JP4629167B2 - Nucleic acid synthesis method - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a nucleic acid synthesis method, and more particularly to a nucleic acid synthesis method by a polymerase chain reaction (hereinafter abbreviated as PCR) method.
[0002]
[Prior art]
The PCR method is intended to multiply the target DNA fragment several hundred thousand times by repeating the dissociation of the DNA strand into a single strand, the binding of a primer across a specific region in the DNA strand, and the DNA synthesis reaction by DNA polymerase. Can be amplified. The PCR method is described in Japanese Patent Laid-Open No. 61-274697 which is an invention of Maris et al.
The PCR method can be used as a high-sensitivity analysis method for nucleic acids in various samples, and can be used particularly for analysis methods for nucleic acids in samples derived from animal body fluids. Therefore, the PCR method is used for diagnosis of infectious diseases, genetic diseases and cancer. Furthermore, the PCR method is also suitable for DNA typing examinations during transplantation and parentage testing. In these cases, peripheral blood is often selected for testing.
[0003]
One disadvantage of the PCR method is that dyes, proteins, sugars or unknown contaminants inhibit the reaction. That is, many DNA polymerases, including Taq DNA polymerase derived from Thermus aquaticus, which is a typical thermostable DNA polymerase, strongly inhibits PCR even if a small amount of contaminants derived from body fluids are mixed in the PCR reaction solution. It is widely known.
[0004]
Therefore, prior to DNA amplification by the PCR method, it is necessary to separate cells, bacteria, viruses, etc. (hereinafter referred to as gene inclusions) from the test substance, and then extract nucleic acids from the gene inclusions. . As the method, a method in which a gene inclusion body is decomposed with an enzyme, a surfactant, a chaotropic agent, etc., and then a nucleic acid is extracted from the decomposition product of the gene inclusion body using phenol or phenol / chloroform is conventionally used. Has been.
[0005]
Recently, in the process of nucleic acid extraction, ion exchange resins, glass filters, glass beads, reagents having a protein aggregating action, and the like are used.
[0006]
[Problems to be solved by the invention]
However, even if nucleic acid in a sample is purified using these methods, it is difficult to completely remove impurities, and the amount of recovered nucleic acid in the sample is often not constant. In particular, it may not be successful if the content of the target nucleic acid in the sample is low. In addition, these purification methods are complicated and require time, and there is a high chance of contamination during operation.
[0007]
Therefore, in order to solve these problems, a simpler and more effective sample pretreatment method is desired.
[0008]
We have previously increased the pH in the PCR reaction solution (Japanese Patent Application No. 8-238112) or added polyamines in the PCR reaction solution (Japanese Patent Application No. 6-146500). It was found that PCR can be performed by mixing the inclusion body or the sample itself with the gene amplification reaction solution. However, even when this method is used, depending on the type of gene inclusion in the sample or the sample itself, the action of the PCR inhibitor cannot be suppressed, and the DNA in the sample may not be efficiently amplified. I found something.
[0009]
Accordingly, the present invention aims to provide a novel method for further amplifying DNA in a sample efficiently by further improving and suppressing the action of a PCR inhibitor regardless of the type of the sample.
[0010]
[Means for Solving the Problems]
In order to solve the above problems, the present invention provides a nucleic acid synthesis method for amplifying a target gene in a gene inclusion body contained in a biological sample or a biological sample, before performing nucleic acid synthesis involving preheating. and characterized by performing a gene amplification reaction solution obtained by adding a biological sample derived from the 5 to 20 minute heat treatment at 70 ° C. to 90 ° C. is a temperature at which thermal stability of heat-resistant enzyme is maintained synthesizing a nucleic acid by primer added To do.
[0011]
In the present invention, a sample refers to a gene inclusion body in a biological sample or a biological sample itself, and the biological sample refers to animal and plant tissue, body fluid, excrement, etc., and a gene inclusion includes cells, bacteria, viruses Etc. Body fluids include blood, saliva, spinal fluid, urine, and milk, and cells include, but are not limited to, white blood cells separated from blood.
[0012]
The gene amplification reaction solution usually contains a pH buffer solution, salts such as MgCl ₂ and KCl, primers, deoxyribonucleotides and a thermostable enzyme. Further, the above salts are used by appropriately changing to other salts. In addition, various substances such as proteins such as gelatin and albumin, dimethyl sulfoxide, and surfactants may be added.
