CN107937493A

CN107937493A - A kind of hair clip Mdification primer for allele PCR

Info

Publication number: CN107937493A
Application number: CN201711277458.1A
Authority: CN
Inventors: 肖君华; 王茂春; 董晶晶
Original assignee: SHANGHAI YIHE APPLICATION BIO-TECH Co Ltd; Donghua University
Current assignee: SHANGHAI YIHE APPLICATION BIO-TECH Co Ltd; Donghua University; National Dong Hwa University
Priority date: 2017-12-06
Filing date: 2017-12-06
Publication date: 2018-04-20
Anticipated expiration: 2037-12-06
Also published as: CN107937493B

Abstract

The present invention relates to a kind of AS PCR primers of hair clip modification, kit and its application in SNP detections.The amplification of genotype identification can be completed in a PCR, is included in the primer that the first two recycles hairpin structure, reduces the generation of multiple dimer, while the presence of hair clip improves the specificity of 3 ' terminal bases identification；And as outside universal fluorescent primer is in the introducing of following cycle, not while homotactic primer identification different genotype purpose fragment, different loci can also be carried out at the same time amplification, i.e., a set of fluorescent primer can carry out Multiple detection, add flux.Invention not only improves the resolution ratio of AS PCR terminal bases mispairing, the sensitivity of reaction is improved, while is also a kind of more convenient operation, price more reasonably method for detecting single nucleotide polymorphism.

Description

A kind of hair clip Mdification primer for allele PCR

Technical field

The present invention relates to biological technical field, and equipotential base is carried out more particularly to a kind of primer modified by hairpin structure Because of specific PCR, and the technology of genetic test is realized by the fluorescent primers of two kinds of different fluorescent markers, applied to bioscience Research and clinical molecular diagnosis.

Background technology

Nucleic acid is the basis of all inhereditary materials, after genetic code is cracked, studies the relation of genotype and phenotype It is exactly the tireless pursuit of geneticist.Researchers constantly explore the relation of genetic mutation and biological function, it is expected In new breakthrough is located to genetic disease, evolutionary source and disease.And single nucleotide polymorphism (SNP) is in genomic level The DNA sequence polymorphism as caused by making a variation single nucleotide acid, is that the mankind can be inherited the most common type in variation, in genetic disease It is important detection target spot on level diagnosis.

Allele-specific PCR (AS-PCR) is that one kind is usually used in analyzing gene pleiomorphism, particularly The method of single nucleotide polymorphism (SNP).In traditional AS-PCR, the 3 ' of the primer of PCR holds last base to be necessary for gene Polymorphic site, holds last base correctly to be matched with template by primer 3 ', and PCR can normally extend to form product bar Band, and the primer of mispairing is reduced to 1/100 to 1/100000, PCR and cannot normally extend to form product band in extension efficiency, So as to detect differentiation mutation type.

Have more at present based on the evolutionary approach in traditional AS-PCR methods, early stage is mainly by avoiding high-elongation Mispairing or by PCR react key component (dNTP, Mg²⁺Deng) being diluted to extremely low concentration i.e. under limited resources, mispairing is drawn Thing extends extremely inefficient the methods of being not easy to expand, and improves its specificity.The two schemes need to grope by multiple condition at present And optimization, it is difficult to large-scale application, the method that a variety of optimizations occurs in the later stage, such as document " Simple allele- again discriminating PCR for cost-effective and rapid genotyping and mapping”(Plant Methods 2009,5(1):1-8), " An improved allele-specific PCR primer designmethod for SNP marker analysis and itsapplication”(Plant Methods 2012,8(34):It is 1-9) etc. logical 3 ' ends are artificially introduced the method for mispairing to improve the scheme of AS-PCR terminal bases mispairing resolving powers when crossing design primer.

The present invention, by a kind of easy easy promotion method different from above technology, carries on the basis of traditional AS-PCR High AS-PCR terminal bases mispairing resolution ratio, improves specificity.

