JPH1070984A - Primer mixture for pcr reaction and amplification of nucleic acid - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject mixture capable of conquering nucleic acid amplification suppression caused by the mismatch of a primer and a template nucleic acid, by using plural primers in which 3' ends of nucleic acid domains to be hybridized on a template DNA. SOLUTION: Plural primers in which 3' ends of nucleic acid domains to be hybridized on a template DNA to be amplified are shifted by three bases, the 3' ends correspond bases except a base constituting a third codon in the template DNA and 5' ends correspond to the same base are combined to give the objective primer mixture for PCR reaction in which the suppression of amplification or reduction in amplification ratio of nucleic acid caused by the mismatch of a primer and a template nucleic acid, especially the mismatch of the 3' end can be conquered in the case of a PCR reaction and an amplification reaction can be efficiently carried out even to the same kind of DNA having an unknown gene sequence.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD [0001] The present invention belongs to the field of genetic engineering. In particular, the present invention belongs to a nucleic acid amplification method.

[0002]

2. Description of the Related Art A method of enzymatically synthesizing a specific DNA base sequence in vitro using two oligonucleotide primers that hybridize to different complementary strands at both ends of a target DNA region. Ie PC
The R method is one of the most innovative technologies recently developed in the field of genetic engineering. This PCR method has been used to detect and diagnose genes derived from viruses and bacteria,
After the discovery of a new type of protein or gene, cloning of the same type of gene or complementary c
It has been used to synthesize DNA and confirm gene expression. However, the virus causes mutations due to repeated infection of the same strain, and bacteria have differences in the nucleotide sequences of genes between closely related strains within a species or genus. However, even when a primer is prepared based on the nucleotide sequence of the gene and PCR is performed, the target DNA is often not amplified.

To overcome this, primers containing inosine (Thomas Ehlen and Louis Dubeau. Biochemical and
Biophysical Research Communications, 160, 441-447 (19
89), Anthony P. Fordham-skelton et al. Mol Gen Gene
t, 221,134-138 (1990)) and degenerate primers (L
ee, CC et al. Science, 239, 1288-1291 (1988), GuoCai
Various improvements in primers have been made, including the use of Huang et al. FEBS Letters, 264, 193-197 (1990). The inosine-containing primer is a primer in which an inosine residue is substituted at a position complementary to an ambiguous base composition on a gene, and is used as a primer for detecting a specific mutation. Although the inosine residue does not form a complementary bond with the base composition of the target DNA, the polymerase reaction can proceed by the complementary bond with the DNA formed by the bases on both sides of the inosine of the primer. However, inosine-containing primers cannot be applied unless the position where the primer is incompatible is specified, and the rigor of hybridization is lost as the number of inosine residues increases. In addition, the primer
At the 3 'end, there was a drawback that inosine could not be used. On the other hand, degenerate primers are used as primers that predict the sequence of a nucleic acid from the amino acid sequence that is commonly conserved between families, and mix the nucleic acids corresponding to the different sites of this sequence in the synthesis process. And used when cloning family genes. However, since the proportion of primers compatible with the template is reduced depending on the mixing ratio of the different bases and the location and number of the bases introduced in each primer, if the effective primer concentration is not reached, the target DNA can be sufficiently reduced. There was a disadvantage that it was not amplified. In addition, when a mix is used at the 3 'end of the primer, there is a disadvantage that efficient amplification becomes difficult. Therefore, it has been desired to establish a PCR method capable of easily overcoming a mismatch between a primer and a template nucleic acid, particularly a mismatch between the 3 'end of the primer and a template. When amplifying an unknown homologous gene based on the sequence information of a known gene, the 3 ′ end of the primer prepared based on the sequence information of the known gene in a single primer is the third codon in the template DNA. The technology to deal with bases other than the bases that make up PCR is already known (PCR
protocols, A guide to Methods and aplications; E
d., Michael A. Innis et al., Academic Press, Inc. P4
1 (1990)).

[0004]

DISCLOSURE OF THE INVENTION The present invention relates to an improved primer set capable of overcoming the inhibition of nucleic acid amplification or the reduction in amplification efficiency due to a mismatch between a primer and a template nucleic acid, particularly a mismatch at the 3 'end, and the primer set. It is an object of the present invention to provide a method for amplifying a nucleic acid using the same.

