositions d'identification de genes exprimes - Google Patents

Procedes et compositions d'identification de genes exprimes Download PDF

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Publication number
WO1998039480A1
WO1998039480A1 PCT/US1998/004094 US9804094W WO9839480A1 WO 1998039480 A1 WO1998039480 A1 WO 1998039480A1 US 9804094 W US9804094 W US 9804094W WO 9839480 A1 WO9839480 A1 WO 9839480A1
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sequence
samples
primer
primers
nucleic acid
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PCT/US1998/004094
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English (en)
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Tariq M. Haqqi
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Haqqi Tariq M
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Priority to AU64446/98A priority Critical patent/AU6444698A/en
Publication of WO1998039480A1 publication Critical patent/WO1998039480A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing

Definitions

  • the present invention relates to the identification of expressed genes, and in particular, methods and compositions for distinguishing between the expression of genes in two or more biological samples.
  • HGP Human Genome Project
  • a subtracted cDNA library contains cDNA clones corresponding to mRNAs present in one sample and not present in another (e.g. present in a particular species, tissue or cell and not present in another species, tissue or cell). See generally, Current Protocols in Molecular Biology, Section 5.8.9 (1990). In the protocol, cDNA containing the gene(s) of interest ["+cDNA”] is prepared with EcoRI ends and the cDNA not containing the gene(s) of interest ["-cDNA”] is prepared with blunt ends.
  • the +cDNA is mixed with a 50-fold excess of -cDNA inserts and the mixture is heated to make the DNA single-stranded. Thereafter, the mixture is cooled to allow for hybridization. Annealed cDNA inserts are ligated to a vector and transfected.
  • the only +cDNA likely to be double-stranded with an ⁇ coRI site at each end are those not hybridized to something in the -cDNA preparation; in other words, where a complementary sequence is in the -cDNA preparation, the sequence will not be transfected.
  • sequences unique to the +cDNA preparation will be cloned and amplified.
  • DDRT-PCR differential display of mRNAs using arbitrarily primed polymerase chain reaction
  • the polymerase chain reaction is described by Mullis, et al., in U.S. Patents Nos. 4.683,195, 4,683,202 and 4,965,188, hereby incorporated by reference.
  • the PCR process consists of introducing a molar excess of two oligonucleotide primers to the DNA mixture containing the desired target sequence. The two primers are complementary to their respective strands of the double-stranded sequence. The mixture is denatured and then allowed to hybridize.
  • the primers are extended with a thermostable DNA polymerase so as to form complementary strands.
  • the steps of denaturation, hybridization, and polymerase extension can be repeated as often as needed to obtain a relatively high concentration of a segment of the desired target sequence.
  • the target is mRNA; the mRNA is, however, treated with reverse transcriptase in the presence of oligo(dT) primers to make cDNA prior to the PCR process.
  • the PCR is carried out with random primers in combination with the oligo(dT) primer used for cDNA synthesis.
  • the amplified products are placed in side-by-side lanes of a gel; following electrophoresis, the products can be compared or "differentially displayed.”
  • the present invention relates to the identification of expressed genes, and in particular, methods and compositions for distinguishing between the expression of genes in two or more biological samples.
  • the present invention employs oligonucleotide primers targeting conserved motifs within each expressed gene.
  • the present invention contemplates first and second oligonucleotide primers, said first oligonucleotide primer specific for the highly conserved Kozak sequence present before the translation initiating first methionine codon and said second oligonucleotide primer containing sequence complementary to a specific restriction endonuclease recognition site.
  • the specificity of the oligonucleotide primers can be enhanced by the presence of degenerate bases 5' and 3' of the target sequence thus allowing for PCR to be performed at a higher annealing temperature which in turn provide sufficient specificity to generate reproducible patterns of bands on a sequencing gel. This reproducibility enables the method of the present invention
  • the present invention contemplates applying the method for the study of functional genomics and for analyzing the differentially expressed genes in various cell types. It is not intended that the present invention be limited by the nature of the sample.
  • sample and “specimen” in the present specification and claims are used in their broadest sense. On the one hand they are meant to include a specimen or culture. On the other hand, they are meant to include both biological and environmental samples. These terms encompasses all types of samples obtained from humans and other animals, including but not limited to, body fluids such as urine, blood, fecal matter, cerebrospinal fluid (CSF), semen, and saliva, cells as well as solid tissue (including both normal and diseased tissue). These terms also refers to swabs and other sampling devices which are commonly used to obtain samples for culture of microorganisms.
  • body fluids such as urine, blood, fecal matter, cerebrospinal fluid (CSF), semen, and saliva
  • CSF cerebrospinal fluid
  • saliva cells as well as solid tissue (including both normal and diseased tissue).
  • solid tissue including both normal and diseased tissue
  • the invention may be desirable to differentiate between normal and cancerous tissue.
  • the present invention may be used to differentiate between cancer tissue that is metastatic and cancer tissue that is non-metastatic.
  • the present invention may be used to detect drug resistance.
  • it may be desirable to simply detect the presence or absence of specific pathogens (or pathogenic variants) in a clinical sample.
  • it may be disirable to distinguish one species or strain from another.
  • the present invention contemplates comparing the expressed genes of two samples suspected to be different species.
  • a species that is suspected to have changed or diverged from the parent species is compared with the parent species.
  • a species or strain of bacteria may develop a different susceptibilities to a drug (e.g. antibiotics) as compared to the parent species: rapid identification of the specific species or subspecies aids diagnosis and allows initiation of appropriate treatment.
  • the present invention contemplates a method of analyzing nucleic acid in a sample, comprising: a) providing: i) a sample containing nucleic acid, ii) a first primer having a sequence of which at least a portion is at least partially complementary to a natural common non-coding sequence on a portion of said nucleic acid of said sample, iii) a second primer having a sequence of which at least a portion is at least partially complementary to a restriction enzyme recognition sequence present on a portion of said nucleic acid of said sample, and iv) a polymerase and PCR reagents; b) preparing said nucleic acid from said sample under conditions so as to produce amplifiable nucleic acid; c) amplifying said nucleic acid with said first and second primers, said polymerase and said
  • said sample comprises eukaryotic cells and said natural common sequence is the Kozak sequence.
  • said sample comprises prokaryotic cells and said natural common sequence is the Shine-Dalgarno sequence.
  • said detecting comprises gel electrophoresis.
  • the present invention can be used with particular success when comparing samples.
  • the present invention contemplates amethod of analyzing expressed genes in biological samples, comprising: a) providing: i) two samples containing mRNA. ii) a first primer having a sequence of which at least a portion is at least partially complementary to a natural common non-coding sequence on at least a portion of said mRNA of said two samples, iii) a second primer having a sequence of which at least a portion is at least partially complementary to a restriction enzyme recognition sequence present on a portion of said mRNA of said two samples, and iv) a polymerase and PCR reagents; b) treating said mRNA of each of said two samples under conditions so as to produce amplifiable DNA from each sample; c) amplifying said DNA from each sample with said first and second primers, said polymerase and said PCR reagents under conditions such that amplified product is generated from each of said two samples; d) detecting said amplified product.
