WO2016104272A1 - Pcr method - Google Patents

Pcr method Download PDF

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Publication number
WO2016104272A1
WO2016104272A1 PCT/JP2015/085184 JP2015085184W WO2016104272A1 WO 2016104272 A1 WO2016104272 A1 WO 2016104272A1 JP 2015085184 W JP2015085184 W JP 2015085184W WO 2016104272 A1 WO2016104272 A1 WO 2016104272A1
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Prior art keywords
pcr
dna polymerase
pcna
amino acid
composition
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PCT/JP2015/085184
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French (fr)
Japanese (ja)
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哲大 小林
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東洋紡株式会社
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Priority to JP2016566150A priority Critical patent/JP6950184B2/en
Publication of WO2016104272A1 publication Critical patent/WO2016104272A1/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)
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • 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

Definitions

  • the present invention relates to the field of nucleic acid amplification, in particular PCR (Polymerase chain reaction). More specifically, the present invention relates to a method for shortening the PCR reaction time.
  • PCR Polymerase chain reaction
  • the PCR method is (1) DNA denaturation by heat treatment (dissociation from double-stranded DNA to single-stranded DNA), (2) primer annealing to template single-stranded DNA, and (3) the above using DNA polymerase
  • This is a method of amplifying a target nucleic acid in a sample by repeating three cycles of three steps of primer extension as one cycle.
  • This method can amplify the target nucleic acid hundreds of thousands of times from a trace amount sample such as several copies, so it can be used not only for research purposes but also in forensic fields such as genetic diagnosis and clinical diagnosis, or for microbial testing in food and the environment. Etc. are also widely used.
  • PCR has high detection sensitivity, it is necessary to carry out a thermal cycle for the reaction, and the problem is that the reaction time is long. Recently, thermal cyclers that perform temperature changes at high speed have been sold, and devices for completing the reaction in a shorter time have been studied. In terms of the reaction, the PCR composition and method have been studied in order to cope with the high-speed cycle.
  • DNA polymerase used for PCR is important for high-speed PCR.
  • DNA polymerase belonging to family B is more suitable for high-speed PCR than polymerase belonging to family A such as Taq DNA polymerase (Patent Document 1).
  • PCNA Proliferating Cell Nuclear Antigen
  • DNA polymerase did not work well in extreme high-speed cycles, and poor amplification was observed.
  • a substance that inhibits PCR may be included in the measurement sample, and high-speed PCR could not be performed under such conditions containing the inhibitor.
  • rapid results reporting is required for diagnostic applications, and a method for DNA synthesis at high speed and high efficiency has been strongly demanded.
  • JP 2010-239880 A International Publication No. 2007/004654 Pamphlet
  • An object of the present invention is to provide a method and a reagent composition for shortening the reaction time in DNA synthesis using PCR.
  • the representative present invention is as follows.
  • Item 1 A composition comprising DNA polymerase belonging to family B, Proliferating Cell Nuclear Antigen (PCNA) and a primer at a concentration of 0.6 ⁇ M or more, and 30 to 50 cycles within 40 minutes
  • a PCR method composition characterized by being.
  • Item 2. The PCR method composition according to Item 1, which is a composition for carrying out 30 to 50 cycles within 20 minutes.
  • Item 3. The PCR method composition according to Item 1 or 2, which is a composition for performing from 30 cycles to 50 cycles within 12 minutes.
  • Item 4. Item 4.
  • Item 6. The PCR method composition according to any one of Items 1 to 5, wherein the PCR method composition is a composition for performing PCR at a rate of temperature increase or decrease of 0.1 ° C / second to 20 ° C / second.
  • Item 7. The PCR method composition according to any one of Items 1 to 6, which is a composition for performing PCR at a rate of temperature increase or decrease of 5 ° C / second to 20 ° C / second.
  • Item 8. Item 8.
  • Item 9. Item 9.
  • Item 14 A PCR reagent kit comprising the PCR reagent according to Item 13.
  • Item 15. Item 13.
  • Item 16. Item 16.
  • Item 17. Item 17.
  • the PCR reaction time can be shortened.
  • the composition and composition in the present invention enables high-speed PCR even under conditions containing an inhibitor such as blood, and is very useful particularly in diagnostic applications that require rapidity.
  • the PCR method in the present invention is characterized in that DNA polymerase belonging to Family B, PCNA and a high concentration primer are included in the reaction solution, and PCR is performed at a high speed.
  • “D143A / D147A” indicates that the 143rd aspartic acid was substituted with alanine and the 147th aspartic acid was substituted with alanine.
  • “mutant” and “mutant” mean that the protein has a different amino acid sequence from a conventionally known protein, and distinguishes whether it is due to an artificial mutation or a mutation in nature. Not what you want.
  • the “142st amino acid corresponding to the amino acid sequence shown in SEQ ID NO: 1” is 142 of SEQ ID NO: 1 in PCNA having an amino acid sequence that is not completely identical to the amino acid sequence shown in SEQ ID NO: 1. It is an expression including the amino acid sequence corresponding to the th.
  • the meaning of “corresponding” in the notation of the same format as described above is the same as in the above example.
  • a position (order) on SEQ ID NO: 1 and a corresponding position are when the primary structure of the sequence is compared (alignment) , A position corresponding to the position of SEQ ID NO: 1.
  • the meaning of “corresponding position” in the notation of the same format as described above is the same as the above example.
  • the PCR in the present invention is a high-speed PCR in which the reaction time is within 40 minutes when 30 thermal cycles are performed, preferably within 30 minutes, more preferably within 20 minutes, and even more preferably 10 minutes. Fast PCR that is within.
  • the reaction time indicates the time of a thermal cycle in which denaturation, annealing, and elongation are repeated. With a thermal cycler setting of a normal thermal cycler, the reaction time rarely falls within 60 minutes. For example, in order to complete the reaction within 60 minutes using PCR system GeneAmp (registered trademark) 9700 (manufactured by Applied Biosystem), it is necessary to extremely shorten the time of each step of the thermal cycle.
  • the reaction time is about 60 minutes. Minutes.
  • a thermal cycler that changes the temperature at high speed, and to further shorten the time of each step of the thermal cycle. For example, when Light cycler (registered trademark) 2.0 (Roche) capable of changing temperature at high speed is used, after the reaction at 94 ° C.
  • reaction time is about 20 minutes.
  • the number of thermal cycles of PCR in the present invention can be set and carried out in the range of 15 to 50 times. Preferably it is 25 to 45 times, more preferably 30 to 40 times.
  • the annealing step it is preferable to shorten the annealing step. This is because, in order to increase the PCR reaction rate, it is necessary to increase the primer concentration. However, generally, when the primer concentration is increased, non-specificity or primer dimer is likely to occur. By shortening the annealing step, non-specificity and primer dimers are less likely to occur even at high concentrations of primers.
  • the preferred annealing step time is 10 seconds. More preferably 5 seconds, particularly preferably 0 seconds.
  • DNA polymerase belonging to family B The DNA polymerase used in the nucleic acid amplification method of the present invention is a DNA polymerase belonging to family B.
  • a DNA polymerase belonging to Family B refers to a DNA polymerase having 3′-5 ′ exonuclease activity and not having 5′-3 ′ exonuclease activity.
  • a DNA polymerase derived from Archaea is preferable.
  • DNA polymerase derived from archaea examples include DNA polymerases isolated from bacteria belonging to the genus Pyrococcus and Thermococcus. Examples of the DNA polymerase derived from the genus Pyrococcus include Pyrococcus furiosus and Pyrococcus sp. Including, but not limited to, DNA polymerases isolated from GB-D, Pyrococcus Wosei, Pyrococcus abyssi, Pyrococcus horikoshii.
  • DNA polymerases derived from the genus Thermococcus include Thermococcus kodakaraensis, Thermococcus gorgonarius, Thermococcus litoralis, Thermococcus sp. JDF-3, Thermococcus sp. 9 degrees North-7 (Thermococcus sp. 9 ° N-7), Thermococcus sp. Including, but not limited to, DNA polymerase isolated from KS-1, Thermococcus celler, or Thermococcus sicili.
  • PCR enzymes using these DNA polymerases are commercially available, such as Pfu (Staragene), KOD (Toyobo), Pfx (Life Technologies), Vent (New England Biolabs), Deep Vent (New England Biobland). Roche) and Pwo (Roche).
  • KOD DNA polymerase excellent in extensibility and thermal stability is preferable.
  • a mutant obtained by modifying the DNA polymerase described later to modify the 3′-5 ′ exonuclease region and / or to have a decreased base analog detection activity may be used.
  • modified DNA polymerase used in the present invention is further added to any one of the amino acid sequences of the 3′-5 ′ exonuclease active region. It may contain at least one amino acid modification.
  • 3'-5 'exonuclease activity refers to the ability to remove incorporated nucleotides from the 3' end of a DNA polymer, and the above 3'-5 'exonuclease region is a DNA polymerase belonging to family B.
  • Thermococcus kodakaraensis DNA polymerase (SEQ ID NO: 1), Pyrococcus furiosus DNA polymerase (SEQ ID NO: 2), Thermococcus gorgonarius DNA polymerase (SEQ ID NO: 3), DNA polymerase derived from Thermococcus litoralis (SEQ ID NO: 4), DNA polymerase derived from Pyrococcus sp GB-D (SEQ ID NO: 5), derived from Thermococcus sp JDF-3 DNA polymerase (SEQ ID NO: 6), DNA polymerase derived from Mococcus sp 9 ° N-7 (SEQ ID NO: 7), DNA polymerase derived from Thermococcus sp KS-1 (SEQ ID NO: 8), DNA polymerase derived from Thermococcus cellar (SEQ ID NO: 9)
  • SEQ ID NO: 10 Thermococcus kodakaraensis
  • the present invention is also applicable to DNA polymerases other than the DNA polymerase specifically presenting the sequence.
  • the 3′- consisting of amino acids 137 to 147, 206 to 222, and 308 to 318 of SEQ ID NO: 1 The region corresponding to the 5 ′ exonuclease region is shown.
  • amino acids corresponding to positions 137 to 147, 206 to 222, and 308 to 318 shown in SEQ ID NO: 1 are DNA polymerases having an amino acid sequence that is not completely identical to the amino acid sequence shown in SEQ ID NO: 1.
  • An expression comprising amino acid sequences corresponding to positions 137 to 147, 206 to 222, and 308 to 318 of SEQ ID NO: 1.
  • the meaning of “corresponding” in the notation of the same format as described above is the same as in the above example.
  • the modification of the 3′-5 ′ exonuclease region can be composed of substitution, deletion, or addition, but is not particularly limited. For example, alterations to amino acids corresponding to positions 137 to 147, 206 to 222, and 308 to 318 in SEQ ID NO: 1 are shown.
  • DNA polymerase in which the 3′-5 ′ exonuclease active region is modified one in which at least one of amino acids corresponding to positions 141, 142, 143, 210, 311 in SEQ ID NO: 1 or SEQ ID NO: 2 is modified is preferable.
  • These modified DNA polymerases are deficient in 3'-5 'exonuclease activity. More preferably, it is a DNA polymerase deficient in 3'-5 'exonuclease activity, wherein the amino acid modification is any one selected from D141A, E143A, D141A / E143A, I142R, N210D, or Y311F.
  • 3'-5 'exonuclease activity-deficient (exo (-)) DNA polymerase includes a complete lack of activity, for example, 0.03%, 0.05% compared to the parent enzyme , 0.1%, 1%, 5%, 10%, 20%, or at most 50% or less of a modified DNA polymerase having exonuclease activity.
  • DNA polymerase in which the 3'-5 'exonuclease active region is modified is any one selected from H147E and H147D in SEQ ID NO: 1 or SEQ ID NO: 2. These modified DNA polymerases have improved PCR efficiency while maintaining exonuclease activity.
  • a method for producing a DNA polymerase with a modified 3′-5 ′ exonuclease active region and a method for analyzing 3′-5 ′ exonuclease activity are known and disclosed in, for example, US Pat. No. 6,946,273. ing.
  • a DNA polymerase with improved PCR efficiency refers to a modified DNA polymerase in which the amount of PCR product is increased compared to the parent enzyme.
  • a method for analyzing whether the amount of the PCR product is increased as compared with the parent enzyme is described in Japanese Patent No. 3891330.
  • the DNA polymerase belonging to family B used in the present invention may be a mutant having reduced base analog detection activity.
  • Base analogs refer to bases other than adenine, cytosine, guanine, and thymine, and include uracil and inosine.
  • a DNA polymerase belonging to Family B binds strongly when a base analog such as uracil or inosine is detected, and inhibits the DNA polymerase function.
  • the base analog detection activity refers to an activity that strongly binds to a base analog and inhibits the DNA polymerase function.
  • a DNA polymerase mutant belonging to family B having reduced base analog detection activity is a DNA polymerase mutant belonging to family B characterized by low binding ability to uracil and inosine.
  • Such a mutant can be prepared by modifying at least one position of the amino acid sequence (uracil binding pocket) relating to uracil binding.
  • uracil-binding pockets formed by archaebacterial DNA polymerase belonging to family B for example, amino acid sequence (SEQ ID NO: 1) of DNA polymerase derived from Thermococcus kodakaraensis KOD1 strain (position No. 1), and positions 78-130 Examples include DNA polymerase mutants that have been modified at least at one site and have reduced ability to bind to uracil or inosine compared to wild-type DNA polymerase.
  • DNA polymerase mutants with low binding ability to uracil and inosine do not show much decrease in DNA polymerase function even in PCR in the presence of dUTP, and the effect of dUTP on the elongation reaction of DNA polymerase is reduced.
  • the amino acid sequence for uracil binding is highly conserved in DNA polymerases from Pyrococcus and DNA polymerases from Thermococcus.
  • DNA polymerase SEQ ID NO: 1 derived from Thermococcus kodakaraensis, it is formed by amino acids 1 to 40 and amino acids 78 to 130.
  • Pyrococcus furiosus SEQ ID NO: 2
  • amino acids 1 to 40 and amino acids 78 to 130 In Thermococcus gorgonarius (SEQ ID NO: 3), it is formed by amino acids 1 to 40 and amino acids 78 to 130.
  • Thermococcus litoralis (SEQ ID NO: 4), it is formed by amino acids 1 to 40 and amino acids 78 to 130.
  • Pyrococcus sp. GB-D (SEQ ID NO: 5), it is formed by amino acids 1 to 40 and amino acids 78 to 130.
  • Thermococcus sp. JDF-3 (SEQ ID NO: 6), it is formed by amino acids 1 to 40 and amino acids 78 to 130.
  • Thermococcus sp 9 ° N-7 (SEQ ID NO: 7), it is formed by amino acids 1-40 and amino acids 78-130.
  • KS-1 (SEQ ID NO: 8), it is formed by amino acids 1 to 40 and amino acids 78 to 130.
  • Thermococcus cellar (SEQ ID NO: 9), it is formed by amino acids 1-40 and amino acids 78-130.
  • Thermococcus cyclis (SEQ ID NO: 10), it is formed by amino acids 1 to 40 and amino acids 78 to 130.
  • More preferred DNA polymerase variants with reduced base analog detection activity for use in the present invention are assumed to be directly related to interaction with uracil 7, 36, 37, 90-97 and 112.
  • An archaeal DNA polymerase variant in which at least one of the 119th amino acids is modified for example, (a) 7, 36, 37, 90 to 97 of the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2; It is an archaeal DNA polymerase variant represented by an amino acid sequence having at least one amino acid modification among amino acids corresponding to positions 112 to 119.
  • the archaeal DNA polymerase mutant may be one represented by the following amino acid sequence (b).
  • the identity or the identity between the amino acid sequence and SEQ ID NO: 2 is 80% or more (preferably 85% or more, more preferably 90% or more, still more preferably 95% or more, particularly preferably 98%. And most preferably 99% or more), and an amino acid sequence encoding a DNA polymerase having reduced base analog detection activity.
  • the archaeal DNA polymerase mutant may be one represented by the following amino acid sequence (c).
  • (C) In the amino acid sequence shown in (a), one or several amino acids are further deleted, substituted or added at sites other than positions 7, 36, 37, 90 to 97 and 112 to 119, and An amino acid sequence encoding a DNA polymerase having reduced base analog detection activity.
  • “several” is not limited as long as “decreased base analog detection activity” is maintained, but is, for example, a number corresponding to less than about 20% of all amino acids, preferably less than about 15%. It is a corresponding number, more preferably a number corresponding to less than about 10%, even more preferably a number corresponding to less than about 5%, and most preferably a number corresponding to less than about 1%. More specifically, the number of amino acid residues to be mutated is, for example, 2 to 160, preferably 2 to 120, more preferably 2 to 80, still more preferably 2 to 40, and particularly preferably. Is 2-5.
  • amino acids corresponding to positions 7, 36, 37, 90 to 97 and 112 to 119 in the amino acid sequence shown in SEQ ID NO: 1 are amino acid sequences that are not completely identical to the amino acid sequence shown in SEQ ID NO: 1.
  • it is an expression including the amino acid sequence relating to the binding of uracil corresponding to positions 7, 36, 37, 90 to 97 and 112 to 119 of SEQ ID NO: 1.
  • a position (order) on SEQ ID NO: 1 in an amino acid sequence that is not completely identical to the amino acid sequence shown in SEQ ID NO: 1 is compared with a position corresponding to a position (sequence) on the primary structure of the sequence, The position corresponds to the position of SEQ ID NO: 1.
  • the meaning of “corresponding” in the notation of the same format as described above is the same as in the above example.
  • a position (order) on SEQ ID NO: 1 in an amino acid sequence that is not completely identical to the amino acid sequence shown in SEQ ID NO: 1 is compared with a position corresponding to a position (sequence) on the primary structure of the sequence, The position corresponds to the position of SEQ ID NO: 1.
  • the meaning of “corresponding position” in the notation of the same format as described above is the same as the above example.
  • the archaeal DNA polymerase mutant having reduced base analog detection activity for use in the present invention is more preferably at least one selected from amino acids corresponding to amino acids Y7, P36, or V93 in SEQ ID NO: 1 or SEQ ID NO: 2.
  • Y7 means a tyrosine (Y) residue that is the seventh amino acid, and one letter of the alphabet represents an abbreviation of a commonly used amino acid.
  • the Y7 amino acid has tyrosine (Y) substituted with a nonpolar amino acid, specifically selected from the group consisting of Y7A, Y7G, Y7V, Y7L, Y7I, Y7P, Y7F, Y7M, Y7W, and Y7C.
  • Y tyrosine
  • the P36 amino acid is substituted with a polar amino acid in which proline (P) is positively charged, specifically P36H, P36K, or P36R.
  • the V93 amino acid is a valine (V) substituted with a positively charged polar amine acid, specifically an amino acid substitution of V93H, V93K, or V93R.
  • the modification is at least one amino acid modification selected from the group consisting of Y7A, P36H, P36K, P36R, V93Q, V93K, and V93R. More preferably, it is P36K, P36R or P36H. Particularly preferred is P36H.
  • the archaeal DNA polymerase mutant having reduced base analog detection activity used in the present invention comprises two or more amino acids selected from amino acids corresponding to amino acids Y7, P36, or V93 in SEQ ID NO: 1 or SEQ ID NO: 2. May be modified. Specific examples include Y7A / V93K, Y7A / P36H, Y7A / P36R, Y7A / V93R, Y7A / V93Q or P36H / V93K, and preferably Y7A / P36H or Y7A / V93K. It is not limited to.
  • mutants can be considered as the modified DNA polymerase used in the present invention.
  • mutants include, but are not limited to, archaeal DNA polymerase mutants having any of the following modifications (1) to (4).
  • the base analog detection activity in the present invention can be evaluated by PCR.
  • the base analog is typically uracil.
  • a dUTP solution is added at a final concentration of 0.5 ⁇ M to 200 ⁇ M to a normal PCR reaction solution containing DNA as a template, buffer material, magnesium, dNTPs, primers, and a DNA polymerase to be evaluated. Cycle. After the reaction, the presence or absence of a PCR product is confirmed by ethidium bromide-stained 1% agarose electrophoresis, and the detection activity of uracil can be evaluated based on an acceptable dUTP concentration.
  • a DNA polymerase having a high uracil detection activity inhibits the extension reaction when a little dUTP is added, and the PCR product cannot be confirmed.
  • DNA polymerase with low uracil detection activity can confirm gene amplification by PCR without any problems even when a high concentration of dUTP is added.
  • An archaeal DNA polymerase mutant having decreased base analog detection activity is a result of optimal thermal cycling using any primer and DNA as a template in an enzyme optimal reaction buffer. Compared with the wild-type without DNA, it means a DNA polymerase in which the extension reaction is not inhibited even when a high concentration of dUTP is added, and the PCR product can be confirmed. However, if it is difficult to compare with the wild type, the archaeal DNA polymerase mutant that can amplify PCR even when dUTP is added at a concentration of 0.5 ⁇ M is reduced compared to the wild type. It is presumed to have the activity of detecting a base analog.
  • the evaluation of the base analog detection activity in the present invention follows the following method. KOD -Plus- Ver. 2 (Toyobo) attached 10 ⁇ PCR Buffer or Pfu DNA Polymerase (Agilent) attached 10 ⁇ PCR Buffer, 1 ⁇ PCR Buffer, 1.5 mM MgSO 4 , 0.2 mM dNTPs (dATP, dTTP, dCTP, dGTP), 15 pmol of the primer described in SEQ ID NOS: 11 and 12 for amplifying about 1.3 kb, 10 ng of human genomic DNA (Roche), and 1 U of each enzyme, 50 ⁇ l of reaction solution containing dUTP (Roche) ) To a final concentration of 0.5, 5, 50, 100, 200 ⁇ M.
  • a PCR system GeneAmp registered trademark 9700 (Applied Biosystem) with a schedule for repeating 30 cycles of 98 ° C., 10 seconds ⁇ 65 ° C., 30 seconds ⁇ 68 ° C., 1 minute 30 seconds Perform PCR.
  • 1% agarose electrophoresis is performed on 5 ⁇ l of the reaction solution, ethidium bromide staining is performed, and the amplified DNA fragment of about 1.3 kb is confirmed under ultraviolet irradiation to determine whether the base analog detection activity is reduced. Can be evaluated.
  • a site-specific mutagenesis method based on the Inverse PCR method can be used.
  • KOD-Plus-Mutageness Kit manufactured by Toyobo
  • Phosphorylation of newly synthesized gene, Ligation (5) A kit that can transform a cyclized gene into Escherichia coli and obtain a transformant having a plasmid introduced with the target mutation.
  • the modified DNA polymerase gene is transferred to an expression vector as necessary, and, for example, E. coli as a host is transformed with the expression vector, and then applied to an agar medium containing a drug such as ampicillin to form colonies.
  • the colony is inoculated into a nutrient medium such as LB medium or 2 ⁇ YT medium and cultured at 37 ° C. for 12 to 20 hours, and then the cells are crushed and the crude enzyme solution is extracted.
  • a vector derived from pBluescript is preferable. Any known method may be used as a method for crushing bacterial cells. For example, ultrasonic treatment, a physical crushing method such as French press or glass bead crushing, or a lytic enzyme such as lysozyme can be used.
  • This crude enzyme solution is heat-treated at 80 ° C. for 30 minutes to inactivate the host-derived DNA polymerase, and the DNA polymerase activity is measured.
  • any method may be used as a method for obtaining purified DNA polymerase from the strain selected by the above method, for example, the following method.
  • the crude enzyme solution is obtained by crushing and extracting by an enzymatic or physical crushing method.
  • the obtained crude enzyme extract is heat-treated, for example, at 80 ° C. for 30 minutes, and then the DNA polymerase fraction is recovered by ammonium sulfate precipitation.
  • This crude enzyme solution can be desalted by a method such as gel filtration using Sephadex G-25 (manufactured by Amersham Pharmacia Biotech). After this operation, it can be separated and purified by heparin sepharose column chromatography to obtain a purified enzyme preparation.
  • the purified enzyme preparation is purified by SDS-PAGE to such an extent that it shows almost a single band.
  • DNA polymerase activity measurement method The activity of the DNA polymerase used in the present invention is measured as follows. If the enzyme activity is strong, samples should be stored in storage buffer (50 mM Tris-HCl (pH 8.0), 50 mM KCl, 1 mM dithiothreitol, 0.1% Tween 20, 0.1% Nonidet P40, 50% glycerin). Dilute and measure. (1) 25 ⁇ l of the following solution A, 5 ⁇ l of solution B, 5 ⁇ l of solution C, 10 ⁇ l of sterilized water, and 5 ⁇ l of enzyme solution are added to a microtube and reacted at 75 ° C. for 10 minutes.
  • storage buffer 50 mM Tris-HCl (pH 8.0), 50 mM KCl, 1 mM dithiothreitol, 0.1% Tween 20, 0.1% Nonidet P40, 50% glycerin.
  • Solution A 40 mM Tris-HCl buffer (pH 7.5), 16 mM magnesium chloride, 15 mM dithiothreitol, 100 ⁇ g / mL BSA (bovine serum albumin)
  • BSA bovine serum albumin
  • Solution B 1.5 ⁇ g / ⁇ l activated calf thymus DNA
  • C 1.5 mM dNTP (250 cpm / pmol [3H] dTTP)
  • Liquid D 20% trichloroacetic acid (2 mM sodium pyrophosphate)
  • E 1 mg / mL calf thymus DNA
  • PCNA (4.1) PCNA used in the present invention is a kind of PCR enhancing factor.
  • the PCNA is not particularly limited, but is preferably a heat-resistant one that can withstand the thermal cycle of PCR, and preferably one that remains active after PCR. More preferably, it is soluble even after heat treatment at 80 ° C. for 30 minutes, and the activity remains at 50% or more, more preferably at least 70%, particularly preferably at least 90%.
  • PCNA examples include PCNA isolated from bacteria of the genus Pyrococcus and Thermococcus.
  • PCNA derived from the genus Pyrococcus includes Pyrococcus furiosus (SEQ ID NO: 13), Pyrococcus sp. Including, but not limited to, PCNA isolated from GB-D, Pyrococcus Wosei, Pyrococcus abyssi or Pyrococcus horikoshii.
  • PCNA derived from the genus Thermococcus includes Thermococcus kodakaaraensis (SEQ ID NO: 14), Thermococcus gorgonaris, Thermococcus literalis, Thermococcus sp.
  • Thermococcus sp. 9 degrees North-7 (Thermococcus sp. 9 ° N-7), Thermococcus sp. Includes, but not limited to, PCNAs isolated from KS-1, Thermococcus celer, or Thermococcus siculi, Methanocladoccus jannaschii (Mja) or Methanobacterium thermoautotropicum (Mth).
  • a gene encoding PCNA can be cloned from an organism having PCNA. It can also be artificially synthesized based on amino acid sequence information and nucleic acid sequence information.
  • the PCNA used in the present invention may be a mutant that alone loads DNA (having DNA polymerase amplification enhancing activity).
  • PCNA usually forms a multimer and has a ring-like structure. Loading to DNA indicates that the DNA is allowed to pass inside the ring structure of the PCNA multimer, and PCNA can be loaded into DNA only in combination with a factor usually called RFC.
  • Mutants that load DNA alone are those that modify the sites involved in PCNA multimer formation and destabilize multimer formation, making it easier to pass DNA into PCNA multimers without RFC. Show.
  • KOD-PCNA and Pfu-PCNA PCNA-related sites for multimer formation are PCNA derived from Thermococcus kodakaraensis (also described as KOD-PCNA) (SEQ ID NO: 13), Pyrococcus furiosus PCNA (also described as Pfu-PCNA) (SEQ ID NO: 14) , Examples include an N-terminal region consisting of amino acids 82, 84 and 109 and a C-terminal region consisting of amino acids 139, 143 and 147. The N-terminal region is positively charged, the C-terminal region is negatively charged, and multimers are formed by interaction.
  • SEQ ID NO: 13 or SEQ ID NO: 14 As an example also applies to PCNAs other than the PCNA specifically presenting the sequences in this specification.
  • a region related to multimer formation consisting of amino acids 82, 84, 109, 139, 143, and 147 of SEQ ID NO: 13 Indicates the corresponding area.
  • the amino acid or region “corresponds” in one of the reference amino acid sequences means that when the primary structure of the amino acid sequences is compared (aligned), The position corresponds to the position.
