JP6387722B2 - Modified heat-resistant RecA protein, nucleic acid molecule encoding the protein, nucleic acid amplification method using the protein, and nucleic acid amplification kit - Google Patents

Description

  The present invention relates to a modified thermostable 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. However, it is difficult to optimize the reaction, and there are problems such as a decrease in amplification efficiency due to non-specific amplification caused by primer misannealing, such as annealing of primers other than the target sequence or annealing of primers. It was. 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.

  Therefore, various attempts to suppress nonspecific amplification have been reported. For example, in PCR nucleic acid amplification reaction, 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-removing protein, the nucleotide is removed from single-stranded or double-stranded DNA strands containing nucleotides that induce mutations such as 8-oxoguanine nucleotides and 2-oxoadenine nucleotides It is. As a result, mutations that can occur during DNA elongation or amplification reaction using the DNA strand as a template are suppressed, and the accuracy of the amplification reaction is improved.

  However, 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.

  In addition, various attempts have been reported to control the reaction so that primer misannealing does not occur at each stage of the amplification cycle. For example, it has been reported that a thermostable RecA protein derived from an extreme thermophile can interact with a template or primer to promote the binding of the primer only to a specific template sequence, thereby suppressing primer misannealing. Yes. Here, 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.

  And, the present inventors previously suppressed the primer misannealing by coexisting the above-mentioned RecA protein and the factor that activates the protein in the PCR nucleic acid amplification reaction. It was reported that it can be amplified more specifically (Patent Documents 2 and 3). Specifically, in the PCR nucleic acid amplification method, a nonspecific PCR product is amplified by adding a homologous recombinant protein such as RecA protein and a protein activator such as ATP-γS to the reaction solution. It has been reported that the desired nucleic acid can be amplified more specifically (Patent Document 2). Furthermore, during PCR 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). And it has been reported that RecA protein is activated by the presence of nucleotide triphosphate, and can maintain its biological function well.

  The techniques of Patent Documents 2 and 3 described above perform PCR nucleic acid amplification reaction using a sequence region to be examined for single nucleotide polymorphism in the 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.

  Therefore, research for solving the above-described problems has been advanced, and a modified thermostable RecA protein that can contribute to highly accurate nucleic acid amplification has been reported (Patent Document 4). The modified thermostable RecA protein of Patent Document 4 includes 13 amino acid sequences of a thermostable RecA protein derived from a 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. It has been reported that such a C-terminal truncated thermostable RecA protein has high homologous recombination activity and contributes to the improvement of the pairing specificity between the primer and the template nucleic acid. When such a C-terminal truncated heat-resistant RecA protein is added to the nucleic acid amplification reaction system, non-specific amplification does not occur, an amplification product specific to the target nucleic acid is obtained, and high-precision nucleic acid amplification can be achieved.

  However, it has been found that 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. For this reason, 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. Combined with this, the protein can be suitably used for single nucleotide polymorphism analysis. However, in order to further improve the accuracy of the 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. 10-32738) 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)

  Accordingly, 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.

  As a result of intensive studies by the present inventors, 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. When such a modified heat-resistant RecA protein is added to the nucleic acid amplification reaction system, 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 and the like. It has been found that highly accurate nucleic acid amplification can be achieved. Furthermore, as a result of repeated studies, high-efficiency nucleic acid amplification was achieved by avoiding the inhibition of amplification as observed in the conventional C-terminal truncated thermostable RecA protein. The present invention has been completed based on these findings.

  In order to achieve the above-mentioned object, an invention comprising the following configurations [1] to [13] is provided.

[1] 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: 1-4 (2) in the amino acid sequence of any of SEQ ID NOS: 1-4, one or several amino acids have been deleted, substituted or added, And a protein consisting of an amino acid sequence in which the amino acid corresponding to position 202 of any one of the amino acid sequences of SEQ ID NOs: 1 to 4 is tryptophan, 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 the amino acid sequence of any one of SEQ ID NOs: 1 to 4 And the amino acid at the position corresponding to position 202 of any one of the amino acid sequences of SEQ ID NOs: 1 to 4 is tryptophan In the nucleic acid amplification reaction system, a mistake in the primer for the template nucleic acid compared to the wild-type heat-resistant RecA protein consisting of the amino acid sequence of SEQ ID NO: 6, 8, 10, or 12 in the nucleic acid amplification reaction system [2] The modified thermostable RecA protein according to [1] above, wherein the protein having a function of reducing annealing [2] is derived from a bacterium belonging to the genus Thermus.
[3] The modified thermostable RecA protein according to [1] or [2] above, wherein the wild type thermostable RecA protein is derived from Thermus thermophilus or Thermus aquaticus.
[4] The modified thermostable RecA protein according to any one of [1] to [3], which comprises the amino acid sequence of any one of SEQ ID NOs: 1 to 3.
[5] The modified heat-resistant RecA protein according to any one of [1] to [3], which comprises the amino acid sequence of SEQ ID NO: 4.

  According to the above-mentioned configurations [1] to [5], 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. In particular, 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. , Pair exactly with complementary sequences. As described below, the effect of reducing misannealing is that 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.

  Furthermore, 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. Has been. 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. On the other hand, 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. This makes it possible for the chain extension reaction to proceed smoothly after pairing, reducing the nucleic acid amplification inhibitory effect observed with the conventional C-terminally truncated thermostable RecA protein, and efficient. Amplification of the target nucleic acid can be realized. Furthermore, the mutant 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.

  According to the configurations of [2] and [3] described above, derived from a genus Thermus having improved ability to contribute to high-precision and high-efficiency amplification of a target nucleic acid in a nucleic acid amplification reaction system, in particular, thermus thermophilus, Alternatively, 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. In addition, 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.

  According to the configurations of [4] and [5] described above, a modified thermostable 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 is provided. 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.

[6] A nucleic acid molecule encoding the modified thermostable RecA protein of any one of [1] to [5].

  According to the above configuration [6], a nucleic acid molecule encoding the modified heat-resistant RecA protein of the present invention is provided. 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 has become possible.