[0005]

Means for Solving the Problems The present inventors have found that even if some of the primers have a 3 ′ end that is not compatible with the template DNA, the 3 ′ end side is a primer that can ensure an effective primer concentration. A plurality of primers were prepared by shifting the nucleic acid region on the template DNA to be hybridized by 2 bases or 3 bases. Also, when comparing the nucleotide sequence of a known structural gene and a structural gene of the same type, focusing on the fact that there is a high possibility that there is a sequence difference at the third codon, the 3 ′ Those whose ends corresponded to bases other than the base constituting the third codon in the template DNA were prepared. Then, when a PCR reaction was performed using the primer mixture, it was found that an amplification reaction was efficiently performed even for DNA of the same type whose gene sequence was unknown.

[0006] When a PCR amplification product is digested with a restriction enzyme and a gene is identified based on the base length of the amplification product, the amplification product must be a fragment of the same length. The 5 'ends of multiple primers were matched. As a result, it was found that the PCR product was efficiently amplified as DNA fragments of the same length.

Specifically, the present invention provides (1) a primer for a PCR reaction comprising a plurality of primers each having a 3 ′ end shifted from a nucleic acid region to be hybridized on a template DNA, and (2) a plurality of primers. 3 'end is the template DNA
Corresponding to other than the base constituting the third codon,
(1) the primer according to (3), a plurality of primers
The primer according to (2), wherein the 3 'end is shifted by 3 bases each, (4) the primer according to any one of (1) to (3), wherein the 5' ends of the plurality of primers correspond to the same base in the template DNA. (5) A method for amplifying a nucleic acid, comprising performing a PCR reaction using the primer according to any one of (1) to (4).

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION As the "plurality of primers having a shifted nucleic acid region to be hybridized on the template DNA" used in the present invention, primers having a shifted 3 'end and a shifted 5' end are used. Besides
An example in which both of these are shifted is given.
There is no particular limitation on this shifting method.
Examples include those shifted by bases, those shifted by 2 bases, and those shifted by 3 bases. When amplifying a specific gene, the 3 ′ end of the primer is Avoiding the third codon in the PCR will increase the success rate of the PCR. For this purpose, for example, it is preferable to use a plurality of primers whose 3 'end is shifted by 3 bases. In addition, in the case of performing restriction enzyme digestion of the amplification product after PCR and comparing the cleavage images of the amplification product to identify a gene, the amplification product needs to be a fragment of the same length. 'It is preferable to use a plurality of primers having matching ends.

[0009] There is no particular limitation on the DNA used as a base for preparing the primer as long as the base sequence is known. The template DNA is not particularly limited as long as it has homology with the nucleotide sequence of the primer or is expected to have homology, and may be used as a base for preparing the primer according to the purpose of PCR. Are determined by the relative relationship of For example, when a new gene is isolated and its structure is elucidated, if the purpose is to isolate a gene of the same species or cognate with the new gene, the nucleotide sequence of the new gene whose nucleotide sequence is known is It is conceivable that a primer is prepared based on the gene and a homologous / cognate gene is used as a template. When there are a plurality of known genes, the PCR success rate is improved by preparing primers based on a region having high conservation between genes.

DNA and template DNA used as primers
Various methods can be considered as a method for extracting and purifying protein from cells.For example, cell components are solubilized with a solution containing a surfactant, treated with protease, and then extracted with an organic solvent to remove protein components and membrane components. Removal and precipitation of the nucleic acid with ethanol. As a method for obtaining a primer, a method using a natural nucleic acid is conceivable in addition to a method using synthesis.

[0011] The reaction conditions for PCR can vary depending on the type of primer, for example, the length of the primer and the GC content, but usually, the double-stranded template DNA is denatured at 90 to 95 ° C and the reaction is performed at 40 to 60 ° C.
A cycle of annealing the primer and the template DNA at a temperature of 70 ° C. followed by a DNA extension reaction at 70 to 75 ° C.
It is effective to repeat about ~ 50 times.

As a method of confirming whether or not the target DNA has been synthesized as a result of the PCR, the electrophoresis using a gel is followed by staining with a stain such as ethidium bromide to detect the band of the target DNA. And the like.

When performing PCR analysis on the amplification product obtained by PCR, first, the amplified DNA is digested with restriction enzymes, and then DNA fragments are subjected to electrophoresis to stain with ethidium bromide or the like. A method of examining the cut image by staining with an agent.

[0014]

The present invention will be described in detail with reference to the following examples, but the present invention is not limited to these examples.