  • each of said two samples comprise eukaryotic cells and said natural common sequence is the Kozak sequence.
  • dissimilar samples can be usefully compared.
  • said two samples comprise prokaryotic cells and said natural common sequence is the Shine-Dalgarno sequence, and said two samples comprises bacterial cells of different species. It is not intended that the present invention be limited by the number of samples compared.
  • the present invention contemplates amethod of analyzing expressed genes in a multiple samples, comprising: a) providing: i) at least two samples containing mRNA, ii) random primers, iii) reverse transcriptase, iv) a first primer having a sequence of which at least a portion is at least partially complementary to a natural common non-coding sequence on a portion of said mRNA of said samples, v) a second primer having a sequence of which at least a portion is at least partially complementary to a restriction enzyme recognition sequence present on a portion of said mRNA of said samples, and vi) a polymerase and PCR reagents: b) extracting mRNA from each of said samples and reverse transcribing said mRNA with said reverse transcriptase and said random primers under conditions such that cDNA is produced: c) amplifying said cDNA from each sample with said first and second primers, said polymerase and said PCR reagents under conditions such that amplified product is generated
  • kits containing these novel compositions.
  • the kit comprises: i) a first primer having a sequence of which at least a portion is at least partially complementary to a natural common non-coding sequence, and ii) a second primer having a sequence of which at least a portion is at least partially complementary to a restriction enzyme recognition sequence.
  • said natural common sequence is the Kozak sequence.
  • said natural common sequence is the Shine-Dalgarno sequence.
  • said restriction enzyme recognition sequence is selected from the group consisting of the sequences set forth in Table 1.
  • primers are contemplated.
  • said first primer is of the general formula:
  • the present invention also contemplates said second primer is of the general formula: 5 X N M0 -X-N
  • the recognition sequences can be selected from a variety of sources, including but not limited to those in Table 1.
  • Nucleic acid sequence and “nucleotide sequence” as used herein refer to an oligonucleotide or polynucleotide, and fragments or portions thereof, and to DNA o RNA of genomic or synthetic origin which may be single- or double-stranded, and represent the sense or antisense strand.
  • the term "recombinant DNA molecule” as used herein refers to a DNA molecule which is comprised of segments of DNA joined together by means of molecular biological techniques.
  • recombinant protein or “recombinant polypeptide” as used herein refers to a protein molecule which is expressed using a recombinant DNA molecule.
  • vector and “vehicle” are used interchangeably in reference to nucleic acid molecules that transfer DNA segment(s) from one cell to another.
  • expression vector or "expression cassette” as used herein refers to a recombinant DNA molecule containing a desired coding sequence and appropriate nucleic acid sequences necessary for the expression of the operably linked coding sequence in a particular host organism.
  • Nucleic acid sequences necessary for expression in prokaryotes usually include a promoter, an operator (optional), and a ribosome binding site, often along with other sequences.
  • Eukaryotic cells are known to utilize promoters, enhancers, and termination and polyadenylation signals.
  • in operable combination refers to the linkage of nucleic acid sequences in such a manner that a nucleic acid molecule capable of directing the transcription of a given gene and/or the synthesis of a desired protein molecule is produced.
  • the term also refers to the linkage of amino acid sequences in such a manner so that a functional protein is produced.
  • transfection refers to the introduction of foreign DNA into cells. Transfection may be accomplished by a variety of means known to the art including calcium phosphate-DNA co-precipitation, DEAE-dextran-mediated transfection, polybrene- mediated transfection. electroporation, microinjection, liposome fusion, lipofection, protoplast fusion, retroviral infection, biolistics (i.e., particle bombardment) and the like. __.
  • the terms “complementary” or “complementarity” are used in reference to “polynucleotides” and “oligonucleotides” (which are interchangeable terms that refer to a sequence of nucleotides) related by the base-pairing rules.
  • sequence “C-A- G-T.” is complementary to the sequence “G-T-C-A.”
  • Complementarity can be “partial” or “total.”
  • Partial complementarity is where one or more nucleic acid bases is not matched according to the base pairing rules.
  • “Total” or “complete” complementarity between nucleic acids is where each and every nucleic acid base is matched with another base under the base pairing rules.
  • the degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands. This is of particular importance in amplification reactions, as well as detection methods which depend upon binding between nucleic acids.
  • nucleotide sequences refer to a degree of complementarity with other nucleotide sequences. There may be partial homology or complete homology (i.e., identity).
  • a nucleotide sequence which is partially complementary, i.e.. “substantially homologous,” to a nucleic acid sequence is one that at least partially inhibits a completely complementary sequence from hybridizing to a target nucleic acid sequence. The inhibition of hybridization of the completely complementary sequence to the target sequence may be examined using a hybridization assay (Southern or Northern blot, solution hybridization and the like) under conditions of low stringency.
  • a substantially homologous sequence or probe will compete for and inhibit the binding (i.e.. the hybridization) of a completely homologous sequence to a target sequence under conditions of low stringency. This is not to say that conditions of low stringency are such that non-specific binding is permitted; low stringency conditions require that the binding of two sequences to one another be a specific (i.e., selective) interaction.
  • the absence of non-specific binding may be tested by the use of a second target sequence which lacks even a partial degree of complementarity (e.g., less than about 30% identity); in the absence of nonspecific binding the probe will not hybridize to the second non-complementary target.
  • Low stringency conditions comprise conditions equivalent to binding or hybridization at 42°C in a solution consisting of 5X SSPE (43.8 g/1 NaCl, 6.9 g/1 NaH 2 PO 4 -H 2 O and 1.85 g/1 EDTA, pH adjusted to 7.4 with NaOH), 0.1% SDS, 5X Denhardt's reagent [50X
  • Denhardt's contains per 500 ml: 5 g Ficoll (Type 400, Pharmacia), 5 g BSA (Fraction V; Sigma)] and 100 ⁇ g/ml denatured salmon sperm DNA followed by washing in a solution comprising 5X SSPE, 0.1% SDS at 42°C when a probe of about 500 nucleotides in length is employed.
  • low stringency conditions factors such as the length and nature (DNA, RNA. base composition) of the probe and nature of the target ( DNA. RNA, base composition, present in solution or immobilized, etc.) and the concentration of the salts and other components (e.g., the presence or absence of formamide, dextran sulfate, polyethylene glycol), as well as components of the hybridization solution may be varied to generate conditions of low stringency hybridization different from, but equivalent to, the above listed conditions.
  • conditions which promote hybridization under conditions of high stringency e.g.. increasing the temperature of the hybridization and/or wash steps, the use of formamide in the hybridization solution, etc.).
  • substantially homologous refers to any probe which can hybridize to either or both strands of the double-stranded nucleic acid sequence under conditions of low stringency as described above.
  • substantially homologous refers to any probe which can hybridize (i.e., it is the complement ol) the single-stranded nucleic acid sequence under conditions of low stringency as described above.
  • hybridization is used in reference to the pairing of complementary nucleic acids using any process by which a strand of nucleic acid joins with a complementary strand through base pairing to form a hybridization complex.