  • PCNA variants that load DNA alone are more preferably involved in PCNA multimer formation, (A) an N-terminal region consisting of amino acids corresponding to positions 82, 84 and 109, or (B) Examples include mutants having at least one modification in the C-terminal region consisting of amino acids corresponding to the 139th, 143rd, and 147th amino acids, loading to DNA without RFC and promoting the elongation reaction of DNA polymerase.
  • the amino acid corresponding to position 143 of SEQ ID NO: 13 is changed to a basic amino acid
  • the positions 82 and 143 are both changed to neutral amino acids
  • the position 147 is changed to a neutral amino acid, or 109 And the like, in which both the 145th and 143rd are modified to neutral amino acids.
  • the neutral amino acid of the present invention include glycine, alanine, valine, leucine, isoleucine, phenylalanine, tyrosine, proline, serine, threonine, cysteine, methionine, asparagine, and glutamine as long as they are natural.
  • the alanine has the smallest influence on the three-dimensional structure of the peripheral site of the substitution site.
  • Examples of basic amino acids include arginine, histidine, lysine and tryptophan if they are natural. Arginine or lysine is preferable.
  • a sequence in which the 147th amino acid residue is replaced with alanine (D147A), the 82nd and 143rd amino acid residues are alanine.
  • the sequence (R109A / D143A or R109A / E143A) in which the 109th and 143rd amino acid residues are changed to alanine It is not limited to.
  • PCNA used in the present invention may be a modified methionine corresponding to position 73 of SEQ ID NO: 13 or SEQ ID NO: 14 in order to increase the expression level. More preferable examples include those modified to M73L, but are not limited thereto.
  • Mja-PCNA PCNA used in the present invention is a PCNA monomer shown in any one of the following (A) to (E).
  • the 142nd amino acid residue in the amino acid sequence of SEQ ID NO: 23 is any one selected from the group consisting of lysine, arginine, histidine, and tryptophan It consists of a polypeptide consisting of an amino acid sequence substituted with an amino acid residue, and any of the following substitutions (a) to (e), deletions, insertions and / or additions (these are also collectively referred to as “mutation”). And a polypeptide having DNA polymerase amplification enhancing activity.
  • the 80th amino acid residue is replaced with one selected from the group consisting of lysine, histidine and tryptophan.
  • the 82nd amino acid residue is selected from the group consisting of arginine, histidine and tryptophan.
  • C the 108th amino acid residue is replaced with any one selected from the group consisting of lysine, histidine and tryptophan
  • d the 138th amino acid residue is replaced with glutamic acid
  • e the 146th amino acid residue In the PCNA monomer represented by (C) (A)
  • the 142nd amino acid residue in the amino acid sequence of SEQ ID NO: 23 is selected from the group consisting of lysine, arginine, histidine, and tryptophan
  • Poly consisting of an amino acid sequence substituted with any amino acid residue 1 to several amino acid residues other than the amino acid residues corresponding to the 80th, 82nd, 108th, 138th, 142nd and 146th amino acids are substituted, deleted, inserted or added.
  • a polypeptide comprising the amino acid sequence having a DNA polymerase amplification enhancing activity In the PCNA monomer represented by (A), any one selected from the group consisting of lysine, arginine, histidine, and tryptophan, in which only the 142nd amino acid residue of the amino acid sequence of SEQ ID NO: 23 is further selected A polypeptide comprising an amino acid sequence substituted with any amino acid residue.
  • the 142nd amino acid residue is one of the amino acid residues involved in PCNA multimer formation. Amino acid residues involved in PCNA multimer formation are present in the N-terminal region and C-terminal region of each monomer. The PCNA multimer is formed by joining the N-terminal region of one monomer and the C-terminal region of the other monomer as an interface. In eukaryotic cells and archaea, PCNA often forms trimers.
  • the N-terminal region corresponds to the position of the group indicated by (a) below
  • the C-terminal region corresponds to the position of the group indicated by (b) below.
  • polypeptide of (2) above one or several amino acid residues are substituted, deleted, inserted and / or added in the amino acid sequence shown in SEQ ID NO: 23 as long as the DNA polymerase amplification enhancing activity is retained. (Hereinafter, these are collectively referred to as “mutation”.)
  • One or several mutations include restriction enzyme treatment, treatment with exonuclease, DNA ligase, etc., site-directed mutagenesis method or random mutagenesis method (Molecular Cloning, Third Edition, Chapter 13, Cold Spring Harbor Laboratory Press, New (York) and the like can be carried out by introducing mutation into DNA encoding the PCNA monomer of the present invention described later.
  • variant PCNA monomer can also be obtained by other methods such as ultraviolet irradiation.
  • variant PCNA monomers also include naturally occurring variants (for example, single nucleotide polymorphisms) such as those based on individual differences in microorganisms holding PCNA, differences in species or genera.
  • the polypeptide of the above [3] is a polypeptide comprising an amino acid sequence having an identity of 80% or more compared to the amino acid sequence shown in SEQ ID NO: 23, as long as it retains DNA polymerase amplification enhancing activity.
  • the identity between the amino acid sequence of the PCNA monomer of the present invention and the amino acid sequence shown in SEQ ID NO: 23 is 85% or more, more preferably 88% or more, still more preferably 90% or more, more More preferably, it is 93% or more, more preferably 95% or more, particularly preferably 98% or more, and most preferably 99% or more.
  • Such a polypeptide comprising an amino acid sequence having a certain identity or more can be prepared based on the known genetic engineering techniques as described above.
  • the PCNA as described in [2] or [3] above preferably includes those to which an affinity tag such as a His tag inserted at the N-terminus is added in order to simplify the purification of PCNA. It is not limited to.
  • the 142nd amino acid residue in SEQ ID NO: 23 is substituted with a basic amino acid residue, but the type of basic amino acid to be substituted is not particularly limited.
  • Examples of basic amino acids include arginine, histidine, lysine and tryptophan if they are natural. Arginine or lysine is preferable.
  • the PCNA gene is transferred to an expression vector as necessary, for example, Escherichia coli as a host, transformed with the expression vector, applied to an agar medium containing a drug such as ampicillin, and colonies are obtained. Let it form. The colony is inoculated into a nutrient medium such as LB medium or 2 ⁇ YT medium and cultured at 37 ° C. for 12 to 20 hours, and then the cells are crushed and the crude enzyme solution is extracted.
  • a vector derived from pBluescript is preferable. Any known method may be used as a method for crushing bacterial cells.
  • ultrasonic treatment a physical crushing method such as French press or glass bead crushing, or a lytic enzyme such as lysozyme can be used.
  • This crude enzyme solution is heat-treated at 80 ° C. for 30 minutes, centrifuged to remove host-derived protein, and subjected to SDS-PAGE, thereby confirming the expression of the target protein.
  • any method may be used as a method for obtaining purified PCNA from the strain selected by the above method, for example, the following method.
  • the crude enzyme solution is obtained by crushing and extracting by an enzymatic or physical crushing method.
  • the obtained crude enzyme extract is heat-treated, for example, at 80 ° C. for 30 minutes, and then the PCNA fraction is recovered by ammonium sulfate precipitation.
  • This crude enzyme solution can be desalted by a method such as gel filtration using Sephadex G-25 (manufactured by Amersham Pharmacia Biotech). After this operation, it can be separated and purified by Q Sepharose column chromatography to obtain a purified enzyme preparation.
  • the purified enzyme preparation is purified by SDS-PAGE to such an extent that it almost shows a single band.
  • PCNA mutant can be loaded into DNA alone (having DNA polymerase amplification enhancing activity) can be evaluated by PCR.
  • PCNA to be evaluated is added to a PCR reaction solution containing DNA as a template, buffer material, magnesium, dNTPs, primers, and DNA polymerase belonging to Family B, amplified with or without the addition of PCNA
  • PCR amplification amount does not change, but rather the amplification amount tends to decrease.
  • a mutant that can be loaded into DNA alone can be evaluated for DNA polymerase amplification enhancing activity by comparing with a mutant without addition of PCNA.
  • the evaluation of “whether the PCNA mutant can be loaded alone into DNA” (evaluation of DNA polymerase amplification enhancing activity) follows the following method. Using 10 ⁇ PCR Buffer (concentrated to 10 times the concentration used in the reaction) attached to KOD Dash (manufactured by Toyobo), 1 ⁇ PCR Buffer, 0.2 mM dNTPs, 15 pmol of the primers set forth in SEQ ID NOs: 15 and 16 that amplify about 3.6 kb, 10 ng of human genomic DNA (Roche's Human Genomic DNA; model number 116111112001), Prepare a reaction solution containing 1U KOD-Plus-polymerase, In a 50 ⁇ l reaction solution, 250 ng of PCNA to be evaluated was added, After the pre-reaction at 94 ° C.
  • PCR is performed on a schedule in which 98 ° C., 10 seconds ⁇ 68 ° C., 30 seconds are repeated 30 cycles.
  • 5% of the reaction solution was subjected to 1% agarose electrophoresis, stained with ethidium bromide, and the amplified DNA fragment of about 3.6 kb under ultraviolet irradiation was compared with the one added with wild type PCNA alone. It can be assessed whether PCNA can be loaded into DNA. The amount of amplification increases with the addition of PCNA that can be loaded into DNA alone.
  • the amplification amount is digitized by using analysis software called Gel Pro Analyzer (Media Cybernetics).
  • the amplification amount when PCNA is added is 1.0 times (preferably 1.2 times, more preferably 1.times.
  • a target that has not been amplified is amplified, it is determined that the PCNA has “DNA polymerase amplification enhancing activity”.
  • the modification of PCNA can be performed in the same manner as the modification of DNA polymerase.
  • the primer concentration in this invention is 0.6 micromol or more. In general, since a high concentration of primer causes non-specificity or primer dimer, it is said to be generally used at 0.1 to 0.5 ⁇ M in normal PCR. Under the high-speed PCR conditions in the present invention, since the annealing step of the cycle is short, it is known that the efficiency of priming is reduced unless the concentration is high. On the other hand, since the annealing step is short under high-speed PCR conditions, non-specificity and primer dimer are unlikely to occur even at high concentration primers.
  • the primer concentration in the present invention is more preferably 0.8 ⁇ M or more, and further preferably 1.0 ⁇ M or more. In addition, it is preferable that it does not exceed 4.0 ⁇ M, preferably 2.0 ⁇ M, more preferably 1.4 ⁇ M.
  • primer concentration when “primer concentration is 0.6 ⁇ M or more”, the forward (F) and reverse (R) primer concentrations in a pair of primer pairs may be the same, or either primer The concentration may exceed the other concentration (primer concentration may be asymmetric).
  • concentration between all primer pairs in addition to the above, the concentration between all primer pairs may be the same, or the concentration may differ between each primer pair.
  • the PCR in the present invention is preferably carried out at a rate of temperature rise or temperature drop in the thermal cycle of 0.1 ° C / second to 20 ° C / second. More preferred is a range of 5 ° C./second to 20 ° C./second, and further preferred is a range of 10 ° C./second to 20 ° C./second.
  • a thermal cycler that can be temperature controlled by heating and cooling with air.
  • the PCR method of the present invention is suitable for carrying out in a state containing an inhibitor.
  • the inhibitory substance in the present invention refers to, for example, a biological sample, but is not particularly limited as long as it is a substance that inhibits PCR.
  • the biological sample is not particularly limited as long as it is a sample collected from a living body.
  • body refers to animal and plant tissues such as body hair, nails, oral mucosa, body fluids such as blood, excrement such as feces and urine, cells, bacteria, viruses and the like.
  • Body fluid includes blood and saliva, and cells include, but are not limited to, leukocytes separated from blood.
  • an antibody having an activity of suppressing the polymerase activity and / or 3′-5 ′ exonuclease activity of a heat-resistant DNA polymerase may be used as necessary.
  • the antibody include a monoclonal antibody and a polyclonal antibody. This reaction composition is particularly effective for increasing the sensitivity of PCR and reducing nonspecific amplification.
  • the reagent for carrying out the PCR of the present invention comprises a DNA polymerase belonging to Family B, PCNA and a primer having a concentration of 0.6 ⁇ M or more in the reaction solution. Other configurations are not particularly limited.
  • the reagent includes a kit form.
  • Example 1 Preparation of KOD DNA polymerase mutant Plasmid containing a modified thermostable DNA polymerase (Y7A / P36H / N210D mutant) gene derived from Thermococcus kodakaraensis KOD1 strain for use in the examples described below was made.
  • a DNA template used for mutagenesis a modified heat-resistant DNA polymerase gene (SEQ ID NO: 17) (pKOD) derived from Thermococcus kodakaraensis KOD1 strain cloned in pBluescript was used. Mutation was introduced using KOD-Plus-Mutageness Kit (manufactured by Toyobo) according to the instruction manual. The mutant was confirmed by decoding the base sequence. Escherichia coli JM109 was transformed with the obtained plasmid and used for enzyme preparation.
  • Example 2 Production of modified thermostable DNA polymerase The cells obtained in Example 1 were cultured as follows. First, 80 mL of TB medium (Molecular cloning 2nd edition, p.A.2) containing sterilized 100 ⁇ g / mL ampicillin was dispensed into a 500 mL Sakaguchi flask. Escherichia cultured at 37 ° C. for 16 hours in 3 mL of LB medium (1% bactotryptone, 0.5% yeast extract, 0.5% sodium chloride; manufactured by Gibco) containing 100 ⁇ g / mL ampicillin in advance.
  • TB medium Molecular cloning 2nd edition, p.A.2
  • LB medium 1% bactotryptone, 0.5% yeast extract, 0.5% sodium chloride; manufactured by Gibco
  • plasmid transformant (plasmid transformant) (using a test tube) was inoculated and cultured at 37 ° C for 16 hours with aeration.
  • the bacterial cells are collected from the culture solution by centrifugation, suspended in 50 mL of disruption buffer (30 mM Tris-HCl buffer (pH 8.0), 30 mM NaCl, 0.1 mM EDTA), and then subjected to sonication. By crushing, a cell lysate was obtained. Next, the cell lysate was treated at 80 ° C. for 15 minutes, and then the insoluble fraction was removed by centrifugation.
  • nucleic acid treatment using polyethyleneimine, ammonium sulfate precipitation, and heparin sepharose chromatography were performed.
  • a storage buffer 50 mM Tris-HCl buffer (pH 8.0), 50 mM potassium chloride, 1 mM dithiothreitol, 0 1% Tween 20, 0.1% Nonidet P40, 50% glycerin
  • the DNA polymerase activity in the purification step was measured according to the above DNA polymerase activity measurement method. When the enzyme activity was high, the sample was diluted for measurement.
  • Example 3 Preparation of KOD-PCNA mutant A plasmid containing a modified thermostable PCNA (M73L / D147A mutant) gene derived from Thermococcus kodakaraensis KOD1 strain was prepared.
  • PCNA SEQ ID NO: 18
  • pKODPCNA a DNA template used for mutagenesis
  • Mutation was introduced using KOD-Plus-Mutageness Kit (manufactured by Toyobo) according to the instruction manual. The mutant was confirmed by decoding the base sequence.
  • Escherichia coli DH5 ⁇ was transformed with the obtained plasmid and used for enzyme preparation.
  • Example 4 Production of modified heat-resistant PCNA
  • the cells obtained in Example 3 were cultured as follows. First, 80 mL of TB medium (Molecular cloning 2nd edition, p.A.2) containing sterilized 100 ⁇ g / mL ampicillin was dispensed into a 500 mL Sakaguchi flask. Escherichia cultivated at 37 ° C. for 16 hours in 3 mL of LB medium (1% bactotryptone, 0.5% yeast extract, 0.5% sodium chloride; Gibco) containing 100 ⁇ g / mL ampicillin in advance in this medium. E. coli DH5 ⁇ (plasmid transformant) (using a test tube) was inoculated and cultured at 37 ° C.
  • TB medium Molecular cloning 2nd edition, p.A.2
  • LB medium 1% bactotryptone, 0.5% yeast extract, 0.5% sodium chloride; Gibco
  • the bacterial cells are collected from the culture solution by centrifugation, suspended in 50 mL of disruption buffer (30 mM Tris-HCl buffer (pH 8.0), 30 mM NaCl, 0.1 mM EDTA), and then subjected to sonication. By crushing, a cell lysate was obtained. Next, the cell lysate was treated at 80 ° C. for 15 minutes, and then the insoluble fraction was removed by centrifugation. Furthermore, the nucleic acid treatment using polyethyleneimine, ammonium sulfate precipitation, and Q sepharose chromatography were performed.
  • a storage buffer 50 mM Tris-HCl buffer (pH 8.0), 50 mM potassium chloride, 1 mM dithiothreitol, 0 1% Tween20, 0.1% Nonidet P40, 50% glycerin
  • a storage buffer 50 mM Tris-HCl buffer (pH 8.0), 50 mM potassium chloride, 1 mM dithiothreitol, 0 1% Tween20, 0.1% Nonidet P40, 50% glycerin
  • PCR Buffer attached to KOD Dash 10 ⁇ PCR Buffer attached to KOD Dash (manufactured by Toyobo) was diluted 10-fold, 1 ⁇ PCR Buffer (containing 1.2 mM MgSO 4 ), 0.2 mM dNTPs, 1 U KOD DNA polymerase mutant, 250 ng KOD PCNA The following (a) or (b) was added to 50 ⁇ l of a reaction solution containing the mutant and SYBR (registered trademark) Green I diluted 1 / 30,000, and the Ct values were compared with each other.
  • SYBR registered trademark
  • Three levels of 0.2, 0.4, and 0.6 ⁇ M were set for the described primers and the human genome primer concentration corresponding to 50 copies.
  • MgSO 4 was added, the Mg concentration was set at five levels of 1.2, 2, 4 , 6, and 8 mM, and combinations with the primer concentration of 3 levels were examined.
  • the PCR was performed using Light cycler 2.0 with a schedule that repeats 50 cycles of a high-speed cycle of 98 ° C, 0 seconds ⁇ 55 ° C, 0 seconds ⁇ 68 ° C, 0 seconds after a pre-reaction of 94 ° C for 30 seconds.
  • the rate of temperature decrease was 20 ° C./second.
  • the reaction time was about 20 minutes.
  • FIG. 1 shows Ct values when amplification is compared by high-speed PCR from 50 copies worth of Salmonella genome or human genome with different primer concentrations and Mg concentrations
  • FIG. 2 shows their melting curves.
  • the primer concentration is 0.2, 0.4, 0.6 ⁇ M
  • the Mg concentration is 1.2, 2, 4, 6, 8 mM
  • non-specific amplification is seen in the melting curve
  • the Ct value is Depending on the amount of non-specific amplification, circled markings (some with target amplification but also non-specific amplification), squares marked (only non-specific amplification is seen).
  • Amplification of Salmonella invA gene tended to improve Ct by increasing the primer and Mg concentrations, respectively.
  • the amplification of the actin gene improved Ct by increasing the primer concentration when the Mg concentration was low, but non-specific amplification appeared when the Mg concentration was increased.
  • concentration of the primer and Mg each of them has a function of increasing the efficiency of PCR, whereas when it is excessively increased, it leads to non-specific amplification.
  • high concentrations of Mg increase the possibility of increasing non-specific amplification, as in normal PCR, but non-specific amplification is less likely to occur with high concentrations of primers. It was.
  • the cycle annealing step is short, so non-specific amplification and primer dimers are unlikely to occur even when using high concentrations of primers, and it may work to improve only PCR efficiency while maintaining specificity. Conceivable.
  • Example 6 Examination of primer concentration As in Example 5, high-speed PCR was performed using the KOD Y7A / P36H / N210D and KOD PCNA D147A obtained above, and the influence of the primer concentration was confirmed. here. It was also confirmed whether the high-speed PCR in the presence of dUTP has an effect on the primer concentration.
  • PCR Buffer attached to KOD Dash 10 ⁇ PCR Buffer attached to KOD Dash (manufactured by Toyobo) was diluted 10-fold, 1 ⁇ PCR Buffer (including 1.2 mM MgSO 4 ), 0.2 mM dNTPs, or 2 mM dNTPs in which dTTP was replaced with dUTP ( dATP, dUTP, dCTP, dGTP), 1U KOD DNA polymerase mutant, 250 ng KOD PCNA mutant, and amplification of about 550 bp of actin gene in 50 ⁇ l reaction solution containing SYBR Green I diluted 1 / 30,000 Primers shown in SEQ ID NOs: 21 and 22 and a human genome corresponding to 50 copies were added, and Ct values were compared with each other.
  • Primer concentrations were 0.2, 0.6, 0.8, 1.0, 1.2, and 1.4 ⁇ M, respectively.
  • the PCR was performed using Light cycler 2.0 with a schedule that repeats 50 cycles of a high-speed cycle of 98 ° C, 0 seconds ⁇ 55 ° C, 0 seconds ⁇ 68 ° C, 0 seconds after a pre-reaction of 94 ° C for 30 seconds.
  • the rate of temperature decrease was 20 ° C./second.
  • the reaction time was about 20 minutes.
  • Table 1 shows Ct values when amplification from 50 copies by high-speed PCR is compared at different primer concentrations
  • FIG. 3 shows a melting curve.
  • Primer concentrations are 0.2, 0.6, 0.8, 1.0, 1.2, 1.4 ⁇ M in the presence of dTTP (ordinary dNTPs) and in the presence of dUTP (dNTPs in which dTTP is replaced with dUTP). It carried out in.
  • Example 7 Confirmation of synergistic effect of PCNA and high concentration primer High-speed PCR was performed using KOD Y7A / P36H / N210D and KOD PCNA D147A obtained above, and the synergistic effect of PCNA and high concentration primer was confirmed. .
  • PCR was also carried out for compositions that did not contain PCNA mutants and compared.
  • the primer concentrations were 0.2 and 1.0 ⁇ M, respectively, and PCR was performed at a temperature of 94 ° C. for 30 seconds, followed by a cycle of 98 ° C., 0 seconds ⁇ 55 ° C., 0 seconds ⁇ 68 ° C., 0 seconds.
  • a high-speed PCR schedule that repeats the cycle and a normal PCR schedule that repeats the cycle of 98 ° C., 10 seconds ⁇ 60 ° C., 10 seconds ⁇ 68 ° C., 30 seconds after the pre-reaction at 94 ° C. for 30 seconds and 50 cycles were compared.
  • the thermal cycler Light cycler (registered trademark) 2.0 was used, and the rate of temperature increase and decrease was 20 ° C./second.
  • FIG. 4 shows the Ct value by comparing the amplification by high-speed PCR from human genomes of various copy numbers in order to confirm the synergistic effect of the primer concentration and the PCNA mutant.
  • the primer concentrations were 0.2 and 1.0 ⁇ M, and Ct was shown in each case with and without PCNA.
  • Ct with and without PCNA in the normal cycle is also shown.
  • circle marking there is amplification of the target gene but also non-specific amplification
  • square marking Only non-specific amplification is seen).
  • Example 8 Examination of high-speed PCR in the presence of PCR inhibitory substance Using the KOD Y7A / P36H / N210D and KOD PCNA D147A obtained above, high-speed PCR was carried out under the condition containing the inhibitory substance.
  • Inhibitors were prepared as follows. Feces: Suspend 2 g of feces in 8 ml of water to make a 20% fecal suspension. After heat treatment at 95 ° C. for 5 minutes and centrifugation at 12,000 rpm for 1 minute, the supernatant was made 20% feces.
  • Urine Urine collected from humans was taken as 100% urine.
  • Oral mucosa The oral mucosa collected with a cotton swab was dropped into 200 ⁇ l of water to make 100% oral mucosa fluid.
  • Blood Blood collected with an EDTA blood collection tube was taken as 100% blood.
  • PCR Buffer containing 1.2 mM MgSO 4 obtained by diluting 10 ⁇ PCR Buffer attached to KOD Dash (manufactured by Toyobo) 10-fold, 0.2 mM dNTPs, 1U KOD DNA polymerase mutant, 250 ng KOD PCNA
  • Primer concentrations were 0.2 and 1.0 ⁇ M.
  • PCR Light cycler (registered trademark) 2.0 is scheduled to repeat 50 cycles of 94 ° C, 30 seconds pre-reaction, 98 ° C, 0 seconds ⁇ 55 ° C, 0 seconds ⁇ 68 ° C, 0 seconds.
  • the rate of temperature increase and decrease was 20 ° C./second.
  • the reaction time was about 20 minutes.
  • FIG. 5 shows Ct when high-speed PCR was performed from a composition containing an inhibitor and 50 copies of Salmonella were amplified.
  • Primer concentrations are 0.2 and 1.0 ⁇ M, with no inhibitor, 2.5, 0.5, 0.1% stool, 5, 1, 0.2, 0.04% urine, 25 The effects of adding 5, 1, 0.2% oral mucosa fluid, or 1.6, 0.32, 0.06% blood were confirmed.
  • circle marking there is amplification of the target gene but also non-specific amplification
  • square marking non-specific amplification Only specific amplification is seen). Samples that are not amplified at all are N.P. D. It was written.
  • This system is a system that does not amplify without PCNA even without an inhibitor.
  • amplification was only up to 0.5% when the primer concentration was 0.2 ⁇ M, but amplification was confirmed even when 2.5% was included by increasing the primer concentration to 1.0 ⁇ M.
  • urine although the rise was delayed when the primer concentration was 0.2 ⁇ M, the rise was improved by raising the primer concentration to 1.0 ⁇ M, and amplification was confirmed even when the concentration was 5%.
  • the oral mucosa amplification was confirmed only up to 5% when the primer concentration was 0.2 ⁇ M, but amplification was confirmed even when 25% was contained by increasing the primer concentration to 1.0 ⁇ M.
  • Example 9 Examination of high-speed PCR in the presence of a PCR inhibitor / dUTP
  • High-speed PCR was carried out using the KOD Y7A / P36H / N210D and KOD PCNA D147A obtained above under the conditions containing the inhibitor and dUTP.
  • Inhibitors were prepared as follows. Feces: 2 g of feces was suspended in 8 ml of water to prepare a 20% fecal suspension. After heat treatment at 95 ° C. for 5 minutes and centrifugation at 12,000 rpm for 1 minute, the supernatant was made 20% feces.
  • PCR Buffer attached to KOD Dash 10 ⁇ PCR Buffer attached to KOD Dash (manufactured by Toyobo) was diluted 10-fold, 1 ⁇ PCR Buffer (including 1.2 mM MgSO 4 ), 2 mM dNTPs (dATP, dUTP, dCTP, dGTP), 1U KOD DNA polymerase mutant, 250 ng KOD PCNA mutant, and SYBR Green I diluted to 1 / 30,000, actin gene, 1.0 ⁇ M Amplifying about 550 bp SEQ ID NO: 21 And the human genome corresponding to 50 copies, and 2.5, 2, 1.5, 1, 0.5, 0.25, 0.1% Ct value in the presence of feces Compared.
  • Taq DNA polymerase was manufactured by Toyobo and mixed with Anti-Taq High (manufactured by Toyobo). The reaction was carried out by mixing Buffer attached to 1 ⁇ BlendTaq, dNTPs (dATP, dUTP, dCTP, dGTP) in which 2 mM dTTP was replaced with dUTP, 0.2, and 1.0 ⁇ M primers (same as above) and antibody. 50 copies of human genome, 2.5, 2, 1.5, 1, 0.5, 0.25, and 0.1% feces were added to a 50 ⁇ l reaction solution containing 5 U of enzyme, and Ct values were compared. .
  • Each PCR was performed using Light cycler 2.0 on a schedule that repeats 50 cycles of high-speed cycles of 94 ° C, 0 seconds ⁇ 55 ° C, 0 seconds ⁇ 68 ° C, 0 seconds after a pre-reaction at 94 ° C for 30 seconds.
  • the rate of temperature and temperature decrease was 20 ° C./second.
  • the reaction time was about 20 minutes.
  • FIG. 6 shows Ct when high-speed PCR was performed from a composition containing feces / dUTP and 50 copies of the human genome were amplified.
  • the primer concentration was 1.0 ⁇ M, and was performed with and without the PCNA mutant. Ct was confirmed in the absence of inhibitor and in 2.5, 2, 1.5, 1, 0.5, 0.25, 0.1% feces.
  • primer concentrations of 0.2 and 1.0 ⁇ M were used, and when no inhibitor was present, 2.5, 2, 1.5, 1, 0.5, 0.25, 0. Ct in 1% feces was confirmed.
  • the present invention is useful in biotechnology-related industries related to DNA synthesis, and particularly useful in diagnostic applications.