[7] A nucleic acid amplification method for performing a nucleic acid amplification reaction by adding the modified heat-resistant RecA protein of any one of [1] to [5] above.
[8] The nucleic acid amplification method according to [7], wherein the amplification reaction is a reaction based on a polymerase chain reaction.

  According to the configuration of [7] and [8] described above, 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 target nucleic acids can be amplified without being affected by background noise, which can contribute to improvement of amplification accuracy, and single nucleotide polymorphism analysis that requires highly accurate nucleic acid amplification, etc. Nucleic acid amplification technology suitable for use in the field. Then, efficient amplification of the target nucleic acid can be realized without causing amplification inhibition or the like in the nucleic acid amplification system. Since the modified thermostable RecA protein of the present invention is a thermostable enzyme, the above effects can be exhibited continuously. 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.

[9] 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.

  According to the configuration of [9] described above, simple and rapid nucleic acid amplification is possible by configuring the components necessary for nucleic acid amplification as a kit.

[10] 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,
In the wild type thermostable 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
Here, the wild-type heat-resistant 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] Furthermore, the method according to [10], wherein the primer misannealing with respect to the template nucleic acid is reduced in the nucleic acid amplification reaction system as compared with the C-terminal truncated heat-resistant RecA protein comprising the amino acid sequence of SEQ ID NO: 13.
[12] The method according to [10] or [11], wherein the protein further reduces inhibition of amplification with respect to the template nucleic acid as compared to a C-terminal truncated heat-resistant RecA protein comprising the amino acid sequence of SEQ ID NO: 13.
[13] Any one of [10] to [12] above, which enhances homologous recombination activity as compared to a wild-type heat-resistant RecA protein consisting of the amino acid sequence of SEQ ID NO: 6, 8, 10, or 12. Method.

  According to the constitutions [10] to [13], a method is provided 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.

  In particular, according to the configurations of [11] and [12], 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. This protein enables smooth progress of the chain extension reaction after pairing, and can reduce the nucleic acid amplification inhibitory effect observed with the conventional C-terminal truncated thermostable RecA protein, which is efficient. It is possible to contribute to the provision of a protein that can realize amplification of a target nucleic acid.

    In particular, according to the configuration of [13], a protein capable of promoting homologous recombination can be constructed. By using the protein constructed by the method of the present invention, efficient gene transfer can be realized in gene recombination experiments. For example, 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.

It is the electrophoretic diagram which shows the result of Example 1 which confirmed the nucleic acid amplification reaction which added RecA protein (TthRecA protein) derived from the wild type Thermus thermophilus, and the schematic diagram of the nucleic acid amplification mode, A is a wild type Amplification result in the presence of 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, and D shows the presence of TthRecA protein shown in A above. , E schematically show the chain extension reaction after annealing of the primer to the template nucleic acid in the nucleic acid amplification reaction system in the presence of the E. coli-derived SSB protein and the E. coli-derived RecA protein shown in B and C above. The electrophoretic diagram which shows the result of 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 TthRecA-Y202W mutant, A is a wild type TthRecA protein, B is a TthRecA-Y202W mutant, C is a Hyper-TthRecA mutation. Body homologous recombination activity. The result of Example 3 which confirmed the homologous recombination activity of the TthRecA-Y202W mutant is shown, and the result of FIG. 3 is quantified and graphed. In the figure, the horizontal axis represents the reaction time (minutes), and the vertical axis represents the D-Loop formation rate (%). It is a graph which shows the result of Example 4 which confirmed the homologous recombination activity of TthRecA-Y202W variant, a horizontal axis is reaction time (minutes), and a vertical axis | shaft is D-Loop formation rate (%). FIG. 6 is a graph showing the results of Example 5 in which the ATPase activity of the TthRecA-Y202W mutant was confirmed, and shows the results of single-stranded DNA-dependent ATPase, in which the vertical axis represents the production rate of ADP from ATP (% ). FIG. 6 is a graph showing the results of Example 5 in which the ATPase activity of the TthRecA-Y202W mutant was confirmed, showing the results of double-stranded DNA-dependent ATPase, where the vertical axis indicates the rate of ADP production from ATP (% ). FIG. 9 is an electrophoretogram showing the results of Example 6 in which the influence of the TthRecA-Y202W mutant on the PCR amplification accuracy was confirmed, where A is the control, B is the amplification result in the presence of the TthRecA-Y202W mutant, and C is The amplification results in the presence of wild type TthRecA protein are shown. It is an electrophoretic diagram which shows the result of Example 7 which confirmed the effect of PCR amplification accuracy of TthRecA-Y202W mutant, A is a control, B is the amplification result in the presence of TthRecA-Y202W mutant, C is The amplification results in the presence of the wild type TthRecA protein, D shows the amplification results in the presence of the TaqRecA protein. FIG. 9 is an electrophoretogram showing the results of Example 8 in which the effect of nucleic acid amplification accuracy of the TthRecA-Y202W mutant was confirmed, where A is the result of amplification with a primer having no mismatched base, and B is the mismatched base. The amplification result with the primer which has is shown. It is an electropherogram which shows the result of Example 9 which confirmed the persistence of the nucleic acid amplification precision improvement effect of a TthRecA-Y202W variant, Comprising: A is an amplification result in 30 cycles, B is an amplification result of 35 cycles. Show.

  Hereinafter, specific embodiments of the present invention will be described. However, these are merely examples of the present invention, and do not limit the present invention.

  Hereinafter, the modified thermostable RecA protein of the present invention may be abbreviated as “modified thermostable RecA protein”. In addition, the wild type thermostable RecA protein may be abbreviated as “wild type thermostable RecA protein”.

  Here, the wild-type 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. Specifically, 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 Thermus bacterium, and particularly preferred is a RecA protein derived from Thermus thermophilus or Thermus aquaticus.

  Of these, 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. Here, 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, and the amino acid sequence. Is disclosed as SEQ ID NO: 8 (GenBank: AAA64935.1). Furthermore, the base 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). Furthermore, 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. Although there are wild-type thermostable RecA proteins derived from Thermus thermophilus and Thermus aquaticus, they have different sequences from those described above, but these should preferably be used as the basis for modification. Can do. However, the present invention is not limited to these, and those having functions such as homologous recombination activity equivalent to the wild type heat-resistant RecA protein and having high sequence identity can be preferably used as a basis for modification.

