CN102660638B - Kit for identifying polymorphic rs2274976 of human MTHFR gene by BsrI - Google Patents

Abstract

The invention discloses a kit for identifying polymorphic rs2274976 of a human MTHFR gene by BsrI. The kit comprises a forward primer and a reverse primer for amplification of a sequence nearby a polymorphic rs2274976 locus of a human MTHFR gene, wherein a reciprocal first basic group of a 3' terminal of the forward primer is adjacent to a rs2274976 polymorphic basic group and a penultimate basicgroup of the 3' terminal of the forward primer is a mismatched basic group C so that in an amplification product, the mismatched basic group C forms a CCAGT structure with a polymorphic A allele. Theforward primer has a sequence number of SEQ ID NO: 1 or SEQ NO: 11. The reverse primer has a sequence number of SEQ ID NO: 2, SEQ ID NO: 7 or SEQ ID NO: 12. The kit also comprises a restriction enzyme BsrI and a specification for implementing identification. The kit can be operated easily, and has a low cost and a wide application scope.

Description

Test kit with BsrI identifier's mthfr gene polymorphism rs2274976

The application is that application number is the dividing an application for the application for a patent for invention of " method of identifier's mthfr gene polymorphism rs2274976 " that " 200910227637.3 ", the applying date be on December 24th, 2009, denomination of invention.

Technical field

The present invention relates to identify the method for single nucleotide polymorphism.More specifically, the present invention relates to the method for identifier's mthfr gene polymorphism rs2274976.

Background technology

SNP (single nucleotide polymorphism) refers to the mutant dna sequence that the change of single Nucleotide causes, comprises the forms such as displacement, insertion and disappearance of single base.SNP now has been widely used in the location of genetic linkage analysis, association analysis and the diseases predisposing gene of simple and complex disease, instructs the tumor susceptibility gene clone.Polymerase chain reaction-restriction fragment length polymorphic (PCR-RFLP) technology is the genotypic classical way of a kind of quick, easy, accurate, low-cost SNP of detection.This method principle is: restriction enzyme is that class identification DNA specific site is (common 4～6bp), and the enzyme that cuts at specific site.The specificity of restriction enzyme site means the same fragment sequence of the allelic complete digestion of specific DNA meeting generation.And the replacement of base or insertion, disappearance can produce or eliminate a specific restriction enzyme site, thereby change size and number that enzyme cutting back produces fragment.The difference of these endonuclease bamhi banding patterns is called restriction fragment length polymorphism.If SNP produces and has eliminated certain restriction endonuclease sites, then can enzyme be cut, electrophoresis is detected by the PCR product is carried out.This method major advantage is easy and simple to handle, and fast, endpoint is accurate.Main drawback is the selection of restriction enzyme site, and not all SNP site can use this method to differentiate, and the part restriction endonuclease is expensive.

5,10-Methylene tetrahydrofolate reductase (5,10-methylenetetrahydrofolate reductase, MTHFR) be a kind of important folic acid metabolism enzyme, catalysis 5 in the body, the 10-methylene tetrahydrofolate is reduced to topmost methyl donor 5-methyl tetrahydrofolate in the body, has the important physical function.Genetic flaw and the arteriosclerotic vascular obstruction disease of discovering this albumen of coding at present are closely related, or the inherited genetic factors of fetal neural tube deformity and cancer generation.People's mthfr gene is positioned karyomit(e) 1P36.3, and this gene the 12 exon A/G nucleotide variation can cause the 594th amino acids variation (Gln/Arg), and it is polymorphic often to be designated as 1793A/G in the document, this polymorphic function that may influence MTHFR.Mthfr gene and polymorphic sequence and relevant information can be consulted by NIH state-run medical library biology information technology center (http://www.ncbi.nlm.nih.gov), this polymorphic rs2274976 that is numbered in snp database.Therefore, common this polymorphic genotype that in the crowd, has three kinds of mthfr genes: GG type (two allelotrope bases of human genome rs2274976 polymorphic site are G), AG type (two allelotrope bases of human genome rs2274976 polymorphic site are respectively G and A) and AA type (two allelotrope bases of human genome rs2274976 polymorphic site are A).

The PCR-RFLP method is often adopted in the evaluation of the polymorphic rs2274976 of people's mthfr gene at present.The polymorphic rs2274976 of identifier's mthfr gene uses restriction endonuclease BsrBI to identify often at present, expensive (can be with reference to the table 1 of back about the reference price of part restriction endonuclease) of this restriction endonuclease, influenced the expense of experiment, be difficult in the laboratory, popularize and use.And because the inherent defect of conventional P CR-RFLP method, those skilled in the art are difficult to select other restriction enzymes that the polymorphic rs2274976 of people's mthfr gene is identified usually.

Therefore, this area exists simple to operate, and cost is low, the demand of the method for the novel detection SNP that use range is wide.

