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WO2016013592A1 - PROTÉINE RecA MODIFIÉE RÉSISTANT À LA CHALEUR, MOLÉCULE D'ACIDE NUCLÉIQUE CODANT POUR LADITE PROTÉINE, PROCÉDÉ D'AMPLIFICATION DE L'ACIDE NUCLÉIQUE AU MOYEN DE LADITE PROTÉINE ET KIT POUR L'AMPLIFICATION DE L'ACIDE NUCLÉIQUE - Google Patents

PROTÉINE RecA MODIFIÉE RÉSISTANT À LA CHALEUR, MOLÉCULE D'ACIDE NUCLÉIQUE CODANT POUR LADITE PROTÉINE, PROCÉDÉ D'AMPLIFICATION DE L'ACIDE NUCLÉIQUE AU MOYEN DE LADITE PROTÉINE ET KIT POUR L'AMPLIFICATION DE L'ACIDE NUCLÉIQUE Download PDF

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WO2016013592A1
WO2016013592A1 PCT/JP2015/070883 JP2015070883W WO2016013592A1 WO 2016013592 A1 WO2016013592 A1 WO 2016013592A1 JP 2015070883 W JP2015070883 W JP 2015070883W WO 2016013592 A1 WO2016013592 A1 WO 2016013592A1
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nucleic acid
protein
amplification
reca protein
amino acid
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Japanese (ja)
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重森康司
柴田武彦
美川務
篠原赳
飯倉ゆかり
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国立研究開発法人理化学研究所
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • 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
    • 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
    • C12N15/09—Recombinant DNA-technology

Definitions

  • the present invention relates to a modified heat-resistant RecA protein, a nucleic acid molecule encoding the protein, a nucleic acid amplification method using the protein, and a nucleic acid amplification kit. Specifically, compared to wild-type heat-resistant RecA protein, modified heat-resistant RecA protein having improved ability to contribute to highly accurate and efficient amplification of template nucleic acid in nucleic acid amplification reaction system, and use thereof Regarding the method.
  • Nucleic acid amplification techniques such as polymerase chain reaction (hereinafter sometimes abbreviated as “PCR”) are epoch-making techniques capable of amplifying a specific target DNA region more than 100,000 times in a short time.
  • PCR polymerase chain reaction
  • An amplification product that is not specific to the template nucleic acid causes a reduction in amplification accuracy, and becomes background noise that hinders subsequent experiments. Therefore, establishment of a highly accurate amplification technique that can suppress non-specific amplification and can specifically and efficiently amplify only the target nucleic acid has been demanded.
  • Patent Document 1 a method for suppressing mutagenesis of amplified nucleic acid by adding a mutagenic nucleotide-removing protein such as MutM and / or MutY protein has been reported (Patent Document 1). Specifically, using a mutagenic nucleotide removal protein, the nucleotide is removed from a single-stranded or double-stranded DNA strand containing a nucleotide that induces a mutation such as 8-oxoguanine nucleotide, 2-oxoadenine nucleotide, etc. It is. This suppresses a mutation that may occur during DNA extension or amplification reaction using the DNA strand as a template, thereby improving the accuracy of the amplification reaction.
  • a mutagenic nucleotide-removing protein such as MutM and / or MutY protein
  • the mutagenic nucleotide removal protein (MutM, MutT) does not have the activity of improving the pairing specificity between the primer and the template nucleic acid. For this reason, the effect of improving the pairing specificity between the primer and the template nucleic acid by the technique of Patent Document 1 is limited, and the primer misannealing cannot be effectively suppressed.
  • thermostable RecA protein derived from a highly thermophilic bacterium can interact with a template and a primer to promote the binding of the primer only to a specific template sequence. It has been reported that this makes it possible to suppress primer misannealing.
  • the RecA protein is a protein that binds cooperatively to a single-stranded nucleic acid, searches for a homologous region between the single-stranded nucleic acid and a double-stranded nucleic acid, and performs homologous recombination of the nucleic acid.
  • the present inventors previously suppressed the primer misannealing by coexisting the aforementioned RecA protein and the factor that activates the protein, and made the desired nucleic acid more specific. It was reported that it can amplify automatically (patent documents 2 and 3). Specifically, in the PCR nucleic acid amplification method, a homologous recombinant protein such as RecA protein and a protein activator such as ATP- ⁇ S are added to the reaction solution. Thus, it has been reported that amplification of non-specific PCR products can be suppressed to a low level, and desired nucleic acids can be amplified more specifically (Patent Document 2).
  • nucleic acid amplification reaction by adding homologous recombinant protein such as RecA protein and nucleotide triphosphate such as ATP to the reaction solution, amplification of non-specific PCR products can be kept low. It has been reported that a desired nucleic acid can be amplified more specifically (Patent Document 3). It has been reported that the presence of nucleotide triphosphate activates RecA protein and retains its biological function well.
  • Patent Documents 2 and 3 perform PCR nucleic acid amplification reaction using a sequence region to be examined for single nucleotide polymorphism in a template nucleic acid as a primer, and detect the presence or absence of single nucleotide polymorphism using the amount of amplification product as an index. It is something that can be done. However, in terms of detection accuracy, it did not fully satisfy market demands. As a factor, it was considered that nonspecific amplification occurred due to insufficient pairing specificity improving activity between the RecA protein primer and the template nucleic acid.
  • the modified thermostable RecA protein of Patent Document 4 includes 13 amino acid sequences of the thermostable RecA protein derived from wild-type Thermus thermophilus (hereinafter referred to as “TthRecA protein”) that constitutes the C-terminal acidic region. This is a C-terminal truncated thermostable RecA protein called “Hyper-TthRecA protein” from which the acidic amino acid residue is deleted.
  • the C-terminal truncated heat-resistant RecA protein of Patent Document 4 causes a nucleic acid amplification reaction inhibition, thereby causing a decrease in amplification efficiency.
  • the reason for this was assumed that even after the protein exhibited homologous recombination activity, it did not dissociate while adhering to the nucleic acid, preventing the subsequent chain extension reaction.
  • the C-terminal truncated heat-resistant RecA protein of Patent Document 4 has room for further improvement in terms of amplification efficiency when used in nucleic acid amplification reactions.
  • the protein can be suitably used for single nucleotide polymorphism analysis.
  • the pairing specificity between the template nucleic acid and the primer is further improved. There was a need to build technology that could contribute.
  • Japanese Patent Application Laid-Open No. 11-225798 Japanese Patent Application No. 10-327378
  • Japanese Patent Application No. 2004/027060 Japanese Patent Application No. 2004-537561
  • JP 2006-174722 Japanese Patent Application No. 2004-368831
  • JP2008-029222 Japanese Patent Application No. 2006-203810
  • the present invention aims to improve the accuracy and efficiency of nucleic acid amplification technology, suppress non-specific amplification, and establish a technology that can amplify only a desired nucleic acid more specifically and efficiently. To do.
  • a modified heat-resistant RecA protein having a modified site in which a specific amino acid is modified in the amino acid sequence of the heat-resistant RecA protein was constructed.
  • Such modified thermostable RecA protein showed high homologous recombination activity.
  • non-specific amplification does not occur, and an amplification product specific to the target nucleic acid is obtained, which can be used for single nucleotide polymorphism analysis, etc. It was found that the nucleic acid amplification can be achieved.
  • a modified thermostable RecA protein selected from the following proteins: (1) a protein comprising any one of the amino acid sequences of SEQ ID NOS: 1 to 4 (2) in the amino acid sequence of any of SEQ ID NOS: 1 to 4, wherein one or several amino acids are deleted, substituted or added, And a protein consisting of an amino acid sequence in which the amino acid at position 202 corresponding to any one of the amino acid sequences of SEQ ID NOs: 1 to 4 is tryptophan, and in a nucleic acid amplification reaction system, SEQ ID NOs: 6, 8, 10, Or a protein having a function of reducing primer misannealing with respect to a template nucleic acid as compared to a wild-type heat-resistant RecA protein consisting of 12 amino acid sequences (3) at least 90% of any of the amino acid sequences of SEQ ID NOs: 1 to 4 And the amino acid at the position corresponding to position 202 in any one of the amino acid sequences of SEQ ID NOs: 1 to 4 is tryptophan
  • a protein
  • thermostable RecA protein according to [1] or [2] above, wherein the wild type thermostable RecA protein is derived from Thermus thermophilus or Thermus aquaticus.
  • a modified heat-resistant RecA protein having improved ability to contribute to high-precision and high-efficiency amplification of a target nucleic acid in a nucleic acid amplification reaction system is provided.
  • it has the activity of improving the pairing specificity between the target nucleic acid and the primer. That is, the primer pairs only with the region on the target nucleic acid that is complementary to the primer.
  • the primer suppresses mis-annealing such as pairing with a non-complementary region or forming a primer dimer between primers, and strictly pairs with a complementary sequence.
  • the modified thermostable RecA protein of the present invention is capable of both pairing a primer with a complementary target nucleic acid, which is a major stage in homologous recombination reaction, and strand exchange. This can be achieved because of the improved activity. Therefore, by adding the modified thermostable RecA protein of the present invention to the nucleic acid amplification system, it is specific and efficient for the target nucleic acid that was insufficient with the wild-type thermostable RecA protein having no modified site. Nucleic acid amplification becomes possible. Therefore, non-specific amplification can be suppressed, and the target nucleic acid can be amplified without being affected by background noise, which can contribute to improvement in amplification accuracy. Therefore, it can be used for single nucleotide polymorphism analysis and the like that require highly accurate nucleic acid amplification, and can provide proteins that can be widely used in various industrial fields, particularly in the field of molecular biology.