  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. Here, 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. Therefore, 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.

  Preferably, as a function of the modified thermostable RecA protein of the present invention, it is required that the target nucleic acid can be dissociated from the nucleic acid (recyclability) in the nucleic acid amplification system after pairing with the target nucleic acid. 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.

  Here, 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. Of the amino acid sequences of SEQ ID NO: 13, 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. However, in the nucleic acid amplification reaction system, it is difficult to dissociate while remaining attached to the nucleic acid even after pairing of the target nucleic acid and the primer. As a result, the undesired property of interfering with the chain extension reaction following pairing and inhibiting nucleic acid amplification was found. On the other hand, according to the modified thermostable RecA protein of the present invention, it is possible to avoid interference with the chain extension reaction, and to reduce the nucleic acid amplification inhibitory effect observed with the Hyper-TthRecA mutant, which is efficient. Amplification of a target nucleic acid can be realized.

  To explain the recyclability, the RecA protein binds to a single-stranded nucleic acid in the presence of NTP such as ATP and searches for a homologous sequence site. 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 to ADP than the Hyper-TthRecA mutant and the wild type thermostable RecA protein. Later, it is highly recyclable to rapidly dissociate from nucleic acid, and does not inhibit nucleic acid amplification.

In the following examples, 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.
(1) Pairing to complementary sequences (homologous pairing)
(2) 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. For example, in PCR and the like described below, after a single-stranded DNA is paired with a complementary double-stranded DNA, 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, but since no improvement in the activity of the strand exchange reaction is observed, the single-stranded DNA and the protein are separated from the double-stranded DNA. There is a tendency to become difficult, and even if there is a mistake in pairing, the tendency does not change. On the other hand, since 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.

  Here, 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.

  Specifically, as 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 | change has produced in the 202th tyrosine of the amino acid sequence of sequence number 12 showing the thermostable RecA protein derived from Thermus aquaticus is illustrated. Here, the modification means deletion, substitution or addition. Preferably, those in which the 202th tyrosine is substituted with an amino acid other than alanine are exemplified. Particularly preferred are those substituted with tryptophan, and examples of amino acid sequences of such modified thermostable RecA proteins are shown in SEQ ID NOs: 1, 2, 3, and 4 in the sequence listing. In addition, when 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. Particularly, in 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.

  That is, as 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. Thus, 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. Further, 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. Are included in the modified thermostable RecA protein. Here, 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.

  Therefore, in the amino acid sequence of SEQ ID NO: 1, 2, 3, or 4, the amino acid at position 202 is tryptophan, so long as it has the above-mentioned function, those having an amino acid sequence containing a modification at other sites Included in modified thermostable RecA protein. 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 amino acid sequence corresponding to position 202 of the amino acid sequence is tryptophan and the amino acid sequence of SEQ ID NO: 1, 2, 3, or 4 and at least 80% or 85%, preferably 90%, particularly preferably 92 %, 95%, or 98% sequence identity and consisting of an amino acid sequence in which the amino acid at the position corresponding to position 202 of the amino acid sequence of SEQ ID NO: 1, 2, 3, or 4 is tryptophan It is included in the modified thermostable RecA protein of the present invention.

  The modified thermostable RecA protein of the present invention can be obtained by a known method. For example, the DNA encoding the wild-type heat-resistant RecA protein, which is the basis of the modification, is modified, and the resulting modified DNA is used to transform a host cell. It can be obtained by collecting RecA protein.

  A DNA encoding a wild-type thermostable RecA protein that is the basis of modification can be obtained using a known gene cloning technique. For example, 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. Here, as genetic information of a thermostable RecA protein suitable as a basis for the modified type of the present invention, as shown above, it is represented by SEQ ID NO: 5 or 7 encoding the thermostable RecA protein derived from the wild type Thermos thermophilus HB8 strain. Base sequence shown in SEQ ID NO: 9 encoding a heat-resistant RecA protein derived from wild type Thermus thermophilus HB27, base shown in SEQ ID NO: 11 encoding a heat-resistant RecA protein derived from wild-type Thermus aquaticus 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. For example, 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. Once the amino acid sequence of the desired modified heat-resistant RecA protein is determined, 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.

  Specifically, 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.

  In order to transform a host cell using the obtained modified gene, a known host / expression vector system such as Escherichia coli can be used. For example, 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. Then, 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. For example, the T7lac promoter is preferably used as a suitable promoter.

  Furthermore, this expression vector may contain other known base sequences. Other known base sequences are not particularly limited. For example, a stability leader sequence that confers stability of the expression product, and a signal sequence that confers secretion of the expression product. Also included are 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.

  As such an expression vector, a commercially available expression vector for Escherichia coli (for example, pET protein expression system: manufactured by Novagen) can be used, and furthermore, an expression vector incorporating a desired sequence is appropriately prepared and used. Is possible.

  The host cells are not limited to E. coli, and Bacillus bacteria, Pseudomonas bacteria, and the like can also be used. Furthermore, eukaryotic cells can be used without being limited to prokaryotes. For example, 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.

  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. From this crude extract, 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.

  Whether 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. For example, an automatic amino acid determination method based on Edman degradation can be used. Furthermore, 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. In addition, 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 amounts of amplification products using primers containing mismatched bases, and specifically by the methods shown in Examples 6 to 8 of the present specification.

  As described above, 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 to the wild type thermostable RecA protein. In particular, 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. 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.

  Since the modified thermostable RecA protein of the present invention has a high homologous recombination function, efficient gene transfer can be realized 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.

  Furthermore, 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. Has been. 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. On the other hand, 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. This makes it possible for the chain extension reaction to proceed smoothly after pairing, reducing the nucleic acid amplification inhibitory effect observed with the conventional C-terminally truncated thermostable RecA protein, and efficient. Amplification of the target nucleic acid can be realized.

  The present invention further provides a nucleic acid amplification method for a template nucleic acid using the modified thermostable RecA protein of the present invention. In the nucleic acid amplification method of the present invention, a nucleic acid amplification reaction is performed by adding the modified heat-resistant RecA protein of the present invention.