Example 1 Amplification of Chlamydia Major Outer Membrane Protein Gene (ompA) by PCR In this example, the sequences of the 5 ′ end and 3 ′ end of the ompA gene are known, such as “C.psittaci”, "C.pneumoniae"
Using a primer prepared based on the highly conserved nucleotide sequence of "C. trachomatis" (Fig. 1), DNA extracted from "C. pecorum Shizuoka strain", whose sequence in the region is unknown, was used as a template. Was performed as PCR. As a control for comparing the amount of gene amplification, the above-mentioned sequence is known, `` C.ps
ittaci MN strain, C.pneumoniae AR39 strain and C.trac
The DNA extracted from the "homatis L2 strain" infected cells was used as a template. Infected cells were centrifuged at 2800 g for 1 hour and incubated at 37 ° C. for 2 hours on monolayer HeLa229 cells washed with these strains with Hanks' solution containing 0.003% (w / v) DEAE-dextran. After culturing at 37 ° C for 48 hours in a culture solution containing 1 μg / ml cycloheximide, the inclusion body formation rate was
It was prepared by subculturing to 80% or more. DNA
Was extracted from infected cells by centrifugation, and the crude cell particles collected by sedimentation were added to a lysis buffer (0.5% Tween2
0.5 mM EDTA, 10 mM Tris-HCl (pH 8.0)) and proteinase K
(Reaction concentration 100 μg / ml), and reacted at 55 ° C. for 2 hours.
Subsequently, proteinase K was inactivated by treatment at 94 ° C. for 10 minutes. This solution is
For PCR.

First, a primer corresponding to the 5 'side of the template DNA starts from a position 12 bases upstream from the translation start point of the gene encoding the major outer membrane protein of Chlamydia and ends at the 3' end with the second codon. Primer consisting of 32 bases (SEQ ID NO: 1 / 5'-TTAGAGGTGAGTATG
AAAAAACTCTTGAAATC-3 ') and two primers (SEQ ID NO: 2 / 5'-TTAG), each having 3 bases shortened by 3 bases.
AGGTGAGTATGAAAAAACTCTTGAA-3 ', SEQ ID NO: 3 / 5'-TTA
GAGGTGAGTATGAAAAAACTCTT-3 ') (3 types in total) was prepared (FIG. 1A). That is, three types of primers were prepared in which the 5 'ends of the primers were identical and the 3' ends were shifted by 3 bases.
On the other hand, as a primer corresponding to the 3 ′ side of the template DNA, a 36-base primer (SEQ ID NO: 4 / 3′-AGAGCTGCTCACGTAAATGCTCAATTCAGAT) which starts at the position of the termination codon of the above gene and ends at 33 bases upstream.
TCTAA-5 ') and two primers (SEQ ID NO: 5 / 3'-GCTGCTCACGTAAATGCTC, each having 3 bases shortened by 3 bases)
AATTCAGATTCTAA-5 ', SEQ ID NO: 6 / 3'-GCTCACGTAAATGC
TCAATTCAGATTCTAA-5 ') (3 types) (Fig. 1)
C).

The obtained primer was subjected to PCR on the extracted template DNA. PCR, 5 μl template DN
A, 0.2 mM dNTPs, 0.4 μM each primer, MgCl ₂ containing 1 ×
In a PCR buffer, 1.2-1.3 units of “Amplitaq ^™ DNA polymerase” (Takara Shuzo), use “Thermal
Sequencer TR-300 "(Iwaki Glass). The reaction conditions were as follows: heat denaturation at 94 ° C for 5 minutes, followed by heat denaturation at 94 ° C for 60 minutes.
A temperature cycle of annealing at 50 ° C. for 120 seconds and extension reaction at 72 ° C. for 180 seconds was repeated 40 times. In order to confirm whether the target DNA was amplified, the PCR product was subjected to agarose electrophoresis and then detected by ethidium bromide staining. As a result, an expected amplification band of about 1.2 kbp was observed. Further analysis of the gene sequence revealed that the ompA gene of the target gene, "C. pecorum", was amplified as an amplification product of equal length to the same extent as the control (FIG. 2, lane 5). Note that lanes 2, 3, and 4 in FIG. 2 are “C. trachomatis L2 strain” and “C.
psittaci MN strain ”and“ C. pneumoniae AR39 strain ”are electrophoresis images of PCR products when DNA extracted from infected cells was used as a template. Lanes 1 and 6 show a pHY marker (manufactured by Takarasha) as a molecular weight marker. 3) is an electrophoresis image.