  • the mutant thermostable RecA protein of the present invention has an improved ability to contribute to highly accurate and highly efficient amplification, compared to the C-terminal truncated thermostable RecA protein previously developed by the present inventors.
  • the C-terminal truncated heat-resistant RecA protein has been conventionally known as having the ability to contribute to highly accurate and highly efficient nucleic acid amplification, but has a problem of inhibition of amplification.
  • the modified heat-resistant RecA protein of the present invention has an enhanced function (recyclability) that can be dissociated from the nucleic acid after pairing the target nucleic acid and the primer in the nucleic acid amplification system.
  • thermostable RecA protein of the present invention can promote homologous recombination, and can realize efficient gene transfer in gene recombination experiments. For example, it is possible to provide proteins that can be used for gene introduction into embryonic stem cells in the production of transgenic animals and can be widely used in the field of molecular biology such as analysis of genetic phenomena.
  • a modified thermostable RecA protein derived from Thermus aquaticus is provided.
  • the sequence information of the RecA protein derived from Thermus thermophilus and Thermus aquaticus is known, and the modified heat-resistant RecA protein of the present invention can be easily obtained.
  • the protein since the protein has excellent heat resistance, it is possible to provide a modified heat-resistant RecA protein that is optimal for use in a nucleic acid amplification system.
  • a modified heat-resistant RecA protein having an improved ability to contribute to highly accurate and highly efficient amplification of a target nucleic acid in a nucleic acid amplification reaction system. That is, such a modified thermostable RecA protein contributes to the construction of a highly accurate nucleic acid amplification technique suitable for use in single nucleotide polymorphism analysis and the like in a nucleic acid amplification system. At the same time, it contributes to the construction of a nucleic acid amplification technique that can efficiently amplify the target nucleic acid without causing amplification inhibition or the like.
  • a nucleic acid molecule encoding the modified heat-resistant RecA protein of the present invention is provided.
  • a nucleic acid molecule By using such a nucleic acid molecule, it is possible to produce a modified heat-resistant RecA protein having the above-mentioned constitutions [1] to [5] in large quantities at low cost and industrially using genetic engineering techniques. It becomes possible.
  • the nucleic acid amplification reaction can be easily controlled, and the target nucleic acid can be specifically and efficiently amplified. Therefore, non-specific amplification can be suppressed, and the target nucleic acid can be amplified without being affected by background noise.
  • a nucleic acid amplification technique that can contribute to improvement of amplification accuracy and is suitable for use in single nucleotide polymorphism analysis and the like that require high-precision nucleic acid amplification. Efficient amplification of the target nucleic acid can be realized without causing amplification inhibition or the like in the nucleic acid amplification system.
  • modified thermostable RecA protein of the present invention is a thermostable enzyme, the above effect can be continuously exhibited. As a result, highly accurate and efficient nucleic acid amplification can be achieved in all cycles of the nucleic acid amplification reaction system including the heating step.
  • a nucleic acid amplification kit for amplifying a nucleic acid comprising a thermostable DNA polymerase and the modified thermostable RecA protein of any one of [1] to [5] above.
  • thermostable RecA protein A method for reducing primer misannealing with respect to a template nucleic acid of a protein compared to a wild-type heat-resistant RecA protein,
  • the method comprises the step of substituting the amino acid at the position corresponding to position 202 of the amino acid sequence of SEQ ID NO: 6, 8, 10, or 12 of the wild type thermostable RecA protein with tryptophan,
  • the wild type thermostable RecA protein is selected from the following proteins (4) to (6): (4) a protein comprising any amino acid sequence of SEQ ID NO: 6, 8, 10, or 12 (5) one or several amino acids in any one of the amino acid sequences of SEQ ID NO: 6, 8, 10, or 12
  • a protein comprising an amino acid sequence deleted, substituted or added (6)
  • a protein comprising an amino acid sequence having at least 90% sequence identity with any amino acid sequence of SEQ ID NO: 6, 8, 10, or 12 [11]
  • the method according to [10] wherein the primer misannealing with respect to the template nucleic acid is reduced in
  • a method which can reduce primer misannealing with respect to a template nucleic acid of a protein compared to a wild-type heat-resistant RecA protein.
  • the protein constructed by the method of the present invention can suppress non-specific amplification, enables amplification of a target nucleic acid that is not affected by background noise, and can contribute to improvement of amplification accuracy.
  • the ability to contribute to high-accuracy and high-efficiency amplification is further improved than the C-terminal truncated heat-resistant RecA protein previously developed by the present inventors.
  • Improved proteins can be constructed.
  • the protein can smoothly proceed with the chain extension reaction after pairing.
  • the inhibitory action of nucleic acid amplification as observed with the conventional C-terminal truncated thermostable RecA protein can be reduced, and it can contribute to the provision of a protein capable of realizing efficient amplification of the target nucleic acid.
  • a protein capable of promoting homologous recombination can be constructed.
  • efficient gene transfer can be realized in gene recombination experiments.
  • it can be used for gene transfer into embryonic stem cells in the production of transgenic animals, and can contribute to the provision of proteins that can be widely used in the field of molecular biology such as analysis of genetic phenomena.
  • Example 1 shows the nucleic acid amplification reaction which added RecA protein (TthRecA protein) derived from a wild type Thermus thermophilus, and the schematic diagram of the nucleic acid amplification mode.
  • A shows the amplification result in the presence of wild type TthRecA protein
  • B shows the amplification result in the presence of E. coli-derived SSB protein
  • C shows the amplification result in the presence of E. coli-derived RecA protein.
  • D is in the presence of the TthRecA protein indicated by A
  • E is the result of annealing of the primer to the template nucleic acid in the nucleic acid amplification reaction system in the presence of the E.
  • Example 2 which prepared the mutant (TthRecA-Y202W mutant) which substituted the tyrosine 202nd of the amino acid sequence of wild type TthRecA protein by tryptophan. It is an electrophoretic diagram which shows the result of Example 3 which confirmed the homologous recombination activity of the TthRecA-Y202W mutant.
  • A shows the wild type TthRecA protein
  • B shows the TthRecA-Y202W mutant
  • C shows the homologous recombination activity of the Hyper-TthRecA mutant.
  • Example 3 which confirmed the homologous recombination activity of TthRecA-Y202W mutant is shown, and the result of FIG. 3 is quantified and graphed.
  • the horizontal axis represents the reaction time (minutes), and the vertical axis represents the D-Loop formation rate (%).
  • the vertical axis represents the D-Loop formation rate (%).
  • Example 4 which confirmed the homologous recombination activity of TthRecA-Y202W variant
  • a horizontal axis is reaction time (minutes)
  • shaft is D-Loop formation rate (%).
  • Example 5 which confirmed the ATPase activity of TthRecA-Y202W mutant.
  • the result of single-stranded DNA-dependent ATPase is shown, and the vertical axis represents the production rate (%) of ADP from ATP. It is a graph which shows the result of Example 5 which confirmed the ATPase activity of TthRecA-Y202W mutant. The result of double-stranded DNA-dependent ATPase is shown, and the vertical axis represents the production rate (%) of ADP from ATP. It is an electrophoretic diagram which shows the result of Example 6 which confirmed the influence on PCR amplification precision of TthRecA-Y202W variant.
  • A shows the control
  • B shows the amplification result in the presence of the TthRecA-Y202W mutant
  • C shows the amplification result in the presence of the wild-type TthRecA protein.
  • A is the control
  • B is the amplification result in the presence of the TthRecA-Y202W mutant
  • C is the amplification result in the presence of the wild-type TthRecA protein
  • D is the amplification result in the presence of the TaqRecA protein.
  • Example 8 It is an electrophoretic diagram which shows the result of Example 8 which confirmed the effect of the nucleic acid amplification accuracy of a TthRecA-Y202W variant.
  • A shows the result of amplification with a primer having no mismatched base
  • B shows the result of amplification with a primer having a mismatched base.
  • A shows the result of amplification in 30 cycles
  • B shows the result of amplification in 35 cycles.
  • modified thermostable RecA protein of the present invention may be abbreviated as “modified thermostable RecA protein”.
  • wild type thermostable RecA protein may be abbreviated as “wild type thermostable RecA protein”.
  • thermostable RecA protein means that the amino acid sequence of the thermostable RecA protein retained in the highly thermophilic bacterium isolated from nature and the base sequence encoding the thermostable RecA protein are intentional or It means that it does not have a modification site where unintentional modification has occurred.
  • the modified thermostable RecA protein of the present invention was based on the wild type thermostable RecA protein for modification.
  • the wild type thermostable RecA protein which is the basis of the modified type, is a protein derived from an extreme thermophile.
  • the highly thermophilic RecA protein suitable for use in the present invention includes RecA protein derived from the genus Thermus, Thermococcus, Pyrococcus, Thermotoga, etc. Is exemplified.
  • Preferred is a RecA protein derived from a bacterium belonging to the genus Thermus (ShigemoriigeY. Et al., Nucleic Acids Res. 2005, 33 (14), e126.; Kato R. et al., J. Biochem. (Tokyo) 1993, 114, 929.; Angov E. et al., J.
  • RecA proteins derived from Thermus thermophilus and Thermus aquaticus are particularly preferred.
  • the wild type thermostable RecA protein derived from Thermus thermophilus HB8 and the wild type thermostable RecA protein derived from Thermophilus HB27 can be preferably used.
  • the base sequence of the wild-type heat-resistant RecA protein derived from Thermus thermophilus HB8 strain is SEQ ID NO: 5
  • the amino acid sequence is SEQ ID NO: 6 (GenBank: BAA04215.1)
  • the base sequence is SEQ ID NO: 7
  • the amino acid sequence Is disclosed as SEQ ID NO: 8 (GenBank: AAA64935.1).
