JP2008178338A - Nucleic acid amplification method in which target nucleic acid in nucleic acid sample mixed with fragmented nucleic acid is amplified, and kit therefor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a nucleic acid amplification method in which a target nucleic acid is specifically and efficiently amplified without being affected by a fragmented nucleic acid, and to provide a kit for nucleic acid amplification. <P>SOLUTION: In the nucleic acid amplification method in which the target nucleic acid in the nucleic acid sample mixed with the fragmented nucleic acid is amplified, the nucleic acid amplification reaction is carried out to the nucleic acid sample mixed with the fragmented nucleic acid in the presence of RecA protein derived from a highly thermophilic bacterium. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a nucleic acid amplification method for amplifying a target nucleic acid in a nucleic acid sample contaminated with fragmented nucleic acid, and a kit thereof. Specifically, the present invention relates to a nucleic acid amplification method for performing a nucleic acid amplification reaction on a nucleic acid sample mixed with a fragmented nucleic acid in the presence of a RecA protein derived from an extreme thermophile, and a kit thereof.

  Nucleic acid amplification technology represented by the polymerase chain reaction (hereinafter sometimes abbreviated as “PCR”) is a revolutionary technology that can amplify a specific target nucleic acid region more than 100,000 times in a short time. It is. However, it is difficult to optimize the reaction, and the target product may not be obtained due to factors attributable to the nucleic acid used as a template. In addition to the case where the target product is not obtained, there are cases where undesired products are generated. One such factor is fragmentation of a part of the template nucleic acid. In some cases, the fragmented nucleic acid in which a part of the template nucleic acid is fragmented works like a primer and the production of the target product is inhibited.

  Fragmentation of the template nucleic acid also occurs in the process of preparing a nucleic acid sample from a subject such as a clinical sample. For example, when a contaminating substance is mixed in a complicated manner, such a contaminating substance becomes an amplification inhibiting factor, and therefore, a nucleic acid sample has been isolated and purified to eliminate this. Sometimes complicated and long processing is required, and fragmentation of nucleic acids has been a serious problem. In addition, during the storage of the specimen before analysis and the prepared nucleic acid, a part of the molecular structure of the nucleic acid is damaged due to oxidation or the like, and fragmentation occurs.

  In the past, when a target product could not be obtained, optimization was attempted by changing parameters constituting the amplification reaction. For example, important parameters include primer design and concentration, magnesium concentration, nucleotide concentration, DNA polymerase type and concentration, and thermocycle. However, parameter optimization requires the skill of an experimenter, and due to sample loss during parameter setting, optimization could not be sufficiently achieved in the case of a small amount of sample. For this reason, there have been cases where it has been necessary to give up nucleic acid amplification.

  Various attempts have also been reported from the aspect of achieving an efficient amplification reaction. For example, it has been reported that nucleic acid amplification efficiency can be improved by performing PCR in the presence of a single-stranded binding protein (hereinafter abbreviated as “SSB”) derived from thermus thermophilus, a DNA binding protein. (For example, see Non-Patent Document 1.) Furthermore, a technique has been reported in which a test sample can be directly applied to PCR without neutralizing and purifying nucleic acid by neutralizing a charged substance that inhibits nucleic acid amplification present in the test sample (for example, , See Non-Patent Document 2.)

  However, in any of the above, no effect of improving the nucleic acid amplification efficiency from a sample containing a fragmented nucleic acid has been reported. In any case, no priming efficiency effect was observed. In particular, the method of neutralizing a charge substance is only neutralization of a charge substance that is an amplification inhibitor contained in a sample. Therefore, establishment of a technique capable of efficiently amplifying a target nucleic acid has been demanded even in a nucleic acid sample in which fragmented nucleic acids are mixed.

Perales et al., “Enhancement of DNA, cDNA synthesis and fidelity at high temperatures by a dimeric single-stranded DNA-binding protein.” Nucleic Acids Research, 2003, Vol. 31, No. 22, pp. 6473-6480 Ampdirect (registered trademark) [online] (searched January 6, 2006), Shimadzu Corporation, Internet <URL: HYPERLINK "http://www.shimadzu-biotech.jp/reagents/amp/" http: // www .shimadzu-biotech.jp / reagents / amp />

  Therefore, in view of the above circumstances, the present invention has an object to provide a nucleic acid amplification method and a nucleic acid amplification kit that can specifically and efficiently amplify a target nucleic acid without being affected by fragmented nucleic acids. To do.

  As a result of diligent research to solve the above problems, the present inventors have conducted a nucleic acid amplification reaction in the presence of a RecA protein derived from a highly thermophilic bacterium on a nucleic acid sample in which fragmented nucleic acids are mixed. As a result, it was found that only the desired nucleic acid can be specifically amplified. Further research has revealed that RecA protein from hyperthermophilic bacteria also has a neutralizing action against amplification inhibitors, and only the desired nucleic acid is specified even when applied to samples with low purity. The knowledge that it can amplify automatically was acquired. Based on these findings, the present invention has been completed.

  That is, in order to achieve the above object, the present invention has the following configurations [1] to [7].

[1] In a nucleic acid amplification method for amplifying a target nucleic acid in a nucleic acid sample contaminated with a fragmented nucleic acid,
A nucleic acid amplification method for amplifying a target nucleic acid in a nucleic acid sample by performing a nucleic acid amplification reaction on a nucleic acid sample contaminated with a fragmented nucleic acid in the presence of a RecA protein derived from an extreme thermophile.
[2] The nucleic acid amplification method according to [1], wherein the nucleic acid sample is a sample selected from biological samples, environmental samples, and foods.
[3] The nucleic acid amplification method according to [1] or [2], wherein the fragmented nucleic acid is a nucleic acid fragment of 100 to 300 bp.
[4] The nucleic acid amplification method according to any one of [1] to [3], wherein the RecA protein derived from the extreme thermophile is derived from Thermus thermophilus or Thermus aquaticus.
[5] The nucleic acid amplification method according to [1] to [4], wherein the nucleic acid amplification reaction is a reaction based on a polymerase chain reaction.

  According to the above configurations [1] to [5], it is possible to efficiently amplify a target nucleic acid from a nucleic acid sample containing a fragmented nucleic acid that has been difficult to amplify by conventional nucleic acid amplification techniques. The function of RecA protein from hyperthermophilic bacteria improves primer priming efficiency and accuracy, and in turn suppresses primer priming to sequences similar to fragmented target nucleic acids and suppresses chimerization of amplification products it can. Moreover, since it acts on a nucleic acid amplification inhibitor at the same time and neutralizes the action, the target nucleic acid can be efficiently amplified from a sample with a low degree of purification. Therefore, it can be widely used in various industrial fields, particularly in the field of molecular biology.