  • Hybridization and the strength of hybridization is impacted by such factors as the degree of complementarity between the nucleic acids, stringency of the conditions involved, the T m of the formed hybrid, and the G:C ratio within the nucleic acids.
  • hybridization complex refers to a complex formed between two nucleic acid sequences by virtue of the formation of hydrogen bonds between complementary G and C bases and between complementary A and T bases; these hydrogen bonds may be further stabilized by base stacking interactions.
  • the two complementary nucleic acid sequences hydrogen bond in an antiparallel configuration.
  • a hybridization complex may be formed in solution (e.g., C 0 t or Rot analysis) or between one nucleic acid __.
  • T m is used in reference to the "melting temperature.”
  • the melting temperature is the temperature at which a population of double-stranded nucleic acid molecules becomes half dissociated into single strands.
  • the equation for calculating the T m of nucleic acids is well known in the art.
  • T m 81.5 + 0.41(% G + C), when a nucleic acid is in aqueous solution at 1 M NaCl [see e.g., Anderson and Young,
  • stringency is used in reference to the conditions of temperature, ionic strength, and the presence of other compounds such as organic solvents, under which nucleic acid hybridizations are conducted. "Stringency” typically occurs in a range from about T m -5°C (5°C below the T m of the probe) to about 20°C to 25°C below T m . As will be understood by those of skill in the art, a stringent hybridization can be used to identify or detect identical polynucleotide sequences or to identify or detect similar or related polynucleotide sequences.
  • amplifiable nucleic acid is used in reference to nucleic acids which may be amplified by any amplification method. It is contemplated that "amplifiable nucleic acid” will usually comprise "sample template.”
  • sample template refers to nucleic acid originating from a sample which is analyzed for the presence of a target sequence of interest. In contrast.
  • background template is used in reference to nucleic acid other than sample template which may or may not be present in a sample. Background template is most often inadvertent. It may be the result of carryover, or it may be due to the presence of nucleic acid contaminants sought to be purified away from the sample. For example, nucleic acids from organisms other than those to be detected may be present as background in a test sample.
  • Amplification is defined as the production of additional copies of a nucleic acid sequence and is generally carried out using polymerase chain reaction technologies well known in the art [Dieffenbach CW and GS Dveksler (1995) PCR Primer, a Laboratory .__
  • PCR polymerase chain reaction
  • PCR With PCR, it is possible to amplify a single copy of a specific target sequence in genomic DNA to a level detectable by several different methodologies (e.g., hybridization with a labeled probe; incorporation of biotinylated primers followed by avidin-enzyme conjugate detection; incorporation of 32 P-labeled deoxynucleotide triphosphates, such as dCTP or dATP. into the amplified segment).
  • any oligonucleotide sequence 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.
  • PCR reagents or "PCR materials”, which herein are defined as all reagents necessary to carry out amplification except the polymerase, primers and template.
  • PCR reagents nomally include nucleic acid precursors (dCTP. dTTP etc.) and buffer.
  • the term "primer” refers to an oligonucleotide. whether occurring naturally as in a purified restriction digest or produced synthetically, which is capable of acting as a point of initiation of synthesis when placed under conditions in which synthesis of a primer extension product which is complementary to a nucleic acid strand is induced, (i.e.. in the presence of nucleotides and an inducing agent such as DNA polymerase and at a suitable temperature and pH).
  • the primer is preferably single stranded for maximum efficiency in amplification, but may alternatively be double stranded. If double stranded, the primer is first treated to separate its strands before being used to prepare extension products.
  • the primer is an oligodeoxyribonucleotide.
  • the primer must be sufficiently long to prime the synthesis of extension products in the presence of the inducing agent. The exact lengths of the primers will depend on many factors, including temperature, source of primer and the use of the method.
  • probe refers to an oligonucleotide (i.e., a sequence of nucleotides), whether occurring naturally as in a purified restriction digest or produced synthetically, recombinantly or by PCR amplification, which is capable of hybridizing to another oligonucleotide of interest.
  • a probe may be single-stranded or double-stranded. Probes are useful in the detection, identification and isolation of particular gene sequences.
  • any probe used in the present invention will be labelled with any "reporter molecule,” so that it is detectable using any detection system, including, but not limited to enzyme (e.g., ELISA, as well as enzyme-based histochemical assays), fluorescent, radioactive, and luminescent systems. It is not intended that the present invention be limited to any particular detection system or label.
  • restriction endonucleases and “restriction enzymes” refer to bacterial enzymes, each of which cut double-stranded DNA at or near a specific nucleotide sequence.
  • DNA molecules are said to have "5' ends” and "3" ends” because mononucleotides are reacted to make oligonucleotides in a manner such that the 5' phosphate of one mononucleotide pentose ring is attached to the 3' oxygen of its neighbor in one direction via a phosphodiester linkage. Therefore, an end of an oligonucleotide is referred to as the "5' end” if its 5 " phosphate is not linked to the 3' oxygen of a mononucleotide pentose ring.
  • an end of an oligonucleotide is referred to as the "3' end” if its 3 " oxygen is not linked to a 5 " phosphate of another mononucleotide pentose ring.
  • a nucleic acid sequence even if internal to a larger oligonucleotide. also may be said to have 5' and 3' ends.
  • discrete elements are referred to as being “upstream” or 5 " of the "downstream” or 3' elements. This terminology reflects the fact that transcription proceeds in a 5 " to 3 " fashion along the DNA strand.
  • the promoter and enhancer elements which direct transcription of a linked gene are generally located 5' or upstream of the coding region. However, enhancer elements can exert their effect even when located 3' of the promoter element and the coding region. Transcription termination and polyadenylation signals are located 3 " or downstream of the coding region.
  • an oligonucleotide having a nucleotide sequence encoding a gene means a nucleic acid sequence comprising the coding region of a gene, i.e. the nucleic acid sequence which encodes a gene product.
  • the coding region may be present in either a cDNA. genomic DNA or RNA form.
  • the oligonucleotide may be single-stranded (i.e., the sense strand) or double-stranded.
  • Suitable control elements such as enhancers/promoters, splice junctions, polyadenylation signals, etc.
  • 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.
  • regulatory element refers to a genetic element which controls some aspect of the expression of nucleic acid sequences.
  • a promoter is a regulatory element which facilitates the initiation of transcription of an operably linked coding region.
  • Other regulatory elements are splicing signals, polyadenylation signals, termination signals, etc.
  • Transcriptional control signals in eukaryotes comprise "promoter” and “enhancer” elements. Promoters and enhancers consist of short arrays of DNA sequences that interact specifically with cellular proteins involved in transcription [Maniatis, T. et al. , Science 236:1237 (1987)]. Promoter and enhancer elements have been isolated from a variety of eukaryotic sources including genes in plant, yeast, insect and mammalian cells and viruses (analogous control elements, i.e., promoters, are also found in prokaryotes). The selection of a particular promoter and enhancer depends on what cell type is to be used to express the protein of interest.
  • Splicing signals mediate the removal of introns from the primary RNA transcript and consist of a splice donor and acceptor site [Sambrook. J. et al.. Molecular Cloning: A Laboratory Manual, 2nd ed.. Cold Spring Harbor
  • a commonly used splice donor and acceptor site is the splice junction from the 16S RNA of SV40.