  Here, the case where the modified thermostable RecA protein of the present invention is applied to PCR will be described. 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.

  Specifically, a PCR reaction solution prepared by containing the modified thermostable RecA protein of the present invention, a target nucleic acid, a thermostable DNA polymerase, a primer, dNTP, and an appropriate buffer 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. Here, 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. In addition, 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.

  The primer is designed to be complementary to a specific sequence of the target nucleic acid. In particular, it is preferable to have a complementary base sequence at both ends of the target sequence to be amplified. In the simplest system, two primers are required, but when performing multiplex PCR, etc., three or more may be used, and it can be suitably used for amplification reaction with 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.

  As the primer, 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. When preparing 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. Moreover, when performing chemical synthesis, it is also possible to use a commercially available automatic synthesizer. Here, 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. Thus, not only perfect complementarity, but also a portion where only a few nucleobases fit along the base-pairing rule as long as the primer and target nucleic acid are sufficient to form a stable duplex structure with each other. Even the perfect complementarity is acceptable. The number of bases must be long enough to specifically recognize the target nucleic acid, but if it is too long, a non-specific reaction is induced, which is not preferable. Accordingly, 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. Preferably, it is 20-50 bases in length.

  Here, the thermostable DNA polymerase used is not particularly limited as long as it is a thermostable DNA polymerase that can be usually used in PCR. For example, Taq polymerase from Thermus aquaticus, Tth polymerase from Thermus thermophilus, Bst polymerase from Bacillus Stearothermophilus, Thermococcus litoralis ) -Derived Vent polymerase, Thermococcus kodakaraensis KOD polymerase, Pyrococcus furiosus-derived Pfu polymerase, and other DNA polymerases derived from thermophilic bacteria.

  As dNTP, four types of deoxynucleotides corresponding to each base of adenine, thymine, guanine and cytosine are used. In particular, a mixture of dGTP, dATP, dTTP, and dCTP is preferably used. Furthermore, 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, 7-deaza-dATP, and the like. For example, they can be used instead of dGTP and dATP, or both can be used together. 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. As the buffer component, 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. Furthermore, potassium salts such as KCl, DMSO, glycerol, betaine, gelatin, Triton and the like can be added as necessary. In addition, 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 μl or less, particularly in the range of 10 to 50 μl. As the 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 μg / μl. The 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. For example, the target nucleic acid is preferably prepared at a concentration of 10 pg to 1 μg per 100 μl, and the primer DNA is preferably prepared at a final concentration of 0.01 to 10 μM, particularly 0.1 to 1 μM. Furthermore, the thermostable DNA polymerase is preferably used at a concentration in the range of 0.1 to 50 units, particularly 1 to 5 units per 100 μl. And dNTP is preferably prepared to a final concentration of 0.1 mM to 1 M. However, it is not limited to this, and is appropriately set as described above.

PCR is performed according to the following steps, which are performed in the presence of the modified thermostable RecA protein of the present invention.
(1) Thermal denaturation of template nucleic acid (2) Primer annealing (3) Extension reaction with thermostable polymerase

  The reaction in which the three-stage temperature change consisting of the above (1) to (3) is one cycle is repeated by repeating the appropriate cycle, so that the primer is the starting point and the target nucleic acid is used as a template. Synthesis of the nucleic acid strand of the book is started. As a result, 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 as a template, but 20 to 40 cycles from the viewpoint of efficient nucleic acid amplification by suppressing non-specific amplification, in particular, 20 It is preferable to carry out in 30 cycles.

Each step will be described in detail below.
(1) Thermal denaturation of template nucleic acid 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. When a long DNA region is amplified, 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.

(2) Primer annealing By lowering the temperature, a hybrid between the template nucleic acid that has been heat-denatured and becomes a single strand in (1) above is formed. 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.

(3) Extension reaction by heat-resistant polymerase The nucleic acid chain extension reaction from the primer is performed at the 3 'end by the heat-resistant polymerase. The extension reaction temperature is appropriately set according to the type of heat-resistant polymerase, but is preferably performed at 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.

  As described above, the general PCR has been described as the nucleic acid amplification method of the present invention. However, the nucleic acid amplification method of the present invention can be applied to various modified PCR methods. For example, 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) And Arbitrary Lily Polymerase Chain Reaction (AP-PCR), RACE, multiplex PCR and the like. However, the present invention is not limited to these, and can be used for any PCR modification.

  By configuring as described above, the nucleic acid amplification reaction is performed in the presence of the addition of the modified heat-resistant RecA protein of the invention, thereby enabling efficient nucleic acid amplification specific to the target nucleic acid. Then, nonspecific amplification that is not related 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. As a result, 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. Thereby, it is possible to provide a nucleic acid amplification technique suitable for use in single nucleotide polymorphism analysis or the like that requires highly accurate nucleic acid amplification. Then, efficient amplification of the target nucleic acid can be realized without causing amplification inhibition or the like in the nucleic acid amplification system. Since the modified 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 thermostable RecA protein of the present invention, the nucleic acid amplification method and kit using the protein can be used for various applications. For example, it can be used for a wide variety of applications such as the medical field, biochemical field, environmental field, and food field. For example, 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. Furthermore, 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.

  Specific examples of uses in the medical field include genetic diagnosis including single nucleotide polymorphism analysis, detection of viruses such as SARS and influenza, and pathogens such as bacteria. In particular, the present invention can be suitably applied to single nucleotide polymorphism analysis. According to the present invention, 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. On the other hand, nucleic acids that do not have such a single nucleotide polymorphism are not amplified or are not suppressed. As a result, a nucleic acid having such a single nucleotide polymorphism can be specifically amplified. Furthermore, 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. In addition, the use in the field of biochemistry includes identification of individuals and identification of biological species.

  And in the utilization in the environmental field, environmental measurements such as pathogen detection such as bacteria and viruses in the environment, search for new useful microorganisms, and the like are exemplified. Furthermore, examples of use in the food field include determination of the inclusion of genetically modified crops, and testing for fake brand foods. However, the present invention is not limited to this. As long as the nucleic acid amplification technique can be applied, the method of the present invention can be applied without limitation.

(Another embodiment 1)
INDUSTRIAL APPLICABILITY 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.