Abstract

Provided are a method for shortening reaction time and a reagent composition for DNA synthesis in which PCR is used. A PCR composition for further increasing the primer concentration and conducting 30 to 50 cycles within 40 minutes by using PCNA and DNA polymerase belonging to family B in a high-speed PCR having a short reaction time.

Description

ＰＣＲ方法PCR method
本発明は、核酸増幅、特にＰＣＲ（Ｐｏｌｙｍｅｒａｓｅ　ｃｈａｉｎ　ｒｅａｃｔｉｏｎ）の分野に関する。さらに詳しくは、ＰＣＲ反応時間を短縮させる方法に関する。 The present invention relates to the field of nucleic acid amplification, in particular PCR (Polymerase chain reaction). More specifically, the present invention relates to a method for shortening the PCR reaction time.
ＰＣＲ法とは、（１）熱処理によるＤＮＡ変性（２本鎖ＤＮＡから１本鎖ＤＮＡへの解離）、（２）鋳型１本鎖ＤＮＡへのプライマーのアニーリング、（３）ＤＮＡポリメラーゼを用いた前記プライマーの伸長、という３ステップを１サイクルとし、このサイクルを繰り返すことによって、試料中の標的核酸を増幅する方法である。この方法により、数コピーといった極微量サンプルから標的核酸を何十万倍に増幅することができるため、研究用途のみならず、遺伝子診断、臨床診断といった法医学分野、あるいは、食品や環境中の微生物検査等においても、広く用いられている。 The PCR method is (1) DNA denaturation by heat treatment (dissociation from double-stranded DNA to single-stranded DNA), (2) primer annealing to template single-stranded DNA, and (3) the above using DNA polymerase This is a method of amplifying a target nucleic acid in a sample by repeating three cycles of three steps of primer extension as one cycle. This method can amplify the target nucleic acid hundreds of thousands of times from a trace amount sample such as several copies, so it can be used not only for research purposes but also in forensic fields such as genetic diagnosis and clinical diagnosis, or for microbial testing in food and the environment. Etc. are also widely used.
ＰＣＲは高い検出感度を持つものの、反応に熱サイクルを実施する必要があり、反応時間が長いことが問題となっている。最近では高速で温度変化を実施するサーマルサイクラーが販売され、より短時間で反応を終了させるための装置の検討が行われている。また、反応の面においても、高速サイクルに対応すべく、ＰＣＲの組成、方法の検討が行われている。 Although PCR has high detection sensitivity, it is necessary to carry out a thermal cycle for the reaction, and the problem is that the reaction time is long. Recently, thermal cyclers that perform temperature changes at high speed have been sold, and devices for completing the reaction in a shorter time have been studied. In terms of the reaction, the PCR composition and method have been studied in order to cope with the high-speed cycle.
反応の面において、高速ＰＣＲにはＰＣＲに用いるＤＮＡポリメラーゼの性能が重要となる。我々は、ファミリーＢに属するＤＮＡポリメラーゼが、Ｔａｑ　ＤＮＡポリメラーゼなどのファミリーＡに属するポリメラーゼより高速ＰＣＲに向いていることを見出している（特許文献１）。またＤＮＡポリメラーゼの補助因子、特にＰＣＮＡ（Ｐｒｏｌｉｆｅｒａｔｉｎｇ　Ｃｅｌｌ　Ｎｕｃｌｅａｒ　Ａｎｔｉｇｅｎ；増殖細胞核抗原）の添加によって、反応時間を短縮できることも報告されている（特許文献２）。 In terms of reaction, the performance of DNA polymerase used for PCR is important for high-speed PCR. We have found that DNA polymerase belonging to family B is more suitable for high-speed PCR than polymerase belonging to family A such as Taq DNA polymerase (Patent Document 1). It has also been reported that the reaction time can be shortened by adding a cofactor of DNA polymerase, particularly PCNA (Proliferating Cell Nuclear Antigen) (Patent Document 2).
しかしながら、これらの検討が進められているに関わらず、極端な高速サイクルではＤＮＡポリメラーゼがうまく作用せず、増幅不良が散見されていた。また診断用途では測定サンプルにＰＣＲを阻害する物質が含まれている場合があり、このような阻害物質が含まれる条件では高速ＰＣＲができなかった。特に、診断用途では迅速な結果報告が要求されており、高速にかつ高効率にＤＮＡ合成する方法が強く求められていた。 However, despite these studies, DNA polymerase did not work well in extreme high-speed cycles, and poor amplification was observed. Moreover, in the diagnostic use, a substance that inhibits PCR may be included in the measurement sample, and high-speed PCR could not be performed under such conditions containing the inhibitor. In particular, rapid results reporting is required for diagnostic applications, and a method for DNA synthesis at high speed and high efficiency has been strongly demanded.
特開２０１０－２３９８８０号公報JP 2010-239880 A 国際公開第２００７／００４６５４号パンフレットInternational Publication No. 2007/004654 Pamphlet
ＰＣＲを用いたＤＮＡ合成において、反応時間を短縮させる方法、及び試薬組成を提供することを目的とする。 An object of the present invention is to provide a method and a reagent composition for shortening the reaction time in DNA synthesis using PCR.
本発明者らは、上記事情に鑑み鋭意研究を行った結果、反応時間の短い高速ＰＣＲにおいて、ファミリーＢに属するＤＮＡポリメラーゼ及びＰＣＮＡを用い、さらにプライマー濃度を高くすることで、より効率的な核酸増幅が得られることを見出し、本発明を完成するに至った。
代表的な本発明は、以下の通りである。 As a result of intensive studies in view of the above circumstances, the present inventors have used DNA polymerase and PCNA belonging to family B in high-speed PCR with a short reaction time, and further increasing the primer concentration, thereby enabling more efficient nucleic acid. It has been found that amplification can be obtained, and the present invention has been completed.
The representative present invention is as follows.
項１．ファミリーＢに属するＤＮＡポリメラーゼ、Ｐｒｏｌｉｆｅｒａｔｉｎｇ　Ｃｅｌｌ　Ｎｕｃｌｅａｒ　Ａｎｔｉｇｅｎ（ＰＣＮＡ；増殖細胞核抗原）、および及び０．６μＭ以上の濃度でプライマーを含み、かつ、３０サイクルから５０サイクルを４０分以内で実施するための組成であることを特徴とするＰＣＲ方法組成。
項２．３０サイクルから５０サイクルを２０分以内で実施するための組成である項1に記載のＰＣＲ方法組成。
項３．３０サイクルから５０サイクルを１２分以内で実施するための組成である項１又は２に記載のＰＣＲ方法組成。
項４．プライマーの濃度が０．８μＭ以上である項１から３のいずれかに記載のＰＣＲ組成方法。
項５．プライマーの濃度が１．０μＭ以上である項１から４のいずれかに記載のＰＣＲ組成方法。
項６．昇温又は降温の速度が０．１℃／秒から２０℃／秒の範囲でＰＣＲを実施するための組成であることを特徴とする項１から５のいずれかに記載のＰＣＲ方法組成。
項７．昇温又は降温の速度が５℃／秒から２０℃／秒の範囲でＰＣＲを実施するための組成である項１から６のいずれかに記載のＰＣＲ方法組成。
項８．昇温又は降温の速度が１０℃／秒から２０℃／秒の範囲でＰＣＲを実施するための組成である項１から７のいずれかに記載のＰＣＲ方法組成。
項９．ファミリーＢに属するＤＮＡポリメラーゼが、古細菌（Ａｒｃｈｅａ）由来のＤＮＡポリメラーゼである項１から８のいずれかに記載のＰＣＲ組成。
項１０．ファミリーＢに属するＤＮＡポリメラーゼが減少した塩基類似体検出活性を有する古細菌ＤＮＡポリメラーゼ変異体である項１から９のいずれかに記載のＰＣＲ組成。
項１１．ＰＣＮＡが古細菌（Ａｒｃｈｅａ）由来のＰＣＮＡである項１から１０のいずれかに記載のＰＣＲ組成。
項１２．ＰＣＮＡがＤＮＡポリメラーゼ増幅増強活性をもつ変異型ＰＣＮＡである項１から１１のいずれかに記載のＰＣＲ組成。
項１３．項１から１２のいずれかに記載のＰＣＲ組成を含むことを特徴とするＰＣＲ用試薬。
項１４．項１３に記載のＰＣＲ用試薬を含むことを特徴とするＰＣＲ用試薬キット。
項１５．項１から１２のいずれかに記載のＰＣＲ組成により、１５回から５０回の熱サイクルによるＰＣＲ方法であって、かつ、ＰＣＲの反応時間が６０分以内であることを特徴とするＰＣＲ方法。
項１６．ＰＣＲの反応時間が３０分以内である項１５に記載のＰＣＲ方法。
項１７．項１から１２のいずれかに記載のＰＣＲ組成を含む反応液中に、生体試料を直接添加することを特徴とする項１５又は１６に記載のＰＣＲ方法。 Item 1. A composition comprising DNA polymerase belonging to family B, Proliferating Cell Nuclear Antigen (PCNA) and a primer at a concentration of 0.6 μM or more, and 30 to 50 cycles within 40 minutes A PCR method composition characterized by being.
Item 2. The PCR method composition according to Item 1, which is a composition for carrying out 30 to 50 cycles within 20 minutes.
Item 3. The PCR method composition according to Item 1 or 2, which is a composition for performing from 30 cycles to 50 cycles within 12 minutes.
Item 4. Item 4. The PCR composition method according to any one of Items 1 to 3, wherein the primer concentration is 0.8 μM or more.
Item 5. Item 5. The PCR composition method according to any one of Items 1 to 4, wherein the primer concentration is 1.0 μM or more.
Item 6. Item 6. The PCR method composition according to any one of Items 1 to 5, wherein the PCR method composition is a composition for performing PCR at a rate of temperature increase or decrease of 0.1 ° C / second to 20 ° C / second.
Item 7. Item 7. The PCR method composition according to any one of Items 1 to 6, which is a composition for performing PCR at a rate of temperature increase or decrease of 5 ° C / second to 20 ° C / second.
Item 8. Item 8. The PCR method composition according to any one of Items 1 to 7, which is a composition for performing PCR at a rate of temperature increase or decrease of 10 ° C./second to 20 ° C./second.
Item 9. Item 9. The PCR composition according to any one of Items 1 to 8, wherein the DNA polymerase belonging to Family B is a DNA polymerase derived from Archaea.
Item 10. Item 10. The PCR composition according to any one of Items 1 to 9, wherein the DNA polymerase belonging to Family B is an archaeal DNA polymerase mutant having reduced base analog detection activity.
Item 11. Item 11. The PCR composition according to any one of Items 1 to 10, wherein the PCNA is PCNA derived from Archaea.
Item 12. Item 12. The PCR composition according to any one of Items 1 to 11, wherein the PCNA is a mutant PCNA having DNA polymerase amplification enhancing activity.
Item 13. Item 13. A PCR reagent comprising the PCR composition according to any one of Items 1 to 12.
Item 14. A PCR reagent kit comprising the PCR reagent according to Item 13.
Item 15. Item 13. A PCR method according to the PCR composition according to any one of Items 1 to 12, wherein the PCR method is based on 15 to 50 thermal cycles, and the PCR reaction time is within 60 minutes.
Item 16. Item 16. The PCR method according to Item 15, wherein the PCR reaction time is within 30 minutes.
Item 17. Item 17. The PCR method according to Item 15 or 16, wherein the biological sample is directly added to the reaction solution containing the PCR composition according to any one of Items 1 to 12.
本発明により、ＰＣＲの反応時間の短縮ができる。本発明における及び組成は血液などの阻害物質が含まれる条件でも、高速ＰＣＲを可能にし、特に迅速性が求められる診断用途において非常に有用である。 According to the present invention, the PCR reaction time can be shortened. The composition and composition in the present invention enables high-speed PCR even under conditions containing an inhibitor such as blood, and is very useful particularly in diagnostic applications that require rapidity.
プライマー濃度、マグネシウム濃度の検討 Ｃｔ値Examination of primer concentration and magnesium concentration Ct value プライマー濃度、マグネシウム濃度の検討 融解曲線Examination of primer concentration and magnesium concentration Melting curve プライマー濃度の検討 融解曲線Examination of primer concentration Melting curve ＰＣＮＡと高濃度プライマーの相乗効果の確認Confirmation of synergistic effect of PCNA and high concentration primer ＰＣＲ阻害物質存在下での高速ＰＣＲの検討Study of high-speed PCR in the presence of PCR inhibitors ＰＣＲ阻害物質・ｄＵＴＰ存在下での高速ＰＣＲの検討Examination of high-speed PCR in the presence of PCR inhibitors and dUTP ＰＣＮＡの配列比較Sequence comparison of PCNA
以下、本発明の実施形態を示しつつ、本発明についてさらに詳説する。
本発明におけるＰＣＲ方法は、ファミリーＢに属するＤＮＡポリメラーゼ、ＰＣＮＡ及び高濃度プライマーを反応液中に含み、高速でＰＣＲを行うことを特徴とする。 Hereinafter, the present invention will be described in more detail while showing embodiments of the present invention.
The PCR method in the present invention is characterized in that DNA polymerase belonging to Family B, PCNA and a high concentration primer are included in the reaction solution, and PCR is performed at a high speed.
（１）アミノ酸の表記
本明細書においては、塩基配列、アミノ酸配列及びその個々の構成因子については、アルファベット表記による簡略化した記号を用いる場合があるが、いずれも分子生物学・遺伝子工学分野における慣行に従う。また、本明細書においては、アミノ酸配列の変異を簡潔に示すため、例えば「Ｄ１４３Ａ」などの表記を用いる。「Ｄ１４３Ａ」は、第１４３番目のアスパラギン酸をアラニンに置換したことを示しており、すなわち、置換前のアミノ酸残基の種類、その場所、置換後のアミノ酸残基の種類を示している。また、配列番号は、特に断らない限り、配列表に記載された配列番号に対応する。また、多重変異体の場合は、上記の表記を「／」でつなげて表す。例えば「Ｄ１４３Ａ／Ｄ１４７Ａ」は、第１４３番目のアスパラギン酸をアラニンに置換し、かつ、第１４７番目のアスパラギン酸をアラニンに置換したことを示す。
また、本明細書において「変異体」、「変異型」とは、従来知られたタンパク質とは異なるアミノ酸配列を備えることを意味するものであり、人為的変異によるか自然界における変異によるかを区別するものではない。 (1) Amino acid notation In this specification, the base sequence, amino acid sequence, and individual constituent factors thereof may be simplified symbols in alphabetical notation, both of which are in the field of molecular biology and genetic engineering. Follow practices. Further, in this specification, in order to simply show the mutation of the amino acid sequence, for example, a notation such as “D143A” is used. “D143A” indicates that the 143rd aspartic acid was substituted with alanine, that is, the type of amino acid residue before substitution, its location, and the type of amino acid residue after substitution. Further, the sequence numbers correspond to the sequence numbers described in the sequence listing unless otherwise specified. In the case of multiple mutants, the above notation is connected by “/”. For example, “D143A / D147A” indicates that the 143rd aspartic acid was substituted with alanine and the 147th aspartic acid was substituted with alanine.
In this specification, “mutant” and “mutant” mean that the protein has a different amino acid sequence from a conventionally known protein, and distinguishes whether it is due to an artificial mutation or a mutation in nature. Not what you want.
なお、「配列番号１に記載のアミノ酸配列における、１４２番目に『相当する』アミノ酸」とは、配列番号１に示されるアミノ酸配列と完全同一ではないアミノ酸配列を有するＰＣＮＡにおいて、配列番号１の１４２番目に対応するアミノ酸配列を含む表現である。本明細書において、前記と同じ形式の表記における「相当する」の意味は、前記の例示と同じである。 The “142st amino acid corresponding to the amino acid sequence shown in SEQ ID NO: 1” is 142 of SEQ ID NO: 1 in PCNA having an amino acid sequence that is not completely identical to the amino acid sequence shown in SEQ ID NO: 1. It is an expression including the amino acid sequence corresponding to the th. In this specification, the meaning of “corresponding” in the notation of the same format as described above is the same as in the above example.
本願明細書において、配列番号１に示されるアミノ酸配列と完全同一ではないアミノ酸配列おける、配列番号１上のある位置（順番）と対応する位置とは、配列の一次構造を比較（アラインメント）したとき、配列番号１の当該位置と対応する位置とする。本明細書において、前記と同じ形式の表記における「対応する位置」の意味は、前記の例示と同じである。 In the present specification, in a amino acid sequence that is not completely identical to the amino acid sequence shown in SEQ ID NO: 1, a position (order) on SEQ ID NO: 1 and a corresponding position are when the primary structure of the sequence is compared (alignment) , A position corresponding to the position of SEQ ID NO: 1. In the present specification, the meaning of “corresponding position” in the notation of the same format as described above is the same as the above example.
配列の一次構造を比較する方法としては、種々の方法が知られている。例えば、市販の又は電気通信回線（インターネット）を通じて利用可能な解析ツールを用いて算出することができる。本明細書では、ＤＮＡ　Ｄａｔａｂａｎｋ　ｏｆ　Ｊａｐａｎ（ＤＤＢＪ）のＣｌｕｓｔａｌＷ（ｈｔｔｐ：／／ｃｌｕｓｔａｌｗ．ｄｄｂｊ．ｎｉｇ．ａｃ．ｊｐ／ｉｎｄｅｘ．ｐｈｐ／ｌａｎｇ＝ｊａ）においてデフォルト（初期設定）のパラメータを用いることにより、配列の一次構造を比較する。 Various methods are known as methods for comparing the primary structures of sequences. For example, it can be calculated using an analysis tool that is commercially available or available through a telecommunication line (Internet). In this specification, by using default (initial setting) parameters in ClustalW (http://clustalw.ddbj.nig.ac.jp/index.php/lang=ja) of DNA Databank of Japan (DDBJ), Compare the primary structure of the sequences.
（２）高速ＰＣＲ
本発明におけるＰＣＲは、３０回の熱サイクルを行う場合に、反応時間が４０分以内である高速ＰＣＲであり、好ましくは３０分以内、さらに好ましくは２０分以内、さらに好ましくは反応時間が１０分以内である高速ＰＣＲが挙げられる。反応時間とは変性、アニーリング、伸長を繰り返す熱サイクルの時間を示す。通常のサーマルサイクラーの熱サイクル設定では反応時間が６０分以内になることはほとんどない。例えば、ＰＣＲ　ｓｙｓｔｅｍ　ＧｅｎｅＡｍｐ（登録商標）９７００（Ａｐｐｌｉｅｄ　Ｂｉｏｓｙｓｔｅｍ製）で６０分以内に反応を終わらせるには熱サイクルの各ステップの時間を極端に短くする必要がある。実際、各ステップの時間を極端に短くし、９４℃、２分の前反応の後、９８℃、０秒→６０℃、０秒→６８℃、０秒を４０サイクル実施すると反応時間は約６０分となる。また、３０分以内の反応時間を達成するには、高速で温度変化を実施するサーマルサイクラーを使用し、さらに熱サイクルの各ステップの時間を短くする必要がある。例えば高速での温度変化が可能なＬｉｇｈｔ　ｃｙｃｌｅｒ（登録商標）２．０（Ｒｏｃｈｅ）を用いた場合、９４℃、２分の前反応の後、９８℃、０秒→５５℃、０秒→６８℃、０分を５０サイクル実施すると反応時間は約２０分となる。本発明におけるＰＣＲの熱サイクル数は、１５回から５０回の範囲で設定して実施することができる。好ましくは２５回から４５回であり、より好ましくは３０回から４０回である。 (2) High-speed PCR
The PCR in the present invention is a high-speed PCR in which the reaction time is within 40 minutes when 30 thermal cycles are performed, preferably within 30 minutes, more preferably within 20 minutes, and even more preferably 10 minutes. Fast PCR that is within. The reaction time indicates the time of a thermal cycle in which denaturation, annealing, and elongation are repeated. With a thermal cycler setting of a normal thermal cycler, the reaction time rarely falls within 60 minutes. For example, in order to complete the reaction within 60 minutes using PCR system GeneAmp (registered trademark) 9700 (manufactured by Applied Biosystem), it is necessary to extremely shorten the time of each step of the thermal cycle. In fact, when the time for each step is extremely shortened and the reaction is performed at 94 ° C. for 2 minutes before the reaction for 98 cycles of 98 ° C., 0 seconds → 60 ° C., 0 seconds → 68 ° C., 0 seconds, the reaction time is about 60 minutes. Minutes. In order to achieve a reaction time of 30 minutes or less, it is necessary to use a thermal cycler that changes the temperature at high speed, and to further shorten the time of each step of the thermal cycle. For example, when Light cycler (registered trademark) 2.0 (Roche) capable of changing temperature at high speed is used, after the reaction at 94 ° C. for 2 minutes, 98 ° C., 0 second → 55 ° C., 0 second → 68 When 50 cycles of 0 ° C. are carried out, the reaction time is about 20 minutes. The number of thermal cycles of PCR in the present invention can be set and carried out in the range of 15 to 50 times. Preferably it is 25 to 45 times, more preferably 30 to 40 times.
本発明における高速の熱サイクル設定においては、アニーリングステップを短くすることが好ましい。なぜなら、ＰＣＲ反応速度を上げるためにはプライマーの濃度を高くする必要があるが、一般に、プライマーの濃度を高くすると非特異やプライマーダイマーが生じやすい。アニーリングステップを短くすることにより、高濃度のプライマーでも非特異やプライマーダイマーが生じにくくなると考えられる。好ましいアニーリングステップの時間は１０秒である。さらに好ましくは５秒、特に好ましくは０秒である。 In the high-speed heat cycle setting in the present invention, it is preferable to shorten the annealing step. This is because, in order to increase the PCR reaction rate, it is necessary to increase the primer concentration. However, generally, when the primer concentration is increased, non-specificity or primer dimer is likely to occur. By shortening the annealing step, non-specificity and primer dimers are less likely to occur even at high concentrations of primers. The preferred annealing step time is 10 seconds. More preferably 5 seconds, particularly preferably 0 seconds.
（３）ファミリーＢに属するＤＮＡポリメラーゼ
本発明の核酸増幅法に用いるＤＮＡポリメラーゼは、ファミリーＢに属するＤＮＡポリメラーゼである。本発明においてファミリーＢに属するＤＮＡポリメラーゼとは、３’－５’エキソヌクレアーゼ活性を有し、５’－３’エキソヌクレアーゼ活性を有さないＤＮＡポリメラーゼをいう。好ましくは古細菌（Ａｒｃｈｅａ）由来のＤＮＡポリメラーゼである。 (3) DNA polymerase belonging to family B The DNA polymerase used in the nucleic acid amplification method of the present invention is a DNA polymerase belonging to family B. In the present invention, a DNA polymerase belonging to Family B refers to a DNA polymerase having 3′-5 ′ exonuclease activity and not having 5′-3 ′ exonuclease activity. A DNA polymerase derived from Archaea is preferable.
（３．１）古細菌由来のＤＮＡポリメラーゼ
ファミリーＢに属する古細菌由来のＤＮＡポリメラーゼとしては、パイロコッカス（Ｐｙｒｏｃｏｃｃｕｓ）属及びサーモコッカス（Ｔｈｅｒｍｏｃｏｃｃｕｓ）属の細菌から単離されるＤＮＡポリメラーゼが挙げられる。パイロコッカス属由来のＤＮＡポリメラーゼとしては、Ｐｙｒｏｃｏｃｃｕｓ　ｆｕｒｉｏｓｕｓ、Ｐｙｒｏｃｏｃｃｕｓ　ｓｐ．ＧＢ－Ｄ、Ｐｙｒｏｃｏｃｃｕｓ　Ｗｏｅｓｅｉ、Ｐｙｒｏｃｏｃｃｕｓ　ａｂｙｓｓｉ、Ｐｙｒｏｃｏｃｃｕｓ　ｈｏｒｉｋｏｓｈｉｉから単離されたＤＮＡポリメラーゼを含むが、これらに限定されない。サーモコッカス属に由来するＤＮＡポリメラーゼとしては、Ｔｈｅｒｍｏｃｏｃｃｕｓ　ｋｏｄａｋａｒａｅｎｓｉｓ、Ｔｈｅｒｍｏｃｏｃｃｕｓ　ｇｏｒｇｏｎａｒｉｕｓ、Ｔｈｅｒｍｏｃｏｃｃｕｓ　ｌｉｔｏｒａｌｉｓ、Ｔｈｅｒｍｏｃｏｃｃｕｓ　ｓｐ．ＪＤＦ－３、Ｔｈｅｒｍｏｃｏｃｃｕｓ　ｓｐ．９ｄｅｇｒｅｅｓ　Ｎｏｒｔｈ－７（Ｔｈｅｒｍｏｃｏｃｃｕｓ　ｓｐ．９°Ｎ－７）、Ｔｈｅｒｍｏｃｏｃｃｕｓ　ｓｐ．ＫＳ－１、Ｔｈｅｒｍｏｃｏｃｃｕｓ　ｃｅｌｅｒ、又はＴｈｅｒｍｏｃｏｃｃｕｓ　ｓｉｃｕｌｉから単離されたＤＮＡポリメラーゼを含むが、これらに限定されない。これらのＤＮＡポリメラーゼを用いたＰＣＲ酵素は市販されており、Ｐｆｕ（Ｓｔａｒａｇｅｎｅ社）、ＫＯＤ（Ｔｏｙｏｂｏ）、Ｐｆｘ（Ｌｉｆｅ　Ｔｅｃｈｎｏｌｏｇｉｅｓ社）、Ｖｅｎｔ（Ｎｅｗ　Ｅｎｇｌａｎｄ　Ｂｉｏｌａｂｓ）、Ｄｅｅｐ　Ｖｅｎｔ（Ｎｅｗ　Ｅｎｇｌａｎｄ　Ｂｉｏｌａｂｓ）、Ｔｇｏ（Ｒｏｃｈｅ）、Ｐｗｏ（Ｒｏｃｈｅ）などがある。 (3.1) DNA polymerase derived from archaea Examples of the DNA polymerase derived from archaea include DNA polymerases isolated from bacteria belonging to the genus Pyrococcus and Thermococcus. Examples of the DNA polymerase derived from the genus Pyrococcus include Pyrococcus furiosus and Pyrococcus sp. Including, but not limited to, DNA polymerases isolated from GB-D, Pyrococcus Wosei, Pyrococcus abyssi, Pyrococcus horikoshii. Examples of DNA polymerases derived from the genus Thermococcus include Thermococcus kodakaraensis, Thermococcus gorgonarius, Thermococcus litoralis, Thermococcus sp. JDF-3, Thermococcus sp. 9 degrees North-7 (Thermococcus sp. 9 ° N-7), Thermococcus sp. Including, but not limited to, DNA polymerase isolated from KS-1, Thermococcus celler, or Thermococcus sicili. PCR enzymes using these DNA polymerases are commercially available, such as Pfu (Staragene), KOD (Toyobo), Pfx (Life Technologies), Vent (New England Biolabs), Deep Vent (New England Biobland). Roche) and Pwo (Roche).
なかでもＰＣＲ効率の観点から、伸長性や熱安定性の優れたＫＯＤ　ＤＮＡポリメラーゼが好ましい。 Among these, from the viewpoint of PCR efficiency, KOD DNA polymerase excellent in extensibility and thermal stability is preferable.
また、前記ＤＮＡポリメラーゼに、後述の３‘－５’エキソヌクレアーゼ領域の改変、及び／又は、減少した塩基類似体検出活性を有するような改変を施した変異体を用いても良い。 In addition, a mutant obtained by modifying the DNA polymerase described later to modify the 3′-5 ′ exonuclease region and / or to have a decreased base analog detection activity may be used.