The content of the invention

Technical problem to be solved

The present invention overcomes the defects of existing AS-PCR terminal bases mispairing conceptual design complexity, detection poor specificity, carry For a kind of easy schemes that 3 ' the end specific amplifications to improve AS-PCR are modified by 3 ' end primer hairpin structures.

Technical solution

The first aspect of the invention is to provide one group of AS-PCR primer, including 3 ' ends are located at the upstream in mutational site and draw There are one section and the sense primer 3 ' in thing and the anti-sense primer specifically complementary with mutational site downstream, described sense primer 5 ' end End, not comprising the 1st mutational site base, the hairpin of 7 to 13 base complementrities of length, and in 3 ' ends and hairpin Between include one section of general upstream sequence；The described end of anti-sense primer 5 ' includes one section of general downstream sequence.

One of preferred embodiment of above-mentioned technical proposal is described general upstream sequence and described general downstream sequence institute The GC base distributions contained are uniformly and length range is 15 to 25bp.

The two of the preferred embodiment of above-mentioned technical proposal are, are designed for different mutated-genotypes different described general The general downstream sequence that upstream sequence and two or more mutated-genotype share.It is described as one of embodiment General upstream and downstream sequence is and random sequence or artificial sequence of the species to be measured without specific pairs.Using mutated-genotype Special general upstream and downstream sequence, can utilize sleeve type PCR to distinguish different genotype.

The three of the preferred embodiment of above-mentioned technical proposal are that described general upstream sequence is according to the general upstream of wild type and dashes forward The general upstream of modification is respectively labeled as different fluorescence, as the general upstream sequence of fluorescence.Described fluorescent marker is selected from following One of fluorophor or a combination thereof：FAM, VIC, HEX, ROX, TaxasRed or CY5.

The four of the preferred embodiment of above-mentioned technical proposal are that described AS-PCR primers further include a pair of and general upstream sequence The upstream and downstream universal primer identical with general downstream sequence base sequence.

The five of the preferred embodiment of above-mentioned technical proposal are that 5 ' ends of described upstream or downstream universal primer have fluorescence mark Note.Especially, described fluorescent marker is selected from one of following fluorophor：FAM、VIC、HEX、ROX、 TaxasRed or CY5.

The second aspect of the invention is to provide a kind of kit for including AS-PCR primers described in claim 1.

One of preferred embodiment of above-mentioned technical proposal is that the kit includes two groups of the different fluorophors of mark AS-PCR primers.

The two of the preferred embodiment of above-mentioned technical proposal are, the kit include two pairs or more for different prominent Become the sense primer and anti-sense primer in site.

The third aspect of the invention is to provide a kind of AS-PCR primers in single nucleotide polymorphism detection Using.

The key of the present invention is the scheme that make use of hairpin structure to transform AS-PCR in the prior art, combines universal sequence To carry out single nucleotide polymorphism detection.

Typically, it is exposed as the leading end of different genotype special primer 3 ' using hair clip special primer in opening rotation Base specific recognition template DNA, hairpin structure is opened, so as to form the product compared with long segment, the fragment products once being formed, Universal sequence on the universal fluorescent primer and then identification long segment of high concentration, amplifies the product compared with short-movie section, causes at the same time The different corresponding fluorescence of genotype Product Labeling, and then the glimmering of diverse location can be collected on 3730 grade fluoroscopic examination platforms Optical signal, distinguishes the gene kenel of different samples.

As one embodiment of the present invention, the design of primers of AS-PCR is used using mutational site as the end of special primer 3 ' End, designs one section of special upstream (section one) in the range of the reasonable Tm recommended in general round pcr handbook, and is held in upstream 5 ' The fixed sequence program (section two) for introducing one section of 15-25bp is used as upstream universal sequence, is re-introduced into one section and 3 ' end number of base sequences The not exposed sequence of complementary pairing (section three) at row complementary pairing but mutating alkali yl, makes the primer in its natural state from figure Into hairpin structure；Anti-sense primer is one section of special primer (section four) positioned at mutational site downstream, in specific implementation, can be made Two kinds of genotype share an anti-sense primer, and 5 ' ends introduce other one section of fixed sequence program (section five) and are used as the general sequence in downstream Row, anti-sense primer is preferably without hairpin structure.Due to the presence of hairpin structure at site, it is not easy to form obvious dimer between primer, And primer 3 ' holds exposed single base only to be substantially reduced with special base pair complementarity, primer mispairing probability, further More accurately distinguish different mutational sites.