[6] A nucleic acid amplification kit for amplifying a nucleic acid sample nucleic acid containing a fragmented nucleic acid containing DNA polymerase and a RecA protein derived from an extreme thermophile.
[7] The nucleic acid amplification kit according to [6], wherein the RecA protein derived from the extreme thermophile is derived from Thermus thermophilus or Thermus aquaticus.

  According to the above configurations [6] to [7], simple and rapid nucleic acid amplification becomes possible by configuring the components necessary for nucleic acid amplification as a kit.

  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.

  The present invention provides a nucleic acid amplification reaction in which a target nucleic acid in a nucleic acid sample in which fragmented nucleic acids are mixed is present in the presence of a RecA protein derived from an extreme thermophile (hereinafter sometimes referred to as an “extremely thermophilic RecA protein”). To provide a method for specifically and efficiently amplifying a target nucleic acid. In conventional nucleic acid amplification reactions, the target nucleic acid could not be amplified due to the presence of fragmented nucleic acid, and it was unavoidable to give up the target product. The target product can be obtained without being affected by

  In the present specification, the nucleic acid amplification reaction is preferably a polymerase chain reaction (hereinafter sometimes referred to as “PCR”) that amplifies a specific nucleic acid region of interest using a thermostable DNA polymerase. It also includes various PCR variants. 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) Arbitrary 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. Furthermore, it 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.

  The target nucleic acid to be amplified in the present invention is not limited to the length and base sequence of the target nucleic acid, 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. 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 target nucleic acids.

  Here, the nucleic acid sample is not particularly limited as long as it is a sample containing the target nucleic acid or possibly containing the target nucleic acid. Therefore, in addition to biological samples, environmental samples, foods, and the like, artificial product samples by DNA synthesis technology and the like are also included. Biological samples such as animal-derived blood, urine, feces, saliva, tissues, cell cultures, and plant-derived roots, stems, leaves, flowers, fruits, etc., environmental samples such as soil, groundwater, river water, lake water, Examples of the food include seawater and meat, eggs, processed foods, and the like.

  The nucleic acid sample may be directly applied to the nucleic acid amplification method of the present invention, or may be applied to the nucleic acid amplification method of the present invention after performing known processes such as nucleic acid extraction and purification. The nucleic acid sample can be prepared by any method. It is carried out by separating cells, protozoa, fungi, bacteria, viruses, etc. from the subject and then extracting the nucleic acid. The nucleic acid can be extracted by disrupting cells with a surfactant such as SDS, enzymes such as lysozyme and proteinase, chaotropic salts, and extracting the nucleic acid with phenol / chloroform or the like. Moreover, you may refine | purify as needed in the case of extraction.

  The nucleic acid sample to be subjected to the nucleic acid amplification method of the present invention is a nucleic acid sample in which fragmented nucleic acid is mixed or possibly mixed. The fragmented nucleic acid may be single-stranded or double-stranded, and in the case of a double-stranded nucleic acid, the form of the end of the nucleic acid may be different from the sticky end and the sticky end. Furthermore, any nucleic acid may be used without any restriction on the base sequence. In particular, it refers to those having a continuous base sequence of at least a partial region of the target nucleic acid as a template, and those having a sequence similar to the continuous base sequence of at least a partial region of the target nucleic acid. The size of the fragmented nucleic acid refers to that having a base length of 1 to n-1 with respect to the polynucleotide (base length n) of the target nucleic acid, in particular 15 to 150 bases, or 300 bases or less. It is long. And what was produced by decomposing | disassembling a part of target nucleic acid, and the fragmented nucleic acid which does not originate in a target nucleic acid are also included. It also includes a nucleic acid in which a nick generated by one cleavage of the nucleic acid backbone is formed. Further, the fragmented nucleic acid is not limited to those generated by physical factors such as shear, enzymatic factors such as degrading enzymes, and chemical factors such as oxidized water decomposition and oxidation.

  Therefore, the present invention can be suitably applied to a sample in which nucleic acid fragmentation occurs due to a target nucleic acid preparation process from a specimen, a specimen storage process, or a dead body or tissue such as a microorganism. For example, a sample derived from a microorganism or a mammal is exemplified. In particular, it can be suitably applied to a sample that is rich in biodiversity, such as a soil sample containing soil microorganisms, and that is expected to be mixed with various nucleic acids. Furthermore, it can be suitably applied to samples that have been difficult to amplify by conventional nucleic acid amplification techniques, such as samples that have been stored for a long time.

Here, the case where it applies to PCR is demonstrated in detail. PCR is a method of amplifying DNA in a chain reaction using a heat-resistant DNA polymerase. The principle of PCR is to amplify a nucleic acid to be amplified (hereinafter sometimes abbreviated as a target nucleic acid) 2 ⁿ times by repeating three cycles of temperature changes in n cycles in the presence of a primer and a heat-resistant DNA polymerase. To do.

  In the present invention, the PCR reaction solution is a target nucleic acid, thermostable DNA polymerase, primer DNA, deoxynucleotide 5′-triphosphate (hereinafter referred to as “dNTP”), and appropriate in the presence of highly thermophilic RecA protein. Prepared with various buffers. Further, it may be prepared containing nucleotide 5'-triphosphate (hereinafter referred to as "NTP") separately from dNTP. Here, the primer DNA 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.

  Here, the hyperthermophilic 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 nucleic acids. is there. The RecA protein used in the present invention is a highly thermophilic bacterium-derived protein. Therefore, use of E. coli-derived RecA protein or the like is not suitable in the present invention. Examples of the highly thermophilic RecA protein suitable for use in the present invention include RecA protein derived from the genus Thermus, Thermococcus, Pyrococcus, Thermotoga and the like. Specifically, RecA protein derived from Thermus thermophilus and Thermus aquaticus is exemplified. In particular, it is preferably derived from Thermus thermophilus.

  These highly thermophilic bacteria RecA proteins include naturally-occurring highly thermophilic bacteria RecA proteins, as well as highly thermophilic bacteria RecA proteins artificially synthesized chemically or genetically. Specifically, those extracted from highly thermophilic bacteria can be used according to a conventional method. Furthermore, it can be easily purified using a known host / expression vector system such as Escherichia coli. For example, host Escherichia coli is transformed and cultured with an expression vector into which a gene encoding the hyperthermophile RecA protein is introduced by a known method, and the hyperthermophile RecA protein is expressed. By crushing the host E. coli and performing heat treatment, E. coli-derived proteins other than the highly thermophilic RecA protein are thermally denatured and thermally aggregated, and thus can be separated and removed by centrifugation or the like. As a result, the highly thermophilic RecA protein that is not heat denatured can be separated from the E. coli-derived protein as a soluble fraction and purified using affinity chromatography or the like.

