JP2001258556A - Method for synthesis of nucleic acid - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a new method for efficiently amplifying DNA in a specimen while suppressing the action of a PCR inhibiting substance. SOLUTION: The action of a PCR inhibiting substance is suppressed in the synthesis of a nucleic acid comprising the amplification of the objective gene in the specimen by including a polyanion and/or its insoluble polymer in the gene amplification reaction liquid.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for synthesizing nucleic acids,
Polymerase Chain Reaction:
(Hereinafter abbreviated as PCR).

[0002]

2. Description of the Related Art In the PCR method, a target DNA fragment is synthesized by repeating dissociation of a DNA strand into single strands, binding of primers sandwiching a specific region of the DNA strand, and a DNA synthesis reaction by the action of a DNA polymerase. This is a method that can be amplified several hundred thousand times. The PCR method is described in Japanese Patent Application Laid-Open No. 61-274697, which is an invention of Maris et al.

[0003] The PCR method can be used as a highly sensitive method for analyzing nucleic acids in various samples, and in particular, can be used for analyzing nucleic acids in samples derived from animal body fluids. Therefore, the PCR method is used for diagnosis and monitoring of infectious diseases and genetic diseases. further,
The PCR method is also suitable for transplantation, paternity testing, and DNA typing tests in medical treatment and the like based on genetic information of individuals. In these cases, peripheral blood is often selected for the test.

One disadvantage of the PCR method is that the reaction is inhibited by dyes, proteins, sugars or unknown contaminants. That is, many DNA polymerases including Taq DNA polymerase derived from Thermus aquaticus, which is a typical heat-resistant DNA polymerase, have a small amount of biologically-derived contaminants mixed in a PCR reaction solution.
It is widely known that the activity is strongly inhibited.

[0005] Therefore, prior to DNA amplification by the PCR method, cells, protozoa, fungi, bacteria, viruses, etc. (hereinafter referred to as gene inclusions) are separated from the test substance, and then nucleic acids are separated from the gene inclusions. An extraction process is required. As the method, a method of decomposing a gene inclusion body with an enzyme, a surfactant, a chaotropic agent, or the like, and then extracting nucleic acid from the decomposition product of the gene inclusion body using phenol or phenol / chloroform has been conventionally used. Have been. Recently, in the process of nucleic acid extraction, ion exchange resins, glass filters, glass beads, reagents having a protein agglutinating action, and the like have been used.

[0006]

However, even if the nucleic acid in the sample is purified using these methods, it is difficult to completely remove the impurities, and the amount of the recovered nucleic acid in the sample is not constant. There are many. Therefore, subsequent nucleic acid synthesis, especially when the content of the target nucleic acid in the sample is low,
In some cases, it cannot be done well. In addition, these purification methods are complicated and time-consuming, and increase the chance of contamination during the operation. Therefore, in order to solve these problems, a simpler and more effective sample pretreatment method is desired.

Accordingly, an object of the present invention is to provide a novel method for suppressing the action of a nucleic acid synthesis inhibitor and efficiently amplifying a nucleic acid in a sample.

[0008]

[0007] Heparin, which is usually used as a blood anticoagulant, is known as a PCR inhibitor,
It is an undesirable substance to be added to the sample. However, as a result of diligent studies of these phenomena, he has found that heparin suppresses the action of PCR inhibitors in biological samples. In addition, it was found that not only heparin but also other sulfated polysaccharides such as dextran sulfate, sulfated polymers such as polyvinyl sulfate, polyanions such as phosphorylated polymers such as DNA, and their insoluble polymers exhibit similar actions. Invented the invention. In order to solve the above problems, the present invention is characterized in that a polyanion and / or an insoluble polymer thereof is added to a nucleic acid amplification reaction solution in a nucleic acid synthesis method for amplifying a target nucleic acid in a sample.

Here, the polyanion means a polymer compound having a repeating structure containing an anion and a salt thereof. Examples of the anion include a sulfate group, a sulfite group,
Examples thereof include a phosphoric acid group, a carboxyl group, and a thiocarboxyl group, but are not limited thereto. Particularly, a polymer compound having a repeating structure containing a sulfate group and a salt thereof (sulfated polymer), a polymer compound having a repeating structure containing a phosphate group and a salt thereof (phosphorylated polymer)
Is preferred.