（３．２）ＤＮＡポリメラーゼの改変（Ｉ）３‘－５’エキソヌクレアーゼ領域の改変
本発明に用いる改変されたＤＮＡポリメラーゼは、さらに３’－５’エキソヌクレアーゼ活性領域のアミノ酸配列のいずれかに少なくとも１つのアミノ酸の改変を含んでいてもよい。３‘－５’ エキソヌクレアーゼ活性とは、取り込まれたヌクレオチドをＤＮＡ重合体の３’末端から除去する能力を指し、上記の３‘－５’エキソヌクレアーゼ領域とは、ファミリーＢに属するＤＮＡポリメラーゼで高度に保存されている部位であり、サーモコッカス・コダカラエンシスに由来するＤＮＡポリメラーゼ（配列番号１）、パイロコッカス・フリオサスに由来するＤＮＡポリメラーゼ（配列番号２）、サーモコッカス・ゴルゴナリウスに由来するＤＮＡポリメラーゼ（配列番号３）、サーモコッカス・リトラリスに由来するＤＮＡポリメラーゼ（配列番号４）、パイロコッカス・エスピーＧＢ－Ｄに由来するＤＮＡポリメラーゼ（配列番号５）、サーモコッカス・エスピーＪＤＦ－３に由来するＤＮＡポリメラーゼ（配列番号６）、サーモコッカス・エスピー９°Ｎ－７に由来するＤＮＡポリメラーゼ（配列番号７）、サーモコッカス・エスピーＫＳ－１に由来するＤＮＡポリメラーゼ（配列番号８）、サーモコッカス・セラーに由来するＤＮＡポリメラーゼ（配列番号９）、又はサーモコッカス・シクリに由来するＤＮＡポリメラーゼ（配列番号１０）においては、１３７～１４７、２０６～２２２、及び３０８～３１８番目のアミノ酸である。本発明は、具体的に配列を提示したＤＮＡポリメラーゼ以外のＤＮＡポリメラーゼにも適用される。また、配列番号１～１０に示されるＤＮＡポリメラーゼ以外のファミリーＢに属する古細菌由来ＤＮＡポリメラーゼにおいては、配列番号１の１３７～１４７、２０６～２２２、及び３０８～３１８番目のアミノ酸からなる３‘－５’エキソヌクレアーゼ領域と対応する領域のことを示す。 (3.2) Modification of DNA polymerase (I) Modification of 3′-5 ′ exonuclease region The modified DNA polymerase used in the present invention is further added to any one of the amino acid sequences of the 3′-5 ′ exonuclease active region. It may contain at least one amino acid modification. 3'-5 'exonuclease activity refers to the ability to remove incorporated nucleotides from the 3' end of a DNA polymer, and the above 3'-5 'exonuclease region is a DNA polymerase belonging to family B. It is a highly conserved site, derived from Thermococcus kodakaraensis DNA polymerase (SEQ ID NO: 1), Pyrococcus furiosus DNA polymerase (SEQ ID NO: 2), Thermococcus gorgonarius DNA polymerase (SEQ ID NO: 3), DNA polymerase derived from Thermococcus litoralis (SEQ ID NO: 4), DNA polymerase derived from Pyrococcus sp GB-D (SEQ ID NO: 5), derived from Thermococcus sp JDF-3 DNA polymerase (SEQ ID NO: 6), DNA polymerase derived from Mococcus sp 9 ° N-7 (SEQ ID NO: 7), DNA polymerase derived from Thermococcus sp KS-1 (SEQ ID NO: 8), DNA polymerase derived from Thermococcus cellar (SEQ ID NO: 9) In the DNA polymerase (SEQ ID NO: 10) derived from Thermococcus cyclis, the amino acids 137 to 147, 206 to 222, and 308 to 318 are used. The present invention is also applicable to DNA polymerases other than the DNA polymerase specifically presenting the sequence. In addition, in the archaeal DNA polymerase belonging to Family B other than the DNA polymerases shown in SEQ ID NOs: 1 to 10, the 3′- consisting of amino acids 137 to 147, 206 to 222, and 308 to 318 of SEQ ID NO: 1 The region corresponding to the 5 ′ exonuclease region is shown.
なお、「配列番号１に示される１３７～１４７、２０６～２２２、及び３０８～３１８番目に相当するアミノ酸」とは、配列番号１に示されるアミノ酸配列と完全同一ではないアミノ酸配列を有するＤＮＡポリメラーゼにおいて、配列番号１の１３７～１４７、２０６～２２２、及び３０８～３１８番目に対応するアミノ酸配列を含む表現である。本発明において、前記と同じ形式の表記における「相当する」の意味は、前記の例示と同じである。 The “amino acids corresponding to positions 137 to 147, 206 to 222, and 308 to 318 shown in SEQ ID NO: 1” are DNA polymerases having an amino acid sequence that is not completely identical to the amino acid sequence shown in SEQ ID NO: 1. , An expression comprising amino acid sequences corresponding to positions 137 to 147, 206 to 222, and 308 to 318 of SEQ ID NO: 1. In the present invention, the meaning of “corresponding” in the notation of the same format as described above is the same as in the above example.
本発明において、配列番号１に示されるアミノ酸配列と完全同一ではないアミノ酸配列における、配列番号１上のある位置（順番）と対応するアミノ酸の位置とは、配列の一次構造を比較（アラインメント）したとき、配列番号１の当該位置と対応する位置とする。本発明において、前記と同じ形式の表記における「対応する位置」の意味は、前記の例示と同じである。 In the present invention, in the amino acid sequence that is not completely identical to the amino acid sequence shown in SEQ ID NO: 1, a certain position (order) on SEQ ID NO: 1 and the corresponding amino acid position were compared (aligned) with the primary structure of the sequence. In this case, the position corresponds to the position of SEQ ID NO: 1. In the present invention, the meaning of “corresponding position” in the notation of the same format as described above is the same as the above example.
配列の一次構造を比較する方法としては、種々の方法が知られている。例えば、市販の又は電気通信回線（インターネット）を通じて利用可能な解析ツールを用いて算出することができる。本発明においては、ＤＮＡ　Ｄａｔａｂａｎｋ　ｏｆ　Ｊａｐａｎ（ＤＤＢＪ）のＣｌｕｓｔａｌＷ（ｈｔｔｐ：／／ｃｌｕｓｔａｌｗ．ｄｄｂｊ．ｎｉｇ．ａｃ．ｊｐ／ｉｎｄｅｘ．ｐｈｐ／ｌａｎｇ＝ｊａ）においてデフォルト（初期設定）のパラメーターを用いることにより、配列の一次構造を比較する。 Various methods are known as methods for comparing the primary structures of sequences. For example, it can be calculated using an analysis tool that is commercially available or available through a telecommunication line (Internet). In the present invention, by using default (initial setting) parameters in ClustalW (http://clustalw.ddbj.nig.ac.jp/index.php/lang=ja) of DNA Databank of Japan (DDBJ), Compare the primary structure of the sequences.
上記の３‘－５’エキソヌクレアーゼ領域の改変とは、置換、欠失、又は付加からなり得るが特に限定されない。例えば、配列番号１における１３７～１４７、２０６～２２２、及び３０８～３１８番目に対応するアミノ酸への改変を示す。 The modification of the 3′-5 ′ exonuclease region can be composed of substitution, deletion, or addition, but is not particularly limited. For example, alterations to amino acids corresponding to positions 137 to 147, 206 to 222, and 308 to 318 in SEQ ID NO: 1 are shown.
前記３’－５’エキソヌクレアーゼ活性領域を改変したＤＮＡポリメラーゼとしては、配列番号１又は配列番号２における１４１、１４２、１４３、２１０、３１１番目に対応するアミノ酸の少なくとも一つを改変したものが好ましい。これらの改変型ＤＮＡポリメラーゼは、３‘－５’エキソヌクレアーゼ活性が欠損している。より好ましくは、アミノ酸の改変がＤ１４１Ａ、Ｅ１４３Ａ、Ｄ１４１Ａ／Ｅ１４３Ａ、Ｉ１４２Ｒ、Ｎ２１０Ｄ、又はＹ３１１Ｆから選択されるいずれか一つである、３‘－５’エキソヌクレアーゼ活性を欠損させたＤＮＡポリメラーゼである。なお、３‘－５’エキソヌクレアーゼ活性を欠損させた（エキソ（－））ＤＮＡポリメラーゼとは、活性の完全な欠如を含み、例えば、親酵素と比較して０．０３％、０．０５％、０．１％、１％、５％、１０％、２０％、又は最大でも５０％以下のエキソヌクレアーゼ活性を有する改変されたＤＮＡポリメラーゼを指す。 As the DNA polymerase in which the 3′-5 ′ exonuclease active region is modified, one in which at least one of amino acids corresponding to positions 141, 142, 143, 210, 311 in SEQ ID NO: 1 or SEQ ID NO: 2 is modified is preferable. . These modified DNA polymerases are deficient in 3'-5 'exonuclease activity. More preferably, it is a DNA polymerase deficient in 3'-5 'exonuclease activity, wherein the amino acid modification is any one selected from D141A, E143A, D141A / E143A, I142R, N210D, or Y311F. In addition, 3'-5 'exonuclease activity-deficient (exo (-)) DNA polymerase includes a complete lack of activity, for example, 0.03%, 0.05% compared to the parent enzyme , 0.1%, 1%, 5%, 10%, 20%, or at most 50% or less of a modified DNA polymerase having exonuclease activity.
前記３’－５’エキソヌクレアーゼ活性領域を改変したＤＮＡポリメラーゼとして、別の好ましい形態は、配列番号１又は配列番号２におけるＨ１４７Ｅ、又はＨ１４７Ｄから選択されるいずれか一つである。これらの改変型ＤＮＡポリメラーゼは、エキソヌクレアーゼ活性を維持したまま、ＰＣＲ効率が向上している。 Another preferred form of the DNA polymerase in which the 3'-5 'exonuclease active region is modified is any one selected from H147E and H147D in SEQ ID NO: 1 or SEQ ID NO: 2. These modified DNA polymerases have improved PCR efficiency while maintaining exonuclease activity.
なお、３‘－５’エキソヌクレアーゼ活性領域を改変したＤＮＡポリメラーゼを生成する方法や、３‘－５’エキソヌクレアーゼ活性を解析する方法は公知であり、例えば、米国特許第６９４６２７３号公報に開示されている。ＰＣＲ効率を向上させたＤＮＡポリメラーゼとは、ＰＣＲ産物の量が親酵素と比較して増加している改変されたＤＮＡポリメラーゼを示す。ＰＣＲ産物の量が親酵素と比較して増加しているかどうかを解析するための方法は、特許第３８９１３３０号公報等に記載されている。 A method for producing a DNA polymerase with a modified 3′-5 ′ exonuclease active region and a method for analyzing 3′-5 ′ exonuclease activity are known and disclosed in, for example, US Pat. No. 6,946,273. ing. A DNA polymerase with improved PCR efficiency refers to a modified DNA polymerase in which the amount of PCR product is increased compared to the parent enzyme. A method for analyzing whether the amount of the PCR product is increased as compared with the parent enzyme is described in Japanese Patent No. 3891330.
（３．３）ＤＮＡポリメラーゼの改変（ＩＩ）減少した塩基類似体検出活性を有するＤＮＡポリメラーゼ変異体を作製する改変
（３．３．１）
本発明に用いるファミリーＢに属するＤＮＡポリメラーゼは、減少した塩基類似体検出活性を有する変異体でもよい。塩基類似体とはアデニンやシトシン、グアニン、チミン以外の塩基を示し、ウラシルやイノシンなどが挙げられる。通常、ファミリーＢに属するＤＮＡポリメラーゼは、塩基類似体であるウラシルやイノシンを検出すると強く結合し、ＤＮＡポリメラーゼ機能を阻害する。塩基類似体検出活性とは、塩基類似体と強く結合し、ＤＮＡポリメラーゼ機能を阻害する活性を示す。減少した塩基類似体検出活性を有するファミリーＢに属するＤＮＡポリメラーゼ変異体とは、ウラシルやイノシンへの結合能力が低いことを特徴とするファミリーＢに属するＤＮＡポリメラーゼ変異体である。 (3.3) Modification of DNA polymerase (II) Modification for producing a DNA polymerase mutant having reduced base analog detection activity (3.3.1)
The DNA polymerase belonging to family B used in the present invention may be a mutant having reduced base analog detection activity. Base analogs refer to bases other than adenine, cytosine, guanine, and thymine, and include uracil and inosine. Usually, a DNA polymerase belonging to Family B binds strongly when a base analog such as uracil or inosine is detected, and inhibits the DNA polymerase function. The base analog detection activity refers to an activity that strongly binds to a base analog and inhibits the DNA polymerase function. A DNA polymerase mutant belonging to family B having reduced base analog detection activity is a DNA polymerase mutant belonging to family B characterized by low binding ability to uracil and inosine.
このような変異体は、ウラシルの結合に関するアミノ酸配列（ウラシル結合ポケット）の少なくとも１か所に改変を加えることにより作製できる。具体的には、ファミリーＢに属する古細菌ＤＮＡポリメラーゼ、例えば、Ｔｈｅｒｍｏｃｏｃｃｕｓ　ｋｏｄａｋａｒａｅｎｓｉｓ　ＫＯＤ１株由来のＤＮＡポリメラーゼのアミノ酸配列（配列番号１）の１～４０番目、及び７８～１３０番目によって形成されるウラシル結合ポケットの少なくとも１か所に改変を加え、野生型のＤＮＡポリメラーゼと比較してウラシルやイノシンへの結合能力を低下させたＤＮＡポリメラーゼ変異体が例示される。ウラシルやイノシンへの結合能力が低いＤＮＡポリメラーゼ変異体は、ｄＵＴＰの存在下のＰＣＲでもＤＮＡポリメラーゼの機能低下があまり見られず、ｄＵＴＰによるＤＮＡポリメラーゼの伸長反応への影響が低減されている。 Such a mutant can be prepared by modifying at least one position of the amino acid sequence (uracil binding pocket) relating to uracil binding. Specifically, uracil-binding pockets formed by archaebacterial DNA polymerase belonging to family B, for example, amino acid sequence (SEQ ID NO: 1) of DNA polymerase derived from Thermococcus kodakaraensis KOD1 strain (position No. 1), and positions 78-130 Examples include DNA polymerase mutants that have been modified at least at one site and have reduced ability to bind to uracil or inosine compared to wild-type DNA polymerase. DNA polymerase mutants with low binding ability to uracil and inosine do not show much decrease in DNA polymerase function even in PCR in the presence of dUTP, and the effect of dUTP on the elongation reaction of DNA polymerase is reduced.
ウラシルの結合に関するアミノ酸配列はパイロコッカス属に由来するＤＮＡポリメラーゼ及びサーモコッカス属に由来するＤＮＡポリメラーゼにおいて高度に保存されている。サーモコッカス・コダカラエンシスに由来するＤＮＡポリメラーゼ（配列番号１）においては、アミノ酸１～４０及びアミノ酸７８～１３０によって形成される。パイロコッカス・フリオサス（配列番号２）においては、アミノ酸１～４０、及びアミノ酸７８～１３０によって形成される。サーモコッカス・ゴルゴナリウス（配列番号３）においては、アミノ酸１～４０、及びアミノ酸７８～１３０によって形成される。サーモコッカス・リトラリス（配列番号４）においては、アミノ酸１～４０、及びアミノ酸７８～１３０によって形成される。パイロコッカス・エスピーＧＢ－Ｄ（配列番号５）においては、アミノ酸１～４０及びアミノ酸７８～１３０によって形成される。サーモコッカス・エスピーＪＤＦ－３（配列番号６）のおいては、アミノ酸１～４０、及びアミノ酸７８～１３０によって形成される。サーモコッカス・エスピー９°Ｎ－７（配列番号７）においては、アミノ酸１～４０及びアミノ酸７８～１３０によって形成される。サーモコッカス・エスピーＫＳ－１（配列番号８）においては、アミノ酸１～４０、及びアミノ酸７８～１３０によって形成される。サーモコッカス・セラー（配列番号９）においては、アミノ酸１～４０及びアミノ酸７８～１３０によって形成される。サーモコッカス・シクリ（配列番号１０）においては、アミノ酸１～４０及びアミノ酸７８～１３０によって形成される。 The amino acid sequence for uracil binding is highly conserved in DNA polymerases from Pyrococcus and DNA polymerases from Thermococcus. In the DNA polymerase (SEQ ID NO: 1) derived from Thermococcus kodakaraensis, it is formed by amino acids 1 to 40 and amino acids 78 to 130. In Pyrococcus furiosus (SEQ ID NO: 2), it is formed by amino acids 1 to 40 and amino acids 78 to 130. In Thermococcus gorgonarius (SEQ ID NO: 3), it is formed by amino acids 1 to 40 and amino acids 78 to 130. In Thermococcus litoralis (SEQ ID NO: 4), it is formed by amino acids 1 to 40 and amino acids 78 to 130. In Pyrococcus sp. GB-D (SEQ ID NO: 5), it is formed by amino acids 1 to 40 and amino acids 78 to 130. In Thermococcus sp. JDF-3 (SEQ ID NO: 6), it is formed by amino acids 1 to 40 and amino acids 78 to 130. In Thermococcus sp 9 ° N-7 (SEQ ID NO: 7), it is formed by amino acids 1-40 and amino acids 78-130. In Thermococcus sp. KS-1 (SEQ ID NO: 8), it is formed by amino acids 1 to 40 and amino acids 78 to 130. In Thermococcus cellar (SEQ ID NO: 9), it is formed by amino acids 1-40 and amino acids 78-130. In Thermococcus cyclis (SEQ ID NO: 10), it is formed by amino acids 1 to 40 and amino acids 78 to 130.
（３．３．２）
本発明に用いる減少した塩基類似体検出活性を有するＤＮＡポリメラーゼ変異体として、より好ましいのは、ウラシルと相互作用に直接関連していると想定されている７、３６、３７、９０～９７及び１１２～１１９番目のアミノ酸のうち少なくとも１つに改変を加えた古細菌ＤＮＡポリメラーゼ変異体、例えば、（ａ）配列番号１又は配列番号２で示されるアミノ酸配列の７、３６、３７、９０～９７及び１１２～１１９番目に相当するアミノ酸のうち、少なくとも１つのアミノ酸の改変を有するアミノ酸配列で示される古細菌ＤＮＡポリメラーゼ変異体である。 (3.3.2)
More preferred DNA polymerase variants with reduced base analog detection activity for use in the present invention are assumed to be directly related to interaction with uracil 7, 36, 37, 90-97 and 112. An archaeal DNA polymerase variant in which at least one of the 119th amino acids is modified, for example, (a) 7, 36, 37, 90 to 97 of the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2; It is an archaeal DNA polymerase variant represented by an amino acid sequence having at least one amino acid modification among amino acids corresponding to positions 112 to 119.
上記の古細菌ＤＮＡポリメラーゼ変異体は、以下の（ｂ）のアミノ酸配列で示されるものであってもよい。
（ｂ）（ａ）で示されるアミノ酸配列においてさらに７、３６、３７、９０～９７及び１１２～１１９番目以外の部位において少なくとも１つのアミノ酸が改変されており、そのアミノ酸配列と配列番号１との同一性又はそのアミノ酸配列と配列番号２との同一性が８０％以上（好ましくは８５％以上であり、より好ましくは９０％以上であり、さらに好ましくは９５％以上であり、特に好ましくは９８％以上であり、最も好ましくは９９％以上である）であり、かつ、減少した塩基類似体検出活性を有するＤＮＡポリメラーゼをコードするアミノ酸配列。 The archaeal DNA polymerase mutant may be one represented by the following amino acid sequence (b).
(B) In the amino acid sequence shown in (a), at least one amino acid is further modified at positions other than the 7, 36, 37, 90 to 97, and 112 to 119th positions. The identity or the identity between the amino acid sequence and SEQ ID NO: 2 is 80% or more (preferably 85% or more, more preferably 90% or more, still more preferably 95% or more, particularly preferably 98%. And most preferably 99% or more), and an amino acid sequence encoding a DNA polymerase having reduced base analog detection activity.
アミノ酸配列の同一性を算出する方法としては、種々の方法が知られている。例えば、市販の又は電気通信回線（インターネット）を通じて利用可能な解析ツールを用いて算出することができる。
本発明においては、全米バイオテクノロジー情報センター（ＮＣＢＩ）の相同性アルゴリズムＢＬＡＳＴ（Ｂａｓｉｃ　ｌｏｃａｌ　ａｌｉｇｎｍｅｎｔ　ｓｅａｒｃｈ　ｔｏｏｌ）ｈｔｔｐ：／／ｗｗｗ．ｎｃｂｉ．ｎｌｍ．ｎｉｈ．ｇｏｖ／ＢＬＡＳＴ／においてデフォルト（初期設定）のパラメーターを用いることにより、アミノ酸配列の同一性を算出する。 Various methods are known as methods for calculating the identity of amino acid sequences. For example, it can be calculated using an analysis tool that is commercially available or available through a telecommunication line (Internet).
In the present invention, the National Biotechnology Information Center (NCBI) homology algorithm BLAST (Basic local alignment search tool) http: // www. ncbi. nlm. nih. The amino acid sequence identity is calculated by using default (initial setting) parameters in gov / BLAST /.
上記の古細菌ＤＮＡポリメラーゼ変異体は、以下の（ｃ）のアミノ酸配列で示されるものであってもよい。
（ｃ）（ａ）で示されるアミノ酸配列において、さらに７、３６、３７、９０～９７及び１１２～１１９番目以外の部位において１もしくは数個のアミノ酸が欠失、置換もしくは付加されており、かつ、減少した塩基類似体検出活性を有するＤＮＡポリメラーゼをコードするアミノ酸配列。 The archaeal DNA polymerase mutant may be one represented by the following amino acid sequence (c).
(C) In the amino acid sequence shown in (a), one or several amino acids are further deleted, substituted or added at sites other than positions 7, 36, 37, 90 to 97 and 112 to 119, and An amino acid sequence encoding a DNA polymerase having reduced base analog detection activity.
ここで「数個」とは、「減少した塩基類似体検出活性」が維持される限り制限されないが、例えば、全アミノ酸の約２０％未満に相当する数であり、好ましくは約１５％未満に相当する数であり、さらに好ましくは約１０％未満に相当する数であり、より一層好ましくは約５％未満に相当する数であり、最も好ましくは約１％未満に相当する数である。より具体的には、変異されるアミノ酸残基の個数は、例えば、２～１６０個、好ましくは２～１２０個、より好ましくは２～８０個、更に好ましくは２～４０個であり、特に好ましくは２～５個である。 Here, “several” is not limited as long as “decreased base analog detection activity” is maintained, but is, for example, a number corresponding to less than about 20% of all amino acids, preferably less than about 15%. It is a corresponding number, more preferably a number corresponding to less than about 10%, even more preferably a number corresponding to less than about 5%, and most preferably a number corresponding to less than about 1%. More specifically, the number of amino acid residues to be mutated is, for example, 2 to 160, preferably 2 to 120, more preferably 2 to 80, still more preferably 2 to 40, and particularly preferably. Is 2-5.
なお、「配列番号１に示されるアミノ酸配列における７、３６、３７、９０～９７及び１１２～１１９番目に相当するアミノ酸」とは、配列番号１に示されるアミノ酸配列と完全同一ではないアミノ酸配列を有するＤＮＡポリメラーゼにおいて、配列番号１の７、３６、３７、９０～９７及び１１２～１１９番目に対応するウラシルの結合に関するアミノ酸配列を含む表現である。 The “amino acids corresponding to positions 7, 36, 37, 90 to 97 and 112 to 119 in the amino acid sequence shown in SEQ ID NO: 1” are amino acid sequences that are not completely identical to the amino acid sequence shown in SEQ ID NO: 1. In the DNA polymerase having, it is an expression including the amino acid sequence relating to the binding of uracil corresponding to positions 7, 36, 37, 90 to 97 and 112 to 119 of SEQ ID NO: 1.
本発明において、配列番号１に示されるアミノ酸配列と完全同一ではないアミノ酸配列おける、配列番号１上のある位置（順番）と対応する位置とは、配列の一次構造を比較（アラインメント）したとき、配列番号１の当該位置と対応する位置とする。本明細書において、前記と同じ形式の表記における「相当する」の意味は、前記の例示と同じである。 In the present invention, a position (order) on SEQ ID NO: 1 in an amino acid sequence that is not completely identical to the amino acid sequence shown in SEQ ID NO: 1 is compared with a position corresponding to a position (sequence) on the primary structure of the sequence, The position corresponds to the position of SEQ ID NO: 1. In this specification, the meaning of “corresponding” in the notation of the same format as described above is the same as in the above example.
本発明において、配列番号１に示されるアミノ酸配列と完全同一ではないアミノ酸配列おける、配列番号１上のある位置（順番）と対応する位置とは、配列の一次構造を比較（アラインメント）したとき、配列番号１の当該位置と対応する位置とする。本明細書において、前記と同じ形式の表記における「対応する位置」の意味は、前記の例示と同じである。 In the present invention, a position (order) on SEQ ID NO: 1 in an amino acid sequence that is not completely identical to the amino acid sequence shown in SEQ ID NO: 1 is compared with a position corresponding to a position (sequence) on the primary structure of the sequence, The position corresponds to the position of SEQ ID NO: 1. In the present specification, the meaning of “corresponding position” in the notation of the same format as described above is the same as the above example.
配列の一次構造を比較する方法としては、種々の方法が知られている。例えば、市販の又は電気通信回線（インターネット）を通じて利用可能な解析ツールを用いて算出することができる。本発明においては、ＤＮＡ　Ｄａｔａｂａｎｋ　ｏｆ　Ｊａｐａｎ（ＤＤＢＪ）のＣｌｕｓｔａｌＷ（ｈｔｔｐ：／／ｃｌｕｓｔａｌｗ．ｄｄｂｊ．ｎｉｇ．ａｃ．ｊｐ／ｉｎｄｅｘ．ｐｈｐ／ｌａｎｇ＝ｊａ）においてデフォルト（初期設定）のパラメーターを用いることにより、配列の一次構造を比較する。 Various methods are known as methods for comparing the primary structures of sequences. For example, it can be calculated using an analysis tool that is commercially available or available through a telecommunication line (Internet). In the present invention, by using default (initial setting) parameters in ClustalW (http://clustalw.ddbj.nig.ac.jp/index.php/lang=ja) of DNA Databank of Japan (DDBJ), Compare the primary structure of the sequences.
（３．３．３）
本発明に用いる減少した塩基類似体検出活性を有する古細菌ＤＮＡポリメラーゼ変異体は、より好ましくは、配列番号１又は配列番号２におけるアミノ酸Ｙ７、Ｐ３６、又はＶ９３に相当するアミノ酸から選択される少なくとも１つのアミノ酸の改変を有する。ここで、例えばＹ７とは、７番目のアミノ酸であるチロシン（Ｙ）残基を意味しており、アルファベット１文字は通用されているアミノ酸の略号を表している。好ましい例において、Ｙ７アミノ酸はチロシン（Ｙ）が非極性アミノ酸に置換されており、具体的にはＹ７Ａ、Ｙ７Ｇ、Ｙ７Ｖ、Ｙ７Ｌ、Ｙ７Ｉ、Ｙ７Ｐ、Ｙ７Ｆ、Ｙ７Ｍ、Ｙ７Ｗ、及びＹ７Ｃからなる群より選ばれるアミノ酸置換である。別の好ましい例において、Ｐ３６アミノ酸はプロリン（Ｐ）が正電荷をもつ極性アミノ酸に置換されており、具体的にはＰ３６Ｈ、Ｐ３６Ｋ、又はＰ３６Ｒのアミノ酸置換である。別の好ましい例において、Ｖ９３アミノ酸はバリン（Ｖ）が、正電荷をもち極性アミン酸に置換されており、具体的にはＶ９３Ｈ、Ｖ９３Ｋ、又はＶ９３Ｒのアミノ酸置換である。 (3.3.3)
The archaeal DNA polymerase mutant having reduced base analog detection activity for use in the present invention is more preferably at least one selected from amino acids corresponding to amino acids Y7, P36, or V93 in SEQ ID NO: 1 or SEQ ID NO: 2. Has one amino acid modification. Here, for example, Y7 means a tyrosine (Y) residue that is the seventh amino acid, and one letter of the alphabet represents an abbreviation of a commonly used amino acid. In a preferred example, the Y7 amino acid has tyrosine (Y) substituted with a nonpolar amino acid, specifically selected from the group consisting of Y7A, Y7G, Y7V, Y7L, Y7I, Y7P, Y7F, Y7M, Y7W, and Y7C. Amino acid substitution. In another preferred example, the P36 amino acid is substituted with a polar amino acid in which proline (P) is positively charged, specifically P36H, P36K, or P36R. In another preferred example, the V93 amino acid is a valine (V) substituted with a positively charged polar amine acid, specifically an amino acid substitution of V93H, V93K, or V93R.