  As the wild type heat-resistant RecA protein, those exemplified as the basis of the mutant heat-resistant RecA protein of the present invention can be preferably used. As other amino acids, 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.

(Another embodiment 2)
1. The modified thermostable RecA protein of the present invention can be applied to enrichment or isolation of target cDNA clones from a DNA library.
Specifically, 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. . Here, in 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. Therefore, by applying 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.

  Here, as the DNA library, a DNA library containing or expected to contain a target DNA region desired to be enriched or isolated is used. The DNA library may be either a genomic library or a cDNA library, but a cDNA library is particularly preferable. In this specification, the term “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. On the other hand, 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. In particular, 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. In addition, 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.

2. 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. Furthermore, the present invention can also be applied to an amplification reaction using synthesized cDNA as a template. Here, in the amplification reaction, it is possible to use a known nucleic acid amplification technique in addition to PCR. Thereby, the synthesis | combination of the non-specific cDNA unrelated to target RNA can be suppressed, and the specific synthesis | combination of cDNA with respect to desired target RNA is attained. Therefore, by applying the modified 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.

  Here, RNA is not particularly limited, such as mRNA, tRNA, rRNA, etc. in addition to total RNA. 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 applicable reaction conditions, and as described above, a random hexamer primer, oligo dT primer, and target gene-specific primer can be used. Here, the target gene-specific primer has a base sequence complementary to a specific template RNA, and preferably the 3 ′ side of a primer used in a general PCR amplification system is used.

  EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples.

  In the following examples, a Thermos thermophilus-derived RecA protein (hereinafter sometimes referred to as “TthRecA protein”) is exemplified as the thermostable RecA protein, but is not limited thereto.

(Example 1) Confirmation of nucleic acid amplification reaction with addition of TthRecA protein
Regarding the influence of TthRecA protein on nucleic acid amplification reaction, E. coli-derived single-stranded binding protein (hereinafter sometimes referred to as “SSB protein”) and other Escherichia coli-derived RecA protein. Was compared with the case of adding.

(Method)
PCR reaction solution (25 μl) is 0.8-50 ng human genomic DNA (Promega, Human Genomic DNA), 0.4 μg wild type TthRecA protein (Storage buffer: 50 mM Tris-HCl pH 7.5, 1.0 mM EDTA, 0.5 mM) DTT, 50% w / v glycerol), 0.8 μM 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.

Here, the following primer set a was used as a primer.
Primer set a:
5′-aacct cacaa ccttg gctga-3 ′ (SEQ ID NO: 14)
5′-ttcac aactt aagat ttggc-3 ′ (SEQ ID NO: 15)

  In addition, the 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). ).

  Subsequently, 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. .

  After amplification, an electrophoresis buffer was added to each amplification reaction 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.

  Then, instead of wild-type TthRecA protein, E. coli-derived SSB protein (Promega, Single-Stranded DNA Binding Protein) and E. coli-derived RecA protein (Epicenter Technologies) were subjected to PCR according to the procedure described above, and then subjected to electrophoresis. .

(result)
The results are shown in FIG.
1A to 1C are diagrams showing the results of electrophoresis, where A is the result of amplification in the presence of wild-type TthRecA protein, B is the result of amplification in the presence of E. coli-derived SSB protein, and C is E. coli-derived. Amplification results in the presence of RecA protein. And lanes 1 to 7 of A to C are the results when the amount of template nucleic acid was 50 ng, 25 ng, 13 ng, 6 ng, 3 ng, 1.5 ng and 0.8 ng, respectively.

  From the results of FIGS. 1A to 1C, when PCR was performed with the addition of the wild-type TthRecA protein, amplification products were confirmed in all template nucleic acid amounts examined this time (lanes 1 to 7 in A). On the other hand, when PCR amplification was performed with the addition of the E. coli SSB protein and when PCR amplification was performed with the addition of the E. coli RecA protein, the amplification efficiency decreased markedly when the template amount was 6 ng or less (B. Lanes 4-7, C lanes 4-7).

  D and E of FIG. 1 are schematic diagrams of the nucleic acid amplification mode. Then, based on the results of FIGS. 1A to 1C, the chain extension reaction after annealing the primer 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.

  From the above results, it was confirmed that when E. coli SSB protein or E. coli RecA protein was added to the PCR amplification reaction, the nucleic acid amplification efficiency was reduced as compared with the case where wild type TthRecA protein was added. This decrease in amplification efficiency is due to the fact that the protein derived from E. coli was denatured in the state of being bound to the template nucleic acid during the high temperature PCR reaction (55 ° C. or higher), or the amplification reaction was inhibited, It can be inferred that the amplification reaction was inhibited because the binding force to the nucleic acid was strong and difficult to dissociate. On the other hand, 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.

(Example 2) Preparation of 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”.

(Method)
(1) Gene cloning The following primer set b was synthesized based on the known sequence information (GenBank: BAA04215.1) of the gene encoding the wild-type TthRecA protein. This is a primer designed to replace the 202th tyrosine of the amino acid sequence (SEQ ID NO: 6) of the wild type TthRecA protein with tryptophan.

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)
5′-tggtc tcggg gttgc cccac gtgac cccca cctt-3 ′ (SEQ ID NO: 17)

Next, mutagenesis was performed by PCR using the above-mentioned primer set b using a wild-type TthRecA protein expression clone as a template. In addition, pET3a was used as an expression vector, and mutation was introduced using a commercially available Quick Change II Site-Directed Mutagenesis Kit. At this time, the PCR reaction solution (50 μl) is 15 ng template nucleic acid, 0.5 μM primer set b, 1.25 U DNA polymerase (pfu Ultra HF polymerase), 0.2 mM dNTPs, 1x 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 one cycle consisting of 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.