As another embodiment of the invention, there is provided fluorescent dye primer during a set of detection applied to AS-PCR. The section two of same site upstream clamp primers has two kinds of different sequences, and two sequence of wild type section is " sequence one ", mutation Two sequence of type section is " sequence two ", the two kinds of fixed sequence programs (general upstream sequence) for different genotype mutation in upstream are respectively It is identical with section five with the sequence of anti-sense primer as the general upstream of fluorescence labeled as different fluorescence.Nest is utilized in PCR The principle of formula PCR, adds fluorescence signal for target area PCR product, it is believed in different platform by detecting fluorescence Number judge different genotype.

As described herein, " GC base distributions are uniform " refers to the two kinds high pairing active forces of G and C in one section of nucleotide sequence Base be avoided as much as it is continuously distributed, such as therebetween be spaced A or T bases or no more than 2, be no more than 3 Continuous G or C.

Beneficial effect

The present invention is by hairpin structure to Modify to primer, there is provided a kind of scheme of AS-PCR design of primers, and monokaryon The detection method of nucleotide polymorphism.Compared to traditional AS-PCR methods, match somebody with somebody present invention improves AS-PCR terminal bases fallibilities Shortcoming, and simplify shell type detection by designing the general upstream and downstream sequence of different mutated-genotypes.In a PCR The amplification of genotype identification is completed, is included in the primer that the first two recycles hairpin structure, reduces the generation of multiple dimer, The presence of hair clip improves the specificity of 3 ' terminal bases identification at the same time；And as outside universal fluorescent primer is in following cycle Introducing, not while homotactic primer identification different genotype purpose fragment, different loci can also be carried out at the same time amplification, i.e., A set of fluorescent primer can carry out Multiple detection, add the flux of AS-PCR.

The general upstream sequence of further fluorescent marker wild type and saltant type, it is possible to achieve fluorescence is detected in different platform Signal judges different genotype.The universal primer of fluorescent marker using the present invention is to when carrying out multiplex PCR, if different loci Product sheet segment length is different, then different mutational sites, which only need to share a set of fluorescent universal primer, can realize the inspection in multiple sites Survey, greatly reduce cost, simplify experimental implementation, be more easy to promote and use.

Brief description of the drawings

The present invention is further described with reference to the accompanying drawings and examples.

Fig. 1 is the primer schematic diagram of one embodiment of the present invention.In figure, 101 and 201 be upstream specific primer sequence, 102 and 202 be downstream specific primer sequences, and 103 be upstream universal primer sequence 1 (exemplified by A types), and 203 be upstream universal primer Sequence 2 (exemplified by G types), than 103 3 bases of difference, but is not limited to 3 bases, and 104 and 204 be upstream universal primer sequence, 105 and 205 is the hairpin structure sequences with 7 to 13 base complementrities in the end of upstream specific primer 3 ', single base only at mutational site Not complementary, 106 be that the upstream universal primer 1,206 that fluorophor 1 marks is the upstream universal primer 2 that fluorophor 2 marks, 107 and 207 be downstream universal primer.

Embodiment

With reference to specific embodiment, the present invention is further explained.It is to be understood that these embodiments are merely to illustrate the present invention Rather than limit the scope of the invention.In addition, it should also be understood that, after reading the content taught by the present invention, people in the art Member can make various changes or modifications the present invention, and such equivalent forms equally fall within the application the appended claims and limited Scope.