より好ましくは、改変がＹ７Ａ、Ｐ３６Ｈ、Ｐ３６Ｋ、Ｐ３６Ｒ、Ｖ９３Ｑ、Ｖ９３Ｋ、及びＶ９３Ｒからなる群より選ばれる、少なくとも１つのアミノ酸の改変である。さらに好ましくはＰ３６Ｋ、Ｐ３６Ｒ又はＰ３６Ｈである。特に好ましくはＰ３６Ｈである。 More preferably, the modification is at least one amino acid modification selected from the group consisting of Y7A, P36H, P36K, P36R, V93Q, V93K, and V93R. More preferably, it is P36K, P36R or P36H. Particularly preferred is P36H.
本発明において用いられる減少した塩基類似体検出活性を有する古細菌ＤＮＡポリメラーゼ変異体は、配列番号１又は配列番号２におけるアミノ酸Ｙ７、Ｐ３６、又はＶ９３に相当するアミノ酸から選択される２つ以上のアミノ酸を改変したものでも良い。具体的には、Ｙ７Ａ／Ｖ９３Ｋ、Ｙ７Ａ／Ｐ３６Ｈ、Ｙ７Ａ／Ｐ３６Ｒ、Ｙ７Ａ／Ｖ９３Ｒ、Ｙ７Ａ／Ｖ９３Ｑ又はＰ３６Ｈ／Ｖ９３Ｋなどが挙げられ、好ましくは、Ｙ７Ａ／Ｐ３６Ｈ又はＹ７Ａ／Ｖ９３Ｋなどが挙げられるが、これらに限定されるものではない。 The archaeal DNA polymerase mutant having reduced base analog detection activity used in the present invention comprises two or more amino acids selected from amino acids corresponding to amino acids Y7, P36, or V93 in SEQ ID NO: 1 or SEQ ID NO: 2. May be modified. Specific examples include Y7A / V93K, Y7A / P36H, Y7A / P36R, Y7A / V93R, Y7A / V93Q or P36H / V93K, and preferably Y7A / P36H or Y7A / V93K. It is not limited to.
上記に例示したＤＮＡポリメラーゼの改変をもとに、本発明に用いる改変されたＤＮＡポリメラーゼとして、種々の変異体が考えられる。そのような変異体として、以下の（１）－（４）のいずれかの改変を有する古細菌ＤＮＡポリメラーゼの変異体が例示されるが、これに限定されるわけではない。
（１）（Ａ）Ｈ１４７Ｅと、（Ｂ）Ｙ７Ａ／Ｖ９３Ｋ、Ｙ７Ａ／Ｖ９３Ｒ、Ｙ７Ａ／Ｖ９３Ｑ、Ｙ７Ａ／Ｐ３６Ｈ、Ｙ７Ａ／Ｐ３６Ｒ、Ｐ３６Ｈ／Ｖ９３Ｋ、Ｐ３６Ｋ、Ｐ３６Ｒ、Ｐ３６Ｈ、Ｖ９３Ｒ又はＶ９３Ｑのいずれか
（２）（Ａ）Ｎ２１０Ｄと、（Ｂ）Ｙ７Ａ／Ｖ９３Ｋ、Ｙ７Ａ／Ｐ３６Ｈ、Ｙ７Ａ／Ｐ３６Ｒ、Ｐ３６Ｋ、Ｐ３６Ｒ、Ｐ３６Ｈ、Ｖ９３Ｑ、Ｖ９３Ｋ又はＶ９３Ｒのいずれか
（３）（Ａ）Ｉ１４２Ｒと、（Ｂ）Ｙ７Ａ／Ｖ９３Ｋ、Ｙ７Ａ／Ｖ９３Ｒ、Ｙ７Ａ／Ｖ９３Ｑ、Ｙ７Ａ／Ｐ３６Ｈ、Ｙ７Ａ／Ｐ３６Ｒ、Ｐ３６Ｒ、Ｐ３６Ｈ、Ｖ９３Ｋ、Ｖ９３Ｒ又はＶ９３Ｑのいずれか
（４）（Ａ）Ｄ１４１Ａ／Ｅ１４３Ａと、（Ｂ）Ｙ７Ａ／Ｖ９３Ｋ、Ｙ７Ａ／Ｐ３６Ｈ、Ｙ７Ａ／Ｐ３６Ｒ、Ｐ３６Ｒ、Ｐ３６Ｈ又はＶ９３Ｋのいずれか Based on the modification of the DNA polymerase exemplified above, various mutants can be considered as the modified DNA polymerase used in the present invention. Examples of such mutants include, but are not limited to, archaeal DNA polymerase mutants having any of the following modifications (1) to (4).
(1) (A) H147E and (B) Y7A / V93K, Y7A / V93R, Y7A / V93Q, Y7A / P36H, Y7A / P36R, P36H / V93K, P36K, P36R, P36H, V93R or V93Q (2 ) (A) N210D, (B) Y7A / V93K, Y7A / P36H, Y7A / P36R, P36K, P36R, P36H, V93Q, V93K or V93R (3) (A) I142R and (B) Y7A / V93K, Y7A / V93R, Y7A / V93Q, Y7A / P36H, Y7A / P36R, P36R, P36H, V93K, V93R, or V93Q (4) (A) D141A / E143A and (B) Y7A / V93K, Y7A / P36H, Y7A / P36R, P36R, P36H or V93 One of the
（３．３．４）塩基類似体検出活性の評価方法
本発明における塩基類似体検出活性はＰＣＲによって評価できる。塩基類似体は、典型的にはウラシルである。本発明においては、鋳型となるＤＮＡ、緩衝材、マグネシウム、ｄＮＴＰｓ、プライマー、及び評価対象のＤＮＡポリメラーゼを含む通常のＰＣＲ反応液に、ｄＵＴＰ溶液を、終濃度０．５μＭ～２００μＭで添加し、熱サイクルを行う。反応後に、エチジウムブロマイド染色１％アガロース電気泳動でＰＣＲ産物の有無を確認し、許容できたｄＵＴＰ濃度によって、ウラシルの検出活性を評価することが出来る。ウラシル検出活性の高いＤＮＡポリメラーゼは少しのｄＵＴＰの添加で伸長反応が阻害され、ＰＣＲ産物が確認できない。また、ウラシルの検出活性の低いＤＮＡポリメラーゼは、高濃度のｄＵＴＰを添加しても問題なくＰＣＲによる遺伝子増幅が確認できる。 (3.3.4) Method for evaluating base analog detection activity The base analog detection activity in the present invention can be evaluated by PCR. The base analog is typically uracil. In the present invention, a dUTP solution is added at a final concentration of 0.5 μM to 200 μM to a normal PCR reaction solution containing DNA as a template, buffer material, magnesium, dNTPs, primers, and a DNA polymerase to be evaluated. Cycle. After the reaction, the presence or absence of a PCR product is confirmed by ethidium bromide-stained 1% agarose electrophoresis, and the detection activity of uracil can be evaluated based on an acceptable dUTP concentration. A DNA polymerase having a high uracil detection activity inhibits the extension reaction when a little dUTP is added, and the PCR product cannot be confirmed. In addition, DNA polymerase with low uracil detection activity can confirm gene amplification by PCR without any problems even when a high concentration of dUTP is added.
減少した塩基類似体検出活性を有する古細菌ＤＮＡポリメラーゼ変異体とは、酵素至適の反応Ｂｕｆｆｅｒ中で、任意のプライマー、及び鋳型となるＤＮＡを用い、至適の熱サイクルを行った結果、変異がない野生型と比較し、高濃度のｄＵＴＰを添加しても伸長反応が阻害されず、ＰＣＲ産物が確認できるＤＮＡポリメラーゼのことをいう。ただし、野生型との比較が困難な場合は、ｄＵＴＰを０．５μＭの濃度で添加してもＰＣＲの増幅ができる古細菌ＤＮＡポリメラーゼ変異体については、当該変異体が野生型と比較して減少した塩基類似体検出活性を有すると推定する。 An archaeal DNA polymerase mutant having decreased base analog detection activity is a result of optimal thermal cycling using any primer and DNA as a template in an enzyme optimal reaction buffer. Compared with the wild-type without DNA, it means a DNA polymerase in which the extension reaction is not inhibited even when a high concentration of dUTP is added, and the PCR product can be confirmed. However, if it is difficult to compare with the wild type, the archaeal DNA polymerase mutant that can amplify PCR even when dUTP is added at a concentration of 0.5 μM is reduced compared to the wild type. It is presumed to have the activity of detecting a base analog.
本発明における塩基類似体検出活性の評価は、以下の方法に従う。
ＫＯＤ　－Ｐｌｕｓ－　Ｖｅｒ．２（Ｔｏｙｏｂｏ製）添付の１０×ＰＣＲ　Ｂｕｆｆｅｒ、又はＰｆｕ　ＤＮＡ　Ｐｏｌｙｍｅｒａｓｅ（Ａｇｉｌｅｎｔ製）添付の１０×ＰＣＲ　Ｂｕｆｆｅｒを用い、１×ＰＣＲ　Ｂｕｆｆｅｒ、及び１．５ｍＭ　ＭｇＳＯ４、０．２ｍＭ　ｄＮＴＰｓ（ｄＡＴＰ、ｄＴＴＰ，ｄＣＴＰ、ｄＧＴＰ）、約１．３ｋｂを増幅する１５ｐｍｏｌの配列番号１１及び１２に記載のプライマー、１０ｎｇのヒトゲノムＤＮＡ（Ｒｏｃｈｅ製）、１Ｕの各酵素を含む５０μｌの反応液中に、ｄＵＴＰ(Ｒｏｃｈｅ製)を終濃度０．５、５、５０、１００、２００μＭになるよう添加する。９４℃、３０秒の前反応の後、９８℃、１０秒→６５℃、３０秒→６８℃、１分３０秒を３０サイクル繰り返すスケジュールでＰＣＲ　ｓｙｓｔｅｍ　ＧｅｎｅＡｍｐ（登録商標）９７００（Ａｐｐｌｉｅｄ　Ｂｉｏｓｙｓｔｅｍ）にてＰＣＲを行う。反応終了後、５μｌの反応液について１％アガロース電気泳動を行い、エチジウムブロマイド染色し、紫外線照射下約１．３ｋｂの増幅ＤＮＡ断片を確認することで、塩基類似体検出活性が減少しているかどうかが評価できる。 The evaluation of the base analog detection activity in the present invention follows the following method.
KOD -Plus- Ver. 2 (Toyobo) attached 10 × PCR Buffer or Pfu DNA Polymerase (Agilent) attached 10 × PCR Buffer, 1 × PCR Buffer, 1.5 mM MgSO 4 , 0.2 mM dNTPs (dATP, dTTP, dCTP, dGTP), 15 pmol of the primer described in SEQ ID NOS: 11 and 12 for amplifying about 1.3 kb, 10 ng of human genomic DNA (Roche), and 1 U of each enzyme, 50 μl of reaction solution containing dUTP (Roche) ) To a final concentration of 0.5, 5, 50, 100, 200 μM. After a pre-reaction at 94 ° C. for 30 seconds, a PCR system GeneAmp (registered trademark) 9700 (Applied Biosystem) with a schedule for repeating 30 cycles of 98 ° C., 10 seconds → 65 ° C., 30 seconds → 68 ° C., 1 minute 30 seconds Perform PCR. After completion of the reaction, 1% agarose electrophoresis is performed on 5 μl of the reaction solution, ethidium bromide staining is performed, and the amplified DNA fragment of about 1.3 kb is confirmed under ultraviolet irradiation to determine whether the base analog detection activity is reduced. Can be evaluated.
（３．４）アミノ酸改変の導入方法
本発明に用いるＤＮＡポリメラーゼを改変する方法は、既に当該技術分野において確立されている。よって、公知の方法に従い改変を行うことが出来、その態様は特に制限されない。 (3.4) Method for Introducing Amino Acid Modification A method for modifying a DNA polymerase used in the present invention has already been established in the art. Therefore, it can modify | change according to a well-known method, The aspect in particular is not restrict | limited.
アミノ酸の改変を導入する方法の一態様として、Ｉｎｖｅｒｓｅ　ＰＣＲ法に基づく部位特異的変異導入法を用いることができる。例えば、ＫＯＤ　－Ｐｌｕｓ－　Ｍｕｔａｇｅｎｅｓｉｓ　Ｋｉｔ（Ｔｏｙｏｂｏ製）は、（１）目的とする遺伝子を挿入したプラスミドを変性させ、該プラスミドに変異プライマーをアニーリングさせ、続いてＫＯＤ　ＤＮＡポリメラーゼを用いて伸長反応を行う、（２）（１）のサイクルを１５回繰り返す、（３）制限酵素ＤｐｎＩを用いて鋳型としたプラスミドのみを選択的に切断する、（４）新たに合成された遺伝子をリン酸化、Ｌｉｇａｔｉｏｎを実施し環化させる、（５）環化した遺伝子を大腸菌に形質転換し、目的とする変異の導入されたプラスミドを保有する形質転換体を取得することのできるキットである。 As one embodiment of the method for introducing amino acid modification, a site-specific mutagenesis method based on the Inverse PCR method can be used. For example, KOD-Plus-Mutageness Kit (manufactured by Toyobo) (1) denatures a plasmid into which the target gene has been inserted, anneals the mutation primer to the plasmid, and then performs an extension reaction using KOD DNA polymerase. (2) Repeat the cycle of (1) 15 times. (3) Selectively cleave only the plasmid as a template using the restriction enzyme DpnI. (4) Phosphorylation of newly synthesized gene, Ligation (5) A kit that can transform a cyclized gene into Escherichia coli and obtain a transformant having a plasmid introduced with the target mutation.
（３．５）
上記改変ＤＮＡポリメラーゼ遺伝子を必要に応じて発現ベクターに移し替え、宿主として例えば大腸菌を、該発現ベクターを用いて形質転換した後、アンピシリン等の薬剤を含む寒天培地に塗布し、コロニーを形成させる。コロニーを栄養培地、例えばＬＢ培地や２×ＹＴ培地に接種し、３７℃で１２～２０時間培養した後、菌体を破砕して粗酵素液を抽出する。ベクターとしては、ｐＢｌｕｅｓｃｒｉｐｔ由来のものが好ましい。菌体を破砕する方法としては公知のいかなる手法を用いても良いが、例えば超音波処理、フレンチプレスやガラスビーズ破砕のような物理的破砕法やリゾチームのような溶菌酵素を用いることができる。この粗酵素液を８０℃、３０分間熱処理し、宿主由来のＤＮＡポリメラーゼを失活させ、ＤＮＡポリメラーゼ活性を測定する。 (3.5)
The modified DNA polymerase gene is transferred to an expression vector as necessary, and, for example, E. coli as a host is transformed with the expression vector, and then applied to an agar medium containing a drug such as ampicillin to form colonies. The colony is inoculated into a nutrient medium such as LB medium or 2 × YT medium and cultured at 37 ° C. for 12 to 20 hours, and then the cells are crushed and the crude enzyme solution is extracted. A vector derived from pBluescript is preferable. Any known method may be used as a method for crushing bacterial cells. For example, ultrasonic treatment, a physical crushing method such as French press or glass bead crushing, or a lytic enzyme such as lysozyme can be used. This crude enzyme solution is heat-treated at 80 ° C. for 30 minutes to inactivate the host-derived DNA polymerase, and the DNA polymerase activity is measured.
上記方法により選抜された菌株から精製ＤＮＡポリメラーゼを取得する方法は、いかなる手法を用いても良いが、例えば下記のような方法がある。栄養培地に培養して得られた菌体を回収した後、酵素的又は物理的破砕法により破砕抽出して粗酵素液を得る。得られた粗酵素抽出液から熱処理、例えば８０℃、３０分間処理し、その後硫安沈殿によりＤＮＡポリメラーゼ画分を回収する。この粗酵素液をセファデックスＧ－２５（アマシャムファルマシア・バイオテク製）を用いたゲル濾過等の方法により脱塩を行うことができる。この操作の後、ヘパリンセファロースカラムクロマトグラフィーにより分離、精製し、精製酵素標品を得ることができる。該精製酵素標品はＳＤＳ－ＰＡＧＥによってほぼ単一バンドを示す程度に純化される。 Any method may be used as a method for obtaining purified DNA polymerase from the strain selected by the above method, for example, the following method. After the cells obtained by culturing in the nutrient medium are collected, the crude enzyme solution is obtained by crushing and extracting by an enzymatic or physical crushing method. The obtained crude enzyme extract is heat-treated, for example, at 80 ° C. for 30 minutes, and then the DNA polymerase fraction is recovered by ammonium sulfate precipitation. This crude enzyme solution can be desalted by a method such as gel filtration using Sephadex G-25 (manufactured by Amersham Pharmacia Biotech). After this operation, it can be separated and purified by heparin sepharose column chromatography to obtain a purified enzyme preparation. The purified enzyme preparation is purified by SDS-PAGE to such an extent that it shows almost a single band.
（３．５．１）ＤＮＡポリメラーゼ活性測定法
本発明に用いるＤＮＡポリメラーゼは、以下のように活性を測定する。酵素活性が強い場合には、保存緩衝液（５０ｍＭ　Ｔｒｉｓ－ＨＣｌ（ｐＨ８．０），５０ｍＭ　ＫＣｌ，１ｍＭ　ジチオスレイトール，０．１％　Ｔｗｅｅｎ２０，０．１％　Ｎｏｎｉｄｅｔ　Ｐ４０，５０％　グリセリン）でサンプルを希釈して測定を行う。（１）下記のＡ液２５μｌ、Ｂ液５μｌ、Ｃ液５μｌ、滅菌水１０μｌ、及び酵素溶液５μｌをマイクロチューブに加えて７５℃にて１０分間反応する。（２）その後氷冷し、Ｅ液５０μｌ、Ｄ液１００μｌを加えて、攪拌後更に１０分間氷冷する。（３）この液をガラスフィルター（ワットマン製ＧＦ／Ｃフィルター）で濾過し、０．１Ｎ　塩酸及びエタノールで十分洗浄する。（４）フィルターの放射活性を液体シンチレーションカウンター（パーキンエルマー製ＴｒｉＣａｒｂ　２８１０ＴＲ）で計測し、鋳型ＤＮＡのヌクレオチドの取り込みを測定する。酵素活性の１単位はこの条件で３０分当りの１０ｎｍｏｌのヌクレオチドを酸不溶性画分（即ち、Ｄ液を添加したときに不溶化する画分）に取り込む酵素量とする。
Ａ液：４０ｍＭ　Ｔｒｉｓ－ＨＣｌ緩衝液（ｐＨ７．５）、１６ｍＭ　塩化マグネシウム、１５ｍＭ　ジチオスレイトール、１００μｇ／ｍＬ　ＢＳＡ（牛血清アルブミン）
Ｂ液：１．５μｇ／μｌ　活性化仔牛胸腺ＤＮＡ
Ｃ液：１．５ｍＭ　ｄＮＴＰ（２５０ｃｐｍ／ｐｍｏｌ　［３Ｈ］ｄＴＴＰ）
Ｄ液：２０％　トリクロロ酢酸（２ｍＭ　ピロリン酸ナトリウム）
Ｅ液：１ｍｇ／ｍＬ仔牛胸腺ＤＮＡ (3.5.1) DNA polymerase activity measurement method The activity of the DNA polymerase used in the present invention is measured as follows. If the enzyme activity is strong, samples should be stored in storage buffer (50 mM Tris-HCl (pH 8.0), 50 mM KCl, 1 mM dithiothreitol, 0.1% Tween 20, 0.1% Nonidet P40, 50% glycerin). Dilute and measure. (1) 25 μl of the following solution A, 5 μl of solution B, 5 μl of solution C, 10 μl of sterilized water, and 5 μl of enzyme solution are added to a microtube and reacted at 75 ° C. for 10 minutes. (2) Then, ice-cool, add 50 μl of E solution and 100 μl of D solution, and stir on ice for another 10 minutes after stirring. (3) This solution is filtered through a glass filter (GF / C filter manufactured by Whatman) and thoroughly washed with 0.1N hydrochloric acid and ethanol. (4) The radioactivity of the filter is measured with a liquid scintillation counter (TriCarb 2810TR manufactured by PerkinElmer), and the incorporation of nucleotides into the template DNA is measured. One unit of enzyme activity is defined as the amount of enzyme that takes 10 nmol nucleotides per 30 minutes into the acid-insoluble fraction (that is, the fraction insolubilized when solution D is added) under these conditions.
Solution A: 40 mM Tris-HCl buffer (pH 7.5), 16 mM magnesium chloride, 15 mM dithiothreitol, 100 μg / mL BSA (bovine serum albumin)
Solution B: 1.5 μg / μl activated calf thymus DNA
Solution C: 1.5 mM dNTP (250 cpm / pmol [3H] dTTP)
Liquid D: 20% trichloroacetic acid (2 mM sodium pyrophosphate)
Solution E: 1 mg / mL calf thymus DNA
（４）ＰＣＮＡ
（４．１）
本発明に用いるＰＣＮＡは、ＰＣＲ増強因子の一種である。前記ＰＣＮＡとしては、特に限定されないが、ＰＣＲの熱サイクルに耐えられる耐熱性のものが望ましく、好ましくはＰＣＲ後も活性が残るものが望まれる。さらに好ましくは８０℃で３０分の熱処理を行っても可溶性であり、活性が５０％以上、さらに好ましくは７０％以上、特に好ましくは９０％以上残っているものが望まれる。 (4) PCNA
(4.1)
PCNA used in the present invention is a kind of PCR enhancing factor. The PCNA is not particularly limited, but is preferably a heat-resistant one that can withstand the thermal cycle of PCR, and preferably one that remains active after PCR. More preferably, it is soluble even after heat treatment at 80 ° C. for 30 minutes, and the activity remains at 50% or more, more preferably at least 70%, particularly preferably at least 90%.
そのようなＰＣＮＡとしては、例えばパイロコッカス（Ｐｙｒｏｃｏｃｃｕｓ）属及びサーモコッカス（Ｔｈｅｒｍｏｃｏｃｃｕｓ）属の細菌から単離されたＰＣＮＡが挙げられる。パイロコッカス属由来のＰＣＮＡとしては、Ｐｙｒｏｃｏｃｃｕｓ　ｆｕｒｉｏｓｕｓ（配列番号１３）、Ｐｙｒｏｃｏｃｃｕｓ　ｓｐ．ＧＢ－Ｄ、Ｐｙｒｏｃｏｃｃｕｓ　Ｗｏｅｓｅｉ、Ｐｙｒｏｃｏｃｃｕｓ　ａｂｙｓｓｉ又はＰｙｒｏｃｏｃｃｕｓ　ｈｏｒｉｋｏｓｈｉｉから単離されたＰＣＮＡを含むが、これらに限定されない。サーモコッカス属に由来するＰＣＮＡとしては、Ｔｈｅｒｍｏｃｏｃｃｕｓ　ｋｏｄａｋａｒａｅｎｓｉｓ（配列番号１４）、Ｔｈｅｒｍｏｃｏｃｃｕｓ　ｇｏｒｇｏｎａｒｉｕｓ、Ｔｈｅｒｍｏｃｏｃｃｕｓ　ｌｉｔｏｒａｌｉｓ、Ｔｈｅｒｍｏｃｏｃｃｕｓ　ｓｐ．ＪＤＦ－３、Ｔｈｅｒｍｏｃｏｃｃｕｓ　ｓｐ．９ｄｅｇｒｅｅｓ　Ｎｏｒｔｈ－７（Ｔｈｅｒｍｏｃｏｃｃｕｓ　ｓｐ．９°Ｎ－７）、Ｔｈｅｒｍｏｃｏｃｃｕｓ　ｓｐ．ＫＳ－１、Ｔｈｅｒｍｏｃｏｃｃｕｓ　ｃｅｌｅｒ、又はＴｈｅｒｍｏｃｏｃｃｕｓ　ｓｉｃｕｌｉ、Ｍｅｔｈａｎｏｃａｌｄｏｃｏｃｃｕｓ ｊａｎｎａｓｃｈｉｉ（Ｍｊａ）又はＭｅｔｈａｎｏｂａｃｔｅｒｉｕｍ ｔｈｅｒｍｏａｕｔｏｔｒｏｐｈｉｃｕｍ（Ｍｔｈ）から単離されたＰＣＮＡを含むが、これらに限定されない。 Examples of such PCNA include PCNA isolated from bacteria of the genus Pyrococcus and Thermococcus. PCNA derived from the genus Pyrococcus includes Pyrococcus furiosus (SEQ ID NO: 13), Pyrococcus sp. Including, but not limited to, PCNA isolated from GB-D, Pyrococcus Wosei, Pyrococcus abyssi or Pyrococcus horikoshii. PCNA derived from the genus Thermococcus includes Thermococcus kodakaaraensis (SEQ ID NO: 14), Thermococcus gorgonaris, Thermococcus literalis, Thermococcus sp. JDF-3, Thermococcus sp. 9 degrees North-7 (Thermococcus sp. 9 ° N-7), Thermococcus sp. Includes, but not limited to, PCNAs isolated from KS-1, Thermococcus celer, or Thermococcus siculi, Methanocladoccus jannaschii (Mja) or Methanobacterium thermoautotropicum (Mth).
ＰＣＮＡをコードする遺伝子は、ＰＣＮＡをもつ生物からクローニングすることができる。また、アミノ酸の配列情報や核酸の配列情報をもとに人工的に合成することもできる。 A gene encoding PCNA can be cloned from an organism having PCNA. It can also be artificially synthesized based on amino acid sequence information and nucleic acid sequence information.
さらに、本発明に用いるＰＣＮＡは単独でＤＮＡにロードする（ＤＮＡポリメラーゼ増幅増強活性のある）変異体であってもよい。ＰＣＮＡは通常、多量体を形成し輪のような構造をとる。ＤＮＡにロードするとは、ＰＣＮＡ多量体の輪の構造内部にＤＮＡを通すことを示し、通常はＲＦＣと呼ばれる因子と共同して初めてＰＣＮＡはＤＮＡにロードすることができる。単独でＤＮＡにロードする変異体とは、ＰＣＮＡの多量体形成に関わる部位を改変し、多量体形成を不安定化することで、ＲＦＣなしでもＤＮＡをＰＣＮＡ多量体内部に通しやすくした変異体を示す。 Furthermore, the PCNA used in the present invention may be a mutant that alone loads DNA (having DNA polymerase amplification enhancing activity). PCNA usually forms a multimer and has a ring-like structure. Loading to DNA indicates that the DNA is allowed to pass inside the ring structure of the PCNA multimer, and PCNA can be loaded into DNA only in combination with a factor usually called RFC. Mutants that load DNA alone are those that modify the sites involved in PCNA multimer formation and destabilize multimer formation, making it easier to pass DNA into PCNA multimers without RFC. Show.
（４．１．１）ＫＯＤ－ＰＣＮＡ及びＰｆｕ－ＰＣＮＡ
ＰＣＮＡが多量体形成に関する部位は、サーモコッカス・コダカラエンシスに由来するＰＣＮＡ（ＫＯＤ－ＰＣＮＡとも記載）（配列番号１３）、パイロコッカス・フリオサスのＰＣＮＡ（Ｐｆｕ－ＰＣＮＡとも記載）（配列番号１４）においては、８２、８４、１０９番目のアミノ酸からなるＮ末端領域と１３９、１４３、１４７番目のアミノ酸からなるＣ末端領域が挙げられる。Ｎ末端領域はプラスに帯電し、Ｃ末端領域はマイナスに帯電し、相互作用することで多量体形成を行う。 (4.1.1) KOD-PCNA and Pfu-PCNA
PCNA-related sites for multimer formation are PCNA derived from Thermococcus kodakaraensis (also described as KOD-PCNA) (SEQ ID NO: 13), Pyrococcus furiosus PCNA (also described as Pfu-PCNA) (SEQ ID NO: 14) , Examples include an N-terminal region consisting of amino acids 82, 84 and 109 and a C-terminal region consisting of amino acids 139, 143 and 147. The N-terminal region is positively charged, the C-terminal region is negatively charged, and multimers are formed by interaction.