Summary of the invention

The invention provides a kind of simple to operately, cost is low, the method for identifier's mthfr gene polymorphism rs2274976 that use range is wide, and it may further comprise the steps:

A) provide human gene group DNA to be measured;

B) forward primer and the reverse primer of sequence near the use amplification people mthfr gene polymorphism rs2274976 site are template with described human gene group DNA to be measured, carry out pcr amplification reaction, obtain amplified production;

C) use restriction enzyme that described amplified production is carried out enzyme and cut, obtain enzyme and cut product; And

D) described enzyme is cut product and carry out electrophoresis, with identifier's mthfr gene polymorphism rs2274976, wherein, its 3 ' terminal bit base last of described forward primer is adjacent with polymorphic rs2274976 base, bit base second from the bottom is base mismatch C, so that (first base C is base mismatch to form CCGG with polymorphic G allelotrope in amplified production, the 3rd bases G is polymorphic allelotrope) or ACCGGT (second base C is base mismatch, the 4th bases G is polymorphic allelotrope) structure, perhaps form CCAGT (first base C is base mismatch, and the 3rd base A is polymorphic allelotrope) structure with polymorphic A allelotrope.

Can near the sequence the SNP be changed into the sequence of the restriction enzyme identification that can be determined in advance by the base mismatch on the primer by method of the present invention, therefore, can overcome the existing defective that needs to adopt expensive restriction endonuclease of existing PCR-RFLP method, and then greatly reduce the cost that detects SNP.Particularly, because bit base C second from the bottom is base mismatch (corresponding position of this base in people's mthfr gene sequence is G) in this forward primer sequence, therefore, sequence changes to ACCGGT or ACCAGT by AGCGGT or AGCAGT (wherein second base is base mismatch near polymorphic in the PCR product after the mispairing, the 4th base is the A/G polymorphic site), so G allelotrope fragment can be identified the restriction enzyme of CCGG sequence or the restriction enzyme identification (being that G allelotrope fragment can be cut by restriction endonuclease) of identification ACCGGT sequence in the amplified production.Sequence changes to CCAGT or CCGGT by GCGGT or GCAGT (wherein first base is base mismatch near polymorphic in the PCR product after the same mispairing, the 4th base is the A/G polymorphic site), so A allelotrope fragment can be identified the restriction enzyme identification (being that A allelotrope fragment can be cut by restriction endonuclease) of CCAGT sequence in the amplified production.And then, can judge polymorphic genotype according to fragment incision situation.More specifically, through sequential analysis as can be known, A/G is polymorphic in the gene original series can be by preceding four kinds of restriction endonucleases identification in the table 1.As by base mispairing PCR the polymorphic site front second bit base G being replaced with C, then this polymorphicly contains the restriction endonuclease identification (as MspI, HpaII) that the CCGG sequence can be identified the CCGG sequence when the G allelotrope behind pcr amplification, contains the restriction endonuclease identification (as BsrFI, BsaWI, AgeI) that the ACCGGT sequence can be identified the ACCGGT sequence.Can and cut by the isoschizomers of above-mentioned restriction endonuclease (isoschizomer, derive from different plant species but can identify the restriction enzyme of same DNA sequence) identification equally, and A allelotrope can not cut.By base mispairing PCR the polymorphic site front second bit base G is replaced with C, then should polymorphicly behind pcr amplification, contain the restriction endonuclease that the CCAGT sequence can be identified ACTGG sequence (complementary sequence is CCAGT) when the A allelotrope and identify (as BsrI) when this is polymorphic.Can and cut by the identification of the isoschizomers of BsrI equally, and G allelotrope can not cut.

The reference of NEB company restriction endonuclease pricing information (www.neb-china.com obtains from http://) as restriction enzyme enzyme recognition site and price is provided in the following table 1.

Table several restriction endonuclease recognition sequences of 1NEB company and price thereof

In one embodiment of the invention, forward primer constitutes by being selected from following nucleotide sequence: SEQ IDNO:1 (ctttgccctg tggattgacc) and SEQ NO:11 (tttgccctgtggattgacc).In another embodiment of the invention, reverse primer constitutes by being selected from following nucleotide sequence: SEQ IDNO:2 (gcagttgtcc agtgggaagt ca), SEQ ID NO:7 (aatgtgtctt ccaccacctgc) and SEQ NO:12 (tctcgcattc tgggtggg).The design of forward and reverse primer considers that mainly primer is to the influence of sensitivity, specific degree and the amplification efficiency of pcr amplification.Usually according to base complementrity pair principle design primer, it is closely complementary that the sequence of primer and template is wanted.Length is 15-30bp, the too short or long poor specificity that causes, and long its elongating temperature that also can cause is unfavorable for the PCR reaction greater than 74 ℃.The definite of forward primer must must be contained the terminal base mismatch C of penultimate except mentioned above principle.Forward primer contains base mismatch C in its 3 ' terminal penultimate, so that (second base C is base mismatch to form ACCGGT with polymorphic G allelotrope in amplified production, the 4th bases G is polymorphic allelotrope) structure, form CCAGT (first base C is base mismatch, and the 3rd base A is polymorphic allelotrope) structure with polymorphic A allelotrope.Thereby can be by corresponding restriction endonuclease identification.The length of amplified production mainly be by reverse primer determine (because forward primer is positioned near the polymorphic site upstream, its position is roughly definite), usually amplified production length is between the 100-200bp, so namely is beneficial to the amplification of PCR product, is beneficial to follow-up enzyme again and cuts evaluation.Otherwise, being unfavorable for amplification if product is too short, fragment length difference was less after long then enzyme was cut, and was unfavorable for differentiating.For example, in one embodiment of the invention, the nucleotide sequence that utilizes SEQ ID NO:1 can obtain following amplified production as the nucleotide sequence of forward primer and SEQ ID NO:2 as the PCR reaction of reverse primer:

ctttgccctg tggattgacc rgtggggaaa gctgtatgag gaggagtccc cgtcccgcac 60

catcatccag tacatccacg acaactactt cctggtcaac ctggtggaca a tgacttccc 120

actggacaac tgc 133(SEQ ID NO：4)

