JPH07132087A - Method for amplifying genetic polymer by pcr method - Google Patents

Abstract

PURPOSE:To dispense with the removal of excessive primer fragments from a PCR product by specifying an amount of each oligonucleotide.primer in a reaction solution to subject DNA or a nucleic acid sequence derived from the DNA to PCR amplification. CONSTITUTION:An amount of each oligonucleotide.primer in a reaction solution to subject DNA or a nucleic acid sequence derived from the DNA to PCR amplification is <=6pmol/mu 1, preferably 2-4pmol/mu 1. Excessive primer fragments do not remain in a PCR product and the PCR product can be fed to the following analysis process.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to an improved P for amplifying DNA or a nucleic acid sequence derived therefrom (hereinafter, these may be collectively referred to as a genetic macromolecule).
It relates to the CR method.

[0002]

2. Description of the Related Art Recent advances in gene manipulation technology in the field of biotechnology have been remarkable, and the structures and functions of genes of all living organisms from prokaryotes to eukaryotes including humans have been comparatively determined. It can be easily analyzed.

Among them, PCR (Polymerase Chain React)
The ion) method is widely used in various research fields because the target genetic polymer region can be easily increased in vitro without a cloning procedure using bacteria or the like. This PCR method is a method for amplifying a genetic polymer using a DNA polymerase reaction. DNA polymerase uses a single-stranded DNA sequence as a template to synthesize a sequence complementary thereto, but to initiate the reaction, template D is used.
A primer of 15 to 30 nucleotides complementary to the partial sequence on the 3'side of NA is required. Then, in the PCR method, by using two kinds of primers sandwiching the amplification target region of the double-stranded DNA serving as a template, the region is doubled in one reaction.

FIG. 1 is a schematic diagram illustrating one reaction cycle of this PCR method, and Table 1 is an example of the composition of the reaction solution. When such a reaction solution is filled in a test tube (or a tube) and the reaction step of FIG. 1 is repeated, the template DNA is amplified 2 ⁿ -n-1 times after n cycles. Usually 20 to 40 cycles are common.

[0005]

[Table 1]

[0006]

In the general PCR method, as shown in the reaction solution composition in Table 1, an excessive amount of primer is used. This is because each primer is efficiently annealed to the single-stranded template DNA. However, when a large amount of the primer is used in this way, a large amount of unused primer fragment remains in addition to the amplified DNA fragment in the finally obtained PCR product.

As a matter of course, what is necessary for the subsequent analysis (for example, sequence analysis) is the amplified DNA fragment, and no primer is necessary. For this reason, conventionally, for example, a primer is labeled in advance with biotin or the like, and an excess primer fragment is adsorbed on a magnetic bead, or a PC is used.
The R fragment had to be removed by, for example, fractionating the R product with a membrane filter.

The present invention has been made in view of the above circumstances, and an object thereof is to provide a new PCR method in which an excess primer fragment does not remain in a PCR product.

[0009]

Means for Solving the Problems The present invention is provided to solve the above problems by subjecting a DNA or a nucleic acid sequence derived therefrom to PCR amplification such that the amount of each oligonucleotide / primer in the reaction solution is 6 pmol or less. A method for amplifying a genetic polymer is provided. This invention also
In a preferred embodiment, the amount of each oligonucleotide primer in the reaction solution is set within the range of 2-4 pmol.

The structure of the present invention will be described in detail below. The method for amplifying a genetic polymer according to the present invention has a general composition shown in Table 1 except that the amount of each oligonucleotide primer used is 6 pmol / μl or less, more preferably 2-4 pmol / μl. The template DNA is amplified using the reaction solution and the reaction cycle shown in FIG.

There are no particular restrictions on the materials constituting the reaction solution, but examples of them are as follows. First, as the template DNA, a nucleic acid sequence such as genomic DNA prepared from cells or tissues of all organisms by a conventional method or cDNA derived therefrom can be used. Also, two types of primers are 15-
A 30 nucleotide sequence can be synthesized and used. However, in that case, it is necessary to select a primer that does not have a complementary or homologous sequence between the two primers and does not have a hairpin-like secondary structure. Furthermore, it is necessary to consider that the sequence is unique to the target DNA and that the two primers have the same melting property in order to prevent annealing with a DNA sequence other than the target. When synthesizing a primer satisfying such conditions, a commercially available search software may be used, or a commercially available primer for amplifying a specific DNA may be used.

