JP2005304354A - Nucleic acid amplification method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a nucleic acid amplification method by which the nucleic acid can be amplified from a small amount of the nucleic acid in a short time in a sufficiently satisfiable state in a specimen whose impurities such as proteins have been reduced in very low concentrations by a sufficient purification treatment or a nucleic acid amplification reaction solution not containing a protein and the like by other production methods. <P>SOLUTION: This nucleic acid amplification method is characterized by adding 0.1 to 5.0 mmol/L of an amine compound to a nucleic acid amplification reaction solution having a protein concentration of ≤0.5 g/L in a nucleic acid amplification reaction by a polymerase chain reaction. Thereby, a nucleic acid amplification rate can largely be increased. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a nucleic acid amplification method, particularly a method for efficiently amplifying a nucleic acid based on a relatively pure nucleic acid obtained by removing impurities from a sample in advance by a nucleic acid purification treatment, particularly a polymerase chain reaction method. It relates to a method of amplification.

As a method for amplifying a nucleic acid, a polymerase chain reaction method (hereinafter referred to as a PCR method) is known.
PCR method includes template nucleic acid (nucleic acid containing the region to be amplified), primer (single-stranded nucleic acid of about 10 to 50 bases complementary to the end of the region to be amplified), deoxynucleotide triphosphate (dNTP ), DNA polymerase (enzyme), etc. are added, and the target region is repeated by repeating the steps of “denaturation of template nucleic acid into single strand → primer binding → primer extension with DNA polymerase (double strand synthesis)”. This is a technique for amplifying the nucleic acid.

The PCR method has been applied in a wide variety of fields, and is particularly used for highly sensitive detection of various viruses and determination of gene types. At that time, for rapid and accurate detection, which is important, further increase in nucleic acid amplification rate and reduction in amplification time have been desired.
As a means to contribute to this, phenol, which efficiently removes impurities from samples, such as blood, urine, and soil, which are often contained as substances other than (template) nucleic acids and are thought to inhibit nucleic acid amplification reactions. A chloroform extraction method is known. However, a sufficient effect of increasing the nucleic acid amplification rate cannot be obtained only by removing such impurities.

On the other hand, a nucleic acid amplification method that makes it difficult to affect nucleic acid amplification by adding polyamine or the like even for a sample containing a large amount of impurities has been proposed (see, for example, Patent Document 1). However, this method can reduce nucleic acid amplification suppression of contaminants by adding polyamines in the presence of contaminants derived from biological samples, and provides a nucleic acid amplification reaction having a sufficient nucleic acid amplification rate. It wasn't something to do.
JP-A-6-277061

As described above, for example, examination of hepatitis B / C virus or AIDS virus using blood as a specimen requires accurate determination in a short time from a small amount of specimen, and nucleic acid amplification conventionally used for these specimens. The method is satisfactory in a short time regardless of the removal of contaminants (even if the sample can be reduced to a very low concentration by sufficient pretreatment) and non-removal (addition of polyamine). No nucleic acid amplification could be performed.
An object of the present invention is to provide a nucleic acid amplification method having a high nucleic acid amplification rate for a sample from which contaminants have been removed by pretreatment or the like and impurities have been reduced.

  In the PCR method, the present inventors use a nucleic acid sample in which impurities are reduced in concentration by sufficient purification treatment as a template nucleic acid, and further contain 0.1 to 5.0 mmol / l amine compound in the reaction solution. Thus, it was found that the nucleic acid amplification rate can be remarkably improved. In addition, when aiming at the same nucleic acid amplification rate, it was found that the reaction time can be reduced or the amount of template nucleic acid can be reduced, and the present invention has been completed.

  That is, according to the present invention, in the nucleic acid amplification reaction by the PCR method, 0.1 to 5.0 mmol / l amine compound is added to the nucleic acid amplification reaction solution having a protein concentration of 0.5 g / l or less. A featured nucleic acid amplification method is provided.

  The present invention can efficiently amplify a nucleic acid, and a nucleic acid can be amplified in a short time even with a small amount of sample.

(Template nucleic acid (sample, specimen))
Samples and specimens containing template nucleic acids for nucleic acid amplification by PCR method include biological samples (animal-derived (skin, hair, saliva, blood, urine, feces), plant-derived samples (roots, stems, leaves, seeds, pulp) , Flowers)) and environmental samples (soil, water, air), and nucleic acids amplified by the PCR method based on them can be used.