  At this time, since the hyperthermophilic RecA protein is derived from the hyperthermophilic bacterium, the structure is stable at room temperature, and also has high stability against organic solvents. Therefore, the purification step can be performed at room temperature.

  The host cell is not limited to E. coli, and eukaryotic cells such as yeast (Saccharomyces cerevisiae) and insects (Sf9 cells) can be used. 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 the RecA protein of hyperthermophilic bacteria can be inserted, and can be expressed in the host cell. Any expression vector can be used as long as it exists. 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, a signal sequence that confers secretion of the expression product, and a neomycin resistance gene, kanamycin resistance gene, chloramphenicol resistance gene, ampicillin resistance gene, hygromycin resistance gene Marking sequences that can confer phenotypic selection in transformed hosts such as 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. Furthermore, an expression vector incorporating a desired sequence can be prepared and used as appropriate.

  Further, it includes a protein having a structure similar to the above-mentioned hyperthermophilic RecA protein and functionally equivalent. Therefore, in addition to RecA-like proteins that exist in nature, it is intended to include proteins that have been modified by artificial mutagenesis or genetic recombination. For example, in the amino acid sequence of the above-mentioned naturally-derived extreme thermophile RecA protein, one or several amino acids have an amino acid sequence substituted, deleted or added, and the above-mentioned extreme thermophile RecA protein and organism Variants that are equivalent in scientific function may be included. The number of mutations in the amino acid sequence is not limited as long as the biological function of the original protein is maintained.

  Here, the modification to the amino acid sequence may be artificially performed on the amino acid sequence to be modified using a known mutation manipulation means such as a site-specific mutagenesis method (Nucleic Acid Res. 10, pp6487, 1982). it can. Furthermore, the modified substance produced by the variation | mutation of the amino acid in nature is also included.

  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, the term “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. As long as it is sufficient to be able to form a stable duplex structure with each other, only a few nucleobases are allowed to have partial complementarity that fits along the base-pairing rules. 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. Therefore, the appropriate length is determined depending on many factors such as sequence information of the target nucleic acid such as GC content, and hybridization reaction conditions such as reaction temperature and salt concentration in the reaction solution. It is 20-50 bases long.

  Here, the DNA polymerase used is not particularly limited as long as it is a heat-resistant DNA polymerase that can be used in PCR. For example, Taq polymerase from Thermus aquaticus, Tth polymerase from Thermus thermophilus, Bst polymerase from Bacillus Stearothermophilus, Thermococcus litoralis And other DNA polymerases derived from thermophilic bacteria such as Vent DNA polymerase derived from thermophilic archaebacteria such as Pfu polymerase derived from Pyrococcus furiosus.

  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 DNA 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.

As the buffer solution, those prepared containing an appropriate buffer component and magnesium salt can be used, and there is no particular limitation. As the buffer component, phosphates such as trisacetic acid, trishydrochloric acid, sodium phosphate and calcium phosphate can be suitably used, and trisacetic acid is particularly preferred. The final concentration of the buffer component is in the range of 5 mM to 100 mM and is prepared in the range of pH 6.0 to 9.5. Further, the magnesium salt is not particularly limited, but magnesium chloride, magnesium acetate and the like can be appropriately used, and magnesium acetate is particularly 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 hyperthermophilic RecA protein 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 hyperthermophilic RecA protein 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 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. The magnesium salt is preferably prepared in a final concentration of 0.1 to 50 mM, particularly 1 to 5 mM.

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

Another reaction having complementarity using the target nucleic acid as a template with the primer as a starting point by repeating the appropriate cycle of the reaction in which the three-stage temperature change consisting of the above (1) to (3) is one cycle. Synthesis of the nucleic acid strand is started. As a result, the target nucleic acid is amplified 2 ⁿ times by the reaction of n cycles. The number of thermocycles is determined according to the type, amount and purity of the target nucleic acid serving as a template, but 20 to 40 cycles, particularly 32, from the viewpoint of efficient nucleic acid amplification by suppressing non-specific amplification. It is preferable to carry out in ~ 36 cycles.

Each process will be explained in detail below. (The following numerical values are general values. Please confirm whether or not they conform to the present invention.
(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 such as genomic DNA is used as a template nucleic acid, only the first heat denaturation can be set longer (about 10 minutes) in order to completely denature the template nucleic acid.

(2) Primer annealing By reducing the temperature, the template nucleic acid that has been heat denatured and becomes a single strand in (1) above and a primer are 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. 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.

  By comprising as mentioned above, it becomes possible to amplify a target nucleic acid efficiently from the sample containing a fragmented nucleic acid by performing PCR in presence of a hyperthermophilic RecA protein. Fragmented nucleic acid acts as a nucleic acid amplification inhibitor, and in the past, it was difficult to amplify target nucleic acid in a nucleic acid sample mixed with fragmented nucleic acid, but the present invention reduces the influence of such nucleic acid amplification inhibition to a minimum. it can. This is because the priming efficiency and accuracy in PCR can be improved by the action of the highly thermophilic bacteria RecA protein. In particular, priming to a sequence similar to the fragmented target nucleic acid can be suppressed, and chimerization of the amplification product can be suppressed. At the same time, since it acts on a nucleic acid amplification inhibitor and neutralizes the action, it is considered that the target nucleic acid can be efficiently amplified from a sample with a low degree of purification.

  The amplification method of the present invention 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. In particular, since the nucleic acid amplification reaction can be easily optimized, it can be suitably applied to a very small amount of sample. For example, it can be used for preparing a large amount of DNA from a small amount of sample for genotyping or preparing a nucleic acid sample for 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 detection of single nucleotide polymorphisms, detection of pathogens such as viruses and bacteria such as SARS and influenza, and the like. In particular, the present invention can be suitably applied to a method for detecting a single nucleotide polymorphism in a clinical sample. According to the present invention, binding of a primer to a nucleic acid other than the target nucleic acid can be particularly suppressed, and primer mispriming 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, since a nucleic acid not having such a single nucleotide polymorphism is not amplified or the amplification is suppressed, a nucleic acid having such a single nucleotide polymorphism can be specifically amplified. Therefore, even for a sample suspected of being contaminated with a fragmented nucleic acid such as a clinical sample, the influence of the fragmented nucleic acid is suppressed to the minimum, and template-specific amplification becomes possible. And it is preferably applied also to the clinical sample etc. which fragmented by long-term storage. 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. In particular, soil is known to have very large biodiversity, but there are limits to the search for useful genes in ordinary nucleic acid amplification techniques due to contamination with fragmented nucleic acids derived from dead bodies of microorganisms. However, since the present invention enables efficient nucleic acid amplification even in the presence of fragmented nucleic acids, its utility value is high. 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. It can be suitably used for a sample suspected of contaminating a fragmented nucleic acid, which has been difficult in the past.