(2) Protein Expression and Purification The TthRecA-Y202W expression clone obtained in (1) was cultured in LB liquid medium containing 100 μg / ml ampicillin at 37 ° C. until OD ₆₀₀ = 0.5. Furthermore, after adding 0.5 mM IPTG and culturing for 4 hours, centrifugation (6,000 × g, 15 minutes) was performed to obtain protein-expressing bacterial cells. Purification of the TthRecA-Y202W mutant was performed by collecting the collected and frozen protein-expressing cells (2 g) in buffer (20 ml) (50 mM Tris-HCl pH 8.0, 25% Sucrose, 5 mM 2-mercapto Ethanol, 0.4% polyoxyethylene cetyl ether). Subsequently, 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. Next, after heat treatment (65 ° C., 1 hour) and 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. Next, after dialysis using PEMG buffer as an external solution, it was applied to a cation-exchange cellulose phosphate chromatography column (Whatman, P11) equilibrated with PEMG buffer (30 ml). After washing with PEMG buffer (50 ml), buffer (PEMG1K: 50 mM potassium phosphate pH 6.5 (30 ml), 1 mM EDTA, 5 mM 2-mercaptoethanol, 10% glycerol, 1 M KCl) The protein elution fraction was fractionated using a gradient. Next, after dialysis using PEMG buffer as an external solution, it was applied to an anion exchange cellulose chromatography column (GE, Resource Q). 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.

  Subsequently, 5 μg equivalent of the obtained TthRecA-Y202W mutant was subjected to SDS-PAGE electrophoresis, and the gel after electrophoresis was stained with CBB. Similarly, for the wild-type TthRecA protein, the wild-type TaqRecA protein, and the Hyper-TthRecA mutant described in Patent Document 4 (Japanese Patent Application Laid-Open No. 2008-029222), which is a prior art document, an amount equivalent to 5 μg of each protein 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. Here, the 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.

(result)
The results are shown in FIG.
FIG. 2 shows the results of electrophoresis. Lane 1 is the wild-type TthRecA protein, Lane 2 is the TthRecA-Y202W mutant, Lane 3 is the wild-type TaqRecA protein, and Lane 4 is the Hyper-TthRecA mutant. It is.

  From the results in FIG. 2, it was confirmed that the TthRecA-Y202W mutant used in the present invention was prepared. Further, as a result of the base sequence analysis, substitution of the codon encoding the 202nd tyrosine of the amino acid sequence was confirmed in the base sequence of the wild type TthRecA protein, and it was confirmed that it encodes tryptophan.

(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.

(Method)
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. 18 μM double-stranded DNA, 0.05 μM single-stranded DNA (5 ′ end labeled with ³³ P), 3.0 μg TthRecA-Y202W mutant, 1.3 mM ATP, reaction buffer (31 mM Tris-HCl pH 7.5, 1.75 mM The mixture was incubated at 37 ° C. for 0 to 20 minutes in DTT, 0.09 mg / ml BSA, 13 mM MgCl ₂ , 4.9 mM CP (creatine phosphate), 7.8 U / μl CK (creatine kinase). After the reaction, a reaction stop solution (0.5% w / vol SDS, 0.7 mg / ml Proteinase K) was added and kept at 0 ° C. for 30 minutes. Next, the 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).

  Then, in place of the TthRecA-Y202W mutant, D-Loop formation was also confirmed according to the above-described procedure for those to which the wild type TthRecA protein and the Hyper-TthRecA mutant were added.

(result)
The results are shown in FIG. 3 and FIG.
FIG. 3 is a diagram showing the results of electrophoresis. In A, B, and C, the bands of the D-Loop product are indicated by arrows. A is a wild-type TthRecA protein, B is a TthRecA-Y202W mutant, and C is a hyper-TthRecA mutant confirmed by D-Loop formation reaction. And the lanes 1-8 of AC are the results of D-Loop formation reaction in 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, TthRecA-Y202W mutant, and Hyper-TthRecA mutant, which are the results of FIG. 3, and the horizontal axis represents the reaction time (minutes). The vertical axis shows the D-Loop formation rate (%).

  3 and 4, the TthRecA-Y202W mutant showed more D-Loop formation than the wild-type TthRecA protein (Comparison between waveforms (1) and (2) in FIG. 4). ). In addition, 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)).

  From these results, it was found that the TthRecA-Y202W mutant had about 4 times higher homologous recombination activity than the wild type TthRecA protein. On the other hand, the maximum value of the homologous recombination activity was equivalent to that of the Hyper-TthRecA mutant. However, compared to the Hyper-TthRecA mutant, the TthRecA-Y202W mutant showed dissociation of D-Loop formation over time, which caused the TthRecA-Y202W mutant to have a RecA protein by hydrolysis of ATP. It was found to be excellent in recyclability for dissociating from nucleic acids.

(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. In this example, 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”.

(Method)
The same double-stranded DNA and single-stranded DNA as in Example 3 were used. 15.5 μM double-stranded DNA, 0.04 μM single-stranded DNA (5 ′ end labeled with ³³ P), 3.0 μg TthRecA-Y202W mutant, 1.1 mM ATP, reaction buffer (25 mM Tris-HCl pH 7.5, 1.46 The cells were incubated at 55 ° C. for 0-20 minutes in mM DTT, 0.08 mg / ml BSA, 10.9 mM MgCl ₂ , 4.1 mM CP (creatine phosphate), 6.5 U / μl CK (creatine kinase). After the reaction, a reaction stop solution (0.5% w / vol SDS, 0.7 mg / ml proteinase K) was added and kept at 0 ° C. for 30 minutes. Next, the 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).

  Then, instead of the TthRecA-Y202W mutant, the addition of the TthRecA-Y202A mutant and the wild-type TthRecA protein was also confirmed to form D-Loop according to the above procedure.

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)

(result)
The results are shown in FIG.
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 in FIG. 4, and the horizontal axis represents the reaction time (minutes). The vertical axis indicates the D-Loop formation rate (%).

  From the results shown in FIG. 5, in the D-Loop formation reaction under the above-mentioned conditions, there is no significant difference in the detection pattern between the wild-type TthRecA protein and the TthRecA-Y202A mutant, and the homologous recombination activity of the TthRecA-Y202A mutant is Similar to wild-type TthRecA protein. In contrast, the TthRecA-Y202W mutant had approximately 2-fold improvement in homologous recombination activity over the wild type TthRecA protein. These results suggest that the aromatic ring of the tryptophan residue, which has a larger area than the tyrosine residue, stably stacks the dissociated DNA, making it easier for pairing reactions of homologous DNA to occur. Is done. In summary, the TthRecA-Y202W mutant showed higher homologous recombination activity than the TthRecA-Y202A mutant and the wild-type TthRecA protein.

