JP2008136421A - Nucleic acid amplification method and nucleic acid amplification kit - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a nucleic acid amplification method, by which a PCR reaction-inhibiting factor is excluded without affecting a normal PCR reaction system and a post process to enhance the efficiency of the amplification, even when an initial amount of a target nucleic acid is rare. <P>SOLUTION: This nucleic acid amplification method for amplifying the target nucleic acid is characterized in that the PCR reaction using the target nucleic acid as a template is performed in the presence of an auxiliary nucleic acid, and the auxiliary nucleic acid does not have complementarity and homology with the target nucleic acid and a primer. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a nucleic acid amplification method using a PCR (Polymerase Chain Reaction) method. More specifically, the present invention relates to a stable and highly efficient amplification method for a small amount of nucleic acid in a test sample.

  With the progress of the genome sequencing project, detection of specific genes on the genome, SNPS analysis, expression analysis, etc. are attracting attention as post-genome issues. Under such circumstances, in recent medical and molecular biology fields, there is an increasing need to amplify and detect a small amount of target nucleic acid from collected specimens.

  Also, the importance of nucleic acid detection is increasing in actual clinical practice. Conventionally, methods such as detection of serum antibodies and pathogen isolation culture from patients have been used as clinical inspection methods for infections caused by viruses and bacteria. However, in the detection method of serum antibodies, detection may not be possible due to the long time from infection to antibody production or because antibody production is not stable. Further, in the case of pathogen isolation culture, there is a problem that long-term culture for about 3 days is required to determine the pathogen. From such a problem, a method has been proposed in which a nucleic acid of a pathogen contained in a specimen collected from a patient is amplified and a specific sequence derived from the pathogen is directly detected.

  As a nucleic acid amplification method, a PCR (Polymerase Chain Reaction) method is generally used. The PCR method is to amplify the desired DNA fragment from several hundred thousand to over one million times in a short time by repeating the DNA synthesis reaction by DNA polymerase using two kinds of primers sandwiching the DNA region to be amplified. Is the method. More specifically, the DNA double strand is separated into a single strand by bringing the reaction solution containing the target nucleic acid, primer, DNA polymerase, various buffers, salts and the like to about 95 ° C. Thereafter, the target sequence and the primer are bound by setting the temperature to about 65 ° C., and the DNA synthesis reaction using the target nucleic acid as a template is promoted from the primer by the DNA polymerase by setting the temperature to about 70 ° C. By repeating this temperature cycle 20 to 40 times, it is amplified to million times in 30 minutes to several hours. Therefore, in principle, the PCR method is a method in which if there is one copy of DNA, it can be amplified to a very large number.

  As a method for detecting and quantifying nucleic acid, a method of staining by agarose gel electrophoresis is common. Also, a method of immobilizing a target nucleic acid on a solid support and hybridizing a labeled probe (see Patent Document 1), or a method of immobilizing a probe on a solid support and hybridizing a labeled target nucleic acid (see Patent Document 2) Etc. have been proposed. These methods are sensitive enough to detect minute target sequences, but generally cannot be detected at the target nucleic acid concentration of the sample as it is, and nucleic acid amplification treatment such as PCR is necessary as a previous step. is there.

  In the future, as genetic analysis progresses, it is expected that the analysis target will become a more detailed and minute target. Further, in the detection of infectious pathogens, the sample contains only a very rare number of pathogens. It is necessary to amplify such a specimen to an amount detectable by the above-described agarose electrophoresis method or hybridization method.

  In principle, in the PCR method, if there is one copy of the target nucleic acid, it can be amplified up to several million times, but generally several hundred to several thousand copies are required as the initial target nucleic acid. If the initial target nucleic acid is less than this, there will be a phenomenon that amplification is not performed at all or the amount of amplification product is not constant.

Various causes are assumed for such a phenomenon. As an example, it can be considered that the amplification efficiency is lowered by binding of primers to each other to form a primer dimer, or by causing a target similar sequence in a sample to react with the primer and inhibiting the binding of normal primers. In addition, it is considered that the PCR reaction is inhibited by the components in the specimen, PCR reagents, reaction instruments, etc. acting on the target nucleic acid or DNA polymerase. For the inhibition factor of the former example, a method of adjusting the reaction solution at a constant temperature (see Patent Document 3) has been proposed. In addition, a method of adding a thermal process, a method of avoiding the factor of the latter example by adjusting pH, filtering, or the like has been proposed (see Patent Document 4).
U.S. Pat. No. 4,358,535 US Pat. No. 5,688,642 JP 05-24495 A Japanese Patent Laid-Open No. 11-113573

  However, even if these methods are used, the inhibition factor is not completely eliminated, and if the initial target nucleic acid amount is scarce, it will be strongly influenced by the remaining inhibition factor, resulting in low amplification efficiency. There is.