Underscore partly is respectively forward, the corresponding sequence of reverse primer in the amplification after product sequence, and the r of 21bp place represents the polymorphic SNP of being of A/G site rs2274976.This amplified production length is 133bp.133bp, 114bp (this fragment downstream sequence is compared upstream sequence and produced two strand bases of sticky end minimizing because the MspI enzyme is cut), three kinds of segments (wherein can not see in the 19bp electrophorogram) of 19bp (this fragment downstream sequence is compared upstream sequence increases by two strand bases because the MspI enzyme is cut the generation sticky end) can appear after the MspI enzyme is cut.Enzyme is cut the back genotype and judged: GG is 114bp, and AG has 133bp and two kinds of fragments of 114bp, and AA is 133bp.

For the evaluation that enzyme is cut product, use agarose gel electrophoresis and polyacrylamide gel electrophoresis usually, consider based on cost and convenience, use agarose gel electrophoresis more common.In the present invention, for the amplified production of above-mentioned 133bp, the condition of agarose gel electrophoresis is that enzyme is cut product in the 2.5%-4% sepharose under the 3-8V/cm condition, electrophoresis 40-80 minute, and the evaluation of under ultraviolet lamp, taking pictures.

In another preferred embodiment of the present invention, described restriction enzyme is selected from MspI, HpaII, BsrFI, BsrI, BsaWI, AgeI and their isoschizomers, and more preferably described restriction enzyme is MspI.This mainly is based on taking all factors into consideration of cost and cutting efficiency.

In the present invention, the condition of pcr amplification reaction is not subjected to special restriction, as long as can obtain amplified production.Significant parameter-annealing temperature of PCR and time, depend on length, based composition and the concentration thereof of primer, also have the length of target sequence.If the PCR annealing temperature is crossed low then is prone to non-special product, the too high amplified production output that then influences finally need be determined with experiment.The PCR time of extending determines that according to product length 20s-60s gets final product usually.In order to improve pcr amplification efficient, and then further improve and identify efficient, in a preferred embodiment of the present invention, the pcr amplification condition that adopts is:

95 ℃ of sex change 3 minutes;

35 circulations: 95 ℃ of sex change 50 seconds, 58 ℃ of annealing 50 seconds, 72 ℃ were extended 50 seconds; And

72 ℃ are extended 10min after above-mentioned 35 loop ends.

For the selection of DNA to be measured, the not special restriction of the present invention.Both can be to obtain genomic dna from the body fluid of human body or tissue, can also be through the genome of degradation treatment in advance.For convenience of implementation, the genomic dna that preferably extracts from blood usually.Wherein said tissue comprises and containing in the human body all or the tissue of mthfr gene at least, and with whether to express this mthfr gene irrelevant.The method of extracting DNA from body fluid and/or tissue is well known to a person skilled in the art, and can be with reference to molecular biology manual commonly used, and for example " molecular cloning " the 2nd edition carries out.

In addition, also provide a kind of test kit for the identification of people's mthfr gene polymorphism rs2274976 in the present invention, comprise: forward primer and the reverse primer of sequence near the amplification people mthfr gene polymorphism rs2274976 site, wherein, described forward primer is adjacent with the polymorphic base of rs2274976 at its 3 ' terminal bit base last, penultimate is base mismatch C, in order in amplified production, form the CCAGT structure with polymorphic A allelotrope, described forward primer is SEQ ID NO:1 or SEQ NO:11, and reverse primer is SEQ ID NO:2, SEQ ID NO:7 or SEQ NO:12; Described test kit also comprises restriction enzyme BsrI; And described test kit also comprises the specification sheets of identifying for implementing.

As previously mentioned, this test kit is simple to operate, and cost is low, and use range is wide.

In one embodiment of the invention, forward primer constitutes by being selected from following nucleotide sequence: SEQID NO:1 (ctttgccctg tggattgacc) and SEQ NO:11 (tttgccctgtggattgacc).In another embodiment of the invention, reverse primer constitutes by being selected from following nucleotide sequence: SEQ IDNO:2 (gcagttgtcc agtgggaagt ca), SEQ ID NO:7 (aatgtgtctt ccaccacctgc) and SEQ NO:12 (tctcgcattc tgggtggg).In another embodiment of the invention, also comprise restriction enzyme and their isoschizomerss such as MspI, HpaII, BsrI, BsrFI, BsaWI, AgeI in the described test kit, like this can be conveniently to the detection of amplified production.Further, preferred described restriction enzyme is BsrI.This mainly is based on the cost of restriction enzyme and taking all factors into consideration of cutting efficiency.Certainly, it will be understood by those skilled in the art that in test kit to comprise other compositions, for example be used for the specification sheets of enforcement evaluation etc.