  • Efficient expression of recombinant DNA sequences in eukaryotic cells requires expression of signals 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 DNA sequence which 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.
  • the poly A signal utilized in an expression vector may be "heterologous" or "endogenous.”
  • An endogenous poly A signal is one that is found naturally at the 3' end of the coding region of a given gene in the genome.
  • a heterologous poly A signal is one which is isolated from one gene and placed 3' of another gene.
  • transfection or "transfected” refers to the introduction of foreign DNA into a cell.
  • nucleic acid molecule encoding As used herein, the terms “nucleic acid molecule encoding.” “DNA sequence encoding.” and “DNA encoding” refer to the order or sequence of deoxyribonucleotides along a strand of deoxyribonucleic acid. The order of these deoxyribonucleotides determines the order of amino acids along the polypeptide (protein) chain. The DNA sequence thus codes for the amino acid sequence.
  • antisense is used in reference to RNA sequences which are complementary to a specific RNA sequence (e.g., mRNA).
  • Antisense RNA may be produced by any method, including synthesis by splicing the gene(s) of interest in a reverse orientation to a viral promoter which permits the synthesis of a coding strand. Once introduced into a cell, this transcribed strand combines with natural mRNA produced by the cell to form duplexes. These duplexes then block either the further transcription of the mRNA or its translation. In this manner, mutant phenotypes may be generated.
  • the term “antisense strand” is used in reference to a nucleic acid strand that is complementary to the "sense” strand.
  • the designation (-) i.e. , "negative" is sometimes used in reference to the antisense strand, with the designation (+) sometimes used in reference to the sense (i.e. , "positive" strand.
  • Southern blot refers to the analysis of DNA on agarose or acrylamide gels to fractionate the DNA according to size, followed by transfer and immobilization of the
  • DNA from the gel to a solid support, such as nitrocellulose or a nylon membrane.
  • the immobilized DNA is then probed with a labeled oligo-deoxyribonucleotide probe or DNA probe to detect DNA species complementary to the probe used.
  • the DNA may be cleaved with restriction enzymes prior to electrophoresis. Following electrophoresis, the DNA may be partially depurinated and denatured prior to or during transfer to the solid support.
  • Southern blots are a standard tool of molecular biologists [J. Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, NY, pp 9.31 -9.58]. __.
  • Northern blot refers to the analysis of RNA by electrophoresis of RNA on agarose gels to fractionate the RNA according to size followed by transfer of the RNA from the gel to a solid support, such as nitrocellulose or a nylon membrane. The immobilized RNA is then probed with a labeled oligo-deoxyribonucleotide probe or DNA probe to detect RNA species complementary to the probe used.
  • Northern blots are a standard tool of molecular biologists [J. Sambrook, J. et al. (1989) supra, pp 7.39-7.52].
  • reverse Northern blot refers to the analysis of DNA by electrophoresis of DNA on agarose gels to fractionate the DNA on the basis of size followed by transfer of the fractionated DNA from the gel to a solid support, such as nitrocellulose or a nylon membrane.
  • a solid support such as nitrocellulose or a nylon membrane.
  • the immobilized DNA is then probed with a labeled oligo-ribonuclotide probe or RNA probe to detect DNA species complementary to the ribo probe used.
  • isolated when used in relation to a nucleic acid, as in “an isolated oligonucleotide” refers to a nucleic acid sequence that is identified and separated from at least one contaminant nucleic acid with which it is ordinarily associated in its natural source. Isolated nucleic acid is nucleic acid present in a form or setting that is different from that in which it is found in nature. In contrast, non-isolated nucleic acids are nucleic acids such as DNA and RNA which are found in the state they exist in nature.
  • the term “purified” or “to purify” refers to the removal of undesired components from a sample.
  • substantially purified refers to molecules, either nucleic or amino acid sequences, that are removed from their natural environment, isolated or separated, and are at least 60% free, preferably 75% free, and most preferably 90% free from other components with which they are naturally associated.
  • An "isolated polynucleotide” is therefore a substantially purified polynucleotide.
  • the term “coding region” when used in reference to a structural gene refers to the nucleotide sequences which encode the amino acids found in the nascent polypeptide as a result of translation of a mRNA molecule.
  • the coding region is bounded, in eukaryotes. on the 5 " side by the nucleotide triplet "ATG” which encodes the initiator methionine and on the 3' side by one of the three triplets which specify stop codons (i.e. , TAA, TAG. TGA).
  • structural gene refers to a DNA sequence coding for RNA or a protein.
  • regulatory genes are structural genes which encode products which control the expression of other genes (e.g., transcription factors).
  • gene means the deoxyribonucleotide sequences comprising the coding region of a structural gene and including sequences located adjacent to the coding region on both the 5' and 3' ends for a distance of about 1 kb on either end such that the gene corresponds to the length of the full-length mRNA.
  • sequences which are located 5 " of the coding region and which are present on the mRNA are referred to as 5' non-translated sequences.
  • the sequences which are located 3' or downstream of the coding region and which are present on the mRNA are referred to as 3' non-translated sequences.
  • the term "gene” encompasses both cDNA and genomic forms of a gene.
  • a genomic form or clone of a gene contains the coding region interrupted with non-coding sequences termed "introns” or “intervening regions” or “intervening sequences.”
  • Introns are segments of a gene which are transcribed into heterogenous nuclear RNA (hnRNA); introns may contain regulatory elements such as enhancers. Introns are removed or "spliced out" from the nuclear or primary transcript: introns therefore are absent in the messenger RNA (mRNA) transcript.
  • mRNA messenger RNA
  • genomic forms of a gene may also include sequences located on both the 5' and 3' end of the sequences which are present on the RNA transcript. 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 which control or influence the transcription of the gene.
  • the 3" flanking region may contain sequences which direct the termination of transcription, posttranscriptional cleavage and polyadenylation.
  • sample as used herein is used in its broadest sense and includes environmental and biological samples.
  • Environmental samples include material from the environment such as soil and water.
  • Biological samples may be animal, including, human, fluid (e.g.. blood, plasma and serum), solid (e.g., stool), tissue, liquid foods (e.g., milk), and solid foods (e.g., vegetables).
  • bacteria and "bacterium” refer to all prokaryotic organisms, including those within all of the phyla in the Kingdom Procaryotae. It is intended that the term encompass all microorganisms considered to be bacteria including Mycoplasma. Chlamydia, Actinomyces, Streptomyces, and Rickettsia. All forms of bacteria are included within this definition including cocci, bacilli, spirochetes, spheroplasts, protoplasts, etc. Also included within this term are prokaryotic organisms which are gram negative or gram positive.
  • Gram negative and gram positive refer to staining patterns with the Gram-staining process which is well known in the art [Finegold and Martin, Diagnostic Microbiology, 6th Ed. (1982), CV Mosby St. Louis, pp 13-15].
  • Gram positive bacteria are bacteria which retain the primary dye used in the Gram stain, causing the stained cells to appear dark blue to purple under the microscope.
  • Gram negative bacteria do not retain the primary dye used in the Gram stain, but are stained by the counterstain. Thus, gram negative bacteria appear red.