  • the nucleotide sequence of the wild-type heat-resistant RecA protein derived from Thermophilus strain HB27 is disclosed as SEQ ID NO: 9, and the amino acid sequence is disclosed as SEQ ID NO: 10 (GenBank: AAK15321.1).
  • the wild type thermostable RecA protein derived from Thermus aquaticus whose sequence information is disclosed as SEQ ID NO: 11 (base sequence) and SEQ ID NO: 12 (amino acid sequence), can be preferably used as a basis for modification.
  • SEQ ID NO: 11 base sequence
  • SEQ ID NO: 12 amino acid sequence
  • the modified thermostable RecA protein of the present invention can contribute to the improvement of the pairing specificity between the target nucleic acid serving as a template and the primer in the nucleic acid amplification reaction system, compared to the above-described wild-type thermostable RecA protein. All thermostable RecA proteins that express the function are included.
  • the above-mentioned “pairing specificity of a target nucleic acid and a primer in a nucleic acid amplification reaction system” means that in the nucleic acid amplification reaction, the primer is paired (paired only to a region on the target nucleic acid complementary to the primer). And does not pair with a non-complementary region or form a primer dimer between primers.
  • the modified thermostable RecA protein of the present invention functions to strictly pair complementary sequences as compared to the wild type thermostable RecA protein. That is, primer misannealing with respect to the target nucleic acid can be further reduced, and even a single base difference can be identified.
  • the modified thermostable RecA protein of the present invention has higher homologous recombination activity than the wild type thermostable RecA protein. Preferably, it has 2 to 4 times the homologous recombination activity as compared with the wild type thermostable RecA protein. With these functions, non-specific amplification can be suppressed, and highly accurate and efficient amplification of the target nucleic acid can be realized.
  • the modified thermostable RecA protein of the present invention is required that the nucleic acid amplification system can be dissociated from the nucleic acid (recyclability) after pairing of the target nucleic acid and the primer. As a result, the chain extension reaction after pairing can proceed smoothly.
  • the modified thermostable RecA protein of the present invention is an improved recycling compared to the Hyper-TthRecA mutant, which is a C-terminal truncated thermostable RecA protein described in Patent Document 4 of the above-mentioned prior art document. It has sex.
  • the Hyper-TthRecA mutant is a C-terminal truncated heat-resistant RecA protein in which the acidic region on the C-terminal side of the wild-type heat-resistant RecA protein is deleted as described above.
  • the amino acid sequence is disclosed.
  • those in which the 151st arginine is substituted with glycine are also exemplified as Hyper-TthRecA mutants as having equivalent functions.
  • Such a Hyper-TthRecA mutant was developed as having higher homologous recombination activity than the wild type thermostable RecA protein.
  • the modified thermostable RecA protein of the present invention can reduce the nucleic acid amplification inhibitory effect observed with the Hyper-TthRecA mutant, and can realize efficient amplification of the target nucleic acid.
  • RecA protein binds to single-stranded nucleic acid in the presence of NTP such as ATP and searches for homologous sequence sites. As a result, the target nucleic acid having the primer and its complementary sequence is paired. Subsequently, in the process of performing the strand exchange reaction, ATP is hydrolyzed to ADP, but under ADP, the affinity of RecA protein for nucleic acid decreases and dissociates from the nucleic acid. This is recyclability.
  • the modified thermostable RecA protein of the present invention has higher ATPase activity to hydrolyze this ATP into ADP than the Hyper-TthRecA mutant and the wild type thermostable RecA protein. Therefore, after exhibiting the pairing activity, the recyclability of rapidly dissociating from the nucleic acid is high, and nucleic acid amplification is not inhibited.
  • homologous recombination activity whose enhancement was confirmed as a function of the modified thermostable RecA protein of the present invention can be broadly divided into the following two stages.
  • Pairing to complementary sequences (homologous pairing)
  • Strand exchange Therefore, both stages (1) and (2) proceed smoothly, thereby enhancing homologous recombination activity and reducing misannealing in the amplification reaction. That is, after pairing to a complementary sequence, if there is a mistake in pairing, it must be removed. Thereby, mis-annealing in the amplification reaction can be prevented.
  • the single-stranded DNA deviates from the double-stranded DNA together with the strand exchange.
  • the Hyper-TthRecA mutant has only the activity (1) as confirmed in Example 3 below. Therefore, pairing of single-stranded DNA to double-stranded DNA proceeds smoothly. However, since no improvement in the activity of the strand exchange reaction is observed, there is a tendency that the single-stranded DNA and the protein are less likely to be detached from the double-stranded DNA, and even if there is a mistake in pairing, the tendency does not change. .
  • the modified thermostable RecA protein of the present invention can enhance homologous recombination activity from the aspects of both stages (1) and (2), it can reduce misannealing in the amplification reaction.
  • the nucleic acid amplification reaction preferably refers to PCR using a thermostable polymerase, but does not exclude various nucleic acid amplification methods using other enzymes having different principles. Therefore, ligase chain reaction (hereinafter sometimes referred to as “LCR”), strand displacement amplification reaction (hereinafter sometimes referred to as “SDA”), rolling cycle amplification reaction (hereinafter sometimes referred to as “RCA”). Etc.) known nucleic acid amplification reactions.
  • LCR ligase chain reaction
  • SDA strand displacement amplification reaction
  • RCA rolling cycle amplification reaction
  • the modified heat-resistant RecA protein of the present invention as long as it has the above-mentioned function, the 202nd amino acid sequence of SEQ ID NO: 6, 8, or 10 representing the heat-resistant RecA protein derived from Thermos thermophilus Examples are those in which tyrosine is altered. Furthermore, the thing which modification
  • the modification means deletion, substitution or addition.
  • those in which the 202th tyrosine is substituted with an amino acid other than alanine are exemplified.
  • thermostable RecA proteins are shown in SEQ ID NOs: 1, 2, 3, and 4 in the sequence listing.
  • a wild-type thermostable RecA protein having an amino acid sequence different from SEQ ID NO: 6, 8, 10, or 12 is used as a basis for modification
  • the 202nd position of SEQ ID NO: 6, 8, 10, or 12 is used.
  • the tyrosine at a position corresponding to is modified in the modified thermostable RecA protein of the present invention.
  • the tyrosine at the position corresponding to the 202nd position of SEQ ID NO: 6, 8, 10, or 12 is included in the present invention in the amino acid sequence of RecA protein derived from the genus Thermus.
  • the modified thermostable RecA protein of the present invention the amino acid sequence of SEQ ID NO: 6, 8, 10, or 12 representing the RecA protein derived from Thermus thermophilus or Thermus aquaticus as long as it has the above-mentioned function.
  • the tyrosine at the position corresponding to the 202nd is modified, and further, in addition to such modification, modification at other sites is also included.
  • a wild-type RecA having at least 80% or 85%, preferably 90%, particularly preferably 92%, 95% or 98% sequence identity with the amino acid sequence of SEQ ID NO: 6, 8, 10, or 12.
  • Those constructed on the basis of protein are also included in the modified thermostable RecA protein of the present invention.
  • the present invention is also constructed based on a wild-type RecA protein having an amino acid sequence in which at least one amino acid is deleted, substituted, or added to the amino acid sequence of SEQ ID NO: 6, 8, 10, or 12.
  • the deletion, substitution and addition of one or more amino acids are preferably deletions, substitutions and additions of one to several amino acids, including combinations thereof.
  • the tyrosine modification at the position corresponding to the 202nd position is preferably a substitution with an amino acid other than alanine, and particularly preferably a substitution with tryptophan.
  • the modified thermostable RecA protein of the present invention includes those having amino acid sequences including modifications at other sites. Specifically, one or several amino acids are deleted, substituted or added in the amino acid sequence of SEQ ID NO: 1, 2, 3, or 4, and the amino acid sequence of SEQ ID NO: 1, 2, 3, or 4
  • the modified heat-resistant RecA protein of the present invention also includes an amino acid sequence in which the amino acid corresponding to position 202 is tryptophan.
  • a modified thermostable RecA protein of the present invention is also composed of an amino acid sequence in which the amino acid corresponding to position 202 of the amino acid sequence of No. 1, 2, 3, or 4 is tryptophan.
  • the modified thermostable RecA protein of the present invention can be obtained by a known method.
  • the DNA encoding the wild-type heat-resistant RecA protein that is the basis of the modification is modified, and host cells are transformed using the obtained modified DNA. It can be obtained by collecting a thermostable RecA protein from a culture of such a transformant.
  • DNA encoding the wild-type heat-resistant RecA protein that is the basis of modification can be obtained using known gene cloning techniques.
  • primers are designed based on gene information that can be obtained by searching known databases such as GenBank, and PCR is performed using genomic DNA extracted from highly thermophilic bacteria capable of producing RecA protein as a template. Can be obtained. It can also be obtained by synthesis by a nucleic acid synthesis method such as a conventional phosphoramidite method based on known gene information.
  • thermostable RecA protein suitable as a basis for the modified type of the present invention is represented by SEQ ID NO: 5 or 7 encoding the thermostable RecA protein derived from the wild type Thermos thermophilus HB8 strain.
  • An array or the like can be preferably used.
  • the method for modifying the DNA encoding the thermostable RecA protein is not particularly limited, and mutagenesis techniques for preparing modified proteins known to those skilled in the art can be used.
  • a known mutagenesis technique such as a site-directed mutagenesis method, a PCR abrupt induction method that introduces a mutation using a PCR method, or a transposon insertion mutagenesis method can be used.