[0010] Examples of the sulfated polymer include sulfated polysaccharides (Sulfat polysaccharides such as heparin and dextran sulfate).
ed polysaccharide) and salts thereof (collectively, sulfated polysaccharides), polyvinyl sulfate and salts thereof, but are not limited thereto. In addition, the salt can include a sodium salt, a potassium salt and the like,
It is not limited to these. The addition amount of the sulfated polymer varies depending on the type thereof.
μg / ml or more, preferably 0.4 μg to 25.6 μm
g / ml, 0.05 for dextran sulfate
μg / ml or more, preferably 0.12 μg to 7.8 μ
g / ml, but not limited to these ranges.

As the phosphorylated polymer, for example, DN
A, RNA, and the like, but are not limited thereto. The amount of the phosphorylated polymer varies depending on the type of the polymer. For example, in the case of DNA, the amount is 0.2 ng or more, preferably 1 to 1000 ng per 50 μl of the reaction solution, but is not limited to these ranges.

The polyanion and / or its insoluble polymer may be added to the nucleic acid amplification reaction solution after being added to the sample.
It may be added in advance to the nucleic acid amplification reaction solution. Further, the polyanion and / or its insoluble polymer is not uniformly contained in the nucleic acid amplification reaction solution (for example, a polyanion and / or its insoluble polymer is added to a sample, and this sample is added to the reaction solution without stirring). Has the same effect. The polyanion and / or its insoluble polymer may be used alone or in combination of several kinds. The insoluble polymer of the polyanion may not be homogeneous, and may be a composite insoluble polymer containing a polyanion or a polyanion bonded to a carrier that does not inhibit the nucleic acid synthesis reaction.

Further, a polyanion may be coated on the inner wall of the reaction vessel in contact with the nucleic acid amplification reaction solution, or the vessel itself may be composed of an insoluble polyanion. The coating may be performed by any method such as spin coating and hot dip, and the thickness of the coating is not particularly limited. When the container itself is made of an insoluble polyanion, it is molded by injection molding or the like, but is not limited to this.

In the present invention, a sample refers to a biological sample or a gene inclusion in the biological sample. The biological sample refers to animal and plant tissues, body fluids, excretions, and the like. , Fungi, bacteria, viruses and the like. Body fluids include blood, cerebrospinal fluid, saliva, and milk, and excretions include, but are not limited to, feces, urine, and sweat. Cells include, but are not limited to, leukocytes and platelets in blood.

The nucleic acid amplification reaction solution usually contains a pH buffer, salts such as MgCl ₂ and KCl, primers, deoxyribonucleotides and a nucleic acid synthase. In addition, the above-mentioned salts are appropriately used after being changed to other salts. Also, gelatin, proteins such as albumin,
Various substances such as dimethyl sulfoxide and surfactant may be added.

The pH buffer is a combination of tris (hydroxymethyl) aminomethane and a mineral acid such as hydrochloric acid, nitric acid, sulfuric acid, and a preferable mineral acid is hydrochloric acid. Also,
Tricine, CAPSO (3-N-Cyclohexylamino-2-hy
Various pH buffers such as a combination of droxypropanesulfonic acid) or CHES (2- (cyclohexylamino) ethanesulfonic acid) with caustic soda and caustic potash can be used. The pH-adjusted buffer is used at a concentration between 10 mM and 100 mM in the nucleic acid amplification reaction solution.

A primer refers to an oligonucleotide that functions as a starting point for synthesis in the presence of a template and an amplification reagent. The primer is preferably single-stranded, but double-stranded may be used. If the primer is double-stranded, it is desirable to make it single-stranded before the amplification reaction.
Primers can be synthesized by known methods, or can be isolated from the living world.