  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 DNA polymerase and highly thermophilic RecA protein. Furthermore, it may be configured by appropriately containing components necessary for PCR such as an appropriate buffer, magnesium, and dNTP salt. 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. Thus, by configuring the components necessary for PCR amplification as a kit, simple and rapid PCR amplification becomes possible.

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

  Various nucleic acid samples were subjected to PCR in the presence of highly thermophilic RecA protein. First, in the examples, the reagents used in the examples will be described.

(reagent)
As the nucleic acid sample, Escherichia coli genomic DNA sample, Bacillus subtilis genomic DNA sample, a mixed sample thereof, and human genomic DNA sample were used.
Specifically, the following was used as an E. coli genomic DNA sample.
E. coli genomic DNA sample 1 was prepared from Escherichia coli W3110 strain according to the method described in Shinsei Kagaku Kogaku Kogyo Nucleic Acid I Separation and Purification Chapter 3 Extraction of E. coli DNA (Tokyo Kagaku Dojin Publishing).
On the other hand, Escherichia coli DNA sample 2 was prepared from Escherichia coli W3110 using DNeasy tissue kit (manufactured by Qiagen).
Bacillus subtilis genomic DNA samples were prepared from 168 strains of Bacillus subtilis using DNeasy tissue kit (Qiagen).
Human genomic DNA (purchased from Promega) was used as the human genomic DNA sample.

    As the DNA polymerase, rTaq DNA Polymerase (Takara-Bio) was used.

  As the hyperthermophilic TthRecA protein, a RecA protein derived from Thermus thermophilus (hereinafter sometimes referred to as “TthRecA protein”) was used. Here, TthRecA protein is described in Masui R, Mikawa T, Kato R, Kuramitsu S., Characterization of the oligomeric states of RecA protein: monomeric RecA protein can form a nucleoprotein filament., Biochemistry. The one prepared by the inventor of the present invention with reference to the description on 37, No. 42, pages 14788 to 14797 was used. At this time, 50 mM Tris-HCl (pH 7.5), 1.5 M KCl, 1.0 mM EDTA, 0.5 mM DTT, and 50% Glycerol were used as stock solutions. TthRecA protein was added and used with such a solution.

The sequence of the primer pair used here is shown below.
Primer vs. BS:
Oligonucleotide 1
5'-AAG CGG ATG ATA TTA TCG GC-3 '(sequence recognition number: 1)
Oligonucleotide 2
5'-ATG TTT TGC AGG TTC GGA TC -3 '(sequence recognition number: 2)
Primer pair BS is a primer designed to amplify a part of the DNA polymerase I gene of Bacillus subtilis genomic DNA.

Primer vs. EC:
Oligonucleotide 3
5'-AAG CGG ACG ACG TTA TCG GT-3 '(sequence recognition number: 3)
Oligonucleotide 4
5'- ATG TTT TGC AGG TTA GGA TC -3 '(sequence recognition number: 4)
Primer pair EC is a primer designed to amplify part of the DNA polymerase I gene of E. coli genomic DNA.

Primer vs. HS:
Oligonucleotide 5
5'-GAC TAC TCT AGC GAC TGT CCA TCT C-3 '(sequence recognition number: 5)
Oligonucleotide 6
5'-GAC AGC CAC CAG ATC CAA TC-3 '(sequence recognition number: 6)

Example 1: Confirmation of effect of highly thermophilic RecA protein in PCR amplification-1
It confirmed about the amplification efficiency improvement effect of highly thermophilic bacteria RecA protein in PCR amplification.

(Method)
In this example, PCR was performed on a nucleic acid sample in which E. coli genomic DNA sample 1 and Bacillus subtilis genomic DNA sample were mixed with primers for amplification of Bacillus subtilis (primer pair BS).

PCR reaction solution (25 μl), 100 ng of E. coli genomic DNA sample 1, 10 ng of Bacillus subtilis genomic DNA sample, 0.5 μg of TthRecA protein, 0.5 μM each of oligonucleotide 1 and oligonucleotide 2 (primer pair BS) (final concentration) ), 0.6 unit of DNA polymerase and 0.2 mM (final concentration) of dNTP mixture were prepared by mixing 10 mM Tris-HCl Buffer (pH 8.3), 50 mM KCl, 1.5 mM MgCl ₂ .

  The PCR reaction solution prepared above was subjected to PCR to obtain an amplification product. PCR was performed after heat denaturation at 94 ° C. for 30 seconds, followed by 35 cycles of amplification reaction with one cycle consisting of 94 ° C. for 10 seconds, 55 ° C. for 30 seconds, and 70 ° C. for 60 seconds. The final reaction was performed in 1 cycle consisting of 7 minutes at 4 ° C and 1 minute at 4 ° C.

  After amplification, an electrophoresis buffer was added to each amplification reaction solution and stirred, and half of the solution 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 TthRecA protein, PCR was carried out in the same manner as described above with addition of 0.15 μl of a TthRecA protein stock solution, followed by electrophoresis. Thereby, the influence with respect to PCR of the preservation | save solution of TthRecA protein can be excluded, and the true effect with respect to PCR of TthRecA protein can be evaluated.

  In addition, PCR was performed in the same procedure as described above without adding any of the TthRecA protein and the TthRecA protein stock solution, and then subjected to electrophoresis, which was used as a control.

(result)
The results are shown in FIG.
In FIG. 1, lane 1 is a control.
In FIG. 1, lane 2 shows the amplification result when PCR was performed by adding a stock solution of TthRecA protein.
In FIG. 1, lane 3 shows the amplification result when PCR was performed by adding the TthRecA protein.

  From the result of FIG. 1, when PCR was performed with the addition of TthRecA protein, an amplification product derived from Bacillus subtilis was confirmed (lane 3). On the other hand, under the addition of the control and the TthRecA protein storage solution alone, the amplification product could not be confirmed (lanes 1 and 2), and it was considered that the amplification reaction was inhibited by some amplification inhibiting factor. From the above results, it was found that the addition of the extreme thermophile RecA protein can reduce the influence of the amplification reaction inhibiting factor and specifically and efficiently amplify the template nucleic acid.