  • a commercially available mutation introduction kit for example, QuikChange (registered trademark) Site-directed Mutagenesis Kit (manufactured by Stratagene) may be used.
  • an appropriate base sequence encoding it can be determined, and the modification of the present invention can be performed using a nucleic acid synthesis technique such as a conventional phosphoramidite method.
  • a DNA encoding a type of thermostable RecA protein can be synthesized.
  • it can be obtained by performing PCR using a DNA encoding a wild-type heat-resistant RecA protein as a template and an oligonucleotide containing a sequence subjected to a desired modification as a primer.
  • a known host / expression vector system such as Escherichia coli can be used.
  • the modified thermostable RecA protein is linked to a DNA vector that can be stably amplified, and introduced into Escherichia coli that can efficiently express the modified thermostable RecA protein.
  • the medium is inoculated into a medium containing a carbon source, a nitrogen source and other essential nutrients, and cultured according to a conventional method to express the modified thermostable RecA protein.
  • the expression vector includes a promoter sequence, a sequence such as a multicloning site having at least one restriction enzyme site into which a gene encoding a thermostable RecA protein can be inserted, and can be expressed in the host cell described above. Any expression vector can be used.
  • the T7lac promoter is preferably used as a suitable promoter.
  • this expression vector may contain other known base sequences.
  • Other known base sequences are not particularly limited.
  • a stability leader sequence that confers stability of the expression product
  • a signal sequence that confers secretion of the expression product.
  • marking sequences that can confer phenotypic selection in transformed hosts such as neomycin resistance gene, kanamycin resistance gene, chloramphenicol resistance gene, ampicillin resistance gene, hygromycin resistance gene, etc. be able to.
  • an expression vector a commercially available expression vector for Escherichia coli (for example, pET protein expression system: Novagen) can be used. Furthermore, an expression vector incorporating a desired sequence can be prepared and used as appropriate.
  • the host cells are not limited to E. coli, and Bacillus bacteria, Pseudomonas bacteria, and the like can also be used.
  • eukaryotic cells can be used without being limited to prokaryotes.
  • yeasts such as Saccharomyces cerevisiae
  • insect cells such as Sf9 cells
  • animal cells such as CHO cells and COS-7 cells, and the like can be suitably used.
  • the collection and purification of the modified thermostable RecA protein of the present invention from the thus obtained transformant culture can be performed using known techniques. Briefly describing an example, microbial cells are collected from a culture of a transformant culture, suspended in an appropriate buffer solution, and microbial cells are crushed by ultrasonication or the like to obtain a microbial cell extract. The crushing is preferably performed in a buffer solution appropriately containing lysozyme and a surfactant. Subsequently, the supernatant is collected by centrifugation, filtration or the like, and subjected to heat treatment to inactivate contaminating proteins derived from the transformant, thereby obtaining a modified heat-resistant RecA protein crude extract of the present invention.
  • the heat treatment is particularly preferably 60 minutes at 65 ° C.
  • the modified thermostable RecA protein of the present invention can be purified using ion exchange chromatography, gel filtration chromatography, hydrophobic interaction chromatography, affinity chromatography, or the like.
  • the purified RecA protein is a modified heat-resistant RecA protein of the present invention having a modified site where a desired modification has occurred can be confirmed by a known amino acid analysis method.
  • a known amino acid analysis method For example, an automatic amino acid determination method based on Edman degradation can be used.
  • the homologous recombination activity is measured by a known D-Loop formation assay or the like to confirm whether the activity is improved as compared with the wild type thermostable RecA protein having no modification site, or D- This can be done by confirming the time-dependent change in Loop forming activity.
  • the D-Loop formation assay can be performed by the methods shown in Examples 3 and 4 herein.
  • it is performed by confirming whether the pairing specificity of the template nucleic acid and the primer is improved as compared with the wild-type heat-resistant RecA protein having no modification site after being subjected to a nucleic acid amplification system such as PCR. be able to. For example, it can be carried out by comparing the amount of amplification product using a primer containing a mismatched base, and specifically by the methods shown in Examples 6 to 8 of the present specification.
  • the modified thermostable RecA protein of the present invention has an improved ability to contribute to the amplification efficiency of the target nucleic acid in the nucleic acid amplification reaction system as compared with the wild type thermostable RecA protein.
  • it has the activity of improving the pairing specificity between the target nucleic acid and the primer. In other words, it suppresses misannealing such as primer pairing only to the region on the target nucleic acid that is complementary to the primer, pairing to a non-complementary region, or forming primer dimer between primers. , Pairing complementary sequences exactly.
  • thermostable RecA protein of the present invention by adding the modified thermostable RecA protein of the present invention to the nucleic acid amplification system, it is specific and efficient for the target nucleic acid that was insufficient with the wild-type thermostable RecA protein having no modified site. Nucleic acid amplification becomes possible. Therefore, non-specific amplification can be suppressed, and the target nucleic acid can be amplified without being affected by background noise, which can contribute to improvement in amplification accuracy. Therefore, it can be used for single nucleotide polymorphism analysis and the like that require highly accurate nucleic acid amplification, and can provide proteins that can be widely used in various industrial fields, particularly in the field of molecular biology.
  • modified thermostable RecA protein of the present invention has a high homologous recombination function, efficient gene transfer can be realized in gene recombination experiments.
  • proteins that can be used for gene introduction into embryonic stem cells in the production of transgenic animals can be widely used in the field of molecular biology such as analysis of genetic phenomena.
  • the modified thermostable RecA protein of the present invention has an improved ability to contribute to high-accuracy and highly efficient amplification compared to the C-terminal truncated thermostable RecA protein previously developed by the present inventors.
  • the C-terminal truncated RecA protein is conventionally known as having the ability to contribute to highly accurate nucleic acid amplification, but has a problem of inhibition of amplification.
  • the modified heat-resistant RecA protein of the present invention has an enhanced function (recyclability) that can be dissociated from the nucleic acid after pairing the target nucleic acid and the primer in the nucleic acid amplification system. As a result, the chain extension reaction after pairing can proceed smoothly.
  • the inhibitory effect on nucleic acid amplification as observed with the conventional C-terminal truncated thermostable RecA protein can be reduced, and efficient amplification of the target nucleic acid can be realized.
  • the present invention further provides a method for amplifying a template nucleic acid using the modified thermostable RecA protein of the present invention.
  • a nucleic acid amplification reaction is performed by adding the modified heat-resistant RecA protein of the present invention.
  • PCR is a method of amplifying DNA in a chain reaction using a heat-resistant DNA polymerase.
  • the principle of PCR is that the nucleic acid to be amplified (hereinafter sometimes abbreviated as a target nucleic acid) is multiplied by 2 to the nth power by repeating three cycles of temperature changes in n cycles in the presence of a primer and a thermostable DNA polymerase. It amplifies.
  • a PCR reaction solution prepared containing the modified thermostable RecA protein of the present invention, target nucleic acid, thermostable DNA polymerase, primer, dNTP, and an appropriate buffer solution is prepared. And it is performed by subjecting to a temperature cycle consisting of heat denaturation, annealing, and elongation reaction. Further, it may be prepared containing nucleotide 5′-triphosphate (hereinafter referred to as “NTP”) separately from dNTP.
  • NTP nucleotide 5′-triphosphate
  • the primer and dNTP may be labeled with an appropriate labeling substance for detection, if necessary. Since such a labeling substance is known, those skilled in the art can select and use it appropriately.
  • the target nucleic acid to be amplified is not limited with respect to its origin, length, base sequence, etc., and may be any nucleic acid, regardless of whether it is single-stranded or double-stranded. Specifically, it may be a genomic DNA of an organism, or a fragment obtained by cleaving the genomic DNA by physical means or restriction enzyme digestion. Furthermore, it can be suitably used for DNA fragments inserted into plasmids, phages and the like. Furthermore, it may be prepared or isolated from a sample that may contain a nucleic acid.
  • a DNA fragment synthesized using an automatic nucleic acid synthesizer or the like commonly used in the technical field, and an artificial product such as a cDNA fragment synthesized using mRNA as a template can be used as the target nucleic acid.
  • Primers are designed to be complementary to a specific sequence of the target nucleic acid.
  • the simplest system requires two primers, but three or more may be used when performing multiplex PCR (Multiplex PCR) or the like. It can also be suitably used for amplification reactions using only one primer.
  • the design of the primer is determined by examining the sequence of the target nucleic acid in advance, except when a random primer is used.
  • a database such as GeneBank or EBI can be suitably used for investigating the base sequence of the target nucleic acid.
  • a chemical synthesis method based on the phosphoramidite method or the like, or a restriction enzyme fragment or the like when a target nucleic acid has already been obtained can be used.
  • a primer based on a chemical synthesis method it is designed based on the sequence information of the target nucleic acid prior to synthesis. After synthesis, the primer is purified by means such as HPLC.
  • complementary means that the primer and the target nucleic acid can specifically bind according to the base pairing rule to form a stable double-stranded structure.
  • the appropriate length is determined depending on many factors such as target nucleic acid sequence information such as GC content, and hybridization reaction conditions such as reaction temperature and salt concentration in the reaction solution. The length is preferably 20 to 50 bases.
  • thermostable DNA polymerase to be used is not particularly limited as long as it is a thermostable DNA polymerase that can be usually used in PCR.
  • a thermostable DNA polymerase that can be usually used in PCR.
  • Taq polymerase from Thermus aquaticus Tth polymerase from Thermus thermophilus
  • Bst polymerase from Bacillus Stearothermophilus Thermococcus ⁇ ⁇ litoralis
  • Thermococcus kodakaraensis KOD polymerase Thermococcus kodakaraensis KOD polymerase
  • Pyrococcus furiosus-derived Pfu polymerase and other DNA polymerases derived from thermophilic bacteria.