The nucleic acid synthase means an enzyme that synthesizes a nucleic acid by adding deoxyribonucleotides, or a chemical synthesis system as described above. Suitable nucleic acid synthases include:
E. FIG. coli DNA polymerase I, E. coli. DNA polymerase Klenow fragment, T4 DNA polymerase, Taq DNA polymerase, T. litoralis DN
A polymerase, Tth DNA polymerase, PfuD
Examples include, but are not limited to, NA polymerase and reverse transcriptase.

In the present invention, a synergistic effect can be obtained by adjusting the pH of the nucleic acid amplification reaction solution. For example, p
H is not less than 8.1, preferably not less than 8.1 at a temperature of 25 ° C.
5 to 9.5.

The procedure of the nucleic acid synthesis method of the present invention is not different from the ordinary method except that a polyanion and / or its insoluble polymer is added to the reaction solution. For example, when the PCR method is used as a nucleic acid synthesis method, first, a target double-stranded DNA fragment to be amplified is converted into a single-stranded DNA by thermal denaturation (a diaturation step). Next, primers sandwiching the region to be amplified are hybridized (annealing step). Next, four types of deoxyribonucleotides (dATP, dGTP, dCTP)
A DNA polymerase is allowed to act in the presence of TP and dTTP) to perform a primer extension reaction (polymerization step).

[0021]

EXAMPLES (Experimental Example 1) In an experimental system in which human plasma was directly added to a PCR reaction solution to carry out PCR, the effect of adding heparin, a kind of sulfated polysaccharide, was examined. In the PCR reaction solution,
10 mM Tris-HCl, 50 mM KCl, 1.5 mM MgCl ₂ , 200 μM dA each
TP, dCTP, dGTP and dTTP, each 0.4μM primer, 1.25unit
s / 50μl of Taq DNA polymerase (TaKaRa Taq: Takara
shuzo, Kyoto, Japan). The mold D
As NA, 3 fg of PUC18 plasmid DNA was used.
PUC18 plasmid DNA as CR primer
Oligonucleotide having the base sequence of the plus strand (RV-
M: SEQ ID NO: 1) and an oligonucleotide having the nucleotide sequence of the minus chain (M13-47: SEQ ID NO: 2) were used. The sequences of the primers are as follows. As a result of PCR using these two types of primers, a 150 bp amplification product can be obtained. RV-M: 5'GAGCGGATAACAAATTT
CACACAGG3 'M13-47: 5' CGCCAGGGTTTTCCCA
GTCACGAC3 '

The PCR was performed at 94 ° C. for 3 minutes, followed by 94 ° C. for 30 seconds, 55 ° C. for 1 minute, and 72 ° C.
Polymerization was performed for 40 cycles under the condition of 1 minute, and finally at 72 ° C. for 7 minutes. After the completion of PCR, use 5 μl of the reaction solution, and add 0.5% agarose,
μg / ml ethidium bromide-added TAE (40 mM Tris-aceta
te, 1mM EDTA) solution for detection.

1 μl of human plasma and various concentrations of heparin sodium salt (Heparin) were added to the PCR reaction solution (50 μl).
Sodium: Wako, Osaka, Japan)
FIG. 1 shows an electrophoretogram of an amplification product when PCR was performed using 8 plasmid DNAs as a template. In the figure, M is a molecular weight marker (250 ng φX174-RF DNA cut with HincII),
1 is without heparin sodium salt, 2 is 0.025 μg / m
1, 3 is 0.05 μg / ml, 4 is 0.1 μg / ml, 5 is 0.2
μg / ml, 6 is 0.4 μg / ml, 7 is 0.8 μg / ml,
8 is 1.6 μg / ml, 9 is 3.2 μg / ml, 10 is 6.4 μg / ml
g / ml, 11 is 12.8 μg / ml, 12 is 25.6 μg / m
1 and 13 show the results when PCR was performed with addition of 51.2 μg / ml and 14 with the addition of 102.4 μg / ml. It has been shown that the addition of heparin results in a PCR amplification product. 0.1 μg / m of heparin sodium salt
1 (lane 4) or more, especially 0.4 μg to 25.6 μg / ml
(Lanes 6 to 12) Good results were obtained when added.