Thermostable DNA polymerase is a commercially available T
Ag polymerase or Vent polymerase can be used, and the reaction buffer may be selected in accordance with them. In addition, four types of dNTP (dATP, dTTP, d
As CTP and dGTP), commercially available products can be used. Further, the PCR cycle as shown in FIG. 1 can be automatically performed using a commercially available program incubator device. The reaction cycle at that time is about 30 to 50 times.

By repeating the PCR cycle using the reaction solution having such a composition, the method of the present invention is such that each oligonucleotide primer used is annealed in sequence to the template DNA throughout the entire reaction cycle and is consumed. Therefore, an extra primer fragment does not remain in the final PCR product, and the PCR product (amplified DNA fragment) can be immediately subjected to the next analysis process.

Next, the method of the present invention will be described in more detail and specifically with reference to Examples, but the present invention is not limited to the following examples.

[0015]

EXAMPLES Example 1 cDNA cloned in λ phage λgt10
It was amplified by the method of the invention using Taq DNA polymerase. (1) Preparation of sample Each well of a microtiter plate (Falcon 3911) contained 10 mM MgCO ₄ in CY-broth medium (casamino acid 10 g; yeast extract 5 g; NaCl 3 g;
KCl 2 g) was dispensed in 100 μl aliquots, λgt10 plaques were inoculated one by one, and sealed at 37 ° C. 4-
The mixture was kept warm for 6 hours until it was dissolved, and then centrifuged at 2200 rpm for 15-20 minutes to collect the supernatant.

In this way, λ phage particles containing cDNA were recovered. (2) Preparation of oligonucleotide primer λ in contact with each of 5 ′ side and 3 ′ side of cDNA insert
Primers 1 and 2 were prepared by synthesizing the base sequence of gt10. (3) Preparation of Reaction Solution Three kinds of reaction solutions (each for 10 samples) having the following compositions were prepared.

Reaction Solution (A) 10 × Buffer (Wako Pure Chemical) 20.0 μl 1.5 mM dNTP 20.0 μl Sterilized Water 60.0 μl 4.5 U / μl Taq DNA Polymerase 4.5 μl (Wako Pure Chemical) Reaction Solution ( B) 10 pmol / μl primer 1 3.0 μl 10 pmol / μl primer 2 3.0 μl sterile water 94.0 μl reaction solution (C) 4.5 U / μl Taq DNA polymerase 1 μl /
1 × PCR buffer containing 100 μl (4) Amplification of cDNA 0.5-1 μl of the culture supernatant of λ phage was dispensed to each well of a thermal cycler PHC-3 (Techne) 96-well microtiter plate, Further, 10 μl of the reaction solution (B) was dispensed, the surface was covered with mineral oil, and the mixture was kept at 95 ° C. for 5 minutes or more to decompose the phage particles.

Then, 10 wells of the reaction solution (A) were added to each well.
Dispense by μl and denature with PHC-3 (95 ° C: 3
0 seconds), annealing (55 ° C: 1 minute), extension (72
15 cycles of denaturation (95 ° C. for 30 seconds),
Annealing (55 ° C: 1 minute), extension (72 ° C: 2 minutes)
Was carried out for 15 cycles. Furthermore, 10 μl of the reaction solution (C) was dispensed into each well to denature (95 ° C .: 30 seconds),
Annealing (55 ° C: 1 minute), extension (72 ° C: 1 minute)
Was carried out for 10 cycles. (5) Sequence analysis of amplified cDNA The nucleotide sequence of the above PCR product was analyzed using the ABL Dye Deoxy Terminator Cycle Sequencing kit (manufactured by Applied Biosystems). As a result, the DNA in the PCR product
The fragment was almost the desired cDNA, and the primer sequence was not contaminated.

As a control, sequence analysis was also carried out in the case where the amount of the primer in the reaction solution (B) was 100 pmol and PCR amplification was carried out in the same manner.
Accurate analysis was impossible because the primer fragment remained in these PCR products.

[0020]

As described in detail above, according to the method of the present invention, it is not necessary to remove excess primer fragments, and the obtained PCR product can be directly used in the next analysis process.

[Brief description of drawings]

FIG. 1 is a schematic view illustrating one reaction cycle of the PCR method.

Claims (2)

[Claims] 1. A method for amplifying a genetic polymer in which the amount of each oligonucleotide primer in a reaction solution is 6 pmol / μl or less when PCR-amplifying a DNA or a nucleic acid sequence derived therefrom. 2. The method for amplifying a genetic polymer according to claim 1, wherein the amount of each oligonucleotide primer in the reaction solution is in the range of 2-4 pmol / μl.