  • dNTP deoxynucleotide
  • four types of deoxynucleotides corresponding to each base of adenine, thymine, guanine and cytosine are used.
  • a mixture of dGTP, dATP, dTTP, and dCTP is preferably used.
  • a derivative of deoxynucleotide can be included in the nucleic acid molecule synthesized and extended by PCR as long as it can be incorporated by a thermostable DNA polymerase. Examples of such derivatives include 7-deaza-dGTP and 7-deaza-dATP.
  • it can be used in place of dGTP and dATP, respectively, or in the presence of both. Therefore, the use of any derivative is not excluded as long as four types corresponding to each base of adenine, thymine, guanine and cytosine necessary for nucleic acid synthesis are included.
  • the buffer solution is generally prepared containing an appropriate buffer component and a magnesium salt.
  • phosphates such as Tris acetic acid, Tris hydrochloric acid, sodium phosphate, and calcium phosphate can be suitably used. In particular, the use of trisacetic acid is preferred.
  • the final concentration of the buffer component is prepared in the range of 5 mM to 100 mM.
  • the pH of the buffer is preferably adjusted within the range of pH 6.0 to 9.5, particularly preferably pH 7.0 to 8.0.
  • the magnesium salt is not particularly limited, and magnesium chloride, magnesium acetate and the like can be used as appropriate. In particular, magnesium acetate is preferable.
  • potassium salts such as KCl, DMSO, glycerol, betaine, gelatin, Triton and the like can be added as necessary.
  • a buffer solution attached to a commercially available heat-resistant DNA polymerase for PCR can be used.
  • the composition of the buffer can be appropriately changed according to the type of DNA polymerase used. In particular, it can be set appropriately in consideration of the effects of ionic strength such as MgCl 2 and KCl, various additives that affect the melting temperature of DNA such as DMSO and glycerol, and their concentrations.
  • the reaction solution is preferably prepared in a volume of 100 ⁇ l or less, particularly in the range of 10 to 50 ⁇ l.
  • concentration of each component the modified thermostable RecA protein of the present invention is preferably used so as to be contained in the reaction solution at a concentration of 0.004 to 0.02 ⁇ g / ⁇ l.
  • concentration of each component other than the modified heat-resistant RecA protein of the present invention can be appropriately set by those skilled in the art since PCR is known.
  • the target nucleic acid is preferably prepared at a concentration of 10 ⁇ g to 1 ⁇ g per 100 ⁇ l
  • primer DNA is preferably prepared at a final concentration of 0.01 to 10 ⁇ M, particularly 0.1 to 1 ⁇ M.
  • thermostable DNA polymerase is preferably used at a concentration in the range of 0.1 to 50 unit, particularly 1 to 5 unit per 100 ⁇ l.
  • dNTP is preferably prepared to a final concentration of 0.1 ⁇ M to 1 ⁇ M.
  • it is not limited to this, and is appropriately set as described above.
  • thermostable RecA protein of the present invention is performed according to the following steps, which are performed in the presence of the modified thermostable RecA protein of the present invention.
  • ⁇ Repeat the appropriate cycle for the reaction consisting of the above three stages of temperature changes (1) to (3).
  • the primer as a starting point, the synthesis of another nucleic acid strand having complementarity is started using the target nucleic acid as a template.
  • the target nucleic acid is amplified to 2 n times in the reaction of n cycles.
  • the number of thermocycles is determined according to the type, amount and purity of the target nucleic acid serving as a template. From the viewpoint of efficient nucleic acid amplification by suppressing nonspecific amplification, 20 to 40 cycles, particularly 20 It is preferable to carry out in 30 cycles.
  • Double-stranded nucleic acid is denatured by heating and dissociated into single strands. Preferably, it is carried out at 92 to 98 ° C. for 10 to 60 seconds.
  • only the first heat denaturation can be set at a low temperature (eg, about 92 ° C.) in order to prevent degradation of the template DNA.
  • Annealing is preferably performed for 30 to 60 seconds.
  • the annealing temperature is preferably estimated from the Tm of the oligonucleotide used for the primer, and this Tm is set as the annealing temperature. It is known that when the annealing temperature is high, the primer-specific binding ability of the primer is improved, but when the annealing temperature is too high, the primer does not bind to the template nucleic acid. Usually, it is carried out at 50 to 70 ° C.
  • the nucleic acid chain extension reaction from the primer is performed at the 3 'end by the heat-resistant polymerase.
  • the elongation reaction temperature is appropriately set according to the type of heat-resistant polymerase, but is preferably 65 to 75 ° C.
  • the extension time is sufficient for about 1 minute when the target sequence is 1 kb or less, and when it is longer, the extension time is preferably increased at a rate of 1 minute per 1 kb.
  • the nucleic acid amplification method of the present invention can be applied to various modified PCR methods.
  • adapter-added PCR allele-specific amplification method (MASA method), asymmetric PCR, reverse PCR (IPCR), reverse transcription PCR (RT-PCR), single-stranded DNA conformation polymorphism PCR (PCR-SSCP method) , Arbitrarily polymerase chain reaction (AP-PCR), RACE, multiplex PCR (multiplex (PCR) and the like.
  • the present invention is not limited to these, and can be used for any PCR modification.
  • the nucleic acid amplification reaction is carried out in the presence of the modified heat-resistant RecA protein of the invention, whereby specific nucleic acid amplification specific to the target nucleic acid becomes possible.
  • Nonspecific amplification unrelated to the target nucleic acid can be suppressed, and nucleic acid amplification that is not affected by background noise becomes possible. That is, it is possible to maintain a state in which the sequence specificity of the primer to the target nucleic acid serving as a template is improved.
  • non-specific amplification due to misannealing such as annealing of the primer other than the target sequence or annealing of the primers can be suppressed. This enables highly accurate nucleic acid amplification.
  • thermostable RecA protein of the present invention is a thermostable enzyme, the above effects can be exhibited continuously. As a result, highly accurate nucleic acid amplification can be achieved in all cycles of the nucleic acid amplification reaction system including the heating step.
  • the present invention also provides a nucleic acid amplification kit for amplifying a nucleic acid by PCR.
  • the nucleic acid amplification kit of the present invention comprises a DNA polymerase and a modified thermostable RecA protein. Furthermore, it may be configured by appropriately containing components necessary for PCR such as an appropriate buffer and dNTP. Moreover, in the case of a kit for detecting a pathogen or the like with a desired nucleic acid, an arbitrary primer or the like specific for desired nucleic acid amplification may be included. By configuring the components necessary for PCR amplification in this way, simple and rapid PCR amplification becomes possible.
  • the modified heat-resistant RecA protein of the present invention and a nucleic acid amplification method and kit using the protein can be used for various applications.
  • it can be used for a wide variety of applications such as the medical field, biochemical field, environmental field, and food field.
  • it can be used when preparing a large amount of DNA from a small amount of sample for genotyping, or when preparing DNA for DNA sequencing.
  • the present invention can be applied to various uses such as preparing DNA for DNA chip fixation from a small amount of sample extracted from animals, plant cells, microorganisms or the like.
  • examples of use in the medical field include genetic diagnosis including single nucleotide polymorphism analysis, detection of pathogens such as viruses and bacteria such as SARS and influenza, and the like.
  • the present invention can be suitably applied to single nucleotide polymorphism analysis.
  • nonspecific binding such as binding of a primer to a nucleic acid other than the target nucleic acid and binding between primers can be suppressed, and primer misannealing can be effectively suppressed. Therefore, by using a primer complementary to a nucleic acid having a desired single nucleotide polymorphism, a nucleic acid having such a single nucleotide polymorphism can be efficiently amplified.
  • nucleic acids that do not have such a single nucleotide polymorphism are not amplified or are not suppressed.
  • a nucleic acid having such a single nucleotide polymorphism can be specifically amplified.
  • the effect is enhanced by the high annealing temperature achieved by the present invention, and single nucleotide polymorphisms can be detected with high sensitivity and efficiency.
  • the use in the field of biochemistry includes identification of individuals and identification of biological species.
  • the present invention can provide a method for reducing primer misannealing with respect to a template nucleic acid of a protein as compared with a wild type thermostable RecA protein. And the said method has the following processes, and is comprised. Replacing the amino acid at the position corresponding to position 202 in the amino acid sequence of SEQ ID NO: 6, 8, 10, or 12 of the wild-type heat-resistant RecA protein with the other wild-type heat-resistant RecA protein.
  • mutant heat-resistant RecA protein those exemplified as the basis of the mutant heat-resistant RecA protein of the present invention can be suitably used.
  • substitution with tryptophan is preferably exemplified, and the substitution of amino acids can be performed by using a known mutation introduction technique for preparing a known modified protein, and such a technique has been described above.
  • the protein constructed by the method of the present invention has the function of the above-described modified thermostable RecA protein of the present invention.
  • the modified thermostable RecA protein of the present invention can be applied to enrichment or isolation of target cDNA clones from a DNA library.
  • the modified thermostable RecA protein of the present invention can be applied when an amplification reaction is performed using a partial sequence of a target cDNA desired to be concentrated or isolated as a primer and a DNA library as a template.
  • the amplification reaction it is possible to use a known nucleic acid amplification reaction system or the like other than PCR. Thereby, non-specific amplification unrelated to the target cDNA can be suppressed, and only the target cDNA can be specifically amplified.
  • the modified thermostable RecA protein of the present invention to a cloning system for a target DNA clone from a DNA library, the desired target cDNA clone can be concentrated and isolated specifically and efficiently. Is possible. Specific and efficient cDNA cloning can greatly contribute to the fields of gene expression, development, analysis of differentiation, etc., and production of useful substances.