(Experimental Example 2) In this example, serially diluted human blood was directly added to the reaction solution under the condition of 0.8 μg / ml heparin sodium salt, which was excellent in amplification efficiency in Experimental Example 1, or was not present. This is an experiment in which PCR was performed. The composition of the PCR reaction solution used for the reaction was the same as in Experiment 1 except for the template DNA and the primer. DNA in blood is used as a template DNA, and an oligonucleotide having a base sequence of a plus chain located in the human β-globin gene region as a primer for PCR (GH20: SEQ ID NO: 3)
And oligonucleotides having the nucleotide sequence of the minus chain (G
H21: SEQ ID NO: 4) was used. The sequences of the primers are as follows.
As a result of the CR, an amplification product of 408 bp can be obtained. GH20: 5'GAAGAGCCAAGGACAGGGT
AC3 'GH21: 5' GGAAAATAGACCAATAGG
The conditions for CAG3 ′ PCR and the conditions for electrophoresis after PCR are the same as in Experimental Example 1.

FIG. 2 shows the experimental results (electropherogram). In the figure, M is a molecular weight marker, 1 and 5 are blood 0.5%, and 2 and 6 are 0.
25%, 3, 7: 0.125% added, 4, 8: PCR without added
Shows the results. In addition, 1-4 are the results of performing PCR in the absence of heparin sodium salt and 5-8 in the presence. This experimental example shows that the effect of the PCR inhibitor is suppressed by heparin, so that amplification products can be detected with high sensitivity even when PCR is performed by directly adding blood to the reaction solution.

(Experimental Example 3) The effect of adding dextran sulfate, a kind of sulfated polysaccharide, was examined in an experimental system in which human serum was directly added to a PCR reaction solution to perform PCR.
The composition of the PCR reaction solution used for the reaction and the conditions for electrophoresis after PCR are the same as in Experimental Example 1. PCR at 94 ° C,
After preheating for 4 minutes and 30 seconds, polymerization was performed for 40 cycles at 94 ° C. for 30 seconds, 58 ° C. for 1 minute and 72 ° C. for 1 minute, and finally at 72 ° C. for 7 minutes.

1 μl of human serum and various concentrations of dextran sulfate sodium salt (average molecular weight 5,500) (Sodium Dextran Sulfate) were added to the PCR reaction solution (50 μl).
ate, Nacalai tesque, Kyoto, Japan)
FIG. 3 shows an electrophoretogram of an amplification product when PCR was performed using C18 plasmid DNA as a template. In the figure, M is a molecular weight marker, 1 is dextran sulfate sodium salt 100 μg, 2 is 25 μg, 3 is 6.3 μg, 4 is 1.6 μg, 5
Are 390ng, 6 is 97ng, 7 is 24ng, 8 is 6.1ng, 9 is 1.52n
g, 10 is 0.38 ng, 11 is 0.095 ng, 12 is 0.023 ng, 13
Shows the results when PCR was performed without addition. 14 is a positive control to which neither human serum nor dextran sulfate sodium salt was added. When 390 ng to 6.1 ng (lane 5-8) of dextran sulfate sodium salt was added and PCR was performed, an amplification product was detected. From the above results, it was revealed that dextran sulfate has the effect of suppressing the action of the PCR inhibitor in serum and restoring the reaction.

The concentration at which the effect was observed in this experimental example was 0.12
It was in the range of μg / ml (6.1 ng / 50 μl) to 7.8 μg / ml (390 ng / 50 μl), but from other experiments, the effective concentration range depends on the molecular weight of dextran sulfate used and the amount of serum added. The concentration has been confirmed to vary, and the effective concentration range is not limited to the above range.