(Contamination)
The sample often contains a lot of contaminants, which is an inhibiting factor for the PCR method, so it is desirable to remove it as much as possible. Examples of contaminants include nucleic acids, proteins, sugar chains, lipids, etc. that are not involved in the reaction. Among them, protein is a typical contaminant, and the protein concentration in the nucleic acid amplification reaction solution needs to be 0.5 g / l or less in order to obtain the effects of the present invention. Further, it is preferably 0.4 g / l or less.

The protein concentration shown in the present invention uses the absorbance at 280 and 260 nm, and the Warburg and Christian derivation formula (Warburg and Christian, Biochemisches Zeitung310, 384 (1941) represented by the formula (1). Reference value) was used.

The nucleic acid amplification reaction solution substantially free of protein referred to in the present invention means that the protein concentration value obtained using the above measurement and formula (1) is 0.00 g / 1 means a nucleic acid amplification reaction solution.

(Purification method)
For the reasons described above, it is preferable to perform a nucleic acid purification treatment for the purpose of extracting nucleic acid from a gene inclusion in a biological sample or the biological sample itself and removing remaining contaminants prior to nucleic acid amplification. .
As a specific purification method, known methods can be used, and examples thereof include phenol / chloroform extraction and ethanol precipitation. Furthermore, using a column using an ion exchange resin, glass beads or the like, a glass filter, or a reagent having a protein aggregating action, the degree of purification can be increased and the protein concentration can be reduced to 0.5 g / l or less. Multiple methods can be used simultaneously or sequentially to further reduce the contaminant concentration.

  In addition, when using nucleic acids that have already been amplified by the so-called PCR method or synthesized nucleic acids, the above-described purification treatment is not necessarily performed because there are no or very few contaminants derived from the sample as described above.

(Amine compound)
The amine compound in the present invention is a general term for compounds having at least one primary, secondary, tertiary amine or quaternary ammonium salt in the same molecule. Examples of amine compounds having primary, secondary, and tertiary amines include branched structures such as ethylenediamine, diethylenetriamine, triethylenetetramine, tetraethylenepentamine, and pentaethylenehexamine as amine compounds having a linear structure. Tris (2-aminoethyl) amine and the like having an amine, piperazine, piperidine, 1- (2-aminoethyl) piperazine, 1- (2-aminoethyl) piperidine, 1,4- Bis (3-aminopropyl) -piperazine, 1,4,10,13, tetraoxa-7,16 diazacyclooctadecane and the like can be mentioned. Examples of amine compounds that are quaternary ammonium salts include tetraethylammonium bromide, tetra-n-butylammonium bromide, cetyltrimethylammonium bromide, and the like.

The addition amount (concentration) of the amine compound is 0.1 to 5.0 mmol / l in the PCR amplification reaction solution, although it varies depending on the type of the amine compound, the amount of impurities contained in the sample, and the origin of the sample. If it is less than 0.1 mmol / l, the target nucleic acid amplification rate is not improved, and if it exceeds 5.0 mmol / l, the nucleic acid amplification reaction is inhibited by the amine compound.
The addition amount of a preferable amine compound is 0.1 to 1.0 mmol / l, more preferably 0.1 to 0.5 mmol / l.

(Nucleic acid amplification reaction solution)
The nucleic acid amplification reaction solution used in the nucleic acid amplification method according to the present invention has a protein concentration of 0.5 g / l or less, and is a generally known nucleic acid except that it contains 0.1 to 5.0 mmol / l of an amine compound. It is not different from the amplification method. Therefore, conditions other than amine compounds, such as template nucleic acid type, primer design guidelines, DNA polymerase selection, deoxynucleotide triphosphate amount, pH, and reaction buffer selection, etc. are the same as in normal nucleic acid amplification methods. You can handle it.

(Specific procedure of nucleic acid amplification method)
The procedure of the nucleic acid amplification method of the present invention is not different from the conventionally known nucleic acid amplification method except that an amine compound is added to the reaction system. As an amplification method, it is preferable to use a PCR method using a heat-resistant DNA polymerase and a thermal cycler because an amplification product can be obtained quickly. As specific examples in the PCR method, a denaturation reaction is performed at 90 to 95 ° C. for 1 second to 1 minute, an association reaction is performed at 40 to 72 ° C. for 1 second to 3 minutes, and an extension reaction is performed at around 72 ° C. for 1 second to 3 seconds. And a procedure for repeating these three reactions about 30 times.