上記及び下記において、配列番号１３又は配列番号１４を例にして説明したことは、本明細書で具体的に配列を提示したＰＣＮＡ以外のＰＣＮＡにも適用される。例えば、図７で示したように配列番号１３及び１４に示されるＰＣＮＡ以外のＰＣＮＡにおいては、配列番号１３の８２、８４、１０９、１３９、１４３、１４７番目のアミノ酸からなる多量体形成に関する領域と対応する領域のことを示す。ここで、異なる２つのアミノ酸配列があるとき、基準となる一方のアミノ酸配列においてアミノ酸や領域が「対応する」とは、アミノ酸配列の一次構造を比較（アラインメント）したとき、基準となる配列の当該位置と対応する位置とする。 What has been described above and below using SEQ ID NO: 13 or SEQ ID NO: 14 as an example also applies to PCNAs other than the PCNA specifically presenting the sequences in this specification. For example, as shown in FIG. 7, in PCNA other than the PCNA shown in SEQ ID NOs: 13 and 14, a region related to multimer formation consisting of amino acids 82, 84, 109, 139, 143, and 147 of SEQ ID NO: 13 Indicates the corresponding area. Here, when there are two different amino acid sequences, the amino acid or region “corresponds” in one of the reference amino acid sequences means that when the primary structure of the amino acid sequences is compared (aligned), The position corresponds to the position.
単独でＤＮＡにロードするＰＣＮＡ変異体は、より好ましくは、ＰＣＮＡの多量体形成に関わる、
（ａ）８２、８４、１０９番目に相当するアミノ酸からなるＮ末端領域、又は、
（ｂ）１３９、１４３、１４７番目に相当するアミノ酸からなるＣ末端領域に少なくともひとつの改変を有し、ＲＦＣがなくともＤＮＡにロードし、ＤＮＡポリメラーゼの伸長反応を促進する変異体が挙げられる。 PCNA variants that load DNA alone are more preferably involved in PCNA multimer formation,
(A) an N-terminal region consisting of amino acids corresponding to positions 82, 84 and 109, or
(B) Examples include mutants having at least one modification in the C-terminal region consisting of amino acids corresponding to the 139th, 143rd, and 147th amino acids, loading to DNA without RFC and promoting the elongation reaction of DNA polymerase.
例えば、配列番号１３の１４３番目に相当するアミノ酸を塩基性アミノ酸に改変したもの、８２番目と１４３番目を共に中性アミノ酸に改変したもの、１４７番目を中性アミノ酸に改変したもの、又は、１０９番目と１４３番目を共に中性アミノ酸に改変したものなどが挙げられる。
本発明の中性アミノ酸としては、天然のものであれば、グリシン、アラニン、バリン、ロイシン、イソロイシン、フェニルアラニン、チロシン、プロリン、セリン、スレオニン、システイン、メチオニン、アスパラギン、グルタミンが挙げられる。好ましくは、置換部位の周辺部位の立体構造に与える影響が最も小さいアラニンである。塩基性アミノ酸としては、天然のものであれば、アルギニン、ヒスチジン、リシン及びトリプトファンが挙げられる。好ましくはアルギニン又はリジンである。 For example, the amino acid corresponding to position 143 of SEQ ID NO: 13 is changed to a basic amino acid, the positions 82 and 143 are both changed to neutral amino acids, the position 147 is changed to a neutral amino acid, or 109 And the like, in which both the 145th and 143rd are modified to neutral amino acids.
Examples of the neutral amino acid of the present invention include glycine, alanine, valine, leucine, isoleucine, phenylalanine, tyrosine, proline, serine, threonine, cysteine, methionine, asparagine, and glutamine as long as they are natural. Preferably, the alanine has the smallest influence on the three-dimensional structure of the peripheral site of the substitution site. Examples of basic amino acids include arginine, histidine, lysine and tryptophan if they are natural. Arginine or lysine is preferable.
より好ましくは、ＷＯ２００７／００４６５４に記載のＰＣＮＡ変異体が例示されるほか、第１４７番目のアミノ酸残基をアラニンに換えた配列（Ｄ１４７Ａ）、第８２番目、及び第１４３番目のアミノ酸残基をアラニンに変えた配列（Ｒ８２Ａ／Ｄ１４３Ａ、もしくはＲ８２Ａ／Ｅ１４３Ａ）、第１０９番目、及び第１４３番目のアミノ酸残基をアラニンに変えた配列（Ｒ１０９Ａ／Ｄ１４３Ａ、もしくはＲ１０９Ａ／Ｅ１４３Ａ）などが挙げられるが、これらに限定されるものではない。 More preferably, in addition to the PCNA variant described in WO2007 / 004654, a sequence in which the 147th amino acid residue is replaced with alanine (D147A), the 82nd and 143rd amino acid residues are alanine. And the sequence (R109A / D143A or R109A / E143A) in which the 109th and 143rd amino acid residues are changed to alanine. It is not limited to.
また、本発明に用いるＰＣＮＡは発現量を増やすため、配列番号１３又は配列番号１４の７３番目に相当するメチオニンを改変したものでもよい。より好ましくはＭ７３Ｌに改変したものが挙げられるが、これに限定されない。 In addition, PCNA used in the present invention may be a modified methionine corresponding to position 73 of SEQ ID NO: 13 or SEQ ID NO: 14 in order to increase the expression level. More preferable examples include those modified to M73L, but are not limited thereto.
（４．１．２）Ｍｊａ－ＰＣＮＡ
また、本発明に用いるＰＣＮＡは、以下の（Ａ）から（Ｅ）のうちいずれかに示されるＰＣＮＡ単量体である。
（Ａ）配列番号２３に記載のアミノ酸配列における１４２番目のアミノ酸残基を、リジン、アルギニン、ヒスチジン、トリプトファンからなる群から選択されるいずれかのアミノ酸残基に置換したアミノ酸配列からなるポリペプチドからなり、かつ、ＤＮＡポリメラーゼ増幅増強活性を有するポリペプチド。
（Ｂ）（Ａ）で示されるＰＣＮＡ単量体において、さらに、配列番号２３に記載のアミノ酸配列における１４２番目のアミノ酸残基を、リジン、アルギニン、ヒスチジン、トリプトファンからなる群がら選択されるいずれかのアミノ酸残基に置換したアミノ酸配列からなるポリペプチドからなり、以下の（ａ）から（ｅ）のいずれかの置換、欠失、挿入及び／又は付加（これらを纏めて「変異」とも表す。）を含んでなり、かつ、ＤＮＡポリメラーゼ増幅増強活性を有するポリペプチド。
（ａ）８０番目のアミノ酸残基をリジン、ヒスチジン及びトリプトファンからなる群から選択されるいずれかに置換
（ｂ）８２番目のアミノ酸残基をアルギニン、ヒスチジン及びトリプトファンからなる群から選択されるいずれかに置換
（ｃ）１０８番目のアミノ酸残基をリジン、ヒスチジン及びトリプトファンからなる群から選択されるいずれかに置換
（ｄ）１３８番目のアミノ酸残基をグルタミン酸に置換
（ｅ）１４６番目のアミノ酸残基をグルタミン酸に置換
（Ｃ）（Ａ）で示されるＰＣＮＡ単量体において、さらに、配列番号２３に記載のアミノ酸配列における１４２番目のアミノ酸残基を、リジン、アルギニン、ヒスチジン、トリプトファンからなる群から選択されるいずれかのアミノ酸残基に置換したアミノ酸配列からなるポリペプチドからなり、８０番目、８２番目、１０８番目、１３８番目、１４２番目及び第１４６番目に相当するアミノ酸残基以外の、１乃至数個のアミノ酸残基が置換、欠失、挿入又は付加されているアミノ酸配列からなり、かつ、ＤＮＡポリメラーゼ増幅増強活性を有するポリペプチド。
（Ｄ）（Ａ）で示されるＰＣＮＡ単量体において、さらに、配列番号２３に記載のアミノ酸配列の１４２番目のアミノ酸残基のみを、リジン、アルギニン、ヒスチジン、トリプトファンからなる群から選択されるいずれかのアミノ酸残基に置換したアミノ酸配列からなるポリペプチド。
（Ｅ）（Ａ）で示されるＰＣＮＡ単量体において、さらに、配列番号２３で示されるアミノ酸配列との相同性が８０％以上であるアミノ酸配列からなり、かつ、ＤＮＡポリメラーゼ増幅増強活性を有するポリペプチド。 (4.1.2) Mja-PCNA
PCNA used in the present invention is a PCNA monomer shown in any one of the following (A) to (E).
(A) From a polypeptide comprising an amino acid sequence in which the 142nd amino acid residue in the amino acid sequence of SEQ ID NO: 23 is substituted with any amino acid residue selected from the group consisting of lysine, arginine, histidine, and tryptophan And a polypeptide having DNA polymerase amplification enhancing activity.
(B) In the PCNA monomer represented by (A), the 142nd amino acid residue in the amino acid sequence of SEQ ID NO: 23 is any one selected from the group consisting of lysine, arginine, histidine, and tryptophan It consists of a polypeptide consisting of an amino acid sequence substituted with an amino acid residue, and any of the following substitutions (a) to (e), deletions, insertions and / or additions (these are also collectively referred to as “mutation”). And a polypeptide having DNA polymerase amplification enhancing activity.
(A) The 80th amino acid residue is replaced with one selected from the group consisting of lysine, histidine and tryptophan. (B) The 82nd amino acid residue is selected from the group consisting of arginine, histidine and tryptophan. (C) the 108th amino acid residue is replaced with any one selected from the group consisting of lysine, histidine and tryptophan (d) the 138th amino acid residue is replaced with glutamic acid (e) the 146th amino acid residue In the PCNA monomer represented by (C) (A), and the 142nd amino acid residue in the amino acid sequence of SEQ ID NO: 23 is selected from the group consisting of lysine, arginine, histidine, and tryptophan Poly consisting of an amino acid sequence substituted with any amino acid residue 1 to several amino acid residues other than the amino acid residues corresponding to the 80th, 82nd, 108th, 138th, 142nd and 146th amino acids are substituted, deleted, inserted or added. A polypeptide comprising the amino acid sequence having a DNA polymerase amplification enhancing activity.
(D) In the PCNA monomer represented by (A), any one selected from the group consisting of lysine, arginine, histidine, and tryptophan, in which only the 142nd amino acid residue of the amino acid sequence of SEQ ID NO: 23 is further selected A polypeptide comprising an amino acid sequence substituted with any amino acid residue.
(E) A PCNA monomer represented by (A), further comprising an amino acid sequence having a homology with the amino acid sequence represented by SEQ ID NO: 23 of 80% or more and having a DNA polymerase amplification enhancing activity peptide.
配列番号２３において、１４２番目のアミノ酸残基はＰＣＮＡの多量体形成に関わるアミノ酸残基のうちの１つである。ＰＣＮＡの多量体形成に関わるアミノ酸残基は、各単量体のＮ末端側領域とＣ末端側領域とに存在する。ＰＣＮＡ多量体は、一方の単量体のＮ末端側領域と他方の単量体のＣ末端側領域とが界面となって接合することにより形成される。真核細胞及び古細菌においては、多くの場合ＰＣＮＡは三量体を形成する。配列番号２３で示されるアミノ酸配列においては、Ｎ末端領域が下記の（ａ）で示される群の位置に該当し、Ｃ末端領域が下記の（ｂ）で示される群の位置に該当する。
（ａ）８０、８２、１０８番目のアミノ酸残基群
（ｂ）１３８、１４２、１４６番目のアミノ酸残基群 In SEQ ID NO: 23, the 142nd amino acid residue is one of the amino acid residues involved in PCNA multimer formation. Amino acid residues involved in PCNA multimer formation are present in the N-terminal region and C-terminal region of each monomer. The PCNA multimer is formed by joining the N-terminal region of one monomer and the C-terminal region of the other monomer as an interface. In eukaryotic cells and archaea, PCNA often forms trimers. In the amino acid sequence represented by SEQ ID NO: 23, the N-terminal region corresponds to the position of the group indicated by (a) below, and the C-terminal region corresponds to the position of the group indicated by (b) below.
(A) 80, 82, 108th amino acid residue group (b) 138, 142, 146th amino acid residue group
上記（２）のポリペプチドは、ＤＮＡポリメラーゼ増幅増強活性を保持する限度で、配列番号２３に示されるアミノ酸配列において、１若しくは数個のアミノ酸配残基が置換、欠失、挿入及び／又は付加（以下、これらを纏めて「変異」とも表す。）されたアミノ酸配列からなるポリペプチドである。一又は数個の変異は、制限酵素処理、エキソヌクレアーゼやＤＮＡリガーゼ等による処理、位置指定突然変異導入法やランダム突然変異導入法（Ｍｏｌｅｃｕｌａｒ　Ｃｌｏｎｉｎｇ，　Ｔｈｉｒｄ　Ｅｄｉｔｉｏｎ，　Ｃｈａｐｔｅｒ　１３　，Ｃｏｌｄ　Ｓｐｒｉｎｇ　Ｈａｒｂｏｒ　Ｌａｂｏｒａｔｏｒｙ　Ｐｒｅｓｓ，　Ｎｅｗ　Ｙｏｒｋ）など公知の手法を利用して、後述する本発明のＰＣＮＡ単量体をコードするＤＮＡに変異を導入することによって実施することが可能である。また、紫外線照射など他の方法によってもバリアントＰＣＮＡ単量体を得ることができる。バリアントＰＣＮＡ単量体には、ＰＣＮＡを保持する微生物の個体差、種や属の違いに基づく場合などの天然に生じるバリアント（例えば、一塩基多型）も含まれる。 In the polypeptide of (2) above, one or several amino acid residues are substituted, deleted, inserted and / or added in the amino acid sequence shown in SEQ ID NO: 23 as long as the DNA polymerase amplification enhancing activity is retained. (Hereinafter, these are collectively referred to as “mutation”.) A polypeptide comprising an amino acid sequence. One or several mutations include restriction enzyme treatment, treatment with exonuclease, DNA ligase, etc., site-directed mutagenesis method or random mutagenesis method (Molecular Cloning, Third Edition, Chapter 13, Cold Spring Harbor Laboratory Press, New (York) and the like can be carried out by introducing mutation into DNA encoding the PCNA monomer of the present invention described later. The variant PCNA monomer can also be obtained by other methods such as ultraviolet irradiation. Variant PCNA monomers also include naturally occurring variants (for example, single nucleotide polymorphisms) such as those based on individual differences in microorganisms holding PCNA, differences in species or genera.
上記［３］のポリペプチドは、ＤＮＡポリメラーゼ増幅増強活性を保持することを限度で、配列番号２３に示されるアミノ酸配列と比較した同一性が８０％以上であるアミノ酸配列からなるポリペプチドである。好ましくは、本発明のＰＣＮＡ単量体が有するアミノ酸配列と配列番号２３に示されるアミノ酸配列との同一性は、８５％以上であり、より好ましくは８８％以上、更に好ましくは９０％以上、より更に好ましくは９３％以上、一層好ましくは９５％以上、特に好ましくは９８％以上、最も好ましくは９９％以上である。このような一定以上の同一性を有するアミノ酸配列からなるポリペプチドは、上述するような公知の遺伝子工学的手法に基づいて作成することができる。 The polypeptide of the above [3] is a polypeptide comprising an amino acid sequence having an identity of 80% or more compared to the amino acid sequence shown in SEQ ID NO: 23, as long as it retains DNA polymerase amplification enhancing activity. Preferably, the identity between the amino acid sequence of the PCNA monomer of the present invention and the amino acid sequence shown in SEQ ID NO: 23 is 85% or more, more preferably 88% or more, still more preferably 90% or more, more More preferably, it is 93% or more, more preferably 95% or more, particularly preferably 98% or more, and most preferably 99% or more. Such a polypeptide comprising an amino acid sequence having a certain identity or more can be prepared based on the known genetic engineering techniques as described above.
上記の［２］又は［３］に記載したようなＰＣＮＡとして、好ましくは、ＰＣＮＡの精製を簡便にすべくＮ末端に挿入したＨｉｓタグなどのアフィニティタグを付加するものが挙げられるが、特にこれに限定されない。 The PCNA as described in [2] or [3] above preferably includes those to which an affinity tag such as a His tag inserted at the N-terminus is added in order to simplify the purification of PCNA. It is not limited to.
上記のＰＣＮＡ単量体においては、配列番号２３における１４２番目のアミノ酸残基が塩基性アミノ酸残基に置換されるが、置換する塩基性アミノ酸の種類は特に限定されない。塩基性アミノ酸としては、天然のものであれば、アルギニン、ヒスチジン、リシン及びトリプトファンが挙げられる。好ましくはアルギニン又はリジンである。 In the PCNA monomer, the 142nd amino acid residue in SEQ ID NO: 23 is substituted with a basic amino acid residue, but the type of basic amino acid to be substituted is not particularly limited. Examples of basic amino acids include arginine, histidine, lysine and tryptophan if they are natural. Arginine or lysine is preferable.
（４．２）
上記ＰＣＮＡを得る方法はＰＣＮＡ遺伝子を必要に応じて発現ベクターに移し替え、宿主として例えば大腸菌を、該発現ベクターを用いて形質転換した後、アンピシリン等の薬剤を含む寒天培地に塗布し、コロニーを形成させる。コロニーを栄養培地、例えばＬＢ培地や２×ＹＴ培地に接種し、３７℃で１２～２０時間培養した後、菌体を破砕して粗酵素液を抽出する。ベクターとしては、ｐＢｌｕｅｓｃｒｉｐｔ由来のものが好ましい。菌体を破砕する方法としては公知のいかなる手法を用いても良いが、例えば超音波処理、フレンチプレスやガラスビーズ破砕のような物理的破砕法やリゾチームのような溶菌酵素を用いることができる。この粗酵素液を８０℃、３０分間熱処理し、遠心することで宿主由来のタンパクを除去し、ＳＤＳ－ＰＡＧＥに供することで、目的タンパク質の発現を確認することができる。 (4.2)
In the method for obtaining PCNA, the PCNA gene is transferred to an expression vector as necessary, for example, Escherichia coli as a host, transformed with the expression vector, applied to an agar medium containing a drug such as ampicillin, and colonies are obtained. Let it form. The colony is inoculated into a nutrient medium such as LB medium or 2 × YT medium and cultured at 37 ° C. for 12 to 20 hours, and then the cells are crushed and the crude enzyme solution is extracted. A vector derived from pBluescript is preferable. Any known method may be used as a method for crushing bacterial cells. For example, ultrasonic treatment, a physical crushing method such as French press or glass bead crushing, or a lytic enzyme such as lysozyme can be used. This crude enzyme solution is heat-treated at 80 ° C. for 30 minutes, centrifuged to remove host-derived protein, and subjected to SDS-PAGE, thereby confirming the expression of the target protein.
上記方法により選抜された菌株から精製ＰＣＮＡを取得する方法は、いかなる手法を用いても良いが、例えば下記のような方法がある。栄養培地に培養して得られた菌体を回収した後、酵素的又は物理的破砕法により破砕抽出して粗酵素液を得る。得られた粗酵素抽出液から熱処理、例えば８０℃、３０分間処理し、その後硫安沈殿によりＰＣＮＡ画分を回収する。この粗酵素液をセファデックスＧ－２５（アマシャムファルマシア・バイオテク製）を用いたゲル濾過等の方法により脱塩を行うことができる。この操作の後、Ｑセファロースカラムクロマトグラフィーにより分離、精製し、精製酵素標品を得ることができる。該精製酵素標品は、ＳＤＳ－ＰＡＧＥによってほぼ単一バンドを示す程度に純化される。 Any method may be used as a method for obtaining purified PCNA from the strain selected by the above method, for example, the following method. After the cells obtained by culturing in the nutrient medium are collected, the crude enzyme solution is obtained by crushing and extracting by an enzymatic or physical crushing method. The obtained crude enzyme extract is heat-treated, for example, at 80 ° C. for 30 minutes, and then the PCNA fraction is recovered by ammonium sulfate precipitation. This crude enzyme solution can be desalted by a method such as gel filtration using Sephadex G-25 (manufactured by Amersham Pharmacia Biotech). After this operation, it can be separated and purified by Q Sepharose column chromatography to obtain a purified enzyme preparation. The purified enzyme preparation is purified by SDS-PAGE to such an extent that it almost shows a single band.
（４．３）ＤＮＡポリメラーゼ増幅増強活性
上記ＰＣＮＡ変異体が単独でＤＮＡにロードできるか（ＤＮＡポリメラーゼ増幅増強活性があるか）どうかは、ＰＣＲによって評価できる。鋳型となるＤＮＡ、緩衝材、マグネシウム、ｄＮＴＰｓ、プライマー、及びファミリーＢに属するＤＮＡポリメラーゼを含むＰＣＲ反応液に、評価するＰＣＮＡを添加し、ＰＣＮＡ添加なしのもの、また野生型ＰＣＮＡ添加のものと増幅量を比較することで、単独でＤＮＡにロードできるかを確認することができる。野生型のＰＣＮＡをはじめ、単独でＤＮＡにロードできないＰＣＮＡは添加しても、ＰＣＲの増幅量は変化せず、むしろ増幅量を減らす傾向がある。一方、単独でＤＮＡにロードできる変異体は、ＰＣＮＡ添加なしのものと比較することでＤＮＡポリメラーゼ増幅増強活性を評価することができる。 (4.3) DNA polymerase amplification enhancing activity Whether the PCNA mutant can be loaded into DNA alone (having DNA polymerase amplification enhancing activity) can be evaluated by PCR. PCNA to be evaluated is added to a PCR reaction solution containing DNA as a template, buffer material, magnesium, dNTPs, primers, and DNA polymerase belonging to Family B, amplified with or without the addition of PCNA By comparing the amounts, it can be confirmed whether the DNA can be loaded alone. Even if wild-type PCNA or other PCNA that cannot be loaded into DNA alone is added, the PCR amplification amount does not change, but rather the amplification amount tends to decrease. On the other hand, a mutant that can be loaded into DNA alone can be evaluated for DNA polymerase amplification enhancing activity by comparing with a mutant without addition of PCNA.
本発明において「ＰＣＮＡ変異体が単独でＤＮＡにロードできるかどうか」の評価（ＤＮＡポリメラーゼ増幅増強活性の評価）は、以下の方法に従う。
ＫＯＤ　Ｄａｓｈ（Ｔｏｙｏｂｏ製）添付の１０×ＰＣＲ　Ｂｕｆｆｅｒ（反応に用いる濃度の１０倍に濃縮されている）を用いて、
１×ＰＣＲ　Ｂｕｆｆｅｒ、
０．２ｍＭ　ｄＮＴＰｓ、
約３．６ｋｂを増幅する１５ｐｍｏｌの配列番号１５及び１６に記載のプライマー、
１０ｎｇのヒトゲノムＤＮＡ（Ｒｏｃｈｅ製Ｈｕｍａｎ　Ｇｅｎｏｍｉｃ　ＤＮＡ；型番１１６９１１１２００１）、
１Ｕ　ＫＯＤ　－Ｐｌｕｓ－　ポリメラーゼ
を含むよう反応液を調製し、
５０μｌの反応液中に、評価するＰＣＮＡを２５０ｎｇ添加し、
９４℃、３０秒の前反応の後、９８℃、１０秒→６８℃、３０秒を３０サイクル繰り返すスケジュールでＰＣＲを行う。反応終了後、５μｌの反応液について１％アガロース電気泳動を行い、エチジウムブロマイド染色し、紫外線照射下約３．６ｋｂの増幅ＤＮＡ断片を、野生型ＰＣＮＡを添加したものと比較することで、単独でＤＮＡにロードできるＰＣＮＡかどうかを評価することができる。単独でＤＮＡにロードできるＰＣＮＡは添加によって増幅量が増加する。 In the present invention, the evaluation of “whether the PCNA mutant can be loaded alone into DNA” (evaluation of DNA polymerase amplification enhancing activity) follows the following method.
Using 10 × PCR Buffer (concentrated to 10 times the concentration used in the reaction) attached to KOD Dash (manufactured by Toyobo),
1 × PCR Buffer,
0.2 mM dNTPs,
15 pmol of the primers set forth in SEQ ID NOs: 15 and 16 that amplify about 3.6 kb,
10 ng of human genomic DNA (Roche's Human Genomic DNA; model number 116111112001),
Prepare a reaction solution containing 1U KOD-Plus-polymerase,
In a 50 μl reaction solution, 250 ng of PCNA to be evaluated was added,
After the pre-reaction at 94 ° C. for 30 seconds, PCR is performed on a schedule in which 98 ° C., 10 seconds → 68 ° C., 30 seconds are repeated 30 cycles. After completion of the reaction, 5% of the reaction solution was subjected to 1% agarose electrophoresis, stained with ethidium bromide, and the amplified DNA fragment of about 3.6 kb under ultraviolet irradiation was compared with the one added with wild type PCNA alone. It can be assessed whether PCNA can be loaded into DNA. The amount of amplification increases with the addition of PCNA that can be loaded into DNA alone.
増幅量の増加を定量的に評価するためには、本発明においては、Ｇｅｌ　Ｐｒｏ　Ａｎａｌｙｚｅｒ（Ｍｅｄｉａ Ｃｙｂｅｒｎｅｔｉｃｓ）という解析ソフトウェアを利用することにより、増幅量を数値化することにより行う。このような方法で増幅量を比較したとき、ＰＣＮＡを添加した場合の増幅量が、ＰＣＮＡを添加しなかった場合の増幅量の１．０倍（好ましくは１．２倍、さらに好ましくは１．５倍、さらに好ましくは２倍、３倍）を超える。もしくは増幅していなかったターゲットが増幅すれば、そのＰＣＮＡが「ＤＮＡポリメラーゼ増幅増強活性を有する」と判断する。 In order to quantitatively evaluate the increase in the amplification amount, in the present invention, the amplification amount is digitized by using analysis software called Gel Pro Analyzer (Media Cybernetics). When the amplification amounts are compared by such a method, the amplification amount when PCNA is added is 1.0 times (preferably 1.2 times, more preferably 1.times. The amplification amount when PCNA is not added. 5 times, more preferably 2 times and 3 times). Alternatively, if a target that has not been amplified is amplified, it is determined that the PCNA has “DNA polymerase amplification enhancing activity”.
ＰＣＮＡの改変についても、ＤＮＡポリメラーゼの改変と同様に行うことができる。 The modification of PCNA can be performed in the same manner as the modification of DNA polymerase.
（５）高濃度プライマー
本発明におけるプライマー濃度は、０．６μＭ以上である。一般に、高濃度のプライマーは非特異やプライマーダイマーの原因になるため、通常のＰＣＲでは０．１～０．５μＭで使用するのが一般的といわれている。本発明における高速ＰＣＲ条件では、サイクルのアニーリングステップが短いため、高い濃度でないとプライミングの効率が低下することがわかっている。逆に、高速ＰＣＲ条件ではアニーリングステップが短いため、高濃度のプライマーでも非特異やプライマーダイマーが生じにくい。本発明におけるプライマー濃度は、より好ましくは０．８μＭ以上、さらに好ましくは１．０μＭ以上である。なお、４．０μＭ、好ましくは２．０μＭ、さらに好ましくは１．４μＭを超えないことが好ましい。 (5) High concentration primer The primer concentration in this invention is 0.6 micromol or more. In general, since a high concentration of primer causes non-specificity or primer dimer, it is said to be generally used at 0.1 to 0.5 μM in normal PCR. Under the high-speed PCR conditions in the present invention, since the annealing step of the cycle is short, it is known that the efficiency of priming is reduced unless the concentration is high. On the other hand, since the annealing step is short under high-speed PCR conditions, non-specificity and primer dimer are unlikely to occur even at high concentration primers. The primer concentration in the present invention is more preferably 0.8 μM or more, and further preferably 1.0 μM or more. In addition, it is preferable that it does not exceed 4.0 μM, preferably 2.0 μM, more preferably 1.4 μM.
本明細書において、「プライマー濃度０．６μＭ以上」と言う場合、一組のプライマー対におけるフォワード（Ｆ）、リバース（Ｒ）それぞれのプライマー濃度が同じであってもよいし、どちらか一方のプライマー濃度が他方の濃度を超えていても（プライマー濃度が非対称であっても）よい。また、マルチプレックスＰＣＲの場合は、前記のほか、すべてのプライマー対間の濃度が同じであってもよいし、各プライマー対間で濃度が異なっていてもよい。 In the present specification, when “primer concentration is 0.6 μM or more”, the forward (F) and reverse (R) primer concentrations in a pair of primer pairs may be the same, or either primer The concentration may exceed the other concentration (primer concentration may be asymmetric). In the case of multiplex PCR, in addition to the above, the concentration between all primer pairs may be the same, or the concentration may differ between each primer pair.