Description of drawings

Fig. 1 has described among the embodiment 1 the polymorphic PCR product of people's mthfr gene (rs2274976) and has cut the rear electrophoresis photo through the MspI enzyme.Wherein M is: dna molecular amount mark; The electrophoresis result of swimming lane 1:GG type sample; The electrophoresis result of swimming lane 2:AA type; The electrophoresis result of swimming lane 3:AG type sample.

Embodiment

Below by the specific embodiment of the present invention technical scheme of the present invention is described in detail.Wherein, need to prove that the present invention also is subject to embodiment described below and embodiment never in any form.

The nucleotide sequence that utilizes SEQ ID NO:1 below describes in detail concept of the present invention as the nucleotide sequence of forward primer and SEQ ID NO:2 as reverse primer as an example.

In one embodiment of the invention, the present invention detects the method for the polymorphic rs2274976 of people's mthfr gene, and step is:

1, extracts human gene group DNA's template to be measured, the human gene group DNA that described human gene group DNA's template obtains for the human body any part;

2, pcr amplification genomic dna carries out pcr amplification to the templet gene group DNA that extracts, and obtains to contain near the PCR product of polymorphic sequence;

3, use restriction enzyme to carry out endonuclease reaction to pcr amplification product, get enzyme and cut product; And

4, the product after enzyme is cut carries out agarose gel electrophoresis, the polymorphic rs2274976 of identifier's mthfr gene.

Below each key step is described in detail

(1) design of primers is with synthetic

Search mthfr gene sequence and SNP information in the state-run medical library biology information technology center http://www.ncbi.nlm.nih.gov of NIH (NCBI) website, determine MTHFR polymorphic site nucleotide variation data;

The portion gene sequence (SEQ ID NO:3) that contains the polymorphic rs2274976 of MTHFR:

cttccctggg cgagagatca tccagcccac cgtagtggat cccgtcagct tcatgttctg 60

gaaggtaaag gagccggggg caagcttgcc ccgcccacct ggaaaaccgt ggggagggat 120

tgggaccaag tcccaagcgt gtgctgaagg ccacactgga cccagccttc agggcacacc 180

cagctctgac tcacccatgt cactgctgat gcagggtgtt tatttctggg caggggtggg 240

aagtgatact ggcagtgggc cttgttctat tccgggaaat gtcctgttga gcagagccct 300

tggagagccc tgttaatctt gcctctgtgt gtgtgtgcat gtgtgcgtgt gtgcgggggt 360

atgtgtgtgt aggacgaggc ctttgccctg tggattgagc rgtggggaaa gctgtatgag 420

gaggagtccc cgtcccgcac catcatccag tacatccacg acaactactt cctggtcaac 480

ctggtggaca atgacttccc actggacaac tgcctctggc aggtggtgga agacacattg 540

gagcttctca acaggcccac ccagaatgcg agagaaacgg aggctccatg accctgcgtc 600

ctgacgccct gcgttggagc cactcctgtc ccgccttcct cctccacagt gctgcttctc 660

ttgggaactc cactctcctt cgtgtctctc ccaccccggc ctccactccc ccacctgaca 720

atggcagcta gactggagtg aggcttccag gctcttcctg g 761

The r of 401bp place represents the polymorphic SNP of being of A/G site rs2274976 in the gene order;

Introduce the primer of base mismatch according to above-mentioned sequences Design, determine position and the length of forward and reverse primer according to the sequence situation, specific as follows:

The forward primer sequence is 5 '-CTTTGCCCTGTGGATTGACC-3 ' (SEQ ID NO:1),

The reverse primer sequence is 5 '-GCAGTTGTCCAGTGGGAAGTCA-3 ' (SEQ ID NO:2);

Wherein bit base C second from the bottom is base mismatch (this base should be G in former sequence) in the forward primer sequence, sequence changes to ACCGGT or ACCAGT by AGCGGT or AGCAGT (wherein the 4th base is that A/G is polymorphic) near polymorphic in the PCR product after the mispairing, so G allelotrope fragment can be identified the restriction enzyme of CCGG sequence or the restriction enzyme identification (being that G allelotrope fragment can be cut by restriction endonuclease) of identification ACCGGT sequence in the amplified production.Containing the restriction endonuclease that CCAGT (the 3rd base A is polymorphic) sequence can be identified ACTGG sequence (complementary sequence is CCAGT) after the same mispairing in the time should polymorphicly being A allelotrope behind the pcr amplification identifies.Judge polymorphic genotype according to fragment incision situation;

According to forward primer sequence and reverse primer sequence synthesized primer thing, synthetic described primer can use method as known in the art, and for example solid-phase synthesis also can be entrusted bio-engineering corporation synthetic and detection forward and reverse primer.It is that 10 μ mol/L concentration are standby that synthetic forward, reverse primer are diluted;

(2) preparation pcr amplification product

Pcr amplification uses the reaction system of 15-100 μ l to be: 2 * PCR MIX consumption is the described human gene group DNA to be detected of 7.5-50 μ l, 50-2000ng and 10 μ mol/L forwards, each 0.1-10 μ l of reverse primer as half of pcr amplification reaction system volume, be supplemented to 15-100 μ l with the sterilization distilled water, mixing namely gets the pcr amplification reaction system;