[0013]
The pH buffer is a combination of tris (hydroxymethyl) aminomethane and a mineral acid such as hydrochloric acid, nitric acid, sulfuric acid, etc., and a desirable mineral acid is hydrochloric acid. In addition, various pH buffer solutions such as a pH buffer solution using a combination of tricine, CAPSO (3-N-Cyclohexylamino-2-hydroxypropanesulfonic acid) or CHES (2- (Cyclohexylamino) ethanesulfonic acid) with caustic soda and caustic potash can be used. The pH-adjusted buffer is used in the gene amplification reaction solution at a concentration between 10 mM and 100 mM.
[0014]
A primer refers to an oligonucleotide that serves as a starting point for synthesis in the presence of a nucleic acid and an amplification reagent. The primer is preferably single-stranded, but double-stranded can also be used. If the primer is double-stranded, it is desirable to make it single-stranded prior to the amplification reaction. The primer can be synthesized by a known method, or can be isolated from the biological world.
[0015]
The thermostable enzyme means an enzyme that synthesizes nucleic acid by adding a primer, or such a chemical synthesis system. Suitable thermoenzymes include E. coli. DNA polymerase I, E. coli Examples include, but are not limited to, the Klenow fragment of Escherichia coli DNA polymerase, T4 DNA polymerase, Taq DNA polymerase, T. litoralis DNA polymerase, Tth DNA polymerase, Pfu DNA polymerase, and reverse transcriptase.
[0016]
The pH of the gene amplification reaction solution is 8.1 or higher, preferably 8.1 to 9.5 under a temperature condition of 25 ° C. More preferably, for example, when PCR is directly performed on leukocytes separated from blood, pH is around 9.0, and when PCR is performed on a blood sample without pretreatment such as hemolysis of red blood cells, pH is about 8.8. When PCR is directly performed from a saliva sample, the pH should be around 9.4.
[0017]
In the present invention, a polyamine may be added to the gene amplification reaction solution. Polyamine is a general term for hydrocarbons having two or more primary or secondary amino groups. Specifically, for example, ethylenediamine, trimethylenediamine, spermine, spermidine, diethylenetriamine, triethylenetetramine, tetraethylenepentamine. Pentaethylenehexamine, 1,4-bis (3-aminopropyl) -piperazine, 1- (2-aminoethyl) piperazine, and the like. The amount of polyamine added varies depending on the type of polyamine, the type of sample, the concentration, etc., but is usually about 10 to 0.01 mM, preferably about 2 to 0.5 mM in the gene amplification reaction solution.
[0018]
The temperature at which the heat stability of the thermostable enzyme is maintained is a temperature at which the activity of the thermostable enzyme is maintained, preferably 70 to 90 ° C, more preferably 72 to 90 ° C. The heating time is 5 to 20 minutes, preferably 5 to 15 minutes, depending on the heat treatment temperature.
[0019]
In the present invention, “before nucleic acid synthesis” refers to both before and after the gene amplification reaction solution is added to the sample, but preferably after the addition of the gene amplification reaction solution.
[0020]
The procedure of the nucleic acid synthesis method (PCR) of the present invention is the same as that of a normal method except that heat treatment is performed before nucleic acid synthesis.
[0021]
【Example】
(Experimental example 1)
2.5 μl of human blood treated with an anticoagulant (EDTA-2K or Heparin-Na) was added to the PCR reaction solution (47.5 μl), heat-treated at 60 ° C., 72 ° C. or 80 ° C., and then PCR was performed. . PCR primers are an oligonucleotide (P1) having a plus strand base sequence located within the HLA-DP gene region and an oligonucleotide (P2) having a minus strand base sequence, and the sequences are as follows. As a result of PCR using these two kinds of primers, an amplification product of 280 bp can be obtained.
[0022]
P1: 5'TCCCCGCAGAGAATTAC3 '
P2: 5 'CACTCACCTCGGGCGC3'
The PCR reaction solution contained 10 mM Tris-HCl, 35 mM KCl, 1.5 mM MgCl ₂ , 200 μM dATP, dCTP, dGTP and dTTP adjusted to pH 8.9, 1.0 μM primer, 1.25 units / 50 μl Taq DNA polymerase ( TaKaRa Taq: Takara shuzo, Kyoto, Japan).
[0023]
PCR was performed at 94 ° C. for 4 minutes 30 seconds, followed by 40 cycles of 94 ° C. for 30 seconds, 58 ° C. for 1 minute, 72 ° C. for 1 minute, and finally 72 ° C. for 7 minutes. After completion of PCR, 5 μl of the reaction mixture was used to perform electrophoresis in 0.5 μg / ml ethidium bromide-added TAE (40 mM Tris-acetate, 1 mM EDTA, pH 8.0) solution containing 2.5% agarose. did.