  In addition, when various chemicals are added or filtered, they may become new inhibitors of PCR reactions, changes in the composition of amplification products due to changes in PCR conditions, nucleic acid purification after PCR There was a possibility of affecting the process.

  An object of the present invention is to overcome these problems and to provide a stable and highly efficient nucleic acid amplification method by eliminating factors that inhibit PCR reactions without affecting normal PCR reaction systems and subsequent processes. is there.

To achieve the above object, the present invention firstly provides a PCR method for amplifying a target nucleic acid,
Performing a PCR reaction using the target nucleic acid as a template in the presence of an auxiliary nucleic acid,
The method for amplifying a target nucleic acid, wherein the auxiliary nucleic acid does not have complementarity and homology with the target nucleic acid and a primer.

The second of the present invention is a PCR reagent kit for amplifying a target nucleic acid,
a) a PCR reagent required for amplification of the target nucleic acid b) a nucleic acid amplification kit comprising at least an auxiliary nucleic acid having no complementarity and homology with the sequence of the target nucleic acid.

  The nucleic acid amplification method of the present invention makes it possible to improve amplification efficiency without affecting the normal PCR reaction system and subsequent processes. Therefore, even a very small amount of the initial target nucleic acid can be efficiently and stably amplified.

  Hereinafter, specific embodiments of the nucleic acid amplification method according to the present invention will be described. However, the present invention is not limited to these.

  In the present invention, the target nucleic acid refers to a nucleic acid having a target sequence amplified by a PCR reaction. Examples of the target nucleic acid include genomic DNA, plasmid DNA, other double-stranded DNA or single-stranded DNA. A nucleic acid sample refers to a sample added to a PCR reaction solution as a sample containing a target nucleic acid. When the nucleic acid specimen is derived from a living body, RNA and other contaminant components may be contained in addition to the target nucleic acid and other living body-derived DNA depending on the conditions of the extraction and purification methods.

  In general, the field in which the present invention is used is amplification treatment using DNA extracted from a biological material as a target nucleic acid, but is not limited thereto, and may be amplification of artificially synthesized nucleic acid. Is available. The biological material refers to, for example, animal and plant tissues, body fluids, excrement, etc., and may be cells, bacteria, viruses, and the like. Body fluids include, but are not limited to blood, saliva, spinal fluid, urine, milk and the like. Moreover, although it does not specifically limit as a sample nucleic acid contained in these biological origin substances, For example, it uses for amplification of a specific gene.

  Any PCR method can be used for the PCR reaction according to the present invention as long as it is a commonly used method. A general PCR method is shown below, but the PCR reaction of the present invention is not limited to these reaction reagent compositions and reaction conditions.

In the PCR method, the temperature reaction cycle is basically performed by heating and cooling the PCR reaction solution. A general PCR reaction solution contains a target nucleic acid (template DNA) having a target sequence to be amplified, a buffer for pH buffer, salts, primers, a substrate such as dNTP, and a DNA polymerase. Further, it may contain a surfactant, gelatin and the like. As the buffer for pH buffer, a buffer having a concentration of about 10 mM to 100 mM such as Tris-HCl is preferably used. The pH is often adjusted to around 8-9. Various DNA polymerases such as Taq DNA Polymerase and T4 DNA Polymerase can be used, and are selected appropriately in consideration of the amplification target and the amplification reaction. In addition, there are methods such as adding various antibodies to DNA polymerase to inactivate them, then detaching the antibodies in the first heating step of PCR and activating DNA polymerase, so select appropriately including reaction conditions . Usually 0.5 to 2.5 units are added to one reaction. As the salt, MgCl ₂ is preferably used, and is usually added at a concentration of about 1.5 mM. Primers are designed according to the target nucleic acid. Design with a length of several tens to several hundred bases on both sides of the region to be amplified. Generally, a sequence that binds to each other or a sequence that has a higher order structure is not used. In addition, it is necessary to design in consideration of tm (dissolution temperature) which is a measure of ease of binding to the target nucleic acid.