The experimental method of actual conditions is not specified in the following example, usually according to normal condition, such as molecular cloning protocols Handbook, or the condition proposed by according to reagent manufacturer.All inorganic chemical reagents and organic solvent are purchased from Shanghai chemistry examination Agent Co., Ltd, Taq archaeal dna polymerases, dNTPs are purchased from Dalian treasured biotech firm, and primer is had by Shanghai belle lattice biotechnology Limit company synthesizes, and people's blood DNA extractings are small using love biotechnology (Hangzhou) Co., Ltd (Axygen companies) blood genome of pursuing progress Measure reagent preparation box.

Embodiment 1

Detect the design of the AS-PCR clamp primers of single nucleotide polymorphism.According to the snp database of NCBI, in mankind's base Because finding out the target zone sequence of 3 sections of carrying SNP sites in group, primer is carried out according to general rule using Oligo 6.0 and set Meter, and manually upstream universal sequence and downstream universal sequence are added respectively at the end of primer upstream and downstream 5 ' of design, then with chemistry Method synthesis upstream and downstream special primer (table 1).

Table 1SNP detects special primer

*：FAM- is modified

#：HEX- is modified

DNA is extracted：Human blood cell uses blood genome Miniprep Kit, is stripped to obtain DNA to specifications, DNA mass and concentration are determined with 0.8% agarose gel electrophoresis.

The AS-PCR detections of unit point base.No. 4 primers in table 1 are chosen (to be used to detect rs1042522 sites C equipotential bases Because of special sense primer), No. 5 primers (are used to detect rs1042522 sites G allele specific sense primers, than No. 1 primer More 3 bases on universal sequence, easy to distinguish), No. 6 primers (be used for the spy for detecting rs1042522 sites C and G allele Different anti-sense primer) specific amplification primer mixture is prepared, it is fluorescent universal primer to choose 7,8, No. 9 primer mixtures, to single Sample carries out PCR reactions.PCR amplification system is specially：The 25ng DNA of each sample are added separately to respective 10 μ L Taq In enzyme system, wherein Taq enzyme system includes 0.1 μM of each primer, 200 μM of dNTPs, 4 μM of MgSO₄, 1 × PCR Buffer, 1U Taq enzymes, and covered using mineral oil；Negative control is set at the same time, response procedures are：98 DEG C of denaturation 2min；94 DEG C of denaturation 30s, 60 DEG C of renaturation 30s, 72 DEG C of extension 30s, 40 circulate.

After amplification, gained PCR product is analyzed on 3730 fluorescence detectors, passes through corresponding two kinds of equipotential bases Because the peak height ratio of fluorescence carries out parting, parting formula is F=H1/ (H1+H2), wherein, H1 represents a kind of genotype (in example Represent c-type), H2 represents another genotype (G types are represented in example).F values represent that CC is homozygous close to 1, and ratio is close to 0 table Show GG homozygosis, CG heterozygous is represented between 0.1-0.9.

It is experimentally confirmed that hairpin structure primer AS-PCR genotyping results are consistent with actual conditions (table 2).

Table 2rs1042522 sites hairpin structure primer AS-PCR result datas

Sample number	Known kenel	C-type peak height H1	G type peak heights H2	F
					1	GG		2323	0
2	CC	3694		1.00
					3	CG	3314	2092	0.61
4	CC	1507		1.00
					5	CG	1339	757	0.63
6	CG	1585	1241	0.56

Embodiment 2

The AS-PCR detections of more site bases.All 1-6 special primers are mixed into specific amplification primer mix in selection table 1, Choose 7-9 primers and be mixed into fluorescent universal primer, multi-PRC reaction is carried out to single sample.PCR amplification system is specially： The 25ng DNA of each sample are added separately in respective 10 μ L Taq enzyme systems, and wherein Taq enzyme system includes 0.05 μM often A special primer, 0.1 μM of each universal fluorescent primer, 200 μM of dNTPs, 4 μM of MgSO₄, 1 × PCR Buffer, 1U Taq Enzyme, and covered using mineral oil；Negative control is set at the same time, response procedures are：98 DEG C of denaturation 2min；94 DEG C of denaturation 30s, 60 DEG C multiple Property 30s, 72 DEG C extension 30s, 40 circulation.