  • the DNA library a DNA library containing or expected to contain a target DNA region desired to be concentrated or isolated is used.
  • the DNA library may be either a genomic library or a cDNA library, but a cDNA library is particularly preferable.
  • genomic library is used as a concept that means an aggregate of cloned DNAs in which total genomic DNA of a specific single species is randomly incorporated into a vector.
  • a cDNA library was used as a concept meaning an aggregate of cDNA fragments prepared by converting mRNAs derived from specific tissues, cells and organisms into cDNAs by reverse transcription reaction and incorporating them into vectors.
  • the primer is usually designed to be complementary to a specific sequence of the target nucleic acid.
  • the target sequence to be amplified preferably has a complementary base sequence at both ends thereof, and a partial sequence of the target cDNA desired to be concentrated or isolated can be suitably used.
  • the design of a primer is well-known, it designs based on the base sequence of cDNA used as a target, and can prepare it by well-known techniques, such as chemical synthesis.
  • the modified thermostable RecA protein of the present invention can be applied to reverse transcription reaction from RNA to DNA. Specifically, when a cDNA synthesis reaction from RNA is performed by reverse transcription using a random hexamer primer, oligo dT primer, and target gene-specific primer in the presence of reverse transcriptase, the modified type of the present invention.
  • the heat-resistant RecA protein can be applied.
  • the present invention can also be applied to an amplification reaction using synthesized cDNA as a template.
  • thermostable RecA protein of the present invention to a reverse transcription reaction system, it is possible to synthesize a cDNA for a desired target RNA specifically and efficiently. Since conversion from RNA to cDNA is an indispensable technique in genetic engineering, its utility value is high, such as gene expression detection and quantification, RNA structure analysis, and cDNA cloning.
  • RNA is not particularly limited, such as total RNA, mRNA, tRNA, rRNA, and the like. It is prepared from a cell or tissue expected to express or express a desired gene using a known method. For example, a guanidine / cesium TFA method, a lithium chloride / urea method, an AGPC method, or the like can be used.
  • the primer is not particularly limited as long as it anneals to the template RNA under the applied reaction conditions. As described above, random hexamer primers, oligo dT primers, and target gene specific primers can be used.
  • the target gene-specific primer has a base sequence complementary to a specific template RNA.
  • the 3 ′ side of a primer used in a general PCR amplification system is used.
  • thermostable RecA protein thermus thermophilus-derived RecA protein
  • TthRecA protein thermus thermophilus-derived RecA protein
  • Example 1 Confirmation of nucleic acid amplification reaction with addition of TthRecA protein Regarding the influence of TthRecA protein on nucleic acid amplification reaction, single-stranded binding protein derived from E. coli, which is another DNA binding protein not derived from thermophilic bacteria (hereinafter, The case where it was called “SSB protein”) and the case where RecA protein derived from E. coli was added were compared.
  • SSB protein single-stranded binding protein derived from E. coli
  • PCR reaction solution (25 ⁇ l) is 0.8-50 ng human genomic DNA (Promega, Human Genomic DNA), 0.4 ⁇ g wild type TthRecA protein (Storage buffer: 50 mM Tris-HCl pH7.5, 1.0 mM EDTA, 0.5 mM) DTT, 50% w / v glycerol), 0.8 ⁇ M primer, 2.0 U DNA polymerase (Takara-Bio, rTaq DNA polymerase), 0.2 mM dNTPs mixture, 10 mM Tris-HCl pH8.3, 50 mM KCl, 1.5 mM MgCl Prepared by mixing to 2 .
  • the amount of template nucleic acid was 50 ng, 25 ng, 13 ng, 6 ng, 3 ng, 1.5 ng, and 0.8 ng, and the amount of amplified product was compared.
  • primer set a 5′-aacct cacaa ccttg gctga-3 ′ (SEQ ID NO: 14) 5′-ttcac aactt aagat ttggc-3 ′ (SEQ ID NO: 15)
  • sequence information of the wild-type TthRecA protein used here and the gene encoding it is shown in SEQ ID NO: 5 (base sequence, GenBank: D17392.1), SEQ ID NO: 6 (amino acid sequence, GenBank: BAA04215.1). ).
  • each PCR reaction solution prepared above was subjected to PCR under the same conditions to obtain an amplification product.
  • PCR was performed by 25 cycles of amplification reaction, with heat denaturation at 92 ° C for 30 seconds, 94 ° C for 10 seconds, 55 ° C for 30 seconds, and 68 ° C for 60 seconds. .
  • an electrophoresis buffer was added to each amplification reaction solution and stirred, and an appropriate amount was taken and subjected to 1.2% agarose gel electrophoresis. The gel after electrophoresis was stained with ethidium bromide to visualize the DNA band.
  • E. coli-derived SSB protein Promega, Single-Stranded DNA Binding Protein
  • E. coli-derived RecA protein Epicenter Technologies
  • FIG. 1A to 1C are diagrams showing the results of electrophoresis.
  • A is the result of amplification in the presence of the wild type TthRecA protein
  • B is the result of amplification in the presence of E. coli-derived SSB protein
  • C is the result of amplification in the presence of E. coli-derived RecA protein.
  • Lanes 1 to 7 in A to C are the results when the template nucleic acid amounts were 50 ng, 25 ng, 13 ng, 6 ng, 3 ng, 1.5 ng, and 0.8 ng, respectively.
  • D and E in FIG. 1 are schematic diagrams of the nucleic acid amplification mode. Based on the results of FIGS. 1A to 1C, the chain extension reaction after the primer annealing to the template nucleic acid is schematically shown. D is the reaction in the presence of the wild-type TthRecA protein indicated by A, and E is the reaction in the presence of the E. coli-derived SSB protein and E. coli-derived RecA protein indicated by B and C above.
  • the wild-type TthRecA protein has heat resistance, and protein denaturation hardly occurs even during a high-temperature PCR reaction (55 ° C. or higher), and as shown in FIG. Since it dissociates, it can be inferred that inhibition of the amplification reaction was difficult to occur.
  • mutant A mutant was prepared by substituting tryptophan for the 202nd tyrosine of the amino acid sequence of the wild-type TthRecA protein. This mutant was named “TthRecA-Y202W mutant”.
  • primer set b The sequence information of primer set b is as follows.
  • Primer set b 5′-aaggt ggggg tcacg tgggg caacc ccgag acca-3 ′ (SEQ ID NO: 16)
  • PCR reaction solution 50 ⁇ l was prepared by using 15 ⁇ ng template nucleic acid, 0.5 ⁇ M primer set b, 1.25 ⁇ U DNA polymerase (pfu Ultra HF polymerase), 0.2 mM mM dNTPs, 1 ⁇ PCR reaction buffer (Takara-Bio, QCII). It was prepared by mixing. Then, the PCR reaction solution was subjected to 18 cycles of amplification reaction with 95 ° C for 30 seconds, 55 ° C for 60 seconds, and 68 ° C for 6 minutes.
  • the obtained amplification product was transformed into BL21 (DE3) pLysS to obtain a TthRecA-Y202W mutant expression clone.
  • the cells are disrupted by sonication, mixed with EDTA (final concentration 5 mM) and KCl (final concentration 2 M), and then centrifuged (4 ° C., 60,000 ⁇ g, 60 minutes) to separate the supernatant. I took it.
  • centrifuged 4 ° C., 60,000 ⁇ g, 60 minutes
  • heat treatment 65 ° C., 1 hour
  • rapid cooling 0. ° C., 10 minutes
  • centrifugation 4 ° C., 60,000 ⁇ g, 20 minutes was performed, and the supernatant was collected.
  • the supernatant was applied to a hydrophobic chromatography column (Tosoh, Butyl Toyopearl 650M).
  • the column equilibration buffer (PEMG2K) was 50 mM potassium phosphate pH 6.5 (200 ml), 1 mM EDTA, 5 mM 2-mercaptoethanol, 10% glycerol, 2 M KCl. After washing with PEM2K buffer (100 ml), the protein was applied with a gradient using buffer solution (PEMG: 50 mM potassium phosphate pH 6.5, 1 mM EDTA, 5 mM 2-mercaptoethanol, 10% glycerol). The eluted fraction was collected.
  • PEMG (20 ml) was used as the equilibration buffer for the column. After washing with PEMG buffer (30 ml), a gradient was applied using PEMG1K buffer (60 ml) to fractionate the protein elution fraction. Finally, dialysis was performed using TEDG (20 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.1 mM DTT, 50% glycerol) buffer as an external solution.
  • TthRecA-Y202W mutant 5 ⁇ g equivalent of the obtained TthRecA-Y202W mutant was subjected to SDS-PAGE electrophoresis, and the gel after electrophoresis was stained with CBB.
  • the equivalent amount of each protein of 5 ⁇ g was subjected to electrophoresis in the same manner.
  • the Hyper-TthRecA mutant used here was prepared based on the description in Patent Document 4 described above.
  • the amino acid information was disclosed as SEQ ID NO: 13 in the sequence listing.
  • sequence information of the TaqRecA protein used was disclosed as SEQ ID NO: 11 (base sequence) and SEQ ID NO: 12 (amino acid sequence) in the sequence listing.
  • FIG. 2 is a diagram showing the results of electrophoresis. Lane 1 shows the results of electrophoresis of the wild type TthRecA protein, Lane 2 shows the TthRecA-Y202W mutant, Lane 3 shows the wild type TaqRecA protein, and Lane 4 shows the result of electrophoresis of the Hyper-TthRecA mutant.