Wherein, the mixed solution of described 2 * PCR MIX PCR reaction usefulness that is 2 times of concentration is made;

With the pcr amplification reaction system after 94-95 ℃ of pre-sex change 3-5 minute; Carry out 30-35 following circulation: 94-95 ℃ of sex change 20-60s, 50-65 ℃ of annealing 20-60s, 72 ℃ are extended 20-60s; 72 ℃ are extended 5-10min after 30-35 the loop ends, namely get pcr amplification product, and its position is corresponding to contain base 381bp-513bp place in the portion gene sequence of the polymorphic rs2274976 of MTHFR;

Amplification after product sequence is (SEQ ID NO:4):

ctttgccctg tggattgacc rgtggggaaa gctgtatgag gaggagtccc cgtcccgcac 60

catcatccag tacatccacg acaactactt cctggtcaac ctggtggaca a tgacttccc 120

Actggacaac tgcThe 133bp sequence

Underscore partly is forward, the corresponding sequence of reverse primer in the amplification after product sequence, and the r of 21bp place represents the polymorphic SNP of being of A/G site rs2274976.

(3) endonuclease reaction

Pcr amplification product cut at 10-30 μ l enzyme carry out endonuclease reaction in the system, the described 10-30 μ l enzyme system of cutting is: restriction enzyme 1-10U and the 10 * enzyme cutting buffering liquid of the pcr amplification product of 2-15 μ l, identification CCGG sequence or identification ACCGGT sequence or identification CCAGT sequence account for 1/10 of cumulative volume, the sterilization distilled water is supplemented to 10-30 μ l, mixing, get enzyme and cut system, enzyme was cut system in 37 ℃ of water-bath 4-16 hours, namely get enzyme and cut product;

The restriction enzyme of described identification CCGG sequence is restriction endonuclease MspI, HpaII and isoschizomers thereof; The restriction enzyme of described identification ACCGGT sequence is restriction endonuclease BsrFI, BsaWI, AgeI and isoschizomers thereof; The restriction enzyme of described identification CCAGT sequence is restriction endonuclease BsrI and isoschizomers thereof; Select MspI or HpaII to carry out enzyme according to price factor and cut evaluation;

Can form after PCR product enzyme was cut when this polymorphic site contained G allelotrope and cut fragment 114bp (this fragment downstream sequence is cut because of enzyme and formed sticky end than two bases of upstream sequence minimizing) sequence (SEQ IDNO:5):

crgtggggaa agctgtatga ggaggagtcc ccgtcccgca ccatcatcca gtacatccac 60

gacaactact tcctggtcaa cctggtggac aa tgacttcc cactggacaa ctgc 114

Enzyme is cut the back and is cut that underscore partly is the corresponding sequence of reverse primer in the fragment 114bp sequence, and the 2bp place represents the polymorphic SNP of being of A/G site rs2274976;

Can form after PCR product enzyme was cut when this polymorphic site contained G allelotrope and cut fragment 19bp (this fragment downstream sequence is cut because of enzyme and formed sticky end than two bases of upstream sequence increase) sequence (SEQ ID NO:6):

ctttgccctg tggattgac 19。

(2) agarose gel electrophoresis, determine gene pleiomorphism:

Enzyme is cut product in 3% sepharose under the 3-8V/cm condition, electrophoresis 40-80 minute, the evaluation of under ultraviolet lamp, taking pictures, wherein, for different genotype, after the amplification of MTHFR site the 133bp segment appears, cut rear electrophoresis through enzyme and 133bp, 114bp, three kinds of segments of 19bp can occur, judge genotype according to having or not of 133bp and 114bp fragment: the GG type is fragment of 114bp, and the AG type has 133bp and two kinds of fragments of 114bp, and the AA type is fragment of 133bp; Identify that through order-checking the result of sequencing result and use prior art detection gained is identical.

Be to implement some embodiment of the present invention below.

Embodiment 1

1 materials and methods

1.1 main agents and instrument

Reagent: 2 * PCR mix (MBI company), restriction enzyme MspI (MBI company), agarose (BBI company), primer is synthetic by Shanghai Sangon company.

Instrument: 9600 type PCR instrument (PE company), miniature electrophoresis chamber (Pharmacia Biotech, EPS1000), Gel Doc 2000 gel imaging instrument (Bio-RAD company).

The order-checking of PCR product is given birth to worker's biotechnology company limited by Shanghai and is finished.

1.2 sequence is searched and design of primers

Search mthfr gene sequence and SNP information in the NCBI website, determine MTHFR polymorphic site nucleotide variation information in conjunction with relevant document, the design primer, specific as follows:

The forward primer sequence is 5 '-CTTTGCCCTGTGGATTGACC-3 ' (SEQ ID NO:1),

The reverse primer sequence is 5 '-GCAGTTGTCCAGTGGGAAGTCA-3 ' (SEQ ID NO:2).