  • K primer partially hybridized to one strand of a denatured double-stranded template.
  • Figure 2 schematically shows one embodiment of the primers of the present invention (an "RE Primer") partially hybridized to the other strand of denatured double-stranded target DNA.
  • Figure 3 is an autoradiograph of PAGE showing differential expression in a variety of human cell types.
  • Figure 4 is an autoradiograph of PAGE showing differential expression in a variety of species of bacteria.
  • Figure 5 is an autoradiograph of PAGE showing differential expression in a variety of human cell types where differentially expressed bands have been obtained and cloned.
  • Figure 6 shows the nucleic acid sequence of one of the cloned transcripts encoding a human mitochondrial hinge protein.
  • Figure 7 shows the sequence of one of the cloned transcripts corresponding to a coactivator gene.
  • Figure 8 is an autoradiograph of PAGE showing differential expression in normal and malignant tissue.
  • the present invention relates to the identification of expressed genes, and in particular, methods and compositions for distinguishing between the expression of genes in two or more biological samples.
  • the description of the invention involves the I) Design of the Primers, II) Preparation of RNA from Samples; and III) Comparing of Biological Samples. .__
  • differentially expressed genes ideally one must be able to identify nearly all of the expressed genes (or at least a significant majority of them) in a cell type, only then a meaningful comparison can be made with a related cell or tissue sample.
  • the present invention contemplates the use of specific primers able to anneal with sequences which are conserved in expressed genes.
  • the present invention contemplates primers directed at the Kozak sequence, a string of non-random nucleotides which are present before the translation initiating first ATG in majority of the mRNAs which are transcribed and translated in an eukarytic cells. See M. Kozak, Cell 44:283-292 (1986).
  • an oligonucleotide primer specific for the Kozak sequence (consensus sequence 5XGCCA/GCCATGG-3') with degenerate bases at its 5 ' and 3" end will provide sufficient specificity to be used in a PCR amplification reaction as an upstream primer.
  • a second primer (an "RE primer”) can be designed. Again, the presence of degenerate bases at the 5' and 3' end of these primers would provide length sufficient to give specificity in a PCR amplification reaction. Since the ability of a primer pair to amplify a transcript is a function of transcript abundance and the specificity of primer-template interactions, the use of K and RE-primers is likely to significantly improve the detection rate of rare mRNAs-an outcome not possible with standard or modified differential display methods because of the use of random primers.
  • M. Kozak performed an analysis of nearly 700 vertebrate mRNAs. See M. Kozak, "An analysis of 5Xnoncoding sequences from 699 vertebrate messenger RNAs, Nucleic Acids
  • the present invention therefore contemplates primers which can specifically hybridize with the nucleotide sequences present around the initiating codon. Collectively, these primers would hybridize with all of the expressed mRNAs although the hydridization of individual primers within an expressed gene pool may vary. This would help in reducing the complexity of the target transcripts by effectively dividing the transcript pool in subsets based on the presence of the nucleotides with reference to the ATG in the mRNA sequence.
  • degenerate bases can be used i) before the consensus Kozak sequence at the 5' end. ii) inside the Kozak sequence (e.g. at position -5) and/or iii) after the ATG at the 3 ' end.
  • the primers are selected from the group consisting of the primers: NNN-X- GCC(A or G)CCATGGNN; NNN-X-GCC(A or G)CCATGANN; NNN-X-GCC(A or G)CCATGCNN; and NNN-X-GCC(A or G)CCATGTNN (wherein X is either a recognition sequence or nothing, and wherein N is either A,T,G,C or nothing).
  • This embodiment contains primers that vary at the +4 position.
  • recognition sequence it is meant that the sequence is a known sequence that can be targeted by a) nucleic acid hybridization (e.g. poly(dT) or poly (dA), b) an enzyme (e.g. a restriction enzyme), or c) a ligand (e.g. biotin or avidin).
  • Preferred primers are those where X is the recognition sequence for a restriction enzyme; introducing this sequence into expressed genes facilitates subsequent manipulation (e.g. cloning).
  • preferred primers are those where X is the recognition sequence for the restriction enzyme BamHl; these primers are selected from the group consisting of NNNGGATCCGCC(A or G)CCATGGNN; NNNGGATCCGCC(A or G)CCATGANN; NNNGGATCCGCC(A or G)CCATGCNN; and NNNGGATCCGCC(A or G)CCATGTNN (wherein N is either A.T,G,C or nothing).
  • Table 1 sets forth, for illustrative purposes, a number of restriction enzyme recognition sequences.
  • "X" can be selected from this list depending on design considerations.
  • Other restriction enzymes from commercially available sources have recognition sequences that can also be employed with success.
  • Primers containing facilitating moieties such as recoginition sequences allow for the introduction of such sequences into the product of the amplification reaction. That is to say, amplification in PCR involves primer extension to make the so-called “long products.” These long products are the template for subsequent cycles of amplification. While it is not intended that the present invention be limited by any understanding of the mechanism whereby the primers of the present invention successfully operate, it is believed that a primer such as NNN-X-GCC(A or G)CCATGGNN will only partially hybridize to one strand of the denatured double-stranded target nucleic acid in the first round as set forth in Figure 1.
  • the present invention contemplates using a lower annealing temperature (discussed more below).
  • a lower annealing temperature discussed more below.
  • the present invention also contemplates isolating the long products via the recognition sequence prior to subsequent cycles.
  • the long products are isolated using an oligo (dT) resin; the long products containing the corresponding recognition sequence bind to the resin, while the background template nucleic acid does not. In this manner, the background template can be removed and subsequent rounds of hybridization are carried out on the long products [with the same primers or with the primers that lack the recognition sequence (but that are otherwise the same)].
  • the primers are selected from the group consisting of the primers: NNN-X-GCC(A or G)CCATGG(C or A)GNN; NNN-X-GCC(A or G)CCATGG(C or A)TNN; NNN-X-GCC(A or G)CCATGG(C or A)ANN; and NNN-X-GCC(A or G)CCATGG (C or A)CNN (wherein X is either a recognition sequence or nothing, and wherein N is either
  • This embodiment contains primers with the concensus sequence extending to the +5 position, but that vary at the +6 position.
  • the present invention contemplates primers where there are many degenerate bases after the ATG at the 3' end (e.g. between three and ten. more preferrably between three and five) as well as where there is only one degenerate base after the ATG at the 3' end.
  • the primers are selected from the group consisting of the primers: GCC(A or G)CCATGN (wherein N is either A,T,G or C). These primers can be linked to a recognition sequence ("X") in the manner described above, if desired.
  • the present invention also contemplates primers where there are a number of degenerate bases at the 5 " end (i.e. prior to the Kozak sequence).
  • the primers are selected from the group consisting of the primers: N 0 GCC(A or G)CCATGGNN; N M0 GCC(A or G)CCATGANN;
  • N M0 GCC(A or G)CCATGCNN; and N, .I0 GCC(A or G)CCATGTNN (wherein N is either A.T,G or C).