(Experimental Example 4) In an experimental system in which human blood was directly added to a PCR reaction solution to perform PCR, the effect of adding dextran sulfate was examined. The composition of the PCR reaction solution used for the reaction was the same as in Experimental Example 2, the PCR conditions were the same as in Experimental Example 3 except that the annealing temperature was set at 55 ° C., and the conditions for electrophoresis after PCR were as in Experimental Example 1. Is the same as

To a PCR reaction solution (50 μl), 0.05 μl of human blood and various concentrations of dextran sulfate sodium salt were added, and the DNA in the blood was used as a template to add P
FIG. 4 shows the electrophoretogram of the amplification product when CR was performed. In the figure, M is a molecular weight marker, 2 is no dextran sulfate sodium salt added, 3 is 10 μg, 4 is 2.5 μg,
5 is 0.625 μg, 6 is 156 ng, 7 is 39 ng, 8 is 9.76 ng, 9
Indicates the results when 2.44 ng was added, and 10 indicates 0.61 ng when PCR was performed. 1 is a negative control to which neither blood nor dextran sulfate sodium salt was added. Dextran sulfate sodium salt 156ng ~ 2.44ng
(Lane 6-9) When PCR was performed with the addition, the amplification product was detected.

From the above results, it has been clarified that dextran sulfate has the effect of suppressing the action of the PCR inhibitor in blood and restoring the reaction. The concentration at which the effect was observed in this experimental example was 0.049 μg / ml (2.44 ng / 50 μl)
From 3.1 μg / ml (156 ng / 50 μl), but from other experiments, it has been confirmed that the effective concentration range varies with the molecular weight of dextran sulfate used and the amount of serum added, The effective concentration range is not limited to the above range.

(Experimental Example 5) In an experimental system for performing PCR by directly adding human plasma to a PCR reaction solution, the effect of adding polyvinyl sulfate, which is a kind of sulfated polymer, was examined. PCR
The composition of the reaction solution and the conditions for electrophoresis after PCR are the same as in Experimental Example 1, and the conditions for PCR are the same as in Experimental Example 3.

Various amounts of human plasma were directly added to the PCR reaction solution (50 μl), and 1 ng of polyvinyl potassium sulfate was added.
(Polyvinylsulfuric Acid Potassium Salt: Nacalai te
sque) in the presence or absence of PUC18 plasmid DNA
FIG. 5 shows an electrophoretogram of the amplification product when PCR was carried out using as a template. In the figure, M is a molecular weight marker, 1 is plasma 2
μl, 2 are 1 μl, and the following 3 to 10 show the results when PCR was performed by directly adding the plasma whose volume was reduced in two-fold steps directly to the reaction solution. Note that 11 is the result when no plasma was added. In the case of adding potassium polyvinylsulfate (A in the figure), the amount of P was not more than 0.5 μl (lane 3).
CR product is detected but not added (B in the figure)
In the case of P, the addition amount of 0.0125 μl (lane 5) or less
A CR product has been detected. This indicates that by adding polyvinyl sulfate to the PCR reaction solution, it is possible to cope with about 4 times the amount of a PCR inhibitor in plasma as compared with the case where polyvinyl sulfate is not added.

(Experimental Example 6) In an experimental system in which human serum was directly added to a PCR reaction solution to perform PCR, the effect of adding DNA, which is a kind of phosphorylated polymer, was examined. The composition of the PCR reaction solution and the conditions for electrophoresis after PCR are the same as in Experimental Example 1, and the conditions for PCR are the same as in Experimental Example 3.

To a PCR reaction solution (50 μl), 1 μl of human serum and various concentrations of human genomic DNA were added.
FIG. 6 shows an electrophoretogram of the amplification product when PCR was performed using PUC18 plasmid DNA as a template. In the figure, M is a molecular weight marker, 1 is 100 ng of DNA, 2 is 10 ng, 3 is 1 ng,
4 shows the results when the PCR was performed with 0.1 ng, 5 with the addition of 0.01 ng, and 6 with no addition. 1 ng of DNA to PCR reaction solution
(Lane 3) When PCR was performed with addition of
Product has been detected. The above results indicate that the effect of the serum-derived PCR inhibitor was suppressed by performing PCR with the addition of DNA.

(Experimental Example 7) In an experimental system in which human plasma was directly added to a PCR reaction solution to carry out PCR, the effect of adding dextran sulfate gel, which is a kind of insoluble polymer of polyanion, was examined. The composition of the PCR reaction solution used for the reaction was the same as in Experiment 1 except for the template DNA and the primer.