(Detection / evaluation method)
The nucleic acid amplified by the nucleic acid amplification method of the present invention is not different from the nucleic acid amplified by the generally known nucleic acid amplification method by the PCR method. Therefore, as a method for detecting / evaluating whether or not the target nucleic acid has been amplified, a generally known nucleic acid detection / evaluation method can be used. For example, nucleic acid amplification products are developed by agarose gel electrophoresis, acrylamide gel electrophoresis, etc., stained with an intercalator such as ethidium bromide or SYBER GREEN, and the target nucleic acid is observed by observing the fluorescence derived from the intercalator. It can be confirmed whether or not is amplified. In addition, when using a primer to which a fluorescent dye such as FITC or Cy5 is bound as a primer, the target nucleic acid is amplified by observing the fluorescence derived from the fluorescent dye after developing the nucleic acid amplification product in the same manner. It can be confirmed whether or not.

(Preparation of cell culture extract)
A cell culture extract was prepared as a nucleic acid sample containing a relatively large amount of impurities. The procedure is as follows.
(I) Escherichia coli transformed with the pUC118 vector was cultured overnight at 37 ° C. in 2 ml of LB medium containing 0.1 g / l ampicillin to obtain a cell culture solution. The bacterial cell culture solution was transferred to an Eppendorf tube and centrifuged at 6000 rpm for 10 minutes to recover the bacterial cells from the bacterial cell culture solution.
(Ii) The collected cells are suspended in 500 μl of 50 mmol / l glucose / 25 mM TrisHCl (pH 8.0) / 10 mmol / l Na 3 · EDTA solution, and further 250 μl of 0.3N NaOH / 2 mass% SDS is added. After resuspension, the mixture was heated at 70 ° C. for 15 minutes.
(Iii) To the heated suspension, 200 μl of neutral phenol / chloroform solution (5 g phenol / 5 ml chloroform / 1 ml Tris-HCl (pH 8.0)) was added and suspended.
(Iv) The suspended solution was centrifuged at 15000 rpm for 10 minutes to separate the organic solvent phase and the aqueous phase, and only the aqueous phase was separated. This aqueous phase was used as a cell culture extract.

(Preparation of nucleic acid solution)
A nucleic acid solution was prepared as a nucleic acid sample containing no impurities. The procedure is as follows.
(I) First, nucleic acid was extracted from human blood using a DNA purification kit (PUREGENE ^™ DNA Purification Blood Kit (manufactured by Gentra Systems)).
(Ii) Next, 1 μl of the extracted nucleic acid was used as a template, and primer A and primer B were used for the first preliminary PCR with a final volume of 100 μl. (Primer A was prepared from an oligonucleotide consisting of 35 bases at a concentration of 10 μmol / l, and Primer B was prepared from an oligonucleotide consisting of 31 bases at a concentration of 10 μmol / l. (The region amplified by these primers was about 750 bases including the primer itself.)
(Iii) Furthermore, 1 μl of the solution after the first preliminary PCR was used as a template, and a second preliminary PCR was performed using primer A and primer B in a final volume of 100 μl.
(Iv) The solution after the second preliminary PCR was diluted 100,000 times with sterilized water to obtain a nucleic acid solution.
By performing two preliminary PCRs, a nucleic acid solution free from contaminants, in particular, substantially free of protein, was obtained. Absence of protein is confirmed by measuring absorbance at 280 nm and 260 nm with a spectrophotometer (Ultraspec 3000 manufactured by Pharmacia Biotech) and calculating the protein concentration (g / l) from the above equation (1). did.

(Preparation of template nucleic acid solution)
The template nucleic acid solutions A, B, and C used in the nucleic acid amplification reactions of the following examples and comparative examples were prepared by mixing the nucleic acid solution and the bacterial cell culture extract at the ratio shown in Table 1. The protein concentration of the obtained template nucleic acid solution was also calculated and described by the following method.
99 μl of sterilized water was added to the template nucleic acid solution A, 98 μl of sterilized water was added to the template nucleic acid solution B, and 96.5 μl of sterilized water was added to the template nucleic acid solution C to dilute the total volume to 100 μl. Thereafter, the absorbance at 280 nm and 260 nm was measured with a spectrophotometer (Ultraspec 3000 manufactured by Pharmacia Biotech), and the protein concentration (g / l) was calculated from the above formula (1).