  • the primers are selected from the group consisting of the primers: CGGGATCCGCC(A or G)CNATGG (hereinafter "Kl” when N is C); CGGGATCCGCC(A or G)CNATGA (hereinafter “K2" when N is C); CGGGATCCGC A or G)CNATGC (hereinafter “K3” when N is C); and CGGGATCCGCC(A or G)CNATGT (hereinafter "K4" when N is C).
  • the primers are selected from the group consisting of the primers: CGGGATCCGCC(A or G)(C or G)NATGG (hereinafter "K-2-1 " when N is C); CGGGATCCGCC(A or G)(C or G)NATGC (hereinafter "K-2-2" when N is C);
  • CGGGATCCGCC(A or G)(C or G)NATGT (hereinafter "K-2-3" when N is C); and CGGGATCCGCC(A or G)CNATGA (hereinafter "K-2-4" when N is C).
  • the primers are selected from the group consisting of the primers: CGGGATCCGCC(A or G)(C or G)NATGGN (hereinafter "K-3-1") when N is C); CGGGATCCGCC(A or G)(C or G)NATGCN (hereinafter "K-3-2"); CGGGATCCGCC(A or
  • N can be A, C, G or T.
  • the primer of the present invention can be only partially complementary to this natural common non-coding sequence.
  • the present invention contemplates linking the ATG triplet to degenerate bases on either side (or both sides).
  • a recognition sequence (“X") can be linked to such a primer on the 5 " end.
  • the primers are of the general formula: 5XN,. 10 X-N,. i() ATGN M0 -3 " (wherein N is A, T,G, C or nothing).
  • X is the recognition sequence for a restriction enzyme; again, introducing this sequence into expressed genes facilitates subsequent manipulation (e.g. cloning).
  • preferred primers are those where X is the recognition sequence for the restriction enzyme BamRl; these primers are selected from the group consisting of NGGATCCNNNATGA; NGGATCCNNNATGC;
  • NGGATCCNNNATGT and NGGATCCNNNATGG (wherein N is either A.T,G,C or nothing).
  • primer extension or PCR of DNA using K primers also contemplates hybridization of the K primers to the corresponding mRNA Kozak sequence: 5XACCAUGG.
  • primers can be made having the ACCAUGG sequence that can be used to hybridize to DNA.
  • the present invention contemplates downstream primers designed with recoginition sequences for common restriction enzymes (hereafter "RE" primers).
  • the RE primers are designed with degeneraate bases on either side (or both sides) of the recognition sequence.
  • the RE primer is designed with 3 degenerate bases at the 5 ' and 2 degenerate bases at the 3' end (5 X N 3 -specific recognition sequence-N 2 -3').
  • the downstream primers of the present invention are primers selected from the group consisting of the primers: 5XX-NNNGATC-3' ( i.e. having the recognition sequence for Mbol); 5'-X-NNNCTAG-3 " (i.e. having the recognition sequence for Bfal); 5 " -X-NNNCCGC-3' (i.e. having the recognition sequence for Acil); 5 " -X-NNNCCGG-3' (i.e. having the recognition sequence for Hpall); and 5'-X- NNNAATT-3 ' (i.e. having the recognition sequence for Tsp 509 I), wherein X is a recognition sequence on the 5 " end that is different from the recognition sequence of the 3' end. or X is nothing).
  • the recognition sequence on the 5 " end of the downstream primers of the present invention is for EcoRI.
  • primers are selected from the group consisting of the primers: GAATTCNNNGATC; GAATTCNNNCTAG; GAATTCNNNCCGC: GAATTCNNNCCGG: GAATTCNNNAATT; GAATTCNNNTTAA: and GAATTCNNNGCGC.
  • the recognition sequence on the 5' end of the downstream primers of the present invention is for BamHI.
  • Such primers are selected from the group consisting of the primers: GGATTCCNNNGATC (hereinafter “Mbol primer”); GGATTCCNNNCTAG (hereinafter “Bfal primer”); GGATTCCNNNCCGC (hereinafter “Acil primer”); GGATTCCNNNCCGG (hereinafter “Hpall primer”); and GGATTCCNNNAATT (hereinafter “Tsp509I primer”).
  • Primers containing facilitating moieties such as 5 " recoginition sequences of the RE primers of the present invention allow for the introduction of such sequences into the product of the amplification reaction.
  • amplification in PCR involves primer extension to make the so-called “long products.” These long products are the template for subsequent cycles of amplification. While it is not intended that the present invention be limited by any understanding of the mechanism whereby the primers of the present invention successfully operate, it is believed that a primer such as X-NNNGATC will only partially hybridize to one strand of the denatured double-stranded target nucleic acid in the first round as set forth in Figure 2.
  • primers of the present invention be limited by the precise sequence of a restriction recognition sequence. Indeed, it is specifically contemplated that the primers of the present invention can be only partially complementary to the recognition sequence.
  • the prokaryotic mRNA ribosome binding site usually contains part or all of a polypurine domain UAAGGAGGU known as the Shine-Dalgarno (SD) sequence found just 5' to the translation initiation codon: mRNA 5'-UAAGGAGGU - N 5 ., 0 - AUG
  • the present invention therefore contemplates primers containing this motif (in a manner similar to the Kozak motif discussed above).
  • An oligonucleotide primer specific for the SD sequence (with or without degenerate bases at its 5' and 3' end) will provide sufficient specificity to be used in a PCR amplification reaction as an upstream primer.
  • Taq DNA polymerase adds an A to the 5 'end of such PCR products and this can be used to clone by virtue of commercially available ligation kits (e.g. from Promega).
  • a second primer (a "RE primer”) can be designed for use with the SD primer. Again, the presence of degenerate bases at the 5" and 3 " end of these primers would provide length sufficient to give specificity in a PCR amplification reaction.
  • the SD primers of the present invention are of the general formula: 5 ' -N O -X-N ( TAAGGAGGN MO -3' (where X is a recognition sequence or nothing, and where N is A, T, G. C or nothing).
  • the recognition sequence (X) is a restriction enzyme recognition sequence; such sequences can be selected from Table 1 or other known lists of such sequences.
  • the recognition sequence can be a region of nucleic acid that can be targeted by hybridization or by a ligand. Such recognition sequences can be used to separate the products of the first cycles of PCR (as discussed above).
  • the recognition sequence is a restriction enzyme recognition sequence
  • a preferred sequence is that for the enzyme EcoRI.
  • the SD primers are selected from the group of the general formula:
  • the present invention contemplates linking a portion of the SD sequence (e.g. AGGAGG) to degenerate bases on either side (or both sides) to create a useful primer. It is also contemplated that the SD primers of the present invention need not hybridize completely to the target nucleic acid. In the manner set forth in Figure 1 for K primers, it is contemplated that the primer can be extended even though portions of the primer are not hybridized.
  • the nucleic acid content of cells consists of deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).
  • DNA contains the genetic blueprint of the cell.
  • RNA is involved as an intermediary in the production of proteins based on the DNA sequence. RNA exists in three forms within cells, structural RNA (i.e., ribosomal RNA "rRNA”), transfer
  • RNA which is involved in translation
  • mRNA messenger RNA
  • the cell's mRNA component at any given time is representative of the physiological state of the cell. In order to study and utilize the molecular biology of the cell, it is therefore important to be able to purify mRNA, including purifying mRNA from the total nucleic acid of a sample.