Example 2: Confirmation of effect of highly thermophilic RecA protein in PCR amplification-2
Following Example 1, the effect of improving the amplification efficiency of the highly thermophilic RecA protein in PCR amplification was confirmed.

(Method)
In this example, PCR was performed on a nucleic acid sample in which E. coli genomic DNA and Bacillus subtilis genomic DNA were mixed using an E. coli genomic DNA amplification primer (primer pair EC) and a Bacillus subtilis genomic DNA amplification primer (primer pair BS). .

Here, the nucleic acid samples subjected to PCR are specifically as follows.
Sample A is a sample containing E. coli genomic DNA sample 1 as an E. coli genomic DNA sample and a Bacillus subtilis genomic DNA sample, and samples A-1 and A-2 differ in the content of the Bacillus subtilis genomic DNA sample. That is,
Sample A-1 is 100 ng of E. coli genomic DNA sample 1, 10 ng of Bacillus subtilis genomic DNA sample,
Sample A-2 contains 100 ng of E. coli genomic DNA sample 1, 5 ng of Bacillus subtilis genomic DNA sample (half of sample A-1).
Sample B is a sample containing E. coli genomic DNA sample 2 as an E. coli E. coli genomic DNA and a Bacillus subtilis genomic DNA sample, and samples B-1 and B-2 differ in the content of the Bacillus subtilis genomic DNA sample. That is,
Sample B-1 is 100 ng of E. coli genomic DNA sample 2, 10 ng of Bacillus subtilis genomic DNA sample,
Sample B-2 contains 100 ng of E. coli genomic DNA sample 2, 5 ng of Bacillus subtilis genomic DNA sample (half of sample B-1).

In order to amplify the above sample by PCR, a PCR reaction solution (25 μl) containing each of the above samples A-1, A-2, B-1, and B-2, 0.5 μg of TthRecA protein, and 0.5 μM of each primer pair (Final concentration), DNA polymerase 0.6 unit, dNTP mixture 0.2 mM (final concentration) was prepared by mixing 10 mM Tris-HCl Buffer (pH 8.3), 50 mM KCl, 1.5 mM MgCl ₂ .

  The PCR reaction solution prepared above was subjected to PCR to obtain an amplification product. PCR was performed after heat denaturation at 94 ° C. for 30 seconds, followed by 35 cycles of amplification reaction with one cycle consisting of 94 ° C. for 10 seconds, 55 ° C. for 30 seconds, and 70 ° C. for 60 seconds. The final reaction was performed in one cycle consisting of 7 minutes at 4 ° C and 1 minute at 4 ° C.

  After amplification, an electrophoresis buffer was added to each amplification reaction solution and stirred, and half of the solution was taken and subjected to 1.2% agarose gel electrophoresis. The gel after electrophoresis was stained with ethidium bromide to visualize the DNA band.

  And about sample A-1 and B-1, it replaced with TthRecA protein, and also about what added 0.15 microliters of preservation | save solutions of TthRecA protein performed the electrophoresis in the procedure similar to the above, and used for electrophoresis. Thereby, the influence with respect to PCR of the preservation | save solution of TthRecA protein can be excluded, and the true effect with respect to PCR of TthRecA protein can be evaluated.

(result)
The results are shown in FIG.
In FIG. 2, lanes 1 to 3 show the results of amplifying sample A containing E. coli genomic DNA sample 1 using primers for amplifying E. coli genomic DNA (primer pair EC). Lane 1 is sample A-1 with the addition of a stock solution of TthRecA protein, lane 2 is sample A-1 with the addition of TthRecA protein, and lane 3 is sample A-2 with the addition of TthRecA protein (B. subtilis). The PCR amplification results for half of the genomic DNA sample are shown.
In FIG. 2, lanes 4 to 6 show results obtained by amplifying sample A containing E. coli genomic DNA sample 1 with a primer for amplifying Bacillus subtilis genomic DNA (primer pair BS). Lane 4 is sample A-1 with the addition of a stock solution of TthRecA protein, lane 5 is sample A-1 with the addition of TthRecA protein, and lane 6 is sample A-2 with the addition of TthRecA protein (B. subtilis). The PCR amplification results for half of the genomic DNA sample are shown.
In FIG. 2, lanes 7 to 9 show the results of amplifying sample B containing E. coli genomic DNA sample 2 with E. coli genomic DNA amplification primers (primer pair EC). Lane 7 is sample B-1 with the addition of the TthRecA protein stock solution, lane 8 is sample B-1 with the addition of TthRecA protein, and lane 9 is sample B-2 with the addition of TthRecA protein (B. subtilis). The PCR amplification results for half of the genomic DNA sample are shown.
In FIG. 2, lanes 10 to 12 show the results of amplifying sample B containing E. coli genomic DNA sample 2 with primers (primer pair BS) for Bacillus subtilis genomic DNA amplification. Lane 10 is sample B-1 with the addition of a stock solution of TthRecA protein, lane 11 is sample B-1 with the addition of TthRecA protein, and lane 12 is sample B-2 with the addition of TthRecA protein. The results of PCR amplification of half of the fungal genomic DNA sample are shown.

  From the results shown in FIG. 2, in sample B, amplification products derived from both E. coli and Bacillus subtilis genomic DNA could be confirmed regardless of whether or not the TthRecA protein was added (lanes 7 to 12). On the other hand, in sample A, when PCR was performed with the addition of TthRecA protein, amplification products derived from both E. coli and Bacillus subtilis genomic DNA could be confirmed (lanes 2, 3, 5, 6). In addition, even when the amount of Bacillus subtilis genomic DNA added as template DNA was reduced, sufficient amplification products could be confirmed (lane 6). On the other hand, in the PCR under the addition of only the TthRecA protein stock solution, the amplification product could not be confirmed (lane 4), or the amount of amplification product was significantly reduced (lane 1). In sample B, since the amplification inhibition as in sample A did not occur, it is considered that the amplification reaction was inhibited by some amplification inhibition factor contained in sample A. Since the difference between sample A and sample B is in the E. coli genomic DNA sample, the E. coli genomic DNA sample 1 contained in sample A contains an amplification inhibiting factor, and the highly thermophilic bacteria RecA protein is the amplification inhibiting factor. It is considered that the influence of the can be reduced. Such results follow the results of Example 1 with nucleic acid samples including E. coli genomic DNA samples as well.

Example 3: Confirmation of properties of E. coli genomic DNA sample 1 and E. coli genomic DNA sample 2 In order to examine the results of Examples 1 and 2 in more detail, the properties of E. coli genomic DNA sample 1 and E. coli genomic DNA sample 2 were examined in terms of molecular weight. Confirmed from.