1.3 extract DNA as DNA to be measured from whole blood sample

The EDTA anticoagulant tube is gathered human peripheral whole blood blood sample 300 μ l, using the RelaxGene RloodDNA System of TIANGEN company poba gene group DNA extraction system (main component is cell pyrolysis liquid CL, Proteinase K, damping fluid FG, elution buffer TB) to extract Whole Blood Genomic DNA is human gene group DNA's template to be measured: 300 μ l peripheral bloods are transferred to centrifuge tube, add 750 μ l cell pyrolysis liquid CL, put upside down mixing 5 times; Centrifugal 20 seconds of 10,000r/min abandons supernatant liquor;

Add the damping fluid mixed solution of 150 μ l in centrifuge tube, the damping fluid mixed solution is to be mixed and made at 100: 1 with volume ratio by damping fluid FG and Proteinase K, and vortex vortex mixer mixing to solution does not have agglomerate;

Then, at 65 ℃ of water-bath 10min, put upside down mixing therebetween 5 times;

The adding mass concentration is 99.9% Virahol 150 μ l, puts upside down abundant mixing to occurring thread or bunch shape genomic dna;

Centrifugal 3 minutes of 10,000r/min abandons supernatant liquor, and centrifuge tube is upside down on the clean thieving paper, guarantees that the DNA throw out is in pipe;

Adding mass concentration is 70% ethanol, 150 μ l, and vortex vibrated for 5 seconds, centrifugal 3 minutes of 10,000r/min, abandon supernatant liquor after, adding mass concentration again is 70% ethanol, 150 μ l, vortex vibrated for 5 seconds, centrifugal 3 minutes of 10,000r/min abandons supernatant liquor;

Centrifuge tube is upside down on the clean thieving paper stopped 5 minutes, guarantee that the DNA throw out is in pipe;

Centrifuge tube is at room temperature left standstill, be dried to all evaporate clean (at least 5 minutes);

Add 200 μ l elution buffer TB again, vortex vibration 5 seconds, 65 ℃ were heated 10 minutes to 1 hour, during heating, flicked hydrotropy for several times, namely got Whole Blood Genomic DNA.

1.4PCR amplification and enzyme are cut evaluation

Human gene group DNA 100ng (preparation method is as mentioned above), each primer 0.2 μ mol/L, 1 * PCRmix, the sterilization distilled water is supplied 25 μ l reaction systems.The PCR reaction conditions is 94 ℃ of pre-sex change 5min at first, and 94 ℃ of sex change 30s get 59 ℃ of annealing 20s according to primer Tm value then, and 72 ℃ are extended 20s, and totally 30 circulations are extended 10min for back 72 ℃.Behind the pcr amplification, get PCR product 10 μ l, add 5U restriction endonuclease and 2 μ l10 * enzyme cutting buffering liquids and form 20 μ l reaction systems with the sterilization distilled water, after enzyme is cut 8h in 37 ℃ of water-baths, observe imaging in the gel imaging instrument behind the electrophoresis, the results are shown among Fig. 1.

2 results

2.1 enzyme is cut the result

Above-mentioned enzyme is cut product under 3% sepharose 8V/cm condition, electrophoresis 40 minutes, the evaluation of under ultraviolet lamp, taking pictures, wherein, the 133bp segment occurring after the amplification of MTHFR site (is as the criterion with upstream sequence, as follows), 133bp, 114bp (downstream sequence is compared upstream sequence and produced two strand bases of sticky end minimizing because the MspI enzyme is cut), three kinds of segments (wherein can not see in the 19bp electrophorogram) of 19bp (downstream sequence is compared upstream sequence increases by two strand bases because the MspI enzyme is cut the generation sticky end) can appear after the MspI enzyme is cut.

As shown in Figure 1.Enzyme is cut the back genotype and judged: the GG type is 114bp, and the AG type has 133bp and two kinds of fragments of 114bp, and the AA type is 133bp.

2.2MTHFR the gene polymorphic result identifies

Detected result finds that GG type, AG type and three kinds of genotype of AA type appear in MTHFR.In addition, to the evaluation of checking order of the polymorphic all types of PCR products of MTHFR, sequencing result and expected results are in full accord.

It is polymorphic that embodiment 2 peripheral blood whole blood samples are measured people's mthfr gene rs2274976:

Basic identical with the step of embodiment 1, just the forward primer that adopts of PCR reaction is SEQ ID NO:1, and reverse primer is SEQ ID NO:7, and annealing temperature is 59 ℃, and the restriction enzyme that adopts is HpaII (NEB company).

The result:

The amplified production sequence that obtains following (159bp, SEQ ID NO:8):

ctttgccctg tggattgacc rgtggggaaa gctgtatgag gaggagtccc cgtcccgcac 60

catcatccag tacatccacg acaactactt cctggtcaac ctggtggaca atgacttccc 120

actggacaac tgcctctggc aggtggtgga agacacatt 159

Can form 140bp product (SEQ ID NO:9) after enzyme is cut during the polymorphic G of the containing allelotrope of MTHFR, sequence is as follows:

crgtggggaa agctgtatga ggaggagtcc ccgtcccgca ccatcatcca gtacatccac 60

gacaactact tcctggtcaa cctggtggac aatgacttcc cactggacaa ctgcctctgg 120

caggtggtgg aagacacatt 140

Can form 19bp product (SEQ ID NO:10, its sequence with SEQ ID NO:6 is consistent) after enzyme is cut during the polymorphic G of the containing allelotrope of MTHFR, sequence is as follows:

ctttgccctg tggattgac 19

Above-mentioned enzyme is cut product under 3% sepharose 6V/cm condition, electrophoresis 50 minutes, the evaluation of under ultraviolet lamp, taking pictures, wherein, the 159bp segment appears after the MTHFR site amplification, through enzyme cut, 159bp appears in electrophoresis, three kinds of segments of 140bp, 19bp, genotype basis for estimation 159 and 140bp fragment have or not judgement: the GG type is 140bp, the AG type has 159bp and two kinds of fragments of 140bp, and the AA type is 159bp.