  Nucleic acid amplification is performed on the PCR reaction solution adjusted to the above composition by repeating the temperature cycle using a commercially available thermal cycler or the like. The temperature cycle includes a step of separating a double-stranded DNA, which is a target nucleic acid, into a single strand (thermal denaturation), a step of binding the single strand to a primer (annealing), and a step of synthesizing DNA from the primer (extension). It is repetition. The heat denaturation step can be performed at about 95 ° C. for about 20 to 60 seconds. Although an annealing process changes with composition of a primer, it is about 50-65 degreeC normally and can be performed in about 10-90 second. The extension step varies depending on the selected DNA polymerase, but is generally about 72 ° C. and generally about 10 to 60 seconds. In addition to such a cycle, a cycle in which the annealing step and the extension step are performed at the same temperature may be used. Also, auxiliary temperature conditions are often given before and after the temperature cycle.

  A feature of the present invention is that the reaction solution for the PCR reaction described above includes an auxiliary nucleic acid having a sequence that is neither complementary nor homologous to the target sequence and the primer sequence. The auxiliary nucleic acid may be extracted from a biological substance or may be artificially synthesized. When the auxiliary nucleic acid is artificially synthesized, it is designed so as not to have complementarity and homology with the target nucleic acid and the primer sequence. When preparing and preparing an auxiliary nucleic acid from a living body, the homology and complementarity of base sequences can be verified using various genome databases published by various public institutions. If there is complementarity to the target sequence, it will be bound to the target sequence in the annealing step, which will inhibit the annealing with the original primer, and should be avoided. In addition, if there is homology to the target sequence, it must be avoided because it binds to the primer or cannot be distinguished from the original amplification product in the subsequent detection step. Furthermore, if the primer is complementary or homologous to the primer, it must be avoided because it binds to the primer or inhibits binding between the target nucleic acid and the primer. As described above, the homology and complementarity of the auxiliary nucleic acid in the present invention is preferably such that it does not hybridize with the target nucleic acid or primer used for detection in the annealing process temperature range.

  When a nucleic acid derived from a living body is used as the auxiliary nucleic acid, it is preferable to select the auxiliary nucleic acid from a kind different from the organism from which the specimen is derived in terms of classification attributes. Here, different types of classification attributes means that they belong to different types when classified based on biological characteristics. For example, from a taxonomic point of view, fields, gates, nets, eyes, Focusing on any hierarchy (classification attribute) selected from the family, genus, and species, it is different from the type to which the specimen-derived organism belongs. For example, when focusing on the world, specimen-derived organisms are classified as animal kingdom, plant kingdom, or fungal kingdom, and auxiliary nucleic acid is genomic DNA of organisms in a world different from the world to which specimen-derived organisms belong or a part thereof. Is preferably used as an auxiliary nucleic acid. The taxonomic hierarchy has a hierarchical structure from the upper order to the lower order in the order described. Which classification attribute is adopted as a criterion for selecting an auxiliary nucleic acid is selected in consideration of the characteristic of the sequence specificity of the target gene to be detected. For example, when targeting a species-specific gene sequence, if the genomic DNA of an organism belonging to a different species or a part thereof is selected as an auxiliary nucleic acid, the target gene and PCR primer sequences have complementarity and homology. Therefore, it is preferable because hybridization can be prevented. Specifically, when the sequence of the target nucleic acid is a gene sequence having a specific sequence for a certain type of fungus, the auxiliary nucleic acid selects genomic DNA of a different type of fungus or a part thereof. Furthermore, even when species-specific genes are targeted, in the higher hierarchy (eg, genus, world) than the species, the target nucleic acid is derived from the genome DNA of a different kind of organism and the classification attribute or a part thereof. More preferably, it is selected. For example, when the target gene is a sequence specific to a certain fungus, it is more preferable to select genomic DNA of an organism belonging to the animal or plant kingdom, or a part thereof, which is different from the fungus. Can be suitably used. Examples of detection that can suitably use human genomic DNA as an auxiliary nucleic acid include detection of microorganisms in blood that is markedly depleted of leukocytes due to leukemia and the like and foods that are subjected to microbial tests, in addition to those described in the examples. Moreover, it is not limited to taxonomic attribute, You may use the classification based on the difference between a eukaryote and a prokaryote, and the genome analysis revealed in recent years.

  In such a method for selecting an auxiliary nucleic acid derived from a living body, the auxiliary nucleic acid sequence has complementarity and homology with the target nucleic acid sequence even for a target nucleic acid whose sequence other than the sequence complementary to the primer sequence is unknown. The reliability of not doing can be ensured. Moreover, as an example of the amplification method of the present invention in which these auxiliary nucleic acid selection methods can be suitably used, PCR amplification performed on a target sequence specific to the biological species for the purpose of detecting the biological species can be mentioned.