As a template DNA, a control RN having a tetracycline resistance gene sequence derived from the pBR322 plasmid was used.
A (Takara shuzo) cDN reverse transcribed from 1000 copies
A was used as a primer for PCR. An oligonucleotide having a base sequence of a plus chain (F-1, Takara shuzo: SEQ ID NO: 5) located in the tetracycline resistance gene region and an oligonucleotide having a base sequence of a minus chain (R- 1. Takara shuzo: SEQ ID NO: 6) was used.
The sequences of the primers are as follows. As a result of PCR using these two types of primers, a 462 bp amplification product can be obtained. F-1: 5'CTCGGTCGCTTCCGCATCTTG
GA3'R-1: 5'CGGCACCTGTCCCTACGAGT
The conditions for TG3 ′ PCR are the same as in Experimental Example 1 except that the annealing temperature was set at 64 ° C., and the conditions for electrophoresis after PCR are the same as in Experimental Example 1.

0.125 μl of human plasma and various amounts of dextran sulfate gel were added to the PCR reaction solution (50 μl).
(Dextran beads, sulfated D5650: SIGMA, Missouri, U
FIG. 7 shows an electropherogram of the amplification product when PCR was performed using cDNA as a template with the addition of SA). Since lactose was added as a stabilizer to this dextran sulfate gel, a gel washed with distilled water and dried was used. In the figure, M is a molecular weight marker, 1 is no dextran sulfate gel added, 2 is 0.2 μg, 3 is 0.4 μg, 4
Is 0.8 μg, 5 is 1.6 μg, 6 is 3.2 μg, 7 is 6.3 μg
g and 8 show the results when PCR was performed with 12.5 μg, 9 with 25 μg, and 10 with 50 μg.

It has been shown that the addition of a dextran sulfate gel results in a PCR amplification product. 1.6 μg or more of dextran sulfate gel (lane 5), especially 3.2 to 25 μg (lane 6 to lane 5)
9) Good results have been obtained when added. The amount of the effect that was observed in this experimental example was 1.6 μg per 50 μl of the reaction solution.
To 50 μg, but due to differences in the introduction rate of sulfate groups to the dextran sulfate gel used, etc.
Since the range of the effective amount varies, the effective amount is not limited to the above range.

(Experimental Example 8) In this example, serially diluted plasma was directly added to the reaction solution in the presence or absence of a 12.5 μg / 50 μl dextran sulfate gel, which was excellent in amplification efficiency in Experimental Example 7. PCR using the cDNA of the tetracycline resistance gene derived from pBR322 as a template
This is an experiment conducted. The composition of the PCR reaction solution used for the reaction and the conditions for PCR were the same as in Experimental Example 7,
The conditions for the subsequent electrophoresis are the same as in Experimental Example 1. 12.5 μg of dextran sulfate gel and serially diluted human plasma were added to the PCR reaction solution (50 μl), and cD
FIG. 8 shows an electrophoretogram of the amplification product when PCR was performed using NA as a template. In the figure, M is a molecular weight marker, 1, 7 is plasma-free, 2, 8 is 0.063%, 3, 9 is 0.125%, 4, 1
0 is 0.25%, 5, 11 is 0.5%, 6, 12 is 1% and PC
The result at the time of performing R is shown. In addition, 1 to 6 are those without dextran sulfate gel, and 7 to 12 are those with dextran sulfate gel.

According to this experimental example, since the action of the PCR inhibitor was suppressed by the dextran sulfate gel, amplification products could be detected with high sensitivity even when PCR was performed by directly adding plasma to the reaction solution. . The results of Experimental Examples 7 and 8 show that the action of the inhibitor can be suppressed not only when the polyanion is added as a solution to the reaction solution but also when an insoluble polyanion is added. This is effective not only when the insoluble polyanion is added as a gel as in this example, but also when the inner wall of the reaction vessel in contact with the reaction solution is coated with the polyanion or when the vessel itself is composed of the insoluble polyanion. is there.

[0042]

Industrial Applicability According to the present invention, a target nucleic acid can be efficiently amplified from a biological sample containing a large amount of a PCR inhibitor such as serum, plasma, blood, etc., without a process of separating and purifying the nucleic acid. It became. Further, according to the present invention, the operation of nucleic acid synthesis can be performed easily and quickly, and the opportunity for contamination can be reduced.