(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.

(Method)
PBluescript SK (-) DNA (Novagen) was used as double-stranded DNA, and M13 ssDNA (TAKARA) was used as single-stranded DNA. 22.6 μM double-stranded DNA or single-stranded DNA, 3.0 μg TthRecA-Y202W mutant, 1.3 mM [α-32P] ATP, reaction buffer (31 mM Tris-HCl pH 7.5, 1.75 mM DTT, 0.09 mg / ml BSA, MgCl ₂ in 13 mM (single-stranded DNA) or 1 mM (double-stranded DNA), 4.9 mM CP (creatine phosphate), 7.8 U / μl CK (creatine kinase)) at 55 ° C. for 30 minutes Keep warm. After the reaction, an equal amount of 25 mM EDTA was added to stop the reaction. After stopping the reaction, the sample was separated into ATP and ADP by thin layer chromatography. After separation, the plate was exposed to an Imaging Plate (IP) for overnight or longer, and the ADP signal generated with ATP was detected with RI Imaging Analyzer BAS2500 (Fuji Film).

  Then, signals were also detected according to the above-described procedure for those added with the wild type TthRecA protein and the Hyper-TthRecA mutant instead of the TthRecA-Y202W mutant.

(result)
The results are shown in 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, and FIG. 7 shows double-stranded DNA-dependent ATPase activity.

  From the results of FIGS. 6 and 7, 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. These results confirm the results of Example 3 in which the TthRecA-Y202W mutant was found to be excellent in recyclability in which the RecA protein dissociates from the nucleic acid 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.

(Method)
The PCR reaction solution (25 μl) was 25 ng of human genomic DNA of Example 1 as a template nucleic acid, 0.5 μg of wild-type TthRecA protein, 0.2 μM of primer (final concentration), DNA polymerase (Takara-Bio, Premix Taq Version 2.0). ) (Standard concentration of the PCR reagent kit).

Here, 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 Each of the PCR reaction solutions was prepared. 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 gtccc-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)

  Each 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. . After amplification, 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.

Then, instead of the wild-type TthRecA protein, PCR was performed according to the above-described procedure for those added with the TthRecA-Y202W mutant, and then subjected to electrophoresis. In addition, PCR was performed according to the above procedure without adding RecA protein, and then subjected to electrophoresis, which was used as a control.

(result)
The results are shown in FIG.
FIG. 8 shows the results of electrophoresis. A is the control, B is the amplification result in the presence of the TthRecA-Y202W mutant, and 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.

  From the results shown in FIG. 8, when PCR was carried out with the addition of the TthRecA-Y202W mutant, a significant decrease in the amount of amplified product was confirmed with primer sets d and e (lanes 2 and 3 in B). On the other hand, when PCR amplification was performed with the addition of the wild-type TthRecA protein, there was no decrease in the amount of amplification product in primer set d, and the amount of amplification product in primer set e was slightly decreased (lanes 2 and 3 in C). In the control, major amplification products were confirmed in all primer sets (lanes 1, 2, and 3 in A).

  From the above results, it was found that by adding the TthRecA-Y202W mutant, the pairing specificity between the primer and the template nucleic acid was improved, that is, the PCR reaction accuracy was improved as compared with the wild type TthRecA protein.

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.

(Method)
The PCR reaction solution (25 μl) was 25 ng of human genomic DNA of Example 1 as a template nucleic acid, 0.5 μg of TthRecA-Y202W mutant, 0.2 μM of primer (final concentration), DNA polymerase (Takara-Bio, Premix Taq Version). 2.0) (standard concentration of the PCR reagent kit).

Here, as a primer, the following 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) Each was prepared using PCR reaction solution. The 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 gtccc-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)

  Each 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. . After amplification, 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.

  Then, instead of the TthRecA-Y202W mutant, PCR was performed according to the above-described 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. Here, the 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.

(result)
The results are shown in FIG.
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, and D is the result. The amplification results in the presence of wild type TaqRecA protein are shown. Lanes 1 and 2 of A to D are amplification results with the primer sets f and g, respectively.

  From the results shown in FIG. 9, when PCR was performed with the addition of the TthRecA-Y202W mutant, the amplification product was confirmed in primer set f, and the amplification product was detected in primer set g containing a single-base mismatch between the primer and template nucleic acid. The amount was significantly reduced (B, lane 2). In addition, when PCR amplification was performed with the addition of wild-type TthRecA protein, the amount of amplification product was only slightly reduced in primer set g (lane 2 of C). On the other hand, when PCR amplification was performed with the addition of the wild-type TaqRecA protein, and in the control, both primer sets f and g had the same amount of amplification product (D lanes 1 and 2).

  From the above results, it was found that by adding the TthRecA-Y202W mutant, the pairing specificity between the primer and the template nucleic acid is improved, that is, the PCR amplification accuracy is improved as compared with the wild type TthRecA protein. This result is consistent with the result of Example 6 described above.

(Example 8) Confirmation of effect of nucleic acid amplification accuracy of TthRecA-Y202W mutant-2
The effect of the TthRecA-Y202W mutant obtained in Example 2 on the amplification accuracy of nucleic acid was compared with the wild type TthRecA protein, the wild type TaqRecA protein, and the Hyper-TthRecA mutant. This was performed by comparing the amount of amplification product in PCR using a primer containing a mismatch between the primer and the template nucleic acid.

(Method)
The PCR reaction solution (25 μl) was 25 ng of human genomic DNA of Example 1 as a template nucleic acid, 0.5 μg of TthRecA-Y202W mutant, 0.2 μM of primer (final concentration), DNA polymerase (Takara-Bio, Premix Taq Version). 2.0) (standard concentration of the PCR reagent kit).

Here, as a primer, the following 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) 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)

  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 94 ° C. for 30 seconds, 94 ° C. for 15 seconds, 55 ° C. for 30 seconds, and 72 ° C. for 60 seconds. . After amplification, 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.