(Preparation of nucleic acid amplification reaction solution)
A nucleic acid amplification reaction solution was prepared in the same manner as a generally known nucleic acid amplification method except that an amine compound was added.
As the template nucleic acid, the aforementioned template nucleic acid solutions A, B, and C were used.
As the amine compound solution, an aqueous solution of pentaethylenehexamine was used, and the final concentrations were 0, 0.1 mmol / l, 0.25 mmol / l, 0.5 mmol / l, and 1.0 mmol / l. As a control, a test plot containing no amine compound was also provided.
Primer 1 is an oligonucleotide composed of 22 bases labeled with FITC, and primer 2 is an oligonucleotide composed of 22 bases (unlabeled and has a different base sequence from primer 1), each at a concentration of 10 μmol / l. Prepared and used. These were added so that final concentration might be set to 0.5 micromol / l. (The region amplified by these primers was 74 bases including the primer itself.)
As a DNA polymerase, GeneTac (manufactured by Gene Japan) was used, and the amount added to the reaction solution was 0.5 μl.
For the buffer solution and deoxynucleotide triphosphate, 10 × PCR Buffer and 2.5 mM dNTP attached to GeneTac were used.
Sterile water was added to the above so that the total amount became 100 μl to obtain a nucleic acid amplification reaction solution. Specifically, it is as shown in Tables 2-4. Unless otherwise noted, the unit of the numbers in the table is μl.

Table 2: Composition of nucleic acid amplification reaction solution when PCR template nucleic acid solution A is used

Table 3: Composition of nucleic acid amplification reaction solution when using template nucleic acid solution B for PCR

Table 4: Composition of nucleic acid amplification reaction solution when using template nucleic acid solution C for PCR

(Examples 1-1 to 4, 2-1 to 4, and 2, Comparative Examples 1, 2, 3, 3-1 to 3-4)
(Nucleic acid amplification reaction conditions)
The prepared nucleic acid amplification reaction solution was heated at 95 ° C. for 3 minutes using a thermal cycler for PCR reaction, and then heated at 95 ° C. for 30 seconds, 60 ° C. for 5 seconds, and 72 ° C. for 1 second. Repeated 30 times.

(Quantification method of nucleic acid amplification reaction products (relative))
The nucleic acid amplification reaction solution after nucleic acid amplification by the PCR method is 0.5 × TBE buffer (10 × TBE buffer (“10 × TBE powder” manufactured by Wako Pure Chemical Industries, Ltd. dissolved in 1 liter of sterilized water)) Buffer solution diluted with distilled water 20 times) was used as a buffer for electrophoresis, and 2.5% agarose gel (Agarose L03 “TAKARA” (manufactured by Takara Bio Inc.) was added to 0.5 × TBE buffer solution. After dissolution at a concentration of 5%, it was gelled). The developed gel was excited at 473 nm using a fluorescence image analyzer (Fuji Photo Film, FLA3000G), and a fluorescence wavelength of 520 nm was observed (FIG. 1). The gel after fluorescence observation was stained with 0.5 mg / l ethidium bromide for 10 minutes, and the stained gel was observed under 312 nm UV irradiation to confirm the base chain length of the developed nucleic acid amplification reaction product. (FIG. 2).

1 and 2, lane 1: molecular weight marker (20 bp DNA ladder (manufactured by Takara Bio Inc.)) from the left
Lanes 2 to 6: Template nucleic acid solution A for nucleic acid amplification reaction is used. (the detail is right below)
(Comparative Example 1) Lane 2: 0 mmol / l of amine compound added (no addition).
(Example 1-1) Lane 3: 0.1 mmol / l of amine compound was added.
(Example 1-2) Lane 4: Add 0.25 mmol / l of amine compound.
(Example 1-3) Lane 5: 0.5 mmol / l of amine compound was added.
(Example 1-4) Lane 6: 1.0 mmol / l of amine compound was added.
Lanes 7 to 11: Template nucleic acid solution B for nucleic acid amplification reaction is used. (the detail is right below)
(Comparative Example 2) Lane 7: 0 mmol / l of amine compound added (no addition).
(Example 2-1) Lane 8: 0.1 mmol / l of amine compound was added.
(Example 2-2) Lane 9: Addition of 0.25 mmol / l of amine compound.
(Example 2-3) Lane 10: 0.5 mmol / l of amine compound was added.
(Example 2-4) Gne 11: Add 1.0 mmol / l of amine compound.
Lanes 12 to 16: Template nucleic acid solution C for nucleic acid amplification reaction is used. (the detail is right below)
(Comparative Example 3) Lane 12: 0 mmol / l of amine compound added (no addition).
(Comparative Example 3-1) Lane 13: 0.1 mmol / l of amine compound was added.
(Comparative Example 3-2) Lane 14: 0.25 mmol / l of amine compound was added.
(Comparative Example 3-3) Lane 15: 0.5 mmol / l of amine compound was added.
(Comparative Example 3-4) Lane 16: 1.0 mmol / l of amine compound was added.