  The target sequence amplified by the PCR method is usually several hundred to several thousand bases long. In consideration of distinguishing from the amplification product in a later step, it is desirable that the auxiliary nucleic acid has a nucleic acid sequence length different from the target sequence. By making the lengths sufficiently different, they can be separated by agarose gel electrophoresis or the like. Actually, the auxiliary nucleic acid has a nucleic acid sequence length longer than that of the target sequence, so that the sequence specificity is increased, which is advantageous for preventing binding with a primer or a target sequence. Although it is possible to use an auxiliary nucleic acid having a short nucleic acid sequence length relative to the target nucleic acid sequence, it is necessary to pay attention to the binding with the primer and the target sequence because the sequence specificity is reduced accordingly. Therefore, the nucleic acid sequence length of the auxiliary nucleic acid is preferably 5 times or more or 1/5 time or less of the sequence length of the target sequence. More preferably, the nucleic acid sequence length of the auxiliary nucleic acid is 10 times or longer than the sequence length of the target sequence.

  By containing the auxiliary nucleic acid in the PCR reaction solution, the auxiliary nucleic acid absorbs various inhibitory factors. On the other hand, the influence of the inhibitory factor on the target nucleic acid, the primer and the like is reduced. Therefore, more inhibitory factors can be absorbed by adding a large amount of auxiliary nucleic acid compared to the target nucleic acid. It is desirable to add the auxiliary nucleic acid to the PCR reaction solution in the range of 20 ng / ml to 8 μg / ml. Since the auxiliary nucleic acid is also composed of the same nucleic acid as the target nucleic acid, unlike the method of adding an originally unnecessary chemical to the PCR method, no new inhibition or problems in the subsequent steps are caused. Therefore, it is possible to eliminate the influence of the inhibition factor in the PCR reaction without affecting the subsequent process, and a highly efficient and stable amplification reaction is possible.

  Auxiliary nucleic acid in a normal PCR reaction solution is contained in a significantly larger amount than the target nucleic acid contained in the sample nucleic acid, but the total amount of nucleic acid contained in the PCR reaction solution is 20 ng / ml or more and 20 μg / ml or less. From the viewpoint of reaction efficiency, it is desirable to add an auxiliary nucleic acid so that it is in the range of.

  Moreover, it is possible to use not only DNA but also RNA as the auxiliary nucleic acid of the present invention.

  The present invention also relates to a nucleic acid amplification kit having a PCR reagent required for amplification of a target nucleic acid and the auxiliary nucleic acid according to the present invention. The reagent contained in the PCR reagent includes at least a pH buffering buffer, salts, and dNTP as a substrate. In addition, PCR primers or DNA polymerase may be included.

Although the Example of the nucleic acid amplification method of this invention is shown below, the content of this invention is not limited by a following example.
(Example 1)
In this example, an example is shown in which a gene of Streptococcus pneumoniae (hereinafter referred to as Sp) is detected from a human sputum. In this example, the rRNA sequence to be detected may be responsible for the main function of protein synthesis, and its base sequence is very well preserved in the species. However, since the homology of the sequence decreases as the types of classification attributes differ, it is often used for species identification and phylogenetic analysis. The human genome used as an auxiliary nucleic acid in this example also contains 28 s, 18 s, 5.8 s, and 5 s rRNA sequences, but there is little homology compared to the 16 s rRNA sequences of prokaryotic bacteria. Few sequences are homologous over bases.

  First, S.M. p. A 1500 base sequence present in 16 srRNA was selected as a specific sequence present in 2 and two types of primers at both ends for specifically amplifying the DNA of the region encoding this sequence were designed. The primer sequences are shown in Table 1.

  2 ml of human sputum was collected, and 200 μl of the nucleic acid was extracted using Dneasy Tissue Kit (manufactured by QIAGEN). The extract volume was 50 μl. At this point, it seems that only a very small amount of nucleic acid was contained, and the presence of the nucleic acid could not be confirmed even by measuring agarose gel electrophoresis or absorbance. The same sputum was cultured in a medium for 3 days. p. From the number of colonies, the initial number of bacteria was found to be about 50 / ml. Therefore, 50 μl of nucleic acid extract may contain as an upper limit genomic DNA corresponding to 10 bacteria.