  • RNA is complicated by the presence of ribonucleases that degrade RNA (e.g.. T. Maniatis et al.. Molecular Cloning, pp. 188-190, Cold Spring Harbor Laboratory [1982]). Furthermore, the preparation of amplifiable RNA is made difficult by the presence of ribonucleoproteins in association with RNA. ( See, R. J. Slater, In:
  • the steps involved in purification of nucleic acid from cells include 1) cell lysis; 2) inactivation of cellular nucleases; and 3) separation of the desired nucleic acid from the cellular debris and other nucleic acid.
  • Cell lysis may be achieved through various methods, including enzymatic, detergent or chaotropic agent treatment.
  • Inactivation of cellular nucleases may be achieved by the use of proteases and/or the use of strong denaturing agents.
  • separation of the desired nucleic acid is typically achieved by extraction of the nucleic acid with phenol or phenol-chloroform; this method partitions the sample into an aqueous phase (which contains the nucleic acids) and an organic phase (which contains other cellular components, including proteins).
  • Commonly used protocols require the use of salts in conjunction with phenol (P. Chomczynski and N. Sacchi, Anal. Biochem. 162:156 [1987]), __.
  • the structure of the mRNA molecule may used to assist in the purification of mRNA from DNA and other RNA molecules. Because the mRNA of higher organisms is usually polyadenylated on its 3' end
  • poly-A tail or "poly-A track”
  • poly-A track one means of isolating RNA from cells has been based on binding the poly-A tail with its complementary sequence (i.e., oligo-dT), that has been linked to a support such as cellulose.
  • oligo-dT its complementary sequence
  • the hybridized mRNA/ oligo-dT is separated from the other components present in the sample through centrifugation or. in the case of magnetic formats, exposure to a magnetic field.
  • the mRNA is usually removed from the oligo-dT. However, for some applications, the mRNA may remain bound to the oligo-dT that is linked to a solid support.
  • RNA Ribonucleic acid
  • mammalian e.g. liver tissue
  • the present invention contemplates the isolation of PolyA+ RNA from extracts, including direct isolation from crude extracts.
  • the present invention may be used to compare normal tissue with cancer tissue, as well as to differentiate between cancer tissue that is metastatic and cancer tissue that is non-metastatic.
  • the present invention may be used to detect drug resistance.
  • metastatic disease it is believed that cancer cells proteolytically alter basement membranes underlying epithelia or the endothelial linings of blood and lymphatic vessels, invade through the defects created by proteolysis, and enter the circulatory or lymphatic systems to colonize distant sites. During this process, the secretion of proteolytic enzymes is coupled with increased cellular motility and altered adhesion. After their colonization of distant sites, metastasizing tumor cells proliferate to establish metastatic nodules.
  • the present invention can be used to compare metastatic cancer tissue with non- metastatic cancer tissue to identify differentially expressed genes as markers of metastatic potential. Thereafter, the present invention can be used to determine the presence or absence of these markers in various clinical cancer isolates.
  • the present invention also contemplates "phenotyping" cancer cells adapted to tissue culture.
  • differentially expressed genes as markers of drug resistance. Thereafter, the present invention can be used to determine the presence or absence of these markers in various clinical cancer isolates.
  • microorganisms recovered from clinical specimens or environmental sources is an important aspect of clinical microbiology, as this information is important to physicians in making decisions related to methods of treatment.
  • reproducible systems for identifying microorganisms are critical.
  • Finegold "The primary purpose of nomenclature of microorganisms is to permit us to know as exactly as possible what another clinician, microbiologist, epidemiologist, or author is referring to when describing an organism responsible for infection of an individual or outbreak" (S. Finegold. "Introduction to summary of current nomenclature, taxonomy, and classification of various microbial agents," Clin. Infect. Dis., 16:597 [1993]).
  • Classification, nomenclature, and identification are three separate, but interrelated aspects of taxonomy. Classification is the arranging of organisms into taxonomic groups (i.e., taxa) on the basis of similarities or relationships. A multitude of prokaryotic organisms has been identified, with great diversity in their types, and many more organisms being characterized and classified on a regular basis. It is a matter of convenience to classify the organisms into groups based upon their similarities. Classification has been used to organize the seemingly chaotic array of individual bacteria into an orderly framework. Through use of a classification framework, a new isolate can be more easily be characterized by comparison with known organisms. The choice of criteria for placement into groups is somewhat arbitrary, although most classifications are based on phylogenetic relationships.
  • rRNA Ribosomal RNA sequence analysis
  • molecular probes and amplification methods e.g., PCR
  • the test DNA is denatured and exposed to denatured DNA of known sequence from a particular organism.
  • the amount of hybridization between the test DNA and known DNA provides an indication of the degree of relatedness between the test and known organisms.
  • An important drawback to this approach is that hybridization between two single DNA strands can occur even when 15% of the sequences are not complementary.
  • Ribosomal RNA analysis is another method by which the relatedness of organisms has been determined. Because ribosomes are critical to cellular function and interact with many other molecules (e.g., mRNA and tRNAs), the core rRNA sequences are highly constrained and well-conserved throughout evolution.
  • rRNA also contains highly variable regions, it is usually possible to identify regions of 20-30 bases that are unique to a particular species. While analyzing sequence differences between the rRNAs of different organisms, this approach is extremely narrow in that it looks at no other differences between organisms.
  • identification of an organism is based on its overall morphological and biochemical patterns observed in culture.
  • numerous organisms associated with disease may not be cultured in vitro. Indeed, some do not grow well in traditional in vivo culture systems, such as cell cultures or embryonated eggs. Nonetheless, their detection and identification is crucial for the appropriate treatment of affected individuals.
  • Genetic testing methods have proven useful for the classification and identification of such organisms. For example, universal ribosomal primers designed to hybridize to and amplify all bacterial rRNA may be used to detect bacteria in any sterile body site (e.g., synovial fluid). Once detected, the organism may then be identified by sequencing and/or amplification methods, and comparing the results with those obtained from known organisms. While this method has led to the identification and classification of various organisms that were historically not cultivable, it is again limited in its focus on rRNA.
  • the present invention can be used to identify genes unique to a particular species, subspecies or strain. Unlike the above-described currently used genetic approaches, the __.
  • present invention is not limited to any particular genes or gene sequences (e.g. rRNA sequences).
  • the present invention contemplates comparing the expressed genes of two samples suspected to be different species.
  • a species that is suspected to have changed or diverged from the parent species is compared with the parent species.
  • a species or strain of bacteria may develop a different susceptibilities to a drug (e.g. antibiotics) as compared to the parent species: rapid identification of the specific species or subspecies aids diagnosis and allows initiation of appropriate treatment.
  • RNAse-free DNAse-1 RQ-1 DNAse, Promega, Madison, WI
  • phenol-Chloroform Sigma Chemical Company. St. Louis, MO
  • cDNA used for the PCR reaction can be made in a variety of ways. However, in the examples below, single stranded cDNA (sscDNA) was synthesized using 1 ⁇ g of total
  • RNA or 100 ⁇ g of mRNA with random primers according to the instructions supplied with a commercially available kit (Superscript, BRL-GIBCO, Gaithersburg. MD).