  • Example 3 Confirmation of Homologous Recombination Activity of TthRecA-Y202W Mutant-1
  • the homologous recombination activity of the TthRecA-Y202W mutant obtained in Example 2 was confirmed by comparing the wild-type TthRecA protein and the Hyper-TthRecA mutant with a D-Loop formation reaction.
  • PBluescript SK (-) DNA (Novagen) is used as double-stranded DNA, and 90 mer oligonucleotide (5'-AAAT AATCT AAAGT ATATA TGAGT AAACT TGGTC TGACA GTTAC CAATG CTTAA TCAGT GAGGC ACCTA TCTCA GCGAT CTGTC TATTT- 3 ′ (SEQ ID NO: 18)) was used as single-stranded DNA.
  • reaction stop solution (0.5% w / vol SDS, 0.7 mg / ml Proteinase K) was added and kept at 0 ° C. for 30 minutes.
  • reaction solution was subjected to 1% agarose gel electrophoresis (electrophoresis buffer solution: 0.5 ⁇ TBE), and then the gel was dried and signal detection was performed with an RI imaging analyzer BAS2500 (Fuji Film).
  • FIG. 3 is a diagram showing the results of electrophoresis.
  • A, B and C D-Loop product bands are indicated by arrows.
  • A is a wild-type TthRecA protein
  • B is a TthRecA-Y202W mutant
  • C is a hyper-TthRecA mutant confirmed by D-Loop formation reaction.
  • Lanes 1 to 8 of A to C are the results of D-Loop formation reactions at 0, 0.5, 1, 1.5, 2.5, 5, 10, and 20 minutes, respectively.
  • FIG. 4 is a graph summarizing the homologous recombination activities of the wild type TthRecA protein, the TthRecA-Y202W mutant, and the Hyper-TthRecA mutant, which are the results of FIG.
  • the horizontal axis represents the reaction time (minutes), and the vertical axis represents the D-Loop formation rate (%).
  • the TthRecA-Y202W mutant showed more D-Loop formation than the wild-type TthRecA protein (Comparison between waveforms (1) and (2) in FIG. 4). ).
  • the TthRecA-Y202W mutant has the same degree of homologous recombination activity as compared with the Hyper-TthRecA mutant, but dissociation of D-Loop formation over time was observed (Fig. 4). Comparison of waveforms (1) and (3)).
  • the TthRecA-Y202W mutant had about 4 times higher homologous recombination activity than the wild type TthRecA protein.
  • the maximum value of the homologous recombination activity was equivalent to that of the Hyper-TthRecA mutant.
  • the TthRecA-Y202W mutant showed dissociation of D-Loop formation over time, compared to the Hyper-TthRecA mutant.
  • the TthRecA-Y202W mutant is excellent in recyclability in which the RecA protein is dissociated from the nucleic acid by ATP hydrolysis.
  • Example 4 Confirmation of homologous recombination activity of TthRecA-Y202W mutant-2
  • the homologous recombination activity of the TthRecA-Y202W mutant obtained in Example 2 was confirmed by comparing the wild-type TthRecA protein and the Hyper-TthRecA mutant with the D-Loop formation reaction following Example 3 above. did.
  • mutants were prepared by substituting the 202th tyrosine of the amino acid sequence of the wild-type TthRecA protein with alanine instead of tryptophan, and the D-Loop forming ability was also compared. This mutant was named “TthRecA-Y202A mutant”.
  • reaction stop solution (0.5% w / vol SDS, 0.7 mg / ml proteinase K) was added and kept at 0 ° C. for 30 minutes.
  • reaction solution was subjected to 1% agarose gel electrophoresis (electrophoresis buffer solution: 0.5 ⁇ TBE), and then the gel was dried and signal was detected with an imaging analyzer (Fuji Film, BAS2500).
  • the TthRecA-Y202A mutant was prepared by the same method as TthRecA-Y202W in Example 2. At this time, the following primers were used. Primer set: 5'-gaagg tgggg gtcac ggccg gcaac cccga gacc -3 '(SEQ ID NO: 19) 5'- ggtct cgggg ttgcc ggccg tgacc cccac cttc -3 '(SEQ ID NO: 20)
  • FIG. 5 is a graph summarizing the homologous recombination activities of the wild-type TthRecA protein, TthRecA-Y202W mutant, and TthRecA-Y202A mutant in the same manner as FIG.
  • the horizontal axis represents the reaction time (minutes), and the vertical axis represents the D-Loop formation rate (%).
  • Example 5 Confirmation of ATPase activity of TthRecA-Y202W mutant The homologous recombination activity of TthRecA-Y202W mutant obtained in Example 2 was compared with wild-type TthRecA protein and Hyper-TthRecA mutant by measuring ATPase activity. It confirmed by examining.
  • FIGS. 6 and 7 are graphs showing the ATPase activity of wild-type TthRecA protein, TthRecA-Y202W mutant, and Hyper-TthRecA mutant, and the vertical axis shows the ADP production rate (%).
  • FIG. 6 shows single-stranded DNA-dependent ATPase activity
  • FIG. 7 shows double-stranded DNA-dependent ATPase activity.
  • the TthRecA-Y202W mutant is higher in both double-stranded DNA and single-stranded DNA-dependent ATPase than the wild-type TthRecA protein and the Hyper-TthRecA mutant. Activity was obtained. Therefore, it is considered that the TthRecA-Y202W mutant is excellent in recyclability for dissociating proteins from nucleic acids accompanied by ATP hydrolysis.
  • Example 6 Confirmation of influence of TthRecA-Y202W mutant on nucleic acid amplification accuracy
  • the influence of the TthRecA-Y202W mutant obtained in Example 2 on nucleic acid amplification precision was compared with that of the wild-type TthRecA protein. Specifically, the amplification product amount in PCR using a primer containing a mismatch of 1 to 5 bases between the primer and the template nucleic acid was compared.
  • the PCR reaction solution (25 ⁇ l) was 25 ng of human genomic DNA of Example 1 as a template nucleic acid, 0.5 ⁇ g of wild-type TthRecA protein, 0.2 ⁇ M of primer (final concentration), DNA polymerase (Takara-Bio, Premix Taq Version 2.0). ) (Standard concentration of the PCR reagent kit).
  • primers are prepared based on the following primer sets c, d, and e (Homo sapiens chromosome 7, ACCESSION NT_079596 REGION: 56196821..56325337, primer set d is 1 base and e is 5 base mismatch
  • primer set d is 1 base
  • e is 5 base mismatch
  • the sequence information of primer sets c, d, and e is as follows. Primer set c does not contain mismatched bases, but primer sets d and e contain mismatched bases, and the bases are shown in capital letters.
  • Primer set c (0 mismatched bases): 5'-gccta aggtc acgtt gtcc-3 '(SEQ ID NO: 21) 5'-gcagg cacca agaac tactg c-3 '(SEQ ID NO: 22)
  • Primer set d (number of mismatched bases 1): 5'-gccta aggtc acgtt gtccc-3 '(SEQ ID NO: 21) 5'-gcagg cacca Ggaac tactg c-3 '(SEQ ID NO: 23)
  • Primer set e (mismatched base number 5): 5'-gccta aggtc acgtt gtccc-3 '(SEQ ID NO: 21) 5′-gcGgg cGcca GgaaG tacGg c-3 ′ (SEQ ID NO: 24)
  • PCR reaction solution prepared above was subjected to PCR under the same conditions to obtain an amplification product.
  • PCR was performed by 25 cycles of amplification reaction, with heat denaturation at 94 ° C. for 30 seconds, 94 ° C. for 15 seconds, 55 ° C. for 30 seconds, and 72 ° C. for 60 seconds. .
  • an electrophoresis buffer was added to each amplification reaction solution and stirred. Half of the solution was taken and subjected to 1.2% agarose gel electrophoresis. The gel after electrophoresis was stained with Vistra green to visualize DNA bands.
  • the TthRecA-Y202W mutant added was subjected to PCR according to the above procedure and then subjected to electrophoresis.
  • PCR was performed according to the above procedure without adding RecA protein, and then subjected to electrophoresis, which was used as a control.
  • FIG. 8 is a diagram showing the results of electrophoresis.
  • A is the control
  • B is the amplification result in the presence of the TthRecA-Y202W mutant
  • C is the amplification result in the presence of the wild-type TthRecA protein.
  • Lanes 1 to 3 of A to C are amplification results with the primer sets c, d, and e, respectively.
  • Example 7 Confirmation of Effect of Nucleic Acid Amplification Accuracy of TthRecA-Y202W Mutant-1
  • the effect of the TthRecA-Y202W mutant on the nucleic acid amplification accuracy was compared with that of the wild-type TaqRecA protein. Specifically, it was carried out by comparing the amount of amplification product in PCR using a primer containing a single base mismatch between the primer and the template nucleic acid.
  • the PCR reaction solution (25 ⁇ l) was 25 ng of human genomic DNA of Example 1 as a template nucleic acid, 0.5 ⁇ g of TthRecA-Y202W mutant, 0.2 ⁇ M of primer (final concentration), DNA polymerase (Takara-Bio, Premix Taq Version). 2.0) (standard concentration of the PCR reagent kit).
  • primer set f or g prepared based on Homo sapiens chromosome 7, ACCESSION NT_079596 REGION: 56196821..56325337, and prepared so that primer set g includes a mismatch of 1 base
  • sequence information of the primer sets f and g is as follows. The primer set f does not contain mismatched bases, but the primer set g contains one base mismatch, and the bases are shown in capital letters.