(Method)
200 ng each of E. coli genomic DNA sample 1 and E. coli genomic DNA sample 2 were labeled with ³² P according to the manufacturer's protocol using a DNA Labeling Kit (Takara-Bio) in a reaction volume of 25 μl. The total amount of the reaction solution was subjected to phenol / chloroform extraction followed by gel filtration (Amersham Biosciences: G-50 Micro-spin column) to stop the reaction. Electrophoresis buffer was added to the reaction solution and stirred, and the entire amount was subjected to 1.2% agarose gel electrophoresis, and the gel after electrophoresis was stained with ethidium bromide to visualize the DNA band.

(result)
The results are shown in FIG.
In FIG. 3, lane 1 shows the result of E. coli genomic DNA sample 1, and in FIG. 3, lane 2 shows the result of E. coli genomic DNA sample 2. From the above results, it was confirmed that the Escherichia coli genomic DNA sample 1 contained a large amount of fragmented DNA having a size of 100 to 300 bp (lane 1). On the other hand, it was confirmed that E. coli genomic DNA sample 2 was a sample containing almost no fragmented DNA (lane 2).

  As a result, in Examples 1 and 2, it was found that the confirmed amplification inhibition factor was due to contamination of fragmented nucleic acids. Further, it was found that the highly thermophilic bacteria RecA protein has the ability to improve the nucleic acid amplification efficiency for nucleic acid samples in which fragmented nucleic acids are mixed.

Example 4: Confirmation of effect of highly thermophilic RecA protein in PCR amplification-3
The effect of improving the amplification efficiency of the highly thermophilic RecA protein in PCR amplification was confirmed by adding a fragmented nucleic acid based on the results of Examples 1-3.

(Method)
It confirmed about the amplification efficiency improvement effect by TthRecA protein addition in the nucleic acid sample in which fragmented nucleic acid was mixed. Specifically, a sample consisting of 10 ng of Bacillus subtilis genomic DNA sample and a sample of 10 ng of Bacillus subtilis genomic DNA added with 100-200 ng of fragmented Escherichia coli genomic DNA was used as a primer for Bacillus subtilis amplification, and TthRecA protein was present. PCR was performed in the absence and absence. Here, the added fragmented E. coli genomic DNA was prepared as follows. E. coli genomic DNA sample 1 was subjected to gel filtration chromatography (S-400 HR: Amersham Pharmacia) in a buffer solution (10 mM Tris-HCl pH 8.0, 1 mM EDTA, 150 mM NaCl) A high molecular weight DNA and a low molecular weight fragmented DNA were separated and used.

Here, the examined nucleic acid samples examined are specifically shown below.
Nucleic acid sample C
10 ng Bacillus subtilis genomic DNA sample Nucleic acid sample D
10 ng Bacillus subtilis genomic DNA sample + 100 ng fragmented E. coli genomic DNA
Nucleic acid sample E
10 ng Bacillus subtilis genomic DNA sample + 150 ng fragmented E. coli genomic DNA
Nucleic acid sample F
10ng Bacillus subtilis genomic DNA sample + 200ng fragmented E. coli genomic DNA

In order to PCR amplify the nucleic acid sample, a PCR reaction solution (25 μl) containing the nucleic acid samples C to F, 0.5 μg of TthRecA protein, and 0.5 μM each of oligonucleotide 1 and oligonucleotide 2 (primer pair BS) (Final concentration), 0.6 unit of DNA polymerase and 0.2 mM (final concentration) of dNTP mixture were prepared by mixing 10 mM Tris-HCl Buffer (pH 8.3), 50 mM KCl, 1.5 mM MgCl ₂ .

  The PCR reaction solution prepared above was subjected to PCR to obtain an amplification product. PCR was performed after heat denaturation at 94 ° C. for 30 seconds, followed by 35 cycles of amplification reaction with one cycle consisting of 94 ° C. for 10 seconds, 55 ° C. for 30 seconds, and 70 ° C. for 60 seconds. The final reaction was performed in one cycle consisting of 7 minutes at 4 ° C and 1 minute at 4 ° C.

  After amplification, an electrophoresis buffer was added to each amplification reaction solution and stirred, and half of the solution 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, each of the nucleic acid samples C to F was attached without adding the TthRecA protein, PCR was performed in the same procedure as described above, and then subjected to electrophoresis, which was used as a control for each sample.

(result)
The results are shown in FIG.
In FIG. 4, lanes 1 and 2 show the results of the nucleic acid sample C, that is, the sample containing only the Bacillus subtilis genomic DNA sample. Lane 1 shows the result in the absence of TthRecA protein (control), and Lane 2 shows the result in the presence of TthRecA protein.
In FIG. 4, lanes 3 to 4 show the results of nucleic acid sample D, that is, a sample containing Bacillus subtilis genomic DNA sample and 100 ng of fragmented E. coli genomic DNA. Lane 3 shows the result in the absence of TthRecA protein (control), and Lane 4 shows the result in the presence of TthRecA protein.
In FIG. 4, lanes 5 to 6 show the results of the nucleic acid sample E, that is, a sample containing Bacillus subtilis genomic DNA sample and 150 ng of fragmented E. coli genomic DNA. Lane 5 shows the result in the absence of TthRecA protein (control), and lane 6 shows the result in the presence of TthRecA protein.
In FIG. 4, lanes 7 to 8 show the results of nucleic acid sample F, that is, a sample containing Bacillus subtilis genomic DNA sample and 200 ng of fragmented E. coli genomic DNA. Lane 7 shows the result in the absence of TthRecA protein (control), and lane 8 shows the result in the addition of TthRecA protein.

  From the results of FIG. 4, when PCR was performed with the addition of TthRecA protein, a specific amplification product derived from Bacillus subtilis genomic DNA was confirmed even when the amount of fragmented DNA was increased (lanes 2, 4). , 6, 8). On the other hand, when PCR was performed without adding the TthRecA protein, an amplified product derived from Bacillus subtilis genomic DNA could not be confirmed when the amount of fragmented DNA increased (lanes 1, 3, 5, 7). The amplification reaction was inhibited. From the above results, it was found that the addition of the highly thermophilic bacterium RecA protein can reduce the influence of fragmented DNA, which is an amplification inhibiting factor, and specifically and efficiently amplify the template nucleic acid.

Example 5: Confirmation of effect of highly thermophilic RecA protein in PCR amplification-4
The amplification efficiency improvement effect of the highly thermophilic bacteria RecA protein in PCR amplification was confirmed with nucleic acid samples mixed with various environmental samples.