  Then, instead of the TthRecA-Y202W mutant, PCR was performed according to the above-described procedure for the wild type TthRecA protein, the wild type TaqRecA protein, and the Hyper-RecA mutant added, and then subjected to electrophoresis.

(result)
The results are shown in FIG.
FIG. 10 is a diagram showing the results of electrophoresis, where A is the amplification result with primer set h, and B is 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.

  From the results shown in FIG. 10, when PCR using the primer set h was performed, amplification products at the same level were added with the addition of the wild type TthRecA protein, the TthRecA-Y202W mutant, the wild type TaqRecA protein, and the Hyper-TthRecA mutant. Check the amount. However, when PCR was performed using primer set i containing a single-base mismatch between the primer and the template nucleic acid, the amount of amplified product decreased with the addition of the TthRecA-Y202W mutant (lane 2 of B). On the other hand, when the wild-type TthRecA protein, the wild-type TaqRecA protein, and the Hyper-TthRecA mutant were added, the amount of the amplified product was not reduced (lanes 1, 3, and 4 in B).

  From the above results, it was confirmed that the addition of the TthRecA-Y202W mutant improves the pairing specificity between the primer and the template nucleic acid, that is, improves the PCR amplification accuracy. This effect was found to be superior to the Hyper-TthRecA mutant previously developed by the present inventors.

(Example 9) Persistence confirmation of improvement effect of nucleic acid amplification accuracy of TthRecA-Y202W mutant From the results of Examples 6 to 8 above, it was confirmed that the TthRecA-Y202W mutant improves the amplification accuracy of nucleic acid. . 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.

(Method)
The PCR reaction solution (25 μl) was 25 ng of human genomic DNA of Example 1 as a template nucleic acid, 0.5 μg of TthRecA-Y202W mutant, 0.2 μM of each primer (final concentration), DNA polymerase (Takara-Bio, Premix Taq Version 2.0) (standard concentration of the PCR reagent kit).

Here, as a primer, a PCR reaction solution was prepared using the following primer set j (based on Homo sapiens chromosome 7, ACCESSION NT_079596 REGION: 56196821..56325337 so as to contain a single base mismatch). . The 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)

  Each 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. After amplification, 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.

  Then, instead of the TthRecA-Y202W mutant, PCR was carried out according to the above-described procedure for those to which the wild type TthRecA protein was added, 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.

(result)
The results are shown in FIG.
FIG. 11 is a diagram showing the results of electrophoresis. A shows the results of amplification for 30 cycles and B shows the results of amplification for 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.

  From the results shown in FIG. 11, when PCR was performed for 30 cycles using primer set j containing a single-base mismatch between the primer and template nucleic acid, compared to the amount of control amplification, the addition of wild-type TthRecA protein The amount of amplification decreased to 1/3, and the amount of amplification decreased to 1/6 when the TthRecA-Y202W mutant was added. On the other hand, when PCR was performed for 35 cycles, the amount of amplification was the same when the wild type TthRecA protein was added and when the TthRecA-Y202W mutant was added, compared to the amount of control amplification.

  From the above results, it was found that the addition 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. In addition, when used for real-time PCR, a decrease in effect due to an increase in PCR cycle is not a big problem. Therefore, 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 techniques useful in the medical field, biochemical field, environmental field, food field and the like

Claims (13)

A modified thermostable RecA protein selected from the following proteins (1) to (3):
(1) a protein comprising any one of the amino acid sequences of SEQ ID NOS: 1-4 (2) in the amino acid sequence of any of SEQ ID NOS: 1-4, one or several amino acids have been deleted, substituted or added, And a protein consisting of an amino acid sequence in which the amino acid corresponding to position 202 of any one of the amino acid sequences of SEQ ID NOs: 1 to 4 is tryptophan, 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 the amino acid sequence of any one of SEQ ID NOs: 1 to 4 And the amino acid at the position corresponding to position 202 of any one of the amino acid sequences of SEQ ID NOs: 1 to 4 is a triplicate. A protein consisting of an amino acid sequence which is a fan, and in a nucleic acid amplification reaction system, a primer error for a template nucleic acid is compared with a wild-type heat-resistant RecA protein consisting of an amino acid sequence of SEQ ID NO: 6, 8, 10, or 12. Protein with function to reduce annealing   The modified heat-resistant RecA protein according to claim 1, wherein the wild-type heat-resistant RecA protein is derived from a bacterium belonging to the genus Thermus.   The modified heat-resistant RecA protein according to claim 1 or 2, wherein the wild-type heat-resistant RecA protein is derived from Thermus thermophilus or Thermus aquaticus.   The modified thermostable RecA protein according to any one of claims 1 to 3, comprising any one of the amino acid sequences of SEQ ID NOs: 1 to 3.   The modified thermostable RecA protein according to any one of claims 1 to 3, comprising the amino acid sequence of SEQ ID NO: 4.   A nucleic acid molecule encoding the modified thermostable RecA protein according to any one of claims 1 to 5.   A nucleic acid amplification method for performing a nucleic acid amplification reaction by adding the modified heat-resistant RecA protein according to any one of claims 1 to 5.   The nucleic acid amplification method according to claim 7, wherein the amplification reaction is a reaction based on a polymerase chain reaction. A nucleic acid amplification kit for amplifying a nucleic acid comprising a thermostable DNA polymerase and the modified thermostable RecA protein according to any one of claims 1 to 5. A method for reducing primer mis-annealing to a template nucleic acid of a protein compared to a wild type thermostable RecA protein,
In the wild type thermostable 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,
Here, the wild-type heat-resistant 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.   The method according to claim 10, further comprising reducing primer misannealing with respect to a template nucleic acid in a nucleic acid amplification reaction system as compared with a C-terminal truncated heat-resistant RecA protein comprising the amino acid sequence of SEQ ID NO: 13.   The method according to claim 10 or 11, wherein the protein further reduces inhibition of amplification with respect to a template nucleic acid as compared to a C-terminal truncated thermostable RecA protein consisting of the amino acid sequence of SEQ ID NO: 13.   Furthermore, the method as described in any one of Claims 10-12 which enhances a homologous recombination activity compared with the wild-type heat-resistant RecA protein which consists of an amino acid sequence of sequence number 6, 8, 10, or 12.