The nucleic acid extracted by the above method was amplified by the nucleic acid amplification method of the present invention. The composition of the reaction solution was as follows: PCR buffer (Applied Biosystems), 4 mM MgCl ₂ , 10 mM dNTP (dATP, dCTP, dUTP, dGTP mixture), 2 units AmpliTaq Gold (Applied Biosystems), and 5 μM each of the above primers. The total volume was 48 μl including 10 μl of the extract. Then, 2 μl of commercially available 100 ng / μl Human Genomic DNA (manufactured by Takara Bio Inc.) was added as an auxiliary nucleic acid to the reaction solution to make the total volume 50 μl. Since the amount of target nucleic acid corresponds to genomic DNA for two bacteria at the upper limit with respect to 200 ng of auxiliary nucleic acid contained in the reaction solution, most of the amount of nucleic acid in the reaction solution was auxiliary nucleic acid. In addition, even when the amount of amplification of the target gene was taken into account from the copy number of the rRNA sequence, the auxiliary nucleic acid occupied the majority of the total amount of nucleic acid throughout the reaction.

  The PCR solution was set in a T3000 thermocycler (manufactured by Biometra), and PCR reaction was performed. As the thermal cycle, primary heating was 95 ° C. for 10 minutes, denaturation 95 ° C. for 45 seconds, annealing 65 ° C. for 45 seconds, and extension 72 ° C. for 45 seconds were repeated 40 times, and then final extension was performed at 72 ° C. for 10 minutes. A similar amplification reaction using the same sample was performed 10 times in total. S. p. The same operation was performed for sputum specimens that were confirmed to be free of sucrose by a culture method.

After the reaction, the presence of the nucleic acid was confirmed by electrophoresis using BioAnalyzer (manufactured by Agilent). The result of electrophoresis is shown in FIG. In the figure, (a) to (j) are S.P. p. (K) is the result of amplifying the DNA extracted from the cocoon containing the S. p. It is the result of amplifying the DNA extracted from the straw that does not contain. As can be seen from the figure, amplification products having a length of 1500 bases are generated in all 10 amplifications of the present invention. In the amplification reaction from the sample not containing nucleic acid, no amplification product was produced. p. It can be confirmed that the desired target sequence is amplified from the nucleic acid derived therefrom. As described above, it was found that even a very small amount of pathogenic bacteria can be stably subjected to genetic testing.
(Comparative Example 1)
Extraction and amplification reactions were carried out in the same manner as in Example 1 except that 2 μl of pure water was added to the reaction solution of the amplification reaction instead of adding Human Genomic DNA to obtain a general PCR reaction composition.

  The same amount of S.I. p. Ten times of amplification reaction was performed on sputum specimens containing. S. p. The same reaction was carried out for specimens that did not contain. After the reaction, the presence of the nucleic acid was confirmed by electrophoresis using BioAnalyzer (manufactured by Agilent).

  The result of electrophoresis is shown in FIG. In the figure, (a) to (j) are S.P. p. (K) is the result of amplifying the DNA extracted from the cocoon containing the S. p. It is the result of amplifying the DNA extracted from the straw that does not contain. As is apparent from the figure, there is a difference in the amount of amplification product in 10 reactions despite the use of the same specimen, and no amplification is performed in half.

As described above, when the nucleic acid amplification method of the present invention is used, even a sample that cannot be amplified by the conventional amplification method or whose amplification amount is unstable can be stably and efficiently amplified.
(Example 2)
In this example, an example is shown in which a gene of Haemophilus influenzae (hereinafter referred to as Hi) is detected from human cerebrospinal fluid.

  First, H. i. A 1000 base sequence present in 16srRNA was selected as a specific sequence present in 2 and two primers at both ends for specifically amplifying the DNA encoding the sequence were designed. Primer sequences are shown in Table 2.

  2 ml of human cerebrospinal fluid was collected, and 1 ml was extracted with MORA-EXTRACT (manufactured by Kyokuto Pharmaceutical). The extract was 50 μl. At this point, only a very small amount of nucleic acid was contained, and the presence of the nucleic acid could not be confirmed even by measuring agarose gel electrophoresis or absorbance. The same cerebrospinal fluid was cultured in a medium for 3 days, and the initial number of bacteria was found to be about 10 per ml of cerebrospinal fluid.

The nucleic acid extracted by the above method was amplified by the nucleic acid amplification method of the present invention. The composition of the reaction solution was PCR buffer (manufactured by Applied Biosystems), 4 mM MgCl ₂ , 10 mM dNTP (mixture of dATP, dCTP, dUTP, dGTP), 2 units of AmpliTaq Gold (manufactured by Applied Biosystems), and 5 μM each of the above primers. The total volume was 48 μl including 20 μl of the extract. Then, 2 μl of commercially available 100 ng / μl Human Genomic DNA (manufactured by Takara Bio Inc.) was added to the reaction solution to make the total volume 50 μl.