As another type of primer corresponding to the 5 'side, a primer consisting of 31 bases starting from a position 10 bases upstream from the translation start point of the gene encoding chlamydia major outer membrane protein (SEQ ID NO: 7 / 5'-AGAGG
TGAGTATGAAAAAACTCTTGAAATCG-3 ') and 2 bases each
Two types of primers with shortened 3 'side (SEQ ID NO: 8 /
5'-AGAGGTGAGTATGAAAAAACTCTTGAAAT-3 ', SEQ ID NO: 9
/ 5'-AGAGGTGAGTATGAAAAAACTCTTGAA-3 '), that is, a total of three types of primers were prepared in which the 5' ends of the primers were matched and the 3 'ends were shifted by 2 bases (FIG. 1B), and the same experiment was performed. As in the case where the primers shifted by 3 bases were used, the ompA gene was amplified to the same extent as the comparative control as an amplification product having the same length (FIG. 3, lane 5). As in FIG. 2, lanes 2, 3, and 4 in FIG.
C.trachomatis L2 strain and C.psittaci MN
Strain "and" C.pneumoniae AR39 strain "extracted from infected cells
It is an electrophoresis image of the PCR product when NA was used as a template. Lanes 1 and 6 show the molecular weight marker pH.
It is an electrophoresis image of a Y marker (manufactured by Takara).

[0019]

Industrial Applicability According to the present invention, the disadvantages of conventional inosine-containing primers and degenerate primers, that is, mismatches between primers and template nucleic acids, particularly mismatches at the 3 'end of primers, are unknown, and the gene sequence is unknown. A primer capable of easily amplifying the same kind of nucleic acid and a PCR method using the primer are provided. Furthermore, by unifying the position of the 5 'end of the primer on the template DNA, it is possible to amplify DNA of the same length,
It is expected to be used for restriction enzyme digestion analysis.

[0020]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 32 Sequence type: Nucleic acid Number of strands: Single stranded Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence TTAGAGGTGA GTATGAAAAA ACTCTTGAAA TC 32 SEQ ID NO: 2 Sequence length : 29 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence TTAGAGGTGA GTATGAAAAA ACTCTTGAA 29 SEQ ID NO: 3 Sequence length: 26 Sequence type: Nucleic acid Number: Single-stranded Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence TTAGAGGTGA GTATGAAAAA ACTCTT 26 SEQ ID NO: 4 Sequence length: 36 Sequence type: Nucleic acid Number of strands: Single-stranded Topology: Linear Type Sequence type: Other nucleic acid Synthetic DNA sequence AATCTTAGAC TTAACTCGTA AATGCACTCG TCGAGA 36 SEQ ID NO: 5 Sequence length: 33 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acids Synthetic DNA sequence AATCTTAGAC TTAACTCGTA AATGCACTC G TCG 33 SEQ ID NO: 6 Sequence length: 30 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid Synthetic DNA sequence AATCTTAGAC TTAACTCGTA AATGCACTCG 30 SEQ ID NO: 7 Length: 31 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence AGAGGTGAGT ATGAAAAAAC TCTTGAAATC G 31 SEQ ID NO: 8 Sequence length: 29 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence AGAGGTGAGT ATGAAAAAAC TCTTGAAAT 29 SEQ ID NO: 9 Sequence length: 27 Sequence type: Nucleic acid Number of strands: Single strand topology : Linear sequence type: Other nucleic acid Synthetic DNA sequence AGAGGTGAGT ATGAAAAAAC TCTTGAA 27

[Brief description of the drawings]

FIG. 1 is a diagram showing complementary positions and sequences of primers used for amplification of a chlamydia main outer membrane protein gene.

FIG. 2 is a diagram showing chlamydia major outer membrane protein genes amplified by PCR using various primers (those shifted by 3 bases).

FIG. 3 is a diagram showing chlamydia major outer membrane protein genes amplified by PCR using various primers (shifted by two bases).

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[Procedure amendment]

[Submission date] December 12, 1996

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] Figure 2

[Correction method] Change

[Correction contents]

FIG. 2 is an electrophoretic image showing chlamydia major outer membrane protein genes amplified by PCR using various primers (shifted by three bases).

[Procedure amendment 2]

[Document name to be amended] Statement

[Correction target item name] Figure 3

[Correction method] Change

[Correction contents]

FIG. 3 is an electrophoretic image showing chlamydia major outer membrane protein genes amplified by PCR using various primers (shifted by two bases).

Claims (5)

[Claims] 1. A primer for a PCR reaction, comprising a plurality of primers each having a 3 ′ end shifted from a nucleic acid region to be hybridized on a template DNA. 2. The method according to claim 2, wherein the 3 ′ ends of the plurality of primers are
2. The primer according to claim 1, wherein the primer corresponds to a base other than the base constituting the third codon. 3. The primer according to claim 2, wherein the 3 ′ ends of the plurality of primers are shifted by 3 bases. 4. The method according to claim 4, wherein the 5 ′ ends of the plurality of primers are
The primer according to any one of claims 1 to 3, which corresponds to the same base in the above. 5. A method for amplifying a nucleic acid, comprising performing a PCR reaction using the primer according to any one of claims 1 to 4.