Based on the results of the fluorescence observation, the relationship between the protein concentration in the nucleic acid amplification reaction solution and the addition amount of the amine compound and the amount of amplified nucleic acid is summarized in Table 5. Unless indicated otherwise, the units of the numbers in the table are LAU / mm ² (fluorescence intensity).

The magnification described in the parentheses on the second line of the data item in Table 5 is the nucleic acid amplification rate in the system (Comparative Example 1) in which protein is not substantially contained and no amine compound is added. The magnification of the nucleic acid amplification rate of each system when 00 is shown.
Moreover, the magnification described in parentheses on the third line of each data item in Table 5 is the nucleic acid amplification rate (Comparative Example 2 and Comparative Example 3) in the amine compound non-addition system in each template nucleic acid solution is 1.00. The magnification of the nucleic acid amplification rate of the amine addition system is shown.

Table 5: Comparison of protein concentration and amount of added amine compound and amount of amplified nucleic acid

When no amine compound is added, the nucleic acid amplification factor increases to 0.59 times and 0.02 times when the protein concentration in the system increases from 0.00 g / l to 0.38 g / l and 2.13 g / l, respectively. It decreased rapidly (Comparative Examples 1 to 3).
Conventionally, it has been known that when an amine compound is added to a system in which the content of contaminants such as proteins is high and nucleic acid amplification is strongly inhibited, inhibition of nucleic acid amplification is reduced. In the system (Comparative Example 3-2) in which the amine compound was added to the system containing 2.13 g / l of protein, the nucleic acid amplification factor was 8.84 times that of Comparative Example 3. The improvement was seen. However, the amount of amplified nucleic acid is small, the protein is not substantially contained (protein concentration 0.00 g / l), and the nucleic acid amplification is only 0.22 times that of the system containing no amine compound (Comparative Example 1). Became a rate.

On the other hand, when an amine compound is added to a system that does not substantially contain a protein or a system containing 0.38 g / l in which the protein concentration is less than 0.5 g / l, compared to a system that does not add an amine compound. Nucleic acid amplification rates of several times or more were obtained in the same time (Examples 1-1 to 1-4, Examples 2-1 to 2-4). In particular, in the system (Example 1-1) in which an amine compound was added to 0.1 mmol / l to a nucleic acid amplification reaction solution having a protein concentration of 0.00 g / l as an example of the present invention, no amine was added. A 7.37-fold increase in nucleic acid amplification rate was observed over the additive system (Comparative Example 1).
That is, when an amine compound is added to a system with less than 0.5 g / l of contaminants, nucleic acid amplification inhibition is not alleviated by the contaminants, but the nucleic acid amplification reaction is clearly promoted by another mechanism. I understand that.

By the nucleic acid amplification reaction in which the polymerase chain reaction according to the nucleic acid amplification method of the present invention was carried out for 30 cycles, the nucleic acid amplification rate in the nucleic acid amplification reaction solution without addition of the amine compound and substantially containing no protein was determined. A nucleic acid amplification rate of 3 times or more could be obtained. The nucleic acid amplification rate was preferably 5 times or more, more preferably 7 times or more.

In addition, the nucleic acid amplification reaction in which the polymerase chain reaction according to the nucleic acid amplification method of the present invention is performed for 30 cycles results in a nucleic acid amplification of 4 times or more by adding an amine compound to the nucleic acid amplification rate before the addition of the amine compound to the nucleic acid amplification reaction solution. I was able to get a rate. The nucleic acid amplification rate was preferably 7 times or more, more preferably 9 times or more.

Example 4
The nucleic acid amplification reaction was performed in the same manner as in Example 2 except that pentaethylenehexamine was changed to 3 mmol / l, 5 mmol / l, 7 mmol / l, and 10 mmol / l as the amine compound. The obtained nucleic acid amplification amount is shown in Table 6. The unit of the numbers in the table is LAU / mm ² (fluorescence intensity).