  • Primer set f (0 mismatched bases): 5'-gccta aggtc acgtt gtcc-3 '(SEQ ID NO: 25) 5'-gcagg cacca agaac tactgc-3 '(SEQ ID NO: 26)
  • Primer set g (number of mismatched bases 1): 5'-gccta aggtc acgtt gtccc-3 '(SEQ ID NO: 25) 5'-gcagg cacca Ggaac tactgc-3 '(SEQ ID NO: 27)
  • PCR reaction solution prepared above was subjected to PCR under the same conditions to obtain an amplification product.
  • PCR was carried out by amplification reaction of 25 cycles, with heat denaturation at 94 ° C for 30 seconds, reaction at 94 ° C for 15 seconds, 55 ° C for 30 seconds and 72 ° C for 60 seconds. .
  • an electrophoresis buffer was added to each amplification reaction solution and stirred. Half of the solution was taken and subjected to 1.2% agarose gel electrophoresis. The gel after electrophoresis was stained with Vistra green to visualize DNA bands.
  • PCR was performed according to the above-mentioned procedure for the wild type TthRecA protein and the wild type TaqRecA protein added, and then subjected to electrophoresis. Furthermore, PCR was performed according to the above procedure without adding any RecA protein or mutant, and then subjected to electrophoresis, which was used as a control.
  • sequence information of the TaqRecA protein used was disclosed as SEQ ID NO: 11 (base sequence) and SEQ ID NO: 12 (amino acid sequence) in the sequence listing.
  • FIG. 9 is a diagram showing the results of electrophoresis.
  • A is the control
  • B is the amplification result in the presence of the TthRecA-Y202W mutant
  • C is the amplification result in the presence of the wild type TthRecA protein
  • D is the amplification result in the presence of the wild type TaqRecA protein.
  • Lanes 1 and 2 of A to D are amplification results with the primer sets f and g, respectively.
  • Example 8 Confirmation of Effect of Nucleic Acid Amplification Accuracy of TthRecA-Y202W Mutant-2
  • the PCR reaction solution (25 ⁇ l) was 25 ng of human genomic DNA of Example 1 as a template nucleic acid, 0.5 ⁇ g of TthRecA-Y202W mutant, 0.2 ⁇ M of primer (final concentration), DNA polymerase (Takara-Bio, Premix Taq Version). 2.0) (standard concentration of the PCR reagent kit).
  • primer set h or i prepared based on Homo sapiens chromosome 7, ACCESSION NT_079596 REGION: 56196821..56325337, and prepared so that primer set i includes a single base mismatch
  • primer set i each was prepared using PCR reaction solution.
  • the sequence information of primer sets h and i is as follows. Primer set h does not contain mismatched bases, but primer set i contains a single base mismatch, and the bases are shown in capital letters.
  • Primer set h (0 mismatched bases): 5'-gccta aggtc acgtt gtccc-3 '(SEQ ID NO: 28) 5'-gcagg cacca agaac tactgc-3 '(SEQ ID NO: 29)
  • Primer set i (mismatched base number 1): 5'-gccta aggtc acgtt gtccc-3 '(SEQ ID NO: 28) 5'-gcagg cacca Ggaac tactgc-3 '(SEQ ID NO: 30)
  • PCR reaction solution prepared above was subjected to PCR under the same conditions to obtain an amplification product.
  • PCR was performed by 25 cycles of amplification reaction, with heat denaturation at 94 ° C. for 30 seconds, 94 ° C. for 15 seconds, 55 ° C. for 30 seconds, and 72 ° C. for 60 seconds. .
  • an electrophoresis buffer was added to each amplification reaction solution and stirred. Half of the solution was taken and subjected to 1.2% agarose gel electrophoresis. The gel after electrophoresis was stained with Vistra green to visualize DNA bands.
  • the wild-type TthRecA protein, the wild-type TaqRecA protein, and the Hyper-RecA mutant were also added to the electrophoresis after performing PCR according to the above procedure.
  • FIG. 10 is a diagram showing the results of electrophoresis.
  • A shows the amplification result with primer set h
  • B shows the amplification result with primer set i.
  • Lanes 1 to 4 of A and B show the results in the presence of the wild type TthRecA protein, the TthRecA-Y202W mutant, the wild type TaqRecA protein, and the Hyper-TthRecA mutant, respectively.
  • Example 9 Persistence confirmation of effect of improving nucleic acid amplification accuracy of TthRecA-Y202W mutant From the results of the above Examples 6 to 8, it was confirmed that the TthRecA-Y202W mutant improves the nucleic acid amplification accuracy. . Next, the persistence of the effect with the progress of the PCR cycle was examined. Specifically, the PCR was carried out by comparing the PCR cycle between 30 times and 35 times, using the amount of amplification product in PCR using a primer containing a mismatch between the primer and the template nucleic acid as an index.
  • the PCR reaction solution (25 ⁇ l) was 25 ng of human genomic DNA of Example 1 as a template nucleic acid, 0.5 ⁇ g of TthRecA-Y202W mutant, 0.2 ⁇ M of each primer (final concentration), DNA polymerase (Takara-Bio, Premix Taq Version 2.0) (standard concentration of the PCR reagent kit).
  • primer set j (based on Homo sapiens chromosome 7, ACCESSION NT_079596 REGION: 56196821..56325337 so as to contain a single base mismatch).
  • sequence information of primer set j is as follows.
  • the primer set j contains a mismatch of 1 base, and the base is shown in capital letters.
  • Primer set j (number of mismatched bases 1): 5'-gccta aggtc acgtt gtccc-3 '(SEQ ID NO: 31) 5'-gcagg cacca Ggaac tactgc-3 '(SEQ ID NO: 32)
  • PCR reaction solution prepared above was subjected to PCR under the same conditions to obtain an amplification product.
  • PCR is performed by 25 or 30 cycles of amplification reaction, with heat denaturation at 94 ° C for 30 seconds followed by 94 ° C for 15 seconds, 55 ° C for 30 seconds and 72 ° C for 60 seconds. went.
  • an electrophoresis buffer was added to each amplification reaction solution and stirred. Half of the solution was taken and subjected to 1.2% agarose gel electrophoresis. The gel after electrophoresis was stained with Vistra green (GE Healthcare) to visualize DNA bands.
  • PCR was performed according to the above-described procedure for the addition of the wild type TthRecA protein, and then subjected to electrophoresis. Furthermore, PCR was performed according to the above procedure without adding RecA protein, and then subjected to electrophoresis, which was used as a control.
  • FIG. 11 shows the results of electrophoresis.
  • A shows an amplification result of 30 cycles
  • B shows an amplification result of 35 cycles.
  • Lanes 1 to 3 of A and B are the results in the presence of the control, the wild type TthRecA protein, and the TthRecA-Y202W mutant, respectively.
  • the addition specificity of the TthRecA-Y202W mutant improves the pairing specificity between the primer and the template nucleic acid, but the effect decreases with repeated PCR cycles. It can be inferred that the decrease in the effect can be reduced by additionally adding the TthRecA-Y202W mutant during the amplification reaction.
  • the function of the TthRecA-Y202W mutant of the present invention can be fully exhibited by appropriately setting the number of cycles, the timing of addition of the TthRecA-Y202W mutant, etc. according to the purpose of use.
  • nucleic acid amplification technique useful in the medical field, biochemical field, environmental field, food field and the like.

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Abstract

La présente invention concerne l'amélioration de la précision et de l'efficacité d'une technique d'amplification d'acide nucléique pour empêcher l'apparition d'une amplification non-spécifique, ce qui permet d'établir une technique permettant d'amplifier uniquement un acide nucléique souhaité de manière plus précise et beaucoup plus efficace. La présente invention concerne une protéine RecA modifiée résistant à la chaleur qui a une fonction de réduction de la fréquence de mauvaise hybridation d'une amorce à un acide nucléique de matrice par rapport à la protéine RecA résistant à la chaleur de type sauvage et comprend une séquence d'acides aminés représentée par l'une quelconque des SEQ ID n° 1 à 4, et un équivalent fonctionnel de la protéine RecA modifiée résistant à la chaleur.
PCT/JP2015/070883 2014-07-24 2015-07-22 PROTÉINE RecA MODIFIÉE RÉSISTANT À LA CHALEUR, MOLÉCULE D'ACIDE NUCLÉIQUE CODANT POUR LADITE PROTÉINE, PROCÉDÉ D'AMPLIFICATION DE L'ACIDE NUCLÉIQUE AU MOYEN DE LADITE PROTÉINE ET KIT POUR L'AMPLIFICATION DE L'ACIDE NUCLÉIQUE WO2016013592A1 (fr)

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WO2019009361A1 (fr) 2017-07-05 2019-01-10 国立研究開発法人科学技術振興機構 Méthode de production d'adn et kit d'assemblage de fragment d'adn
WO2020027110A1 (fr) 2018-07-30 2020-02-06 オリシロジェノミクス株式会社 Procédé d'édition d'adn dans un système acellulaire
WO2024170684A1 (fr) 2023-02-15 2024-08-22 Sanofi Criblage de séquences nucléotiques avec optimisation des codons

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JP2006174722A (ja) * 2004-12-21 2006-07-06 Aisin Seiki Co Ltd 核酸増幅反応における高度好熱菌由来タンパク質の活性化方法およびその利用
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Publication number Priority date Publication date Assignee Title
WO2019009361A1 (fr) 2017-07-05 2019-01-10 国立研究開発法人科学技術振興機構 Méthode de production d'adn et kit d'assemblage de fragment d'adn
WO2020027110A1 (fr) 2018-07-30 2020-02-06 オリシロジェノミクス株式会社 Procédé d'édition d'adn dans un système acellulaire
WO2024170684A1 (fr) 2023-02-15 2024-08-22 Sanofi Criblage de séquences nucléotiques avec optimisation des codons

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