[Sequence list]

 <110> shimadzu corp. <120> Method for synthesis of nucleic acids <130> K1000157 <160> 6 <210> 1 <211> 24 <212> DNA <213> Artificial Sequence <400> 1 gagcggataacaatttcacacagg <210> 2 < 211> 24 <212> DNA <213> Artificial Sequence <400> 2 cgccagggttttcccagtcacgac <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <400> 3 gaagagccaaggacaggtac <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <400> 4 ggaaaatagaccaataggcag <210> 5 <211> 21 <212> DNA <213> Artificial Sequence <400> 5 ctcgtcgcttcgcatcttgga <210> 6 <211> 21 <212> DNA <213> Artificial Sequence <400> 6 cggcacctgtcctacgagttg

[Brief description of the drawings]

FIG. 1: 1 μl of human plasma and a different concentration of heparin sodium salt were added to a PCR reaction solution (50 μl), and PC
Electrophoresis diagram with R

FIG. 2 shows 0.8 μg / ml, which was excellent in amplification efficiency in Experimental Example 1.
Electrophoretogram of PCR in which human blood was serially diluted and added to the reaction solution, with and without the addition of heparin sodium salt to the reaction solution

FIG. 3 shows an electrophoretogram obtained by adding 1 μl of human serum and dextran sulfate sodium salt having different concentrations to a PCR reaction solution (50 μl) and performing PCR.

FIG. 4 is an electrophoretogram obtained by adding 0.05 μl of human blood and dextran sulfate sodium salt having different concentrations to a PCR reaction solution (50 μl) and performing PCR.

FIG. 5: Various amounts of human plasma and 1 ng of polyvinyl potassium sulfate were added to a PCR reaction solution (50 μl), and PC
Electrophoresis diagram with R

FIG. 6 is an electrophoretogram obtained by adding 1 μl of human serum and human genomic DNA having different concentrations to a PCR reaction solution (50 μl) and performing PCR.

FIG. 7: An electrophoretogram obtained by adding 0.125 μl of human plasma and dextran sulfate gels having different amounts to a PCR reaction solution (50 μl) and performing PCR.

FIG. 8 shows that the amplification efficiency was 12.5 μg /
FIG. 9 is an electrophoretogram showing PCR in which human plasma was serially diluted and added to the reaction solution, with and without addition of dextran sulfate gel to the reaction solution.

 ──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Naoyuki Nishimura 1 Nishinokyo Kuwaharacho, Nakagyo-ku, Kyoto F-term in Shimadzu Corporation 4B024 AA11 AA20 CA01 CA11

Claims (7)

[Claims] In a nucleic acid synthesis method for amplifying a target nucleic acid in a sample, a nucleic acid amplification reaction solution contains a polymer compound having an anion-containing repeating structure (polyanion) and a salt thereof (hereinafter collectively referred to as polyanion). And / or an insoluble polymer thereof is added. 2. The nucleic acid synthesis method according to claim 1, wherein the polyanion is a polymer compound having a repeating structure containing a sulfate group (polysulfate) and a salt thereof (hereinafter collectively referred to as a sulfated polymer). 3. The nucleic acid synthesis method according to claim 1, wherein the polyanion is a polymer compound having a repeating structure containing a phosphate group (polyphosphate) and a salt thereof (hereinafter collectively referred to as a phosphorylated polymer). 4. The method according to claim 2, wherein the sulfated polymer is polyvinyl sulfate or a salt thereof. 5. The phosphorylated polymer is DNA or RNA.
The method for synthesizing nucleic acid according to the above. 6. A nucleic acid synthesis method for amplifying a target nucleic acid in a sample, wherein the inner wall of the reaction vessel in contact with the nucleic acid amplification reaction solution is coated with a polyanion, or the vessel itself is composed of an insoluble polyanion. Nucleic acid synthesis method. 7. The nucleic acid synthesis method according to claim 1, wherein the sample is a biological sample or a gene inclusion in the biological sample, and the sample is directly added to the nucleic acid amplification reaction solution.