Table 6: Comparison of protein concentration, amine compound addition amount and nucleic acid amplification amount

When evaluated together with the results of Example 2, when the protein concentration is 0.5 g / l or less, the addition of the amine compound in the range of 0.1 to 5.0 mmol / l dramatically promotes the nucleic acid amplification rate. It became clear. This means that the process time required for amplification can be greatly shortened when a nucleic acid amplification rate comparable to that of the conventional method is sufficient. This knowledge has not been obtained so far, and at the same time, it can be said that the effect is tremendous for genetic testing that requires rapid testing in small quantities.

It is considered that the nucleic acid amplification method of the present invention can increase the nucleic acid amplification rate in one cycle by adding an amine compound, and increase the nucleic acid amplification rate as a result of performing multiple cycles of amplification.
The ratio of the final nucleic acid amplification rate before and after the addition of the amine can be expressed as the formula (2) by the ratio of the nucleic acid amplification rate in one cycle before and after the addition of the amine and the number of PCR amplification cycles.

However,
2r ₀ : Nucleic acid amplification rate in one cycle before amine compound addition 2r ₁ : Nucleic acid amplification rate in one cycle after amine compound addition
K: Number of PCR amplification cycles
It is.

In both the examples and comparative examples, the nucleic acid amplification rate is the result of 30 PCR amplification cycles, that is, K = 30.
Based on the results obtained in Table 5, the ratio (r ₁ / r ₀ ) of the nucleic acid amplification rate in one cycle in each system relative to the amine-free system was determined from Equation (2) and summarized in Table 7.

このように、上記の式（２）で定義される１サイクルにおける核酸増幅率を、アミンの添加により１．０５倍以上増加させることができることがわかる。好ましい場合には１．０６倍以上、更に好ましくは１．０７倍以上にすることが可能となる。
タンパク質濃度が２．１３ｇ／ｌと高い系においても１サイクルにおける核酸増幅率の比（ｒ_１／ｒ_０）は増加しているが、アミン無添加系（比較例３）での核酸増幅率の低減が大きいため、十分な核酸増幅量（比較例１の０．２２倍程度以下）が得られず、本発明の課題である十分な核酸増幅量を得るに至らない。

Thus, it can be seen that the nucleic acid amplification rate in one cycle defined by the above formula (2) can be increased by 1.05 times or more by the addition of amine. In the preferred case, it can be made 1.06 times or more, more preferably 1.07 times or more.
Even in a system where the protein concentration is as high as 2.13 g / l, the ratio of the nucleic acid amplification rate in one cycle (r ₁ / r ₀ ) is increased, but the nucleic acid amplification rate in the amine-free system (Comparative Example 3) is increased. Since the reduction is large, a sufficient amount of nucleic acid amplification (about 0.22 times or less of Comparative Example 1) cannot be obtained, and a sufficient amount of nucleic acid amplification, which is a problem of the present invention, cannot be obtained.

There is a strong demand in the medical field and the like for rapid examination of hepatitis B / C virus and AIDS virus. The present invention can provide a technique useful for such rapid gene analysis / diagnosis.

The nucleic acid amplification reaction solution after PCR is developed by agarose gel electrophoresis, and excited at 473 nm using a fluorescence image analyzer (Fuji Photo Film, FLA3000G), and observed with a fluorescence wavelength around 520 nm. . The agarose gel in FIG. 1 was stained with 0.5 mg / l ethidium bromide for 10 minutes, and the stained gel was observed under 312 nm ultraviolet irradiation.

Claims (4)

In a nucleic acid amplification reaction by a polymerase chain reaction method, 0.1 to 5.0 mmol / l amine compound is added to a nucleic acid amplification reaction solution having a protein concentration of 0.5 g / l or less. Amplification method. In the nucleic acid amplification reaction in which the polymerase chain reaction is carried out for 30 cycles, the nucleic acid amplification reaction solution is substantially free of protein and has a nucleic acid amplification rate of 3 times or more with respect to the nucleic acid amplification rate without addition of an amine compound. The nucleic acid amplification method according to claim 1. In the nucleic acid amplification reaction in which the polymerase chain reaction is carried out for 30 cycles, the nucleic acid amplification rate after addition of the amine compound is 4 times or more of the nucleic acid amplification rate before addition of the amine compound to the nucleic acid amplification reaction solution. 2. The nucleic acid amplification method according to 2. The addition of 0.1 to 5.0 mmol / l amine compound to the nucleic acid amplification reaction solution increases the nucleic acid amplification rate in one cycle of the polymerase chain reaction by 1.05 times or more. The nucleic acid amplification method described.

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