It is polymorphic that embodiment 3 peripheral blood blood clot samples are measured people's mthfr gene rs2274976:

Basic identical with the step of embodiment 1, just adopt in the following method and extract DNA as DNA to be measured from peripheral blood blood clot sample.In addition, the forward primer that the PCR reaction is adopted is SEQ NO:11 (tttgccctgtggattgacc), and reverse primer is SEQ NO:12 (tctcgcattc tgggtggg), and annealing temperature is 58 ℃.Restriction enzyme is MspI (NEB company).

Extract DNA:

General blood collection tube is gathered human peripheral 400 μ l, and genomic dna is human gene group DNA's template to be measured in the use TIANGEN RelaxGene Rlood DNASystem of the company poba gene group DNA extraction system extraction peripheral blood blood clot;

Treat that serum is separated out the back separation of serum in the heparin tube, follow these steps to then carry out:

Sludged blood in the above-mentioned heparin tube is ground to the homogenate shape is placed in the centrifuge tube, add 750 μ l cell pyrolysis liquid CL, put upside down mixing 5 times;

Centrifugal 20 seconds of 10,000r/min abandons supernatant liquor;

Add 150 μ l damping fluid mixed solutions, the damping fluid mixed solution is to be mixed and made at 100: 1 with volume ratio by damping fluid FG and Proteinase K, and vortex vortex mixer mixing to solution does not have agglomerate;

Then, at 65 ℃ of water-bath 10min, put upside down mixing therebetween 6 times;

The adding mass concentration is 99.9% Virahol 150 μ l, puts upside down abundant mixing to occurring thread or bunch shape genomic dna;

Centrifugal 3 minutes of 10,000r/min abandons supernatant liquor, and centrifuge tube is upside down on the clean thieving paper, guarantees that the DNA throw out is in pipe;

Add mass concentration and be 70% ethanol 150 μ l, vortex vibrated for 5 seconds, centrifugal 3 minutes of 10,000r/min, abandon supernatant liquor after, add mass concentration again and be 70% ethanol 150 μ l, vortex vibrated for 5 seconds, centrifugal 3 minutes of 10,000r/min abandons supernatant liquor;

Centrifuge tube is upside down on the clean thieving paper stopped at least 5 minutes, guarantee that the DNA throw out is in pipe;

Centrifuge tube is at room temperature left standstill, be dried to all evaporate clean (at least 5 minutes);

Add 200 μ l elution buffer TB, vortex vibration 5 seconds, 65 ℃ were heated 10 minutes to 1 hour, during heating, flicked hydrotropy for several times, namely got genomic dna in the peripheral blood blood clot.

The result:

The amplified production sequence that obtains following (192bp, SEQ ID NO:13):

tttgccctgt ggattgaccr gtggggaaag ctgtatgagg aggagtcccc gtcccgcacc 60

atcatccagt acatccacga caactacttc ctggtcaacc tggtggacaa tgacttccca 120

ctggacaact gcctctggca ggtggtggaa gacacattgg agcttctcaa caggcccacc 180

cagaatgcga ga 192

Can form 174bp product (SEQ ID NO:14) after enzyme is cut during the polymorphic G of the containing allelotrope of MTHFR, sequence is as follows:

crgtggggaa agctgtatga ggaggagtcc ccgtcccgca ccatcatcca gtacatccac 60

gacaactact tcctggtcaa cctggtggac aatgacttcc cactggacaa ctgcctctgg 120

caggtggtgg aagacacatt ggagcttctc aacaggccca cccagaatgc gaga 174

Can form 18bp product (SEQ ID NO:15) after enzyme is cut during the polymorphic G of the containing allelotrope of MTHFR, sequence is as follows:

tttgccctgt ggattgac 18

Above-mentioned enzyme is cut product under 3% sepharose 5V/cm condition, electrophoresis 60 minutes, the evaluation of under ultraviolet lamp, taking pictures, wherein, the 192bp segment appears after the MTHFR site amplification, through enzyme cut, 192bp appears in electrophoresis, three kinds of segments of 174bp, 18bp, genotype basis for estimation 192 and 174bp fragment have or not judgement: the GG type is 174bp, the AG type has 192bp and two kinds of fragments of 174bp, and the AA type is 192bp.

It is polymorphic that embodiment 4 human lung tissue's samples are measured people's mthfr gene rs2274976

Basic identical with the step of embodiment 1, just adopt in the following method and extract DNA as DNA to be measured from lung tissue sample.