  The PCR solution was set in a T3000 thermocycler (manufactured by Biometra), and PCR reaction was performed. As the thermal cycle, primary heating was 95 ° C. for 10 minutes, denaturation 95 ° C. for 45 seconds, annealing 65 ° C. for 45 seconds, and extension 72 ° C. for 45 seconds were repeated 40 times, and then final extension was performed at 72 ° C. for 10 minutes. The same amplification reaction was performed 10 times. H. i. The same operation was performed on the cerebrospinal fluid specimen that was confirmed by the culture method to be free of.

After the reaction, the presence of the nucleic acid was confirmed by electrophoresis using BioAnalyzer (manufactured by Agilent). The result of electrophoresis is shown in FIG. In the figure, (a) to (j) are H.264. i. (K) is the result of amplifying the DNA extracted from the cerebrospinal fluid containing H. i. It is the result of amplifying DNA extracted from cerebrospinal fluid that does not contain. As can be seen from the figure, amplification products of 1000 base length are generated in all 10 amplifications of the present invention. In the amplification reaction from the sample not containing nucleic acid, no amplification product is produced. i. It can be confirmed that the desired target sequence is amplified from the nucleic acid derived therefrom. As described above, it was found that even a very small amount of pathogenic bacteria can be stably subjected to genetic testing.
(Comparative Example 2)
Extraction and amplification reactions were carried out in the same manner as in Example 2 except that 2 μl of pure water was added to the reaction solution of the amplification reaction instead of adding Human Genomic DNA to obtain a general PCR reaction composition.

  H. of the same amount as in Example 2. i. Ten times of amplification reaction was performed on sputum specimens containing. H. i. The same reaction was carried out for specimens that did not contain. After the reaction, the presence of the nucleic acid was confirmed by electrophoresis using BioAnalyzer (manufactured by Agilent).

  The result of electrophoresis is shown in FIG. In the figure, (a) to (j) are H.264. i. (K) is the result of amplifying the DNA extracted from the cerebrospinal fluid containing H. i. It is the result of amplifying DNA extracted from cerebrospinal fluid that does not contain. As is apparent from the figure, there are differences in the amount of amplification product in 10 reactions despite using the same specimen. Only (a) and (e) are barely amplified, and amplification is not performed at all in the subsequent 8 reactions.

  Thus, when the nucleic acid amplification method of the present invention is used, even a sample that cannot be amplified by the conventional amplification method or whose amplification amount is unstable can be stably and efficiently amplified.

It is the photograph of the electrophoresis result which shows the result of the preferable Example of this invention. It is the photograph of the electrophoresis result which shows the amplification result by the conventional nucleic acid amplification method. It is the photograph of the electrophoresis result which shows the result of the preferable Example of this invention. It is the photograph of the electrophoresis result which shows the amplification result by the conventional nucleic acid amplification method.

Claims (8)

A PCR method for amplifying a target nucleic acid comprising:
Performing a PCR reaction using the target nucleic acid as a template in the presence of an auxiliary nucleic acid,
The target nucleic acid amplification method, wherein the auxiliary nucleic acid does not have complementarity and homology with the target nucleic acid and the primer.   The nucleic acid amplification method according to claim 1, wherein the amount of the auxiliary nucleic acid contained in the reaction solution of the PCR reaction is larger than the amount of the target nucleic acid. The nucleic acid amplification method according to claim 1 or 2, wherein the nucleic acid sequence length of the auxiliary nucleic acid is different from the nucleic acid sequence length of the amplification product generated by the PCR reaction.   The nucleic acid amplification method according to any one of claims 1 to 3, wherein the auxiliary nucleic acid is human genomic DNA.   The nucleic acid amplification method according to any one of claims 1 to 3, wherein the auxiliary nucleic acid is a synthetic DNA. A PCR reagent kit for amplifying a target nucleic acid,
a) a PCR reagent required for amplification of the target nucleic acid b) a nucleic acid amplification kit comprising at least an auxiliary nucleic acid having no complementarity and homology with the sequence of the target nucleic acid.   The nucleic acid amplification kit according to claim 6, wherein the auxiliary nucleic acid is human genomic DNA.   The nucleic acid amplification kit according to claim 6, wherein the auxiliary nucleic acid is a synthetic DNA.