After amplification, gained PCR product is analyzed on 3730, passes through corresponding two kinds of equipotentials for each site The peak height ratio of gene by fluorescence, judges genotype, the results are shown in Table 3.

Table site more than 3 hairpin structure primer AS-PCR result datas

SEQUENCE LISTING

<110>Donghua University of Shanghai Yihe Application Bio-Tech Co., Ltd.

<120>A kind of hair clip Mdification primer for allele PCR

<130> 20171108

<160> 9

<170> PatentIn version 3.5

<210> 1

<211> 53

<212> DNA

<213>It is artificial synthesized

<400> 1

ggtctcaccg agagtgcact gctgtcgtgt ggagcagtgt atcggtgaga cca 53

<210> 2

<211> 56

<212> DNA

<213>It is artificial synthesized

<400> 2

ggtctcaccg actgtcctgt ggtcagcaca cctgctagag tgtatcggtg agaccg 56

<210> 3

<211> 38

<212> DNA

<213>It is artificial synthesized

<400> 3

gtgtcctgac gagtgctcac cagcccagtg gcacgctg 38

<210> 4

<211> 47

<212> DNA

<213>It is artificial synthesized

<400> 4

ggggagcagg agtgcactgc tgtcgtgtgg agcgaggctg ctccccc 47

<210> 5

<211> 50

<212> DNA

<213>It is artificial synthesized

<400> 5

ggggagcagc tgtcctgtgg tcagcacacc tgctaggagg ctgctccccg 50

<210> 6

<211> 38

<212> DNA

<213>It is artificial synthesized

<400> 6

gtgtcctgac gagtgctcac cagcgtagct gccctggt 38

<210> 7

<211> 23

<212> DNA

<213>It is artificial synthesized

<400> 7

gagtgcactg ctgtcgtgtg gag 23

<210> 8

<211> 23

<212> DNA

<213>It is artificial synthesized

<400> 8

ctgtcctgtg gtcagcacac ctg 23

<210> 9

<211> 23

<212> DNA

<213>It is artificial synthesized

<400> 9

gtgtcctgac gagtgctcac cag 23

Claims

1. one group of AS-PCR primer, including 3 ' ends are located at the sense primer in mutational site and specifically mutual with mutational site downstream The anti-sense primer of benefit, it is characterised in that the described end of sense primer 5 ' has one section to be held, not comprising the 1st with the sense primer 3 ' Mutational site base, 7 to 13 base complementrities of length hairpin, and it is logical comprising one section between 3 ' ends and hairpin Use upstream sequence；The described end of anti-sense primer 5 ' includes one section of general downstream sequence.

2. AS-PCR primers according to claim 1, it is characterised in that described general upstream sequence and described general GC base distributions contained by downstream sequence are uniformly and length range is 15 to 25bp.

3. AS-PCR primers according to claim 1, it is characterised in that different for different mutated-genotype designs The general downstream sequence that described general upstream sequence and two or more mutated-genotype shares.

4. AS-PCR primers according to claim 3, it is characterised in that described general upstream sequence leads to according to wild type Different fluorescence is respectively labeled as with upstream and the general upstream of saltant type, as the general upstream sequence of fluorescence.

5. AS-PCR primers according to claim 1, it is characterised in that described AS-PCR primers further include it is a pair of with it is logical With the upstream sequence upstream and downstream universal primer identical with general downstream sequence base sequence.

6. AS-PCR primers according to claim 1, it is characterised in that described upstream or 5 ' ends of downstream universal primer There is fluorescent marker.

A kind of 7. kit for including AS-PCR primers described in claim 1.

8. kit according to claim 7, it is characterised in that the kit, which includes, marks different fluorophors Two groups of AS-PCR primers.

9. kit according to claim 7, it is characterised in that the kit is comprising two pairs or more for not The sense primer and anti-sense primer in same mutational site.

A kind of 10. application of the AS-PCR primers in single nucleotide polymorphism detection described in claim 1.