（６）熱サイクルの速度
本発明におけるＰＣＲにおいては、熱サイクルにおける昇温又は降温の速度が０．１℃／秒から２０℃／秒で実施することが好ましい。より好ましくは５℃／秒から２０℃／秒の範囲、さらに好ましくは１０℃／秒から２０℃／秒の範囲である。このような昇温又は降温の速度の熱サイクルを実施するためには、空気による加熱及び冷却により温度制御することができるようなサーマルサイクラーを用いることが特に好ましい。 (6) Speed of thermal cycle The PCR in the present invention is preferably carried out at a rate of temperature rise or temperature drop in the thermal cycle of 0.1 ° C / second to 20 ° C / second. More preferred is a range of 5 ° C./second to 20 ° C./second, and further preferred is a range of 10 ° C./second to 20 ° C./second. In order to carry out a thermal cycle at such a rate of temperature increase or decrease, it is particularly preferable to use a thermal cycler that can be temperature controlled by heating and cooling with air.
（７）阻害物質を含んだ状態でのＰＣＲ
本発明のＰＣＲ方法は、阻害物質を含んだ状態で実施することに適している。本発明における阻害物質とは、例えば生体試料を示すが、ＰＣＲを阻害する物質であれば特に限定されない。生体試料は、生体から採取された試料であれば特に限定されない。例えば、体毛、爪、口腔粘膜などの動植物組織、血液などの体液、糞便や尿などの***物、細胞、細菌、ウイルス等をいう。体液には血液や唾液が含まれ、細胞には血液から分離した白血球が含まれるが、これらに限定されるものではない。 (7) PCR with inhibitors
The PCR method of the present invention is suitable for carrying out in a state containing an inhibitor. The inhibitory substance in the present invention refers to, for example, a biological sample, but is not particularly limited as long as it is a substance that inhibits PCR. The biological sample is not particularly limited as long as it is a sample collected from a living body. For example, it refers to animal and plant tissues such as body hair, nails, oral mucosa, body fluids such as blood, excrement such as feces and urine, cells, bacteria, viruses and the like. Body fluid includes blood and saliva, and cells include, but are not limited to, leukocytes separated from blood.
（８）抗体
本発明においては、必要に応じて、さらに耐熱性ＤＮＡポリメラーゼのポリメラーゼ活性及び／又は３’－５’エキソヌクレアーゼ活性を抑制する活性を有する抗体を用いても良い。前記抗体としては、モノクローナル抗体、ポリクローナル抗体などが挙げられる。本反応組成は、ＰＣＲの感度上昇、非特異的増幅の軽減に特に有効である。 (8) Antibody In the present invention, an antibody having an activity of suppressing the polymerase activity and / or 3′-5 ′ exonuclease activity of a heat-resistant DNA polymerase may be used as necessary. Examples of the antibody include a monoclonal antibody and a polyclonal antibody. This reaction composition is particularly effective for increasing the sensitivity of PCR and reducing nonspecific amplification.
（９）核酸増幅法を実行するための試薬
本発明のＰＣＲを実行するための試薬は、ファミリーＢに属するＤＮＡポリメラーゼ、及びＰＣＮＡ及び０．６μＭ以上の濃度のプライマーを反応液中に含み、それ以外の構成は特に限定されない。なお、前記試薬にはキットの形態も含まれる。 (9) Reagent for carrying out the nucleic acid amplification method The reagent for carrying out the PCR of the present invention comprises a DNA polymerase belonging to Family B, PCNA and a primer having a concentration of 0.6 μM or more in the reaction solution. Other configurations are not particularly limited. The reagent includes a kit form.
以下、実施例に基づき本発明をより具体的に説明する。もっとも、本発明は、下記実施例により、特に限定されるものではない。 Hereinafter, based on an Example, this invention is demonstrated more concretely. However, the present invention is not particularly limited by the following examples.
（実施例１）
ＫＯＤ　ＤＮＡポリメラーゼ変異体の作製
後述の実施例に用いるために、サーモコッカス・コダカラエンシス　ＫＯＤ１株由来の改変型耐熱性ＤＮＡポリメラーゼ（Ｙ７Ａ／Ｐ３６Ｈ／Ｎ２１０Ｄ変異体）遺伝子を含有するプラスミドを作製した。変異導入に使用されるＤＮＡ鋳型は、ｐＢｌｕｅｓｃｒｉｐｔにクローニングされたサーモコッカス・コダカラエンシス　ＫＯＤ１株由来の改変型耐熱性ＤＮＡポリメラーゼ遺伝子（配列番号１７）（ｐＫＯＤ）を用いた。
変異導入にはＫＯＤ　－Ｐｌｕｓ－　Ｍｕｔａｇｅｎｅｓｉｓ　Ｋｉｔ（Ｔｏｙｏｂｏ製）を用いて、方法は取扱い説明書に準じて行った。なお、変異体の確認は塩基配列の解読で行った。得られたプラスミドによりエシェリシア・コリＪＭ１０９を形質転換し、酵素調製に用いた。 (Example 1)
Preparation of KOD DNA polymerase mutant Plasmid containing a modified thermostable DNA polymerase (Y7A / P36H / N210D mutant) gene derived from Thermococcus kodakaraensis KOD1 strain for use in the examples described below Was made. As a DNA template used for mutagenesis, a modified heat-resistant DNA polymerase gene (SEQ ID NO: 17) (pKOD) derived from Thermococcus kodakaraensis KOD1 strain cloned in pBluescript was used.
Mutation was introduced using KOD-Plus-Mutageness Kit (manufactured by Toyobo) according to the instruction manual. The mutant was confirmed by decoding the base sequence. Escherichia coli JM109 was transformed with the obtained plasmid and used for enzyme preparation.
（実施例２）
改変型耐熱性ＤＮＡポリメラーゼの作製
実施例１で得られた菌体の培養は以下のようにして実施した。まず、滅菌処理した１００μｇ／ｍＬのアンピシリンを含有するＴＢ培地（Ｍｏｌｅｃｕｌａｒ　ｃｌｏｎｉｎｇ　２ｎｄ　ｅｄｉｔｉｏｎ、ｐ．Ａ．２）８０ｍＬを、５００ｍＬ坂口フラスコに分注した。この培地に、予め１００μｇ／ｍＬのアンピシリンを含有する３ｍＬのＬＢ培地（１％バクトトリプトン、０．５％酵母エキス、０．５％塩化ナトリウム；ギブコ製）で３７℃、１６時間培養したエシェリシア・コリＪＭ１０９（プラスミド形質転換株）（試験管使用）を接種し、３７℃にて１６時間通気培養した。培養液より菌体を遠心分離により回収し、５０ｍＬの破砕緩衝液（３０ｍＭ　Ｔｒｉｓ－ＨＣｌ緩衝液（ｐＨ８．０）、３０ｍＭ　ＮａＣｌ、０．１ｍＭ　ＥＤＴＡ）に懸濁後、ソニケーション処理により菌体を破砕し、細胞破砕液を得た。次に、細胞破砕液を８０℃にて１５分間処理した後、遠心分離にて不溶性画分を除去した。更に、ポリエチレンイミンを用いた除核酸処理、硫安塩析、ヘパリンセファロースクロマトグラフィーを行い、最後に保存緩衝液（５０ｍＭ　Ｔｒｉｓ－ＨＣｌ緩衝液（ｐＨ８．０）、５０ｍＭ　塩化カリウム、１ｍＭ　ジチオスレイトール、０．１％　Ｔｗｅｅｎ２０、０．１％　ノニデットＰ４０、５０％　グリセリン）に置換し、改変型耐熱性ＤＮＡポリメラーゼを得た。
上記精製工程のＤＮＡポリメラーゼ活性測定は、上記のＤＮＡポリメラーゼ活性測定法に従い行った。また、酵素活性が高い場合はサンプルを希釈して測定を行った。 (Example 2)
Production of modified thermostable DNA polymerase The cells obtained in Example 1 were cultured as follows. First, 80 mL of TB medium (Molecular cloning 2nd edition, p.A.2) containing sterilized 100 μg / mL ampicillin was dispensed into a 500 mL Sakaguchi flask. Escherichia cultured at 37 ° C. for 16 hours in 3 mL of LB medium (1% bactotryptone, 0.5% yeast extract, 0.5% sodium chloride; manufactured by Gibco) containing 100 μg / mL ampicillin in advance. -Coli JM109 (plasmid transformant) (using a test tube) was inoculated and cultured at 37 ° C for 16 hours with aeration. The bacterial cells are collected from the culture solution by centrifugation, suspended in 50 mL of disruption buffer (30 mM Tris-HCl buffer (pH 8.0), 30 mM NaCl, 0.1 mM EDTA), and then subjected to sonication. By crushing, a cell lysate was obtained. Next, the cell lysate was treated at 80 ° C. for 15 minutes, and then the insoluble fraction was removed by centrifugation. Further, nucleic acid treatment using polyethyleneimine, ammonium sulfate precipitation, and heparin sepharose chromatography were performed. Finally, a storage buffer (50 mM Tris-HCl buffer (pH 8.0), 50 mM potassium chloride, 1 mM dithiothreitol, 0 1% Tween 20, 0.1% Nonidet P40, 50% glycerin) to obtain a modified thermostable DNA polymerase.
The DNA polymerase activity in the purification step was measured according to the above DNA polymerase activity measurement method. When the enzyme activity was high, the sample was diluted for measurement.
（実施例３）
ＫＯＤ－ＰＣＮＡ変異体の作製
サーモコッカス・コダカラエンシス　ＫＯＤ１株由来の改変型耐熱性ＰＣＮＡ（Ｍ７３Ｌ／Ｄ１４７Ａ変異体）遺伝子を含有するプラスミドを作製した。変異導入に使用されるＤＮＡ鋳型は、ｐＢｌｕｅＳｃｒｉｐｔにクローニングされたサーモコッカス・コダカラエンシス　ＫＯＤ１株由来のＰＣＮＡ（配列番号１８）（ｐＫＯＤＰＣＮＡ）を用いた。変異導入にはＫＯＤ　－Ｐｌｕｓ－　Ｍｕｔａｇｅｎｅｓｉｓ　Ｋｉｔ（Ｔｏｙｏｂｏ製）を用いて、方法は取扱い説明書に準じて行った。なお、変異体の確認は塩基配列の解読で行った。得られたプラスミドによりエシェリシア・コリＤＨ５αを形質転換し、酵素調製に用いた。 (Example 3)
Preparation of KOD-PCNA mutant A plasmid containing a modified thermostable PCNA (M73L / D147A mutant) gene derived from Thermococcus kodakaraensis KOD1 strain was prepared. As a DNA template used for mutagenesis, PCNA (SEQ ID NO: 18) (pKODPCNA) derived from Thermococcus kodakaraensis KOD1 strain cloned in pBlueScript was used. Mutation was introduced using KOD-Plus-Mutageness Kit (manufactured by Toyobo) according to the instruction manual. The mutant was confirmed by decoding the base sequence. Escherichia coli DH5α was transformed with the obtained plasmid and used for enzyme preparation.
（実施例４）
改変型耐熱性ＰＣＮＡの作製
実施例３で得られた菌体の培養は、以下のようにして実施した。まず、滅菌処理した１００μｇ／ｍＬのアンピシリンを含有するＴＢ培地（Ｍｏｌｅｃｕｌａｒ　ｃｌｏｎｉｎｇ　２ｎｄ　ｅｄｉｔｉｏｎ、ｐ．Ａ．２）８０ｍＬを、５００ｍＬ坂口フラスコに分注した。この培地に予め１００μｇ／ｍＬのアンピシリンを含有する３ｍＬのＬＢ培地（１％　バクトトリプトン、０．５％　酵母エキス、０．５％　塩化ナトリウム；ギブコ製）で３７℃、１６時間培養したエシェリシア・コリＤＨ５α（プラスミド形質転換株）（試験管使用）を接種し、３７℃にて１６時間通気培養した。培養液より菌体を遠心分離により回収し、５０ｍＬの破砕緩衝液（３０ｍＭ　Ｔｒｉｓ－ＨＣｌ緩衝液（ｐＨ８．０）、３０ｍＭ　ＮａＣｌ、０．１ｍＭ　ＥＤＴＡ）に懸濁後、ソニケーション処理により菌体を破砕し、細胞破砕液を得た。次に細胞破砕液を８０℃にて１５分間処理した後、遠心分離にて不溶性画分を除去した。更に、ポリエチレンイミンを用いた除核酸処理、硫安塩析、Ｑセファロースクロマトグラフィーを行い、最後に保存緩衝液（５０ｍＭ　Ｔｒｉｓ－ＨＣｌ緩衝液（ｐＨ８．０）、５０ｍＭ　塩化カリウム、１ｍＭ　ジチオスレイトール、０．１％　Ｔｗｅｅｎ２０、０．１％　ノニデットＰ４０、５０％　グリセリン）に置換し、改変型耐熱性ＰＣＮＡを得た。 Example 4
Production of modified heat-resistant PCNA The cells obtained in Example 3 were cultured as follows. First, 80 mL of TB medium (Molecular cloning 2nd edition, p.A.2) containing sterilized 100 μg / mL ampicillin was dispensed into a 500 mL Sakaguchi flask. Escherichia cultivated at 37 ° C. for 16 hours in 3 mL of LB medium (1% bactotryptone, 0.5% yeast extract, 0.5% sodium chloride; Gibco) containing 100 μg / mL ampicillin in advance in this medium. E. coli DH5α (plasmid transformant) (using a test tube) was inoculated and cultured at 37 ° C. for 16 hours with aeration. The bacterial cells are collected from the culture solution by centrifugation, suspended in 50 mL of disruption buffer (30 mM Tris-HCl buffer (pH 8.0), 30 mM NaCl, 0.1 mM EDTA), and then subjected to sonication. By crushing, a cell lysate was obtained. Next, the cell lysate was treated at 80 ° C. for 15 minutes, and then the insoluble fraction was removed by centrifugation. Furthermore, the nucleic acid treatment using polyethyleneimine, ammonium sulfate precipitation, and Q sepharose chromatography were performed. Finally, a storage buffer (50 mM Tris-HCl buffer (pH 8.0), 50 mM potassium chloride, 1 mM dithiothreitol, 0 1% Tween20, 0.1% Nonidet P40, 50% glycerin) to obtain modified heat-resistant PCNA.
（実施例５）
プライマー濃度、マグネシウム濃度の検討
上記で得られたＫＯＤ　Ｙ７Ａ／Ｐ３６Ｈ／Ｎ２１０Ｄ、及びＫＯＤ　ＰＣＮＡ　Ｄ１４７Ａを用いて、高速ＰＣＲを実施し、プライマー濃度の影響、及びマグネシウム濃度の影響を確認した。
ＰＣＲにはＫＯＤ　Ｄａｓｈ（Ｔｏｙｏｂｏ製）添付の１０×ＰＣＲ　Ｂｕｆｆｅｒを１０倍希釈した　１×ＰＣＲ　Ｂｕｆｆｅｒ（１．２ｍＭ　ＭｇＳＯ４を含む）に、０．２ｍＭ　ｄＮＴＰｓ、１Ｕ　ＫＯＤ　ＤＮＡポリメラーゼ変異体、２５０ｎｇ　ＫＯＤ　ＰＣＮＡ変異体、及び、１／３０，０００に希釈したＳＹＢＲ（登録商標）　ＧｒｅｅｎＩを含む５０μｌの反応液中に、以下の（ａ）又は（ｂ）を添加し、それぞれでＣｔ値を比較した。
（ａ）サルモネラのｉｎｖＡ遺伝子（約７００ｂｐ）を増幅する配列番号１９及び２０に記載のプライマー、及び、５０コピー相当のサルモネラ菌ゲノム（ｂ）アクチン遺伝子（約５５０ｂｐ）を増幅する配列番号２１及び２２に記載のプライマー、及び、５０コピー相当のヒトゲノムプライマー濃度は０．２、０．４、０．６μＭの３水準を設定した。また、ＭｇＳＯ４を添加して、Ｍｇ濃度を１．２、２、４、６、８ｍＭの５水準で設定し、前記プライマー濃度３水準との組合せをそれぞれ検討した。
ＰＣＲは、９４℃、３０秒の前反応の後、９８℃、０秒→５５℃、０秒→６８℃、０秒の高速サイクルを５０サイクル繰り返すスケジュールでＬｉｇｈｔ　ｃｙｃｌｅｒ２．０を用いて、昇温及び降温の速度を２０℃／秒で行った。反応時間は約２０分で実施された。 (Example 5)
Examination of primer concentration and magnesium concentration High-speed PCR was performed using the KOD Y7A / P36H / N210D and KOD PCNA D147A obtained above, and the effects of the primer concentration and the magnesium concentration were confirmed. .
For PCR, 10 × PCR Buffer attached to KOD Dash (manufactured by Toyobo) was diluted 10-fold, 1 × PCR Buffer (containing 1.2 mM MgSO 4 ), 0.2 mM dNTPs, 1 U KOD DNA polymerase mutant, 250 ng KOD PCNA The following (a) or (b) was added to 50 μl of a reaction solution containing the mutant and SYBR (registered trademark) Green I diluted 1 / 30,000, and the Ct values were compared with each other.
(A) the primers of SEQ ID NOs: 19 and 20 that amplify the Salmonella invA gene (about 700 bp), and the SEQ ID NOs: 21 and 22 that amplify the Salmonella genome corresponding to 50 copies (b) the actin gene (about 550 bp) Three levels of 0.2, 0.4, and 0.6 μM were set for the described primers and the human genome primer concentration corresponding to 50 copies. In addition, MgSO 4 was added, the Mg concentration was set at five levels of 1.2, 2, 4 , 6, and 8 mM, and combinations with the primer concentration of 3 levels were examined.
The PCR was performed using Light cycler 2.0 with a schedule that repeats 50 cycles of a high-speed cycle of 98 ° C, 0 seconds → 55 ° C, 0 seconds → 68 ° C, 0 seconds after a pre-reaction of 94 ° C for 30 seconds. The rate of temperature decrease was 20 ° C./second. The reaction time was about 20 minutes.
図１は、プライマー濃度、Ｍｇ濃度を変えて、５０コピー相当のサルモネラ菌ゲノム又はヒトゲノムから高速ＰＣＲで増幅を比較した場合のＣｔ値、図２はそれらの融解曲線を示す。プライマー濃度は、０．２、０．４、０．６μＭ、Ｍｇ濃度は１．２、２、４、６、８ｍＭで実施し、融解曲線で非特異的な増幅が見られる場合、Ｃｔ値は非特異増幅の量に応じて、丸で囲いマーキング（目的増幅もあるが非特異的増幅もある）、四角で囲いマーキングした（非特異的増幅のみ見られる）。 FIG. 1 shows Ct values when amplification is compared by high-speed PCR from 50 copies worth of Salmonella genome or human genome with different primer concentrations and Mg concentrations, and FIG. 2 shows their melting curves. When the primer concentration is 0.2, 0.4, 0.6 μM, the Mg concentration is 1.2, 2, 4, 6, 8 mM, and non-specific amplification is seen in the melting curve, the Ct value is Depending on the amount of non-specific amplification, circled markings (some with target amplification but also non-specific amplification), squares marked (only non-specific amplification is seen).
サルモネラ菌ｉｎｖＡ遺伝子の増幅は、プライマー、Ｍｇ濃度をそれぞれ高めることで、Ｃｔが改善される傾向が見られた。
これに対して、アクチン遺伝子の増幅は、Ｍｇ濃度が低い場合は、プライマー濃度を高めることでＣｔが改善されたが、Ｍｇ濃度を上げると非特異増幅が出現する結果となった。プライマー、Ｍｇは濃度を上げると、それぞれＰＣＲの効率を高める働きがある、一方、あまり上げすぎると非特異的増幅につながることが知られている。しかし、今回のような高速ＰＣＲにおいては、高濃度のＭｇは通常のＰＣＲと同様、非特異的増幅を増やす可能性が高まるものの、高濃度のプライマーでは非特異的増幅は生じにくくする結果になった。
高速ＰＣＲ条件では、サイクルのアニーリングステップが短いため、高濃度のプライマーを用いても非特異的増幅やプライマーダイマーが生じにくく、特異性を保ったまま、ＰＣＲ効率のみを向上させる働きがあることが考えられる。 Amplification of Salmonella invA gene tended to improve Ct by increasing the primer and Mg concentrations, respectively.
In contrast, the amplification of the actin gene improved Ct by increasing the primer concentration when the Mg concentration was low, but non-specific amplification appeared when the Mg concentration was increased. It is known that when the concentration of the primer and Mg is increased, each of them has a function of increasing the efficiency of PCR, whereas when it is excessively increased, it leads to non-specific amplification. However, in high-speed PCR such as this, high concentrations of Mg increase the possibility of increasing non-specific amplification, as in normal PCR, but non-specific amplification is less likely to occur with high concentrations of primers. It was.
Under high-speed PCR conditions, the cycle annealing step is short, so non-specific amplification and primer dimers are unlikely to occur even when using high concentrations of primers, and it may work to improve only PCR efficiency while maintaining specificity. Conceivable.
（実施例６）
プライマー濃度の検討
実施例５と同様、上記で得られたＫＯＤ　Ｙ７Ａ／Ｐ３６Ｈ／Ｎ２１０Ｄ、及びＫＯＤ　ＰＣＮＡ　Ｄ１４７Ａを用いて、高速ＰＣＲを実施し、プライマー濃度の影響を確認した。
ここでは。ｄＵＴＰ存在下の高速ＰＣＲでもプライマー濃度の影響があるかについても確認した。 (Example 6)
Examination of primer concentration As in Example 5, high-speed PCR was performed using the KOD Y7A / P36H / N210D and KOD PCNA D147A obtained above, and the influence of the primer concentration was confirmed.
here. It was also confirmed whether the high-speed PCR in the presence of dUTP has an effect on the primer concentration.
ＰＣＲにはＫＯＤ　Ｄａｓｈ（Ｔｏｙｏｂｏ製）添付の１０×ＰＣＲ　Ｂｕｆｆｅｒを１０倍希釈した　１×ＰＣＲ　Ｂｕｆｆｅｒ（１．２ｍＭ　ＭｇＳＯ４を含む）に、０．２ｍＭ　ｄＮＴＰｓ、又はｄＴＴＰをｄＵＴＰに置換した２ｍＭ ｄＮＴＰｓ（ｄＡＴＰ、ｄＵＴＰ，ｄＣＴＰ、ｄＧＴＰ）、１Ｕ　ＫＯＤ　ＤＮＡポリメラーゼ変異体、２５０ｎｇ　ＫＯＤ　ＰＣＮＡ変異体、及び、１／３０，０００に希釈したＳＹＢＲ　ＧｒｅｅｎＩを含む５０μｌの反応液中に、アクチン遺伝子　約５５０ｂｐを増幅する配列番号２１及び２２に記載のプライマー、ならびに５０コピー相当のヒトゲノムを添加し、それぞれでＣｔ値を比較した。プライマー濃度はそれぞれ０．２、０．６、０．８、１．０、１．２、１．４μＭで実施した。ＰＣＲは、９４℃、３０秒の前反応の後、９８℃、０秒→５５℃、０秒→６８℃、０秒の高速サイクルを５０サイクル繰り返すスケジュールでＬｉｇｈｔ　ｃｙｃｌｅｒ２．０を用いて、昇温及び降温の速度を２０℃／秒で行った。反応時間は約２０分で実施された。 For PCR, 10 × PCR Buffer attached to KOD Dash (manufactured by Toyobo) was diluted 10-fold, 1 × PCR Buffer (including 1.2 mM MgSO 4 ), 0.2 mM dNTPs, or 2 mM dNTPs in which dTTP was replaced with dUTP ( dATP, dUTP, dCTP, dGTP), 1U KOD DNA polymerase mutant, 250 ng KOD PCNA mutant, and amplification of about 550 bp of actin gene in 50 μl reaction solution containing SYBR Green I diluted 1 / 30,000 Primers shown in SEQ ID NOs: 21 and 22 and a human genome corresponding to 50 copies were added, and Ct values were compared with each other. Primer concentrations were 0.2, 0.6, 0.8, 1.0, 1.2, and 1.4 μM, respectively. The PCR was performed using Light cycler 2.0 with a schedule that repeats 50 cycles of a high-speed cycle of 98 ° C, 0 seconds → 55 ° C, 0 seconds → 68 ° C, 0 seconds after a pre-reaction of 94 ° C for 30 seconds. The rate of temperature decrease was 20 ° C./second. The reaction time was about 20 minutes.
表１は、プライマー濃度を変えて、高速ＰＣＲによる５０コピーからの増幅を比較した場合のＣｔ値、図３は融解曲線を示す。ｄＴＴＰ存在下（通常のｄＮＴＰｓ）とｄＵＴＰ存在下（ｄＴＴＰをｄＵＴＰに置換したｄＮＴＰｓ）で、プライマー濃度は、０．２、０．６、０．８、１．０、１．２、１．４μＭで実施した。 Table 1 shows Ct values when amplification from 50 copies by high-speed PCR is compared at different primer concentrations, and FIG. 3 shows a melting curve. Primer concentrations are 0.2, 0.6, 0.8, 1.0, 1.2, 1.4 μM in the presence of dTTP (ordinary dNTPs) and in the presence of dUTP (dNTPs in which dTTP is replaced with dUTP). It carried out in.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
結果、ｄＴＴＰ存在下、ｄＵＴＰ存在下共にプライマー濃度を０．６μＭ以上に上げることで、Ｃｔ値が改善することが示された。また、この実験では非特異的増幅やプライマーダイマーはまったく見られなかった。 As a result, it was shown that the Ct value was improved by increasing the primer concentration to 0.6 μM or more in the presence of dTTP and dUTP. In this experiment, no nonspecific amplification or primer dimer was observed.
（実施例７）
ＰＣＮＡと高濃度プライマーの相乗効果の確認
上記で得られたＫＯＤ　Ｙ７Ａ／Ｐ３６Ｈ／Ｎ２１０Ｄ及びＫＯＤ　ＰＣＮＡ　Ｄ１４７Ａを用いて、高速ＰＣＲを実施し、ＰＣＮＡと高濃度プライマーの相乗効果を確認した。ＰＣＲにはＫＯＤ　Ｄａｓｈ（Ｔｏｙｏｂｏ製）添付の１０×ＰＣＲ　Ｂｕｆｆｅｒを１０倍希釈した　１×ＰＣＲ　Ｂｕｆｆｅｒ（１．２ｍＭ　ＭｇＳＯ４を含む）に、０．２ｍＭ　ｄＮＴＰｓ、１Ｕ　ＫＯＤ　ＤＮＡポリメラーゼ変異体、２５０ｎｇ　ＫＯＤ　ＰＣＮＡ変異体、及び、１／３０，０００に希釈したＳＹＢＲ　ＧｒｅｅｎＩを含む５０μｌの反応液中に、アクチン遺伝子、約５５０ｂｐを増幅する配列番号２１及び２２に記載のプライマー、ならびに様々な濃度のヒトゲノムを添加し、それぞれでＣｔ値を比較した。
ヒトゲノムは、１０４、１０３、１０２、１０１コピー、ノンテンプレートコントロール（ＮＴＣ）の５サンプルで行った。今回、ＰＣＮＡ変異体を含まない組成でもＰＣＲを実施し、比較した。プライマー濃度はそれぞれ０．２、１．０μＭで実施し、ＰＣＲは、９４℃、３０秒の前反応の後、９８℃、０秒→５５℃、０秒→６８℃、０秒のサイクルを５０サイクル繰り返す高速ＰＣＲスケジュールと、９４℃、３０秒の前反応の後、９８℃、１０秒→６０℃、１０秒→６８℃、３０秒のサイクルを５０サイクル繰り返す通常ＰＣＲスケジュールの２種類で比較した。サーマルサイクラーにはＬｉｇｈｔ　ｃｙｃｌｅｒ（登録商標）２．０を用いて、昇温及び降温の速度を２０℃／秒で行った。 (Example 7)
Confirmation of synergistic effect of PCNA and high concentration primer High-speed PCR was performed using KOD Y7A / P36H / N210D and KOD PCNA D147A obtained above, and the synergistic effect of PCNA and high concentration primer was confirmed. . For PCR, 10 × PCR Buffer attached to KOD Dash (manufactured by Toyobo) was diluted 10-fold, 1 × PCR Buffer (containing 1.2 mM MgSO 4 ), 0.2 mM dNTPs, 1 U KOD DNA polymerase mutant, 250 ng KOD PCNA The actin gene, the primers set forth in SEQ ID NOs: 21 and 22 that amplify about 550 bp, and various concentrations of the human genome are added to a 50 μl reaction solution containing SYBR Green I diluted to 1 / 30,000. The Ct values were compared with each other.