(Method)
We confirmed the amplification efficiency improvement effect by adding TthRecA protein in the presence of various environmental samples. Specifically, human genomic DNA was examined by PCR amplification with contamination in environmental samples.

  Here, the examined environmental samples are specifically shown in Table 1 below. Samples 1-6 and 8-11 are common in that they are soil samples, but were collected from various lands with different climates and properties.

PCR reaction solution (25 μl), 0.5 μl each of the above environmental samples 1-11 having the nucleic acid concentrations shown in Table 1, 10 ng human genomic DNA, 0.5 μg TthRecA protein, oligonucleotide 5 and oligonucleotide 6 (primer pair) HS) 0.5 μM each (final concentration), DNA polymerase 0.6 unit, dNTP mixture 0.2 mM (final concentration) mixed in 10 mM Tris-HCl Buffer (pH 8.3), 50 mM KCl, 1.5 mM MgCl ₂ It was prepared by doing.

  The PCR reaction solution prepared above was subjected to PCR to obtain an amplification product. PCR was performed after heat denaturation at 94 ° C. for 30 seconds, followed by 35 cycles of amplification reaction with one cycle consisting of 94 ° C. for 10 seconds, 55 ° C. for 30 seconds, and 70 ° C. for 60 seconds. The final reaction was performed in one cycle consisting of 7 minutes at 4 ° C and 1 minute at 4 ° C.

  After amplification, an electrophoresis buffer was added to each amplification reaction solution and stirred, and half of the solution 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, without adding TthRecA protein, human genomic DNA was subjected to PCR in the same manner as described above in a mixture of environmental samples 1 to 11, and then subjected to electrophoresis, which was used as a control for each sample. At the same time, the results of PCR performed in the same manner without adding any environmental sample are also shown.

(result)
The results are shown in FIG.
In FIG. 5, lanes 1 and 13 show the results of PCR amplification of human genomic DNA in the presence of environmental sample 1. Lane 1 shows the result in the absence of TthRecA protein (control), and Lane 13 shows the result in the presence of TthRecA protein.
In FIG. 5, lanes 2 and 14 show the results of PCR amplification of human genomic DNA in the presence of environmental sample 2. Lane 2 shows the results in the absence of TthRecA protein (control), and lane 14 shows the results with the addition of TthRecA protein.
In FIG. 5, lanes 3 and 15 show the results of PCR amplification of human genomic DNA in the presence of environmental sample 3. Lane 3 shows the result in the absence of TthRecA protein (control), and Lane 15 shows the result in the presence of TthRecA protein.
In FIG. 5, lanes 4 and 16 show the results of PCR amplification of human genomic DNA in the presence of environmental sample 4. Lane 4 shows the results in the absence of TthRecA protein (control), and Lane 16 shows the results in the presence of TthRecA protein.
In FIG. 5, lanes 5 and 17 show the results of PCR amplification of human genomic DNA in the presence of environmental sample 5. Lane 5 shows the results in the absence of TthRecA protein (control), and Lane 17 shows the results in the presence of TthRecA protein.
In FIG. 5, lanes 6 and 18 show the results of PCR amplification of human genomic DNA in the presence of environmental sample 6. Lane 6 shows the results in the absence of TthRecA protein (control), and lane 18 shows the results with the addition of TthRecA protein.
In FIG. 5, lanes 7 and 19 show the results of PCR amplification of human genomic DNA in the presence of environmental sample 7. Lane 7 shows the result in the absence of TthRecA protein (control), and Lane 19 shows the result in the presence of TthRecA protein.
In FIG. 5, lanes 8 and 20 show the results of PCR amplification of human genomic DNA in the presence of environmental sample 8. Lane 8 shows the results in the absence of TthRecA protein (control), and Lane 20 shows the results in the presence of TthRecA protein.
In FIG. 5, lanes 9 and 21 show the results of PCR amplification of human genomic DNA in the presence of environmental sample 9. Lane 9 shows the result in the absence of TthRecA protein (control), and Lane 21 shows the result in the presence of TthRecA protein.
In FIG. 5, lanes 10 and 22 show the results of PCR amplification of human genomic DNA in the presence of environmental sample 10. Lane 10 shows the result in the absence of TthRecA protein (control), and Lane 22 shows the result in the presence of TthRecA protein.
In FIG. 5, lanes 11 and 23 show the results of PCR amplification of human genomic DNA in the presence of environmental sample 11. Lane 11 shows the result in the absence of TthRecA protein (control), and Lane 23 shows the result in the presence of TthRecA protein.
In FIG. 5, lanes 12 and 24 show the results of PCR amplification without contamination with environmental samples. Lane 12 shows the results in the absence of TthRecA protein (control), and lane 24 shows the results in the presence of TthRecA protein.

  From the results of FIG. 5, when PCR was performed with the addition of TthRecA protein, specific amplification products derived from human genomic DNA were confirmed in any environmental sample, but non-specific No amplification product was confirmed (lanes 13-24). However, when PCR was performed without adding the TthRecA protein, there were samples in which no amplification product was obtained (lanes 1, 2, and 4), and even when the amplification product was obtained, non-specific Amplification products were confirmed (lanes 3, 5-12). From this, it is understood that some amplification inhibiting factors are included in the environmental sample, and the template-specific nucleic acid amplification is inhibited by the mixing of such amplification inhibiting factors. And it became clear that the influence of an amplification inhibitory factor can be reduced by addition of highly thermophilic bacteria RecA protein.

  In the environmental sample, it is assumed that fragmented DNA is caused by the dead bodies of microorganisms. As in the results of Examples 1 to 4, the TthRecA protein reduces the influence of fragmented DNA that causes amplification inhibition and reduces the template. It is understood that the nucleic acid is specifically and efficiently guided. It is also noteworthy that template-specific nucleic acid amplification was confirmed for any of various environmental samples under the addition of TthRecA. This is because template nucleic acid-specific amplification is achieved by the addition of TthRecA protein, even though environmental samples are expected to contain a wide variety of amplification inhibitors (eg, sugars, dyes, proteins). Yes. Therefore, it is understood that the highly thermophilic bacteria RecA protein has an effect of reducing the influence of a variety of amplification inhibitors, including fragmented nucleic acids, and can be applied to nucleic acid amplification of crude samples.

Example 6: Confirmation of effect of highly thermophilic RecA protein in PCR amplification-5
The amplification efficiency improvement effect of the highly thermophilic bacteria RecA protein in PCR amplification was confirmed with a nucleic acid sample mixed with blood samples.