  • the reverse transcriptase enzyme was killed by heating at 94°C for 15 min. __.
  • PCR conditions can vary depending on desired outcome. Nonetheless, unless otherwise indicated, the conditions used were as follows. First, the amount of cDNA used in each PCR amplification reaction was empirically determined; 2-5 ng of sscDNA give satisfactory results. Second, the PCR reactions were setup in precooled 0.2ml thin-walled tubes on ice and contained, 50mM TrisHCl (pH 8.5), 50mM KC1, 1.5 mM MgCl 2 , ImM of each dNTP. 2-5 ng of sscDNA, lOpmoles of a K-primer, lOpmoles of an RE-primer, 0.5 ⁇ l of a ⁇ -P 33 dCTP (10 ⁇ Ci/ ⁇ l. Amersham) and water to 20 ⁇ l.
  • the mixture can be subjected to PCR cycles in different ways.
  • the first cycle (or even the first few cycles) involves a lower annealing temperature than the annealing temperature in subsequent cycles.
  • an annealing temperature of between approximately 34°C and approximately 44°C, and more preferrably between approximately 36°C and approximately 40°C, and most preferrably approximately 38°C (for approximately 30 seconds), can be used for the first cycle (or even the first few cycles).
  • the subsequent cycles of denaturation, annealing and extension can involve a higher temperature.
  • annealing temperature is between approximately 40°C and approximately 60"C, more preferrably between approximately 44°C and approximately 54°C, and most preferrably approximately 48°C (for approximately 30 sec).
  • the annealing temperature is approximately the same temperature for all cycles.
  • the above-described mixture is subjected to 35 cycles of denaturation.
  • annealing and extension wherein the annealing temperature is between approximately 38°C and approximately 40°C (for approximately 30 seconds).
  • RNA available commercially from Clontech
  • the total RNA was reverse transcribed using 6-mer random primers (available from Pharmacia).
  • the resultant cDNA was subjected to thirty-five cycles of PCR (in the presence of a radioactive precursor) using a mixture of two anchor primers ("K2" for Figure 3A and "K3" for Figure 3B) and restriction enzyme-based primers [for this experiment, the recognition sequence on the 5' end of the RE downstream primers was for EcoRI; the primer sequences were: GAATTCNNNGT(A or C)(G or T)AC (lanes 1-4); GAATTCNNNCGGC (lanes 5-8); GAATTCNNN(A or G)GCGC(C or T) (lanes 9-12); GAATTCNNNTTAA (lanes 13-16)].
  • the PCR products were analyzed by PAGE using 6% sequencing gels (BRL) and visualized by autoradiography. The results show a large number of bands (see Figures 3A and 3B). Importantly, there is differential expression of transcripts in the various cell types.
  • Bacterial DNA was prepared by standard methods. 10-50 ng of genomic DNA from E coli and P. stuarti (in the first and second lane, respectively, of each two lane group in Figure 4) was subjected to thirty-five cycles of PCR (in the presence of a radioactive precursor) using a mixture of anchor primers (SD-primers: 5'- GGAATTCNNN-TAAGGAGG-3') and restriction enzyme-based primers (RE-primers: 5'- GGATTC-CNNNGATC (this "Mbol primer” was used in lanes 1 and 2 of
  • GGATTCCNNNCTAG this "Bfal primer” was used in lanes 3 and 4 of Figure 4
  • GGATTCCNNNCCGC this "Acil primer” was used in lanes 5 and 6 in Figure 4
  • GGATTCCNNNCCGG this "Hpall primer” was used in lanes 7 and 8 in Figure 4
  • GGATTCCNNNAATT this "Tsp509I primer” was used in lanes 9 and 10 in Figure 4).
  • the PCR products were analyzed by PAGE using 6% sequencing gels (BRL) and visualized by autoradiography.
  • EXAMPLE 3 This example describes the cloning and sequencing of expressed transcripts. Briefly,
  • DNA bands representing differently expressed transcripts were identified by visual scanning of the autoradiograph and marked (Figure 5. which represents a different exposure of the experiment run in Figure 3A).
  • the film was then used as a template and the marked bands were cut out and eluted in water, precipitated with 0.3M sodium acetate. pH 6.0. and 2.5 vol of ethanol, pelleted by centrifugation (12,000 x g, 20 min), washed 2X with 70% ethanol, air dried and dissolved in 10 ⁇ l of nuclease free water.
  • Half of the sample was then used for reamplification using the same primer combination and PCR conditions. Amplified material was resolved on a 2% agarose gel and the size of the amplified fragments was determined with reference to DNA size standards ( 100 bp ladder. BRL) and the amplified DNA fragments were gel purified using a commercially available kit
  • NCBI National Center for Biotechnology Information
  • This example describes the comparison of normal and malignant tissues.
  • a variety of cell types were studied: 1) normal human keratinocytes, 2) normal human skin, and 3-5) three squamous cell carcinoma samples from patients (in the first, second, third, fourth and fifth lane of each five lane group in Figure 8).
  • the total RNA was reverse transcribed using 6- mer random primers (available from Pharmacia).
  • the resultant cDNA was subjected to thirty-five cycles (all cycles were performed using annealing temperatures between 38 and 42 degrees) of PCR (in the presence of a radioactive precursor) using a mixture of two anchor primers ("Kl") and restriction enzyme-based primers (RE-primers: 5 * - GGATTCCNNNGATC (this "Mbo I primer” was used in the reactions represented by lanes 1 through 5 of Figure 8); GGATTCCNNNCTAG (this "Bfal primer” was used in the reactions represented by lanes 6 through 10 of Figure 8); GGATTCCNNNCCGC (this "Acil primer” was used in reactions represented by lanes 11 through 15 in Figure 8); GGATTCCNNNCCGG (this "Hpall primer” was used in reactions represented by lanes 16 through 20 in Figure 8); and GGATTCCNNNAATT (this "Tsp509I primer” was used in the reactions represented in lanes 21 through 25 in Figure 8).
  • the PCR products were analyzed by PAGE using 6% sequencing
  • the present invention provides a convenient method for distinguishing between the expression of genes in two or more biological samples. Importantly, the method also promotes followup analysis once a gene of interest is indentified.

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Abstract

L'invention porte sur un procédé et des compositions permettant de distinguer entre les gènes exprimés par deux ou plus de deux échantillons biologiques. Ladite invention recourt à des motifs de ciblage d'amorces d'oligonucléotides conservés à l'intérieur chaque gène exprimé. Les amorces et procédés de l'invention permettent d'identifier avec précision le ou les gènes exprimés différentiellement dans différents types de cellules.
PCT/US1998/004094 1997-03-03 1998-03-03 Procedes et compositions d'identification de genes exprimes WO1998039480A1 (fr)

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US5599672A (en) * 1992-03-11 1997-02-04 Dana-Farber Cancer Institute, Inc. Method of differential display of exposed mRNA by RT/PCR
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