The human genome, 10 4, 10 3, 10 2, 10 1 copies, were carried out in 5 samples of non-template control (NTC). In this study, PCR was also carried out for compositions that did not contain PCNA mutants and compared. The primer concentrations were 0.2 and 1.0 μM, respectively, and PCR was performed at a temperature of 94 ° C. for 30 seconds, followed by a cycle of 98 ° C., 0 seconds → 55 ° C., 0 seconds → 68 ° C., 0 seconds. A high-speed PCR schedule that repeats the cycle and a normal PCR schedule that repeats the cycle of 98 ° C., 10 seconds → 60 ° C., 10 seconds → 68 ° C., 30 seconds after the pre-reaction at 94 ° C. for 30 seconds and 50 cycles were compared. . As the thermal cycler, Light cycler (registered trademark) 2.0 was used, and the rate of temperature increase and decrease was 20 ° C./second.
図４は、プライマー濃度とＰＣＮＡ変異体の相乗効果を確認するため、様々なコピー数のヒトゲノムから高速ＰＣＲで増幅を比較し、Ｃｔ値を示したものになる。プライマー濃度は、０．２、１．０μＭで実施し、ＰＣＮＡがある場合とない場合とのそれぞれにおけるＣｔを示した。またプライマー０．２μＭにおいて、通常のサイクルでのＰＣＮＡがある場合とない場合とのそれぞれにおけるＣｔも示した。ここでは融解曲線で非特異的な増幅が見られる場合、非特異的増幅の量に応じて、丸で囲いマーキング（目的遺伝子の増幅もあるが非特異的増幅もある）、四角で囲いマーキングした（非特異的増幅のみ見られる）。 FIG. 4 shows the Ct value by comparing the amplification by high-speed PCR from human genomes of various copy numbers in order to confirm the synergistic effect of the primer concentration and the PCNA mutant. The primer concentrations were 0.2 and 1.0 μM, and Ct was shown in each case with and without PCNA. In addition, in the primer 0.2 μM, Ct with and without PCNA in the normal cycle is also shown. Here, when non-specific amplification is seen in the melting curve, depending on the amount of non-specific amplification, circle marking (there is amplification of the target gene but also non-specific amplification), square marking (Only non-specific amplification is seen).
プライマー濃度が０．２μＭの場合、通常のサイクルではＰＣＮＡの添加で大きな差は見られなかった。通常のサイクルでは、ＰＣＮＡの添加の大きな効果は見られないことが示される。一方、高速サイクルでは、ＰＣＮＡの添加なしでは大きくＣｔが遅れるものの、ＰＣＮＡの添加でＣｔが改善する結果となった。しかし、プライマー濃度が０．２μＭではＰＣＮＡを添加しても通常サイクルに比べ、Ｃｔは劣る結果となっている。プライマー濃度が１．０μＭの場合、ＰＣＮＡなしでもＣｔは改善するものの、やはり通常サイクルにはＣｔが１程度遅れている。そこへＰＣＮＡを添加した場合は通常サイクルほぼ同等のＣｔが得られた。高速サイクルを通常サイクルと同等の効率にするには、プライマー濃度を上げることと、ＰＣＮＡを添加することの両方が必要であることが示された。 When the primer concentration was 0.2 μM, no significant difference was observed in the normal cycle with the addition of PCNA. It is shown that in the normal cycle, the great effect of adding PCNA is not seen. On the other hand, in the high-speed cycle, Ct was greatly delayed without addition of PCNA, but Ct was improved with addition of PCNA. However, when the primer concentration is 0.2 μM, Ct is inferior to the normal cycle even when PCNA is added. When the primer concentration is 1.0 μM, Ct improves even without PCNA, but Ct is delayed by about 1 in the normal cycle. When PCNA was added thereto, Ct equivalent to the normal cycle was obtained. It was shown that both increasing the primer concentration and adding PCNA were necessary to make the fast cycle as efficient as the normal cycle.
（実施例８）
ＰＣＲ阻害物質存在下での高速ＰＣＲの検討
上記で得られたＫＯＤ　Ｙ７Ａ／Ｐ３６Ｈ／Ｎ２１０Ｄ、及びＫＯＤ　ＰＣＮＡ　Ｄ１４７Ａを用いて、阻害物質を含む条件で高速ＰＣＲを実施した。
阻害物質は以下のように調製した。
糞便：８ｍｌの水に２ｇの糞便を懸濁し、２０％糞便懸濁液を作製する。９５℃、５分熱処理し、１２，０００ｒｐｍ、１分の遠心後、上清を２０％糞便とした。
尿：ヒトから採取した尿を１００％尿とした。
口腔粘膜：２００μｌの水に綿棒で採取した口腔粘膜を落とし、１００％口腔粘膜液とした。
血液：ＥＤＴＡ採血管で採取した血液を１００％血液とした。 (Example 8)
Examination of high-speed PCR in the presence of PCR inhibitory substance Using the KOD Y7A / P36H / N210D and KOD PCNA D147A obtained above, high-speed PCR was carried out under the condition containing the inhibitory substance.
Inhibitors were prepared as follows.
Feces: Suspend 2 g of feces in 8 ml of water to make a 20% fecal suspension. After heat treatment at 95 ° C. for 5 minutes and centrifugation at 12,000 rpm for 1 minute, the supernatant was made 20% feces.
Urine: Urine collected from humans was taken as 100% urine.
Oral mucosa: The oral mucosa collected with a cotton swab was dropped into 200 μl of water to make 100% oral mucosa fluid.
Blood: Blood collected with an EDTA blood collection tube was taken as 100% blood.
ＰＣＲにはＫＯＤ　Ｄａｓｈ（Ｔｏｙｏｂｏ製）添付の１０×ＰＣＲ　Ｂｕｆｆｅｒを１０倍希釈した１×ＰＣＲ　Ｂｕｆｆｅｒ（１．２ｍＭ　ＭｇＳＯ４を含む）に、０．２ｍＭ　ｄＮＴＰｓ、１Ｕ　ＫＯＤ　ＤＮＡポリメラーゼ変異体、２５０ｎｇ　ＫＯＤ　ＰＣＮＡ変異体、及び、１／３０，０００に希釈したＳＹＢＲ　ＧｒｅｅｎＩを含む５０μｌの反応液中に、サルモネラ菌ｉｎｖＡ遺伝子、約７００ｂｐを増幅する配列番号１９及び２０に記載のプライマー、ならびに５０コピー相当のサルモネラ菌ゲノムを添加し、それぞれでＣｔ値を比較した。
プライマー濃度は０．２、１．０μＭで実施した。阻害物質には上記で調製したサンプルを用い、２．５、０．５、０．１％　糞便、５、１、０．２、０．０４％　尿、２５、５、１、０．２％　口腔粘膜、１．６、０．３２、０．０６％　血液を添加した。ＰＣＲは、９４℃、３０秒の前反応の後、９８℃、０秒→５５℃、０秒→６８℃、０秒の高速サイクルを５０サイクル繰り返すスケジュールでＬｉｇｈｔ　ｃｙｃｌｅｒ（登録商標）２．０を用いて、昇温及び降温の速度を２０℃／秒で行った。反応時間は約２０分で実施された。 For PCR, 1 × PCR Buffer (containing 1.2 mM MgSO 4 ) obtained by diluting 10 × PCR Buffer attached to KOD Dash (manufactured by Toyobo) 10-fold, 0.2 mM dNTPs, 1U KOD DNA polymerase mutant, 250 ng KOD PCNA The Salmonella invA gene, the primer set forth in SEQ ID NOs: 19 and 20 for amplifying about 700 bp, and the 50-copy equivalent Salmonella genome in a 50 μl reaction solution containing SYBR Green I diluted to 1 / 30,000 Were added, and the Ct values were compared with each other.
Primer concentrations were 0.2 and 1.0 μM. For the inhibitor, use the sample prepared above, 2.5, 0.5, 0.1% feces, 5, 1, 0.2, 0.04% urine, 25, 5, 1, 0.2% Oral mucosa, 1.6, 0.32, 0.06% Blood was added. In PCR, Light cycler (registered trademark) 2.0 is scheduled to repeat 50 cycles of 94 ° C, 30 seconds pre-reaction, 98 ° C, 0 seconds → 55 ° C, 0 seconds → 68 ° C, 0 seconds. The rate of temperature increase and decrease was 20 ° C./second. The reaction time was about 20 minutes.
図５は、阻害物質を含む組成から高速ＰＣＲを実施し、サルモネラ菌５０コピーを増幅した場合のＣｔを示す。プライマー濃度は０．２、１．０μＭで実施し、阻害物質がない場合と、２．５、０．５、０．１％糞便、５、１、０．２、０．０４％尿、２５、５、１、０．２％口腔粘膜液、又は１．６、０．３２、０．０６％血液をそれぞれ添加した場合の影響を確認した。融解曲線で非特異的な増幅が見られる場合、非特異的増幅の量に応じて、丸で囲いマーキング（目的遺伝子の増幅もあるが非特異的増幅もある）、四角で囲いマーキングした（非特異的増幅のみ見られる）。また、まったく増幅しないサンプルはＮ．Ｄ．と表記した。 FIG. 5 shows Ct when high-speed PCR was performed from a composition containing an inhibitor and 50 copies of Salmonella were amplified. Primer concentrations are 0.2 and 1.0 μM, with no inhibitor, 2.5, 0.5, 0.1% stool, 5, 1, 0.2, 0.04% urine, 25 The effects of adding 5, 1, 0.2% oral mucosa fluid, or 1.6, 0.32, 0.06% blood were confirmed. When non-specific amplification is seen in the melting curve, depending on the amount of non-specific amplification, circle marking (there is amplification of the target gene but also non-specific amplification), square marking (non-specific amplification) Only specific amplification is seen). Samples that are not amplified at all are N.P. D. It was written.
この系は、阻害物質がなくてもＰＣＮＡなしでは増幅しない系となる。
糞便に関しては、プライマー濃度が０．２μＭでは０．５％までしか増幅しないところ、プライマー濃度を１．０μＭに上げることで、２．５％含まれていても増幅が確認できた。
尿に関しては、プライマー濃度が０．２μＭでは立ち上がりが遅れるものの、プライマー濃度を１．０μＭに上げることで、立ち上がりが改善され、５％の持込でも増幅が確認された。口腔粘膜に関しては、プライマー濃度が０．２μＭでは５％までしか増幅しないところ、プライマー濃度を１．０μＭに上げることで、２５％含まれていても増幅が確認できた。
血液に関しては、プライマー濃度が０．２μＭでは０．３２％までしか増幅しないところ、プライマー濃度を１．０μＭに上げることで、１．６％含まれていても増幅が確認できた。
ＰＣＮＡの添加に加え、プライマー濃度を高めることで阻害物質が含まれていても高速ＰＣＲが可能になったと考えられる。 This system is a system that does not amplify without PCNA even without an inhibitor.
Regarding feces, amplification was only up to 0.5% when the primer concentration was 0.2 μM, but amplification was confirmed even when 2.5% was included by increasing the primer concentration to 1.0 μM.
As for urine, although the rise was delayed when the primer concentration was 0.2 μM, the rise was improved by raising the primer concentration to 1.0 μM, and amplification was confirmed even when the concentration was 5%. Regarding the oral mucosa, amplification was confirmed only up to 5% when the primer concentration was 0.2 μM, but amplification was confirmed even when 25% was contained by increasing the primer concentration to 1.0 μM.
As for blood, amplification was only up to 0.32% at a primer concentration of 0.2 μM, but amplification was confirmed even when 1.6% was included by increasing the primer concentration to 1.0 μM.
In addition to the addition of PCNA, it is considered that high-speed PCR became possible even if an inhibitor was included by increasing the primer concentration.
（実施例９）
ＰＣＲ阻害物質・ｄＵＴＰ存在下での高速ＰＣＲの検討
上記で得られたＫＯＤ　Ｙ７Ａ／Ｐ３６Ｈ／Ｎ２１０Ｄ、及びＫＯＤ　ＰＣＮＡ　Ｄ１４７Ａを用いて、阻害物質及びｄＵＴＰを含む条件で高速ＰＣＲを実施した。
阻害物質は以下のように調製した。
糞便：８ｍｌの水に２ｇの糞便を懸濁し、２０％糞便懸濁液を作製した。９５℃、５分熱処理し、１２，０００ｒｐｍ、１分の遠心後、上清を２０％糞便とした。 Example 9
Examination of high-speed PCR in the presence of a PCR inhibitor / dUTP High-speed PCR was carried out using the KOD Y7A / P36H / N210D and KOD PCNA D147A obtained above under the conditions containing the inhibitor and dUTP. .
Inhibitors were prepared as follows.
Feces: 2 g of feces was suspended in 8 ml of water to prepare a 20% fecal suspension. After heat treatment at 95 ° C. for 5 minutes and centrifugation at 12,000 rpm for 1 minute, the supernatant was made 20% feces.
ＰＣＲにはＫＯＤ　Ｄａｓｈ（Ｔｏｙｏｂｏ製）添付の１０×ＰＣＲ　Ｂｕｆｆｅｒを１０倍希釈した　１×ＰＣＲ　Ｂｕｆｆｅｒ（１．２ｍＭ　ＭｇＳＯ４を含む）に、ｄＴＴＰをｄＵＴＰに置換した２ｍＭ ｄＮＴＰｓ（ｄＡＴＰ、ｄＵＴＰ，ｄＣＴＰ、ｄＧＴＰ）、１Ｕ　ＫＯＤ　ＤＮＡポリメラーゼ変異体、２５０ｎｇ　ＫＯＤ　ＰＣＮＡ変異体、及び、１／３０，０００に希釈したＳＹＢＲ　ＧｒｅｅｎＩを含む５０μｌの反応液中にアクチン遺伝子、１．０μＭ　約５５０ｂｐを増幅する配列番号２１及び２２に記載のプライマー、ならびに、５０コピー相当のヒトゲノムを添加し、２．５、２、１．５、１、０．５、０．２５、０．１％　糞便存在下でのＣｔ値を比較した。
コントロールにＴａｑの反応系でも同様の検討を実施した。Ｔａｑ　ＤＮＡポリメラーゼはＴｏｙｏｂｏ製のものを用い、Ａｎｔｉ－Ｔａｑ　Ｈｉｇｈ（Ｔｏｙｏｂｏ製）と混合したものを用いた。反応は１×ＢｌｅｎｄＴａｑに添付のＢｕｆｆｅｒ、２ｍＭ　ｄＴＴＰをｄＵＴＰに置換したｄＮＴＰｓ（ｄＡＴＰ、ｄＵＴＰ，ｄＣＴＰ、ｄＧＴＰ）、０．２、及び１．０μＭのプライマー（上記と同様）、抗体と混合した２．５Ｕの酵素を含む５０μｌの反応液に５０コピー相当のヒトゲノム、２．５、２、１．５、１、０．５、０．２５、０．１％糞便を添加し、Ｃｔ値を比較した。ＰＣＲはそれぞれ、９４℃、３０秒の前反応の後、９８℃、０秒→５５℃、０秒→６８℃、０秒の高速サイクルを５０サイクル繰り返すスケジュールでＬｉｇｈｔ　ｃｙｃｌｅｒ２．０を用いて、昇温及び降温の速度を２０℃／秒で行った。反応時間は約２０分で実施された。 For PCR, 10 × PCR Buffer attached to KOD Dash (manufactured by Toyobo) was diluted 10-fold, 1 × PCR Buffer (including 1.2 mM MgSO 4 ), 2 mM dNTPs (dATP, dUTP, dCTP, dGTP), 1U KOD DNA polymerase mutant, 250 ng KOD PCNA mutant, and SYBR Green I diluted to 1 / 30,000, actin gene, 1.0 μM Amplifying about 550 bp SEQ ID NO: 21 And the human genome corresponding to 50 copies, and 2.5, 2, 1.5, 1, 0.5, 0.25, 0.1% Ct value in the presence of feces Compared.
For the control, the same examination was carried out in the Taq reaction system. Taq DNA polymerase was manufactured by Toyobo and mixed with Anti-Taq High (manufactured by Toyobo). The reaction was carried out by mixing Buffer attached to 1 × BlendTaq, dNTPs (dATP, dUTP, dCTP, dGTP) in which 2 mM dTTP was replaced with dUTP, 0.2, and 1.0 μM primers (same as above) and antibody. 50 copies of human genome, 2.5, 2, 1.5, 1, 0.5, 0.25, and 0.1% feces were added to a 50 μl reaction solution containing 5 U of enzyme, and Ct values were compared. . Each PCR was performed using Light cycler 2.0 on a schedule that repeats 50 cycles of high-speed cycles of 94 ° C, 0 seconds → 55 ° C, 0 seconds → 68 ° C, 0 seconds after a pre-reaction at 94 ° C for 30 seconds. The rate of temperature and temperature decrease was 20 ° C./second. The reaction time was about 20 minutes.
図６は、糞便・ｄＵＴＰを含む組成から高速ＰＣＲを実施し、ヒトゲノム５０コピーを増幅した場合のＣｔを示す。ＫＯＤ変異体を用いた系ではプライマー濃度は１．０μＭで実施し、ＰＣＮＡ変異体がある場合とない場合とのそれぞれで実施した。阻害物質がない場合と２．５、２、１．５、１、０．５、０．２５、０．１％糞便でのＣｔを確認した。Ｔａｑ　ＤＮＡポリメラーゼを用いた系では０．２、１．０μＭのプライマー濃度で、同様に阻害物質がない場合と２．５、２、１．５、１、０．５、０．２５、０．１％　糞便でのＣｔを確認した。融解曲線で非特異的な増幅が見られる場合、非特異的増幅の量に応じて、丸で囲いマーキング（目的遺伝子の増幅もあるが非特異的増幅もある）、四角で囲いマーキングした（非特異的増幅のみ見られる）。また、まったく増幅しないサンプルは「Ｎ．Ｄ．」と表記した。 FIG. 6 shows Ct when high-speed PCR was performed from a composition containing feces / dUTP and 50 copies of the human genome were amplified. In the system using the KOD mutant, the primer concentration was 1.0 μM, and was performed with and without the PCNA mutant. Ct was confirmed in the absence of inhibitor and in 2.5, 2, 1.5, 1, 0.5, 0.25, 0.1% feces. In the system using Taq DNA polymerase, primer concentrations of 0.2 and 1.0 μM were used, and when no inhibitor was present, 2.5, 2, 1.5, 1, 0.5, 0.25, 0. Ct in 1% feces was confirmed. When non-specific amplification is seen in the melting curve, depending on the amount of non-specific amplification, circle marking (there is amplification of the target gene but also non-specific amplification), square marking (non-specific amplification) Only specific amplification is seen). A sample that was not amplified at all was denoted as “ND”.
検査の現場では、糞便１％存在下で増幅を確認することが必要になる。Ｔａｑの系での結果、糞便１％存在下では、プライマー濃度が高くても、そもそも高速ＰＣＲで増幅が見られなかった。一方、ＫＯＤの系では、ＰＣＮＡを添加しなくても、０．５％まで増幅は確認できたが、０．５％で阻害が生じ始め、非特異的増幅が出現することが示された。ＰＣＮＡを入れた系では１．５％糞便を入れても増幅が確認された。これらの結果から、ファミリーＢに属するＤＮＡポリメラーゼが高速ＰＣＲに向いていることがわかる。しかし、高濃度プライマーとファミリーＢに属するＤＮＡポリメラーゼを用いてもＣｔの立ち上がりはあまりよくなく、１％以下の糞便でも大きく影響を受ける結果となった。やはり高濃度プライマーとファミリーＢに属するＤＮＡポリメラーゼ、ＰＣＮＡを組み合わせた系が最も効率的であり、１％糞便を含んだ高速ＰＣＲにおいては、この組み合わせでないと増幅ができない結果が得られた。 At the site of the test, it is necessary to confirm amplification in the presence of 1% feces. As a result of the Taq system, no amplification was observed in the high-speed PCR even in the presence of 1% stool even if the primer concentration was high. On the other hand, in the KOD system, amplification could be confirmed up to 0.5% without adding PCNA, but inhibition began to occur at 0.5%, indicating that non-specific amplification appeared. In the system containing PCNA, amplification was confirmed even when 1.5% stool was added. From these results, it can be seen that DNA polymerase belonging to Family B is suitable for high-speed PCR. However, even when a high-concentration primer and a DNA polymerase belonging to Family B were used, the rise of Ct was not very good, and even 1% or less of stool was greatly affected. The combination of high-concentration primers, DNA polymerase belonging to Family B, and PCNA is the most efficient, and high-speed PCR containing 1% stool yields results that cannot be amplified without this combination.
本発明は、ＤＮＡ合成に関わるバイオテクノロジー関連産業において有用であり、特に診断用途において有用である。 The present invention is useful in biotechnology-related industries related to DNA synthesis, and particularly useful in diagnostic applications.

Claims (17)

  1. ファミリーＢに属するＤＮＡポリメラーゼ、Ｐｒｏｌｉｆｅｒａｔｉｎｇ　Ｃｅｌｌ　Ｎｕｃｌｅａｒ　Ａｎｔｉｇｅｎ（ＰＣＮＡ；増殖細胞核抗原）、および及び０．６μＭ以上の濃度でプライマーを含み、かつ、３０サイクルから５０サイクルを４０分以内で実施するための組成であることを特徴とするＰＣＲ方法組成。 It contains DNA polymerase belonging to Family B, Proliferating Cell Nuclear Antigen (PCNA; proliferating cell nuclear antigen), and a primer at a concentration of 0.6 μM or more, and has a composition for performing 30 to 50 cycles within 40 minutes. A PCR method composition characterized by being.
  2. ３０サイクルから５０サイクルを２０分以内で実施するための組成である請求項1に記載のＰＣＲ方法組成。 2. The PCR method composition according to claim 1, which is a composition for carrying out 30 to 50 cycles within 20 minutes.
  3. ３０サイクルから５０サイクルを１２分以内で実施するための組成である請求項１又は２に記載のＰＣＲ方法組成。 The PCR method composition according to claim 1 or 2, which is a composition for carrying out 30 to 50 cycles within 12 minutes.
  4. プライマーの濃度が０．８μＭ以上である請求項１から３のいずれかに記載のＰＣＲ組成方法。 The PCR composition method according to any one of claims 1 to 3, wherein the primer concentration is 0.8 µM or more.
  5. プライマーの濃度が１．０μＭ以上である請求項１から４のいずれかに記載のＰＣＲ組成方法。 The PCR composition method according to any one of claims 1 to 4, wherein the primer concentration is 1.0 µM or more.
  6. 昇温又は降温の速度が０．１℃／秒から２０℃／秒の範囲でＰＣＲを実施するための組成であることを特徴とする請求項１から５のいずれかに記載のＰＣＲ方法組成。 The PCR method composition according to any one of claims 1 to 5, wherein the PCR method composition is a composition for performing PCR at a rate of temperature increase or decrease of 0.1 ° C / second to 20 ° C / second.
  7. 昇温又は降温の速度が５℃／秒から２０℃／秒の範囲でＰＣＲを実施するための組成である請求項１から６のいずれかに記載のＰＣＲ方法組成。 The PCR method composition according to any one of claims 1 to 6, which is a composition for carrying out PCR in a temperature rising or cooling rate range of 5 ° C / second to 20 ° C / second.
  8. 昇温又は降温の速度が１０℃／秒から２０℃／秒の範囲でＰＣＲを実施するための組成である請求項１から７のいずれかに記載のＰＣＲ方法組成。 The PCR method composition according to any one of claims 1 to 7, which is a composition for carrying out PCR at a rate of temperature increase or decrease of 10 ° C / second to 20 ° C / second.
  9. ファミリーＢに属するＤＮＡポリメラーゼが、古細菌（Ａｒｃｈｅａ）由来のＤＮＡポリメラーゼである請求項１から８のいずれかに記載のＰＣＲ組成。 The PCR composition according to any one of claims 1 to 8, wherein the DNA polymerase belonging to Family B is an archaea-derived DNA polymerase.
  10. ファミリーＢに属するＤＮＡポリメラーゼが減少した塩基類似体検出活性を有する古細菌ＤＮＡポリメラーゼ変異体である請求項１から９のいずれかに記載のＰＣＲ組成。 The PCR composition according to any one of claims 1 to 9, wherein the DNA polymerase belonging to family B is an archaeal DNA polymerase mutant having reduced base analog detection activity.
  11. ＰＣＮＡが古細菌（Ａｒｃｈｅａ）由来のＰＣＮＡである請求項１から１０のいずれかに記載のＰＣＲ組成。 The PCR composition according to any one of claims 1 to 10, wherein the PCNA is PCNA derived from Archaea.
  12. ＰＣＮＡがＤＮＡポリメラーゼ増幅増強活性をもつ変異型ＰＣＮＡである請求項１から１１のいずれかに記載のＰＣＲ組成。 The PCR composition according to any one of claims 1 to 11, wherein the PCNA is a mutant PCNA having DNA polymerase amplification enhancing activity.
  13. 請求項１から１２のいずれかに記載のＰＣＲ組成を含むことを特徴とするＰＣＲ用試薬。 A PCR reagent comprising the PCR composition according to claim 1.
  14. 請求項１３に記載のＰＣＲ用試薬を含むことを特徴とするＰＣＲ用試薬キット。 A PCR reagent kit comprising the PCR reagent according to claim 13.
  15. 請求項１から１２のいずれかに記載のＰＣＲ組成により、１５回から５０回の熱サイクルによるＰＣＲ方法であって、かつ、ＰＣＲの反応時間が６０分以内であることを特徴とするＰＣＲ方法。 The PCR method according to any one of claims 1 to 12, wherein the PCR method is based on 15 to 50 thermal cycles, and the PCR reaction time is within 60 minutes.
  16. ＰＣＲの反応時間が３０分以内である請求項１５に記載のＰＣＲ方法。 The PCR method according to claim 15, wherein the PCR reaction time is within 30 minutes.
  17. 請求項１から１２のいずれかに記載のＰＣＲ組成を含む反応液中に、生体試料を直接添加することを特徴とする請求項１５又は１６に記載のＰＣＲ方法。
          The PCR method according to claim 15 or 16, wherein the biological sample is directly added to the reaction solution containing the PCR composition according to any one of claims 1 to 12.
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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005111209A1 (en) * 2004-05-18 2005-11-24 Toyo Boseki Kabushiki Kaisha High-speed pcr using high-speed dna polymerase
JP2006507012A (en) * 2002-10-25 2006-03-02 ストラタジーン カリフォルニア DNA polymerase with reduced base analog detection activity
WO2007004654A1 (en) * 2005-07-04 2007-01-11 Celestar Lexico-Sciences, Inc. Mutant pcna
JP2010104304A (en) * 2008-10-30 2010-05-13 Hitachi Ltd Dna polymerase variant having enhanced exonuclease activity
JP2015050995A (en) * 2013-08-06 2015-03-19 東洋紡株式会社 Mutant pcna

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7118860B2 (en) 1999-10-29 2006-10-10 Stratagene California Methods for detection of a target nucleic acid by capture
US20030228616A1 (en) 1999-10-29 2003-12-11 Stratagene DNA polymerase mutants with reverse transcriptase activity
CA2519309A1 (en) * 2003-03-25 2004-10-14 Stratagene California Dna polymerase fusions and uses thereof
JP2009148245A (en) * 2007-06-12 2009-07-09 Toyobo Co Ltd Quick detection method of nucleic acid
JP5593582B2 (en) * 2007-06-12 2014-09-24 東洋紡株式会社 Rapid detection method of nucleic acid
JP2010239880A (en) * 2009-04-02 2010-10-28 Toyobo Co Ltd High speed real time pcr using high speed dna polymerase

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2006507012A (en) * 2002-10-25 2006-03-02 ストラタジーン カリフォルニア DNA polymerase with reduced base analog detection activity
WO2005111209A1 (en) * 2004-05-18 2005-11-24 Toyo Boseki Kabushiki Kaisha High-speed pcr using high-speed dna polymerase
WO2007004654A1 (en) * 2005-07-04 2007-01-11 Celestar Lexico-Sciences, Inc. Mutant pcna
JP2010104304A (en) * 2008-10-30 2010-05-13 Hitachi Ltd Dna polymerase variant having enhanced exonuclease activity
JP2015050995A (en) * 2013-08-06 2015-03-19 東洋紡株式会社 Mutant pcna

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