[0024]
FIG. 1 shows an electrophoretogram of amplification products when PCR is performed by subjecting a PCR reaction solution directly added with anticoagulant-treated human blood (2 samples) to various temperatures before PCR for 15 minutes. In the figure, A ₁ or A ₂ is Heparin-Na treated blood, B ₁ or B ₂ is EDTA-2K treated blood (1, 2 is the sample number), 1 is 60 ° C, 2 is 72 ° C, 3 is 80 ° C 4 shows the result of electrophoresis of the PCR product when the heat treatment was performed before PCR at the temperature of 4 and when the heat treatment was not performed. M indicates a size marker (250 ng of φX174-RF DNA cut with HincII).
[0025]
As a result, in the case where the heat treatment at 60 ° C. was performed, only a slight increase in the PCR amplification product was observed in one case compared to the case where the heat treatment was not performed (A ₂ −1 vs A ₂ −4). ). On the other hand, in the heat treatment at 72 ° C. and 80 ° C., a large amount of specific PCR amplification product was observed as compared with the case where the heat treatment was not performed.
[0026]
(Experimental example 2)
This example is an experiment in which PCR was performed after heat treatment was performed for 5 minutes, 15 minutes, and 30 minutes at a heat treatment temperature of 80 ° C. and 90 ° C., which had the highest amplification efficiency in Experimental Example 1. The samples used were A ₁ and B ₁ blood used in Experimental Example 1. The composition of the PCR reaction solution used, the conditions for PCR, and the conditions for electrophoresis after PCR are the same as in Experimental Example 1.
[0027]
FIG. 2 shows an electrophoretogram of the PCR product when PCR was performed by heat treatment at various temperatures and times. In the figure, I is the heat treatment temperature of 80 ° C., II is the heat treatment temperature of 90 ° C., 1 is 15 minutes, 2 is 10 minutes, 3 is heat treatment for 5 minutes, and 4 is the PCR product electrophoresis when no heat treatment is performed. Results are shown.
[0028]
As a result, when Heparin-Na-treated blood (A ₁ ) was used, PCR amplification products could not be detected when heat treatment was not performed (A ₁ -4), but when heat treatment was performed, 80 ° C. A clear PCR product could be detected even when heat-treated at 90 ° C. for any time for 5 to 15 minutes. On the other hand, when EDTA-2K-treated blood (B ₁ ) is used, when heat treatment is performed, compared with the case where heat treatment is not performed (B ₁ -4), when heat treatment is performed at any temperature and at any time The increase in PCR amplification product was also observed in.
[0029]
【The invention's effect】
According to the present invention, it is possible to efficiently amplify a target DNA directly from a sample containing a large amount of a PCR-inhibiting substance such as blood without going through a process of separating and purifying nucleic acid. In addition, according to the present invention, nucleic acid synthesis can be performed simply and quickly, and the chance of contamination can be reduced.
[0030]
[Sequence Listing]
SEQ ID NO: 1
Sequence length: 17
Sequence type: Number of nucleic acid strands: Single strand topology: Type of linear sequence: Other nucleic acid Synthetic DNA
Antisense: No
Sequence: TCCCCGCAGAGAATTAC
[0031]
SEQ ID NO: 2
Sequence length: 15
Sequence type: Number of nucleic acid strands: Single strand topology: Type of linear sequence: Other nucleic acid Synthetic DNA
Antisense: No
Sequence: CACTCACCCTGGGCGC
[Brief description of the drawings]
[Fig. 1] Electrophoretic diagram of amplification products when PCR reaction solution directly added with anticoagulant-treated human blood (2 samples) is heat-treated at various temperatures for 15 minutes before PCR. [Fig. Electrophoretic diagram of PCR products when PCR is performed by heat treatment at various temperatures and times

Claims (1)

In a nucleic acid synthesis method for amplifying a target gene in a gene inclusion body or a biological sample contained in a biological sample, a gene amplification reaction solution to which the biological sample is added before performing nucleic acid synthesis with preheating a nucleic acid synthesis method which is characterized in that 5 to 20 minutes heat treatment at 70 ° C. to 90 ° C. is a temperature at which thermal stability of heat-resistant enzyme is maintained synthesizing a nucleic acid with primers added.

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