Use the excision lung tissue, it is human gene group DNA's template to be measured that phenol-chloroform method extracts the lung tissue genomic dna:

The lung tissue piece is thawed, and with physiological saline flush away blood stains, clip 0.1g, glass homogenizer grind first centrifuge tube of putting into 1.5ml after the broken lung tissue;

In first centrifuge tube, add 1ml DNA extraction damping fluid (10mmol/LtrisCl ₂, 0.1mol/LEDTA, 20 μ g/ml Pancreatic RNases, 0.5% sodium lauryl sulphate) mixing adds 50 μ l mass concentrations and is 10% sodium lauryl sulphate again, and concentration is the Proteinase K 5.0 μ l of 20mg/ml, fully behind the mixing, in 56 ℃ of insulation 5h, every 2h shakes 1 time;

Be placed into room temperature, add saturated phenol 500 μ l, put upside down mixing, the centrifugal 10min of 10000r/min, water phase separated and organic phase are drawn second centrifuge tube that water that the upper strata contains nucleic acid places 1.5ml;

The isopyknic phenol of the water that contains nucleic acid in second centrifuge tube in adding and the pipe: chloroform: the mixed solution of primary isoamyl alcohol, wherein, phenol: chloroform: the mixed solution of primary isoamyl alcohol is by phenol: chloroform: primary isoamyl alcohol is mixed and made into volume ratio at 25: 24: 1, put upside down mixing, centrifugal 10 minutes of 10000r/min gets supernatant liquid and transfers in the 3rd centrifuge tube of 1.5ml;

In the 3rd centrifuge tube, add and the isopyknic chloroform of liquid in pipe: primary isoamyl alcohol is put upside down mixing with the mixed solution that volume ratio is mixed and made at 24: 1, and centrifugal 10 minutes of 10000r/min gets supernatant liquid in the 4th centrifuge tube of 1.5ml;

Add the dehydrated alcohol of-20 ℃ of precoolings of 2.5 times of volumes of clear liquid in the pipe in the 4th centrifuge tube, deposit D NA gets the DNA throw out;

With the DNA throw out, centrifugal 10 minutes of 12000r/min abandons ethanol;

The mass concentration that adds-20 ℃ again is 75% washing with alcohol, and centrifugal 5 minutes of 10000r/min removes ethanol, and is dry under 55 ℃, gets dry DNA throw out;

Add the dry DNA throw out of 100 μ l sterilization distilled water dissolving ,-20 ℃ of preservations namely get the lung tissue genomic dna, and are standby.

The result:

The gained enzyme is cut product under 3% sepharose 4V/cm condition, electrophoresis 70 minutes, the evaluation of under ultraviolet lamp, taking pictures, wherein, the 133bp segment appears after the MTHFR site amplification, through enzyme cut, 133bp appears in electrophoresis, three kinds of segments of 114bp, 19bp, genotype basis for estimation 133bp and 114bp fragment have or not judgement: the GG type is fragment of 114bp, the AG type has 133bp and two kinds of fragments of 114bp, and the AA type is fragment of 133bp; Identify that through order-checking the result of sequencing result and use prior art detection gained is identical.

From above-described embodiment as can be seen, the present invention is directed to the shortcoming of using the polymorphic rs2274976 of PCR-RFLP identification of M THFR, namely must use identification former polymorphic near the restriction endonuclease of sequence, factor such as the restriction endonuclease choice is little, and is expensive.The present invention overcomes this shortcoming by the mispairing primer that design comprises restriction enzyme site.This method can be thought the special applications of PCR-RFLP, its principle is the base alternative case design PCR primer according to the single base mutation site, wherein a primer is according to the contiguous sequences Design in mutational site, the artificial base mismatch of introducing, make a kind of mutant of primer 3 ' end and single base mutation form a restriction enzyme site behind pcr amplification, its PCR product can be analyzed with the method for similar PCR-RFLP.Because this method has been used primer 3 ' end mispairing technology, the PCR product carries out can carrying out genotypic evaluation work behind the restriction enzyme digestion and electrophoresis, therefore in practice, has very big handiness, and detection method is simple, cost is low, enzyme is cut the result and is easily differentiated, and is a kind of better method of carrying out genotype identification.The present invention redesigns this polymorphic sequence of new primer amplification by improving experimental technique, makes near sequence change this gene polymorphic in the amplified production, can use other restriction endonucleases and identify, thereby can select more cheap restriction endonuclease to finish detection.

The present invention is described in detail, it is to be noted, above-mentioned only is embodiments of the invention, be not that the present invention is done any pro forma restriction, those skilled in the art are not in breaking away from the technical solution of the present invention scope, when utilizing above-mentioned disclosed technology contents to modify the technique effect that acquisition is equal to, be the content that does not break away from technical solution of the present invention in every case, and then still belong in the scope of technical solution of the present invention.

Claims (1)

1. test kit for the identification of people's mthfr gene polymorphism rs2274976, comprise: forward primer and the reverse primer of sequence near the amplification people mthfr gene polymorphism rs2274976 site, wherein, described forward primer is adjacent with the polymorphic base of rs2274976 at its 3 ' terminal bit base last, penultimate is base mismatch C, in order to form the CCAGT structure with polymorphic A allelotrope in amplified production, described test kit also comprises restriction enzyme BsrI; And described test kit also comprises the specification sheets of identifying for implementing,

Wherein said forward primer is that SEQ ID NO:1 and reverse primer are SEQ ID NO:2; Perhaps described forward primer is that SEQ ID NO:1 and reverse primer are SEQ ID NO:7; Perhaps described forward primer is that SEQ NO:11 and reverse primer are SEQ NO:12.

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