(Method)
The effect of increasing amplification efficiency by adding TthRecA protein in a mixture of blood stored for a long time was confirmed. Specifically, human genomic DNA was examined by subjecting it to PCR in a blood sample stored for a long time.

PCR reaction solution (25 μl) was mixed with the above blood sample, human genomic DNA 0, 1, or 100 ng, TthRecA protein 0.5 μg, oligonucleotide 5 and oligonucleotide 6 (primer pair HS) each 0.5 μM (final concentration), It was prepared by mixing 0.6 unit of DNA polymerase and 0.2 mM (final concentration) of dNTP mixture in 10 mM Tris-HCl Buffer (pH 8.3), 50 mM KCl, 1.5 mM MgCl ₂ .

  The PCR reaction solution prepared above was subjected to PCR to obtain an amplification product. PCR was performed after heat denaturation at 94 ° C. for 30 seconds, followed by 35 cycles of amplification reaction with one cycle consisting of 94 ° C. for 10 seconds, 55 ° C. for 30 seconds, and 70 ° C. for 60 seconds. The final reaction was performed in one cycle consisting of 7 minutes at 4 ° C and 1 minute at 4 ° C.

  After amplification, an electrophoresis buffer was added to each amplification reaction solution and stirred, and half of the solution was taken and subjected to 1.2% agarose gel electrophoresis. The gel after electrophoresis was stained with ethidium bromide to visualize the DNA band.

  The sample was subjected to PCR in the same procedure as above without adding the TthRecA protein, and then subjected to electrophoresis.

(result)
The results are shown in FIG.
In FIG. 6, lanes 1 and 2 show the results of PCR amplification of 100 ng of human genomic DNA in the presence of a blood sample. Lane 1 shows the result in the absence of TthRecA protein (control), and Lane 2 shows the result in the presence of TthRecA protein.
In FIG. 6, lanes 3 to 4 show the results of PCR amplification of 1 ng of human genomic DNA in the presence of a blood sample. Lane 3 shows the result in the absence of TthRecA protein (control), and Lane 4 shows the result in the presence of TthRecA protein.
In FIG. 6, lanes 5 to 6 show results obtained by PCR amplification of 0 ng of human genomic DNA, that is, only a blood sample. Lane 5 shows the result in the absence of TthRecA protein (control), and lane 6 shows the result in the presence of TthRecA protein.

  From the results shown in FIG. 6, when PCR was performed with the addition of TthRecA protein, specific amplification products derived from human genomic DNA were confirmed by mixing with blood samples, and non-specific amplification products were confirmed. None (lanes 2, 4). However, when PCR was performed without adding the TthRecA protein, an amplification product was obtained with 100 ng of human genomic DNA (lane 1), but at the same time, the generation of a nonspecific amplification product was also confirmed. On the other hand, when the human genomic DNA was 1 ng, the amount of amplified product was remarkably reduced as compared with the case where PCR was carried out with the addition of TthRecA protein (comparison of lanes 3 and 4). From this, it is understood that some amplification inhibitory substance is contained in the blood sample, and the template-specific nucleic acid amplification is inhibited by mixing of the amplification inhibitory substance. And it became clear that the influence of an amplification inhibitory substance can be reduced by addition of highly thermophilic RecA protein.

  Since nucleic acid samples such as blood samples may be denatured and fragmented due to oxidation, etc. over time, it is assumed that fragmented DNA is mixed in the blood sample used in this example. The Therefore, similarly to the results of Examples 1 to 5, it is understood that the TthRecA protein leads to specific and efficient amplification of the template nucleic acid by reducing the influence of the fragmented DNA that becomes an amplification inhibiting factor. In addition, as with the environmental sample examined in Example 5, the blood sample was added with the TthRecA protein even though a wide variety of amplification inhibitors (eg, sugars, dyes, proteins) were supposed to be mixed. Nucleic acid specific amplification has been achieved. Therefore, it is understood that the highly thermophilic bacteria RecA protein has an effect of reducing the influence of a variety of amplification inhibitors, including fragmented nucleic acids, and can be applied to nucleic acid amplification of crude samples.

  A nucleic acid amplification method useful in the medical field, biochemical field, environmental field, food field and the like is provided.

The figure which shows the result of Example 1 (E. coli genomic DNA sample 1 + Bacillus subtilis genomic DNA sample) which confirmed the effect of the extreme thermophile RecA protein in PCR amplification The figure which shows the result of Example 2 (E. coli genomic DNA sample 1 + Bacillus subtilis genomic DNA sample, E. coli genomic DNA sample 2+ Bacillus subtilis genomic DNA sample) which confirmed the effect of the extreme thermophilic bacteria RecA protein in PCR amplification The figure which shows the result of Example 3 which confirmed the property of E. coli genomic DNA sample 1 and E. coli genomic DNA sample 2 The figure which shows the result of Example 4 (Bacillus subtilis genome DNA sample + fragmented DNA) which confirmed the effect of the hyperthermophilic bacteria RecA protein in PCR amplification The figure which shows the result of Example 5 (human genomic DNA + environmental sample) which confirmed the effect of the hyperthermophilic RecA protein in PCR amplification The figure which shows the result of Example 6 (human genomic DNA + blood sample) which confirmed the effect of the hyperthermophilic RecA protein in PCR amplification

Claims (7)

In a nucleic acid amplification method for amplifying a target nucleic acid in a nucleic acid sample contaminated with a fragmented nucleic acid,
A nucleic acid amplification method for amplifying a target nucleic acid in a nucleic acid sample by performing a nucleic acid amplification reaction on a nucleic acid sample contaminated with a fragmented nucleic acid in the presence of a RecA protein derived from an extreme thermophile.   The nucleic acid amplification method according to claim 1, wherein the nucleic acid sample is a sample selected from a biological sample, an environmental sample, and a food.   The nucleic acid amplification method according to claim 1 or 2, wherein the fragmented nucleic acid is a nucleic acid fragment of 100 to 300 bp.   The nucleic acid amplification method according to any one of claims 1 to 3, wherein the RecA protein derived from the extreme thermophile is derived from Thermus thermophilus or Thermus aquaticus.   The nucleic acid amplification method according to any one of claims 1 to 4, wherein the nucleic acid amplification reaction is a reaction based on a polymerase chain reaction. DNA polymerase;
A nucleic acid amplification kit for amplifying a target nucleic acid in a nucleic acid sample contaminated with a fragmented nucleic acid containing a highly thermophilic bacterium-derived RecA protein. The nucleic acid amplification kit according to claim 6, wherein the RecA protein derived from the extreme thermophile is derived from Thermus thermophilus or Thermus aquaticus.