CN102660639A - Kit for identifying polymorphic rs2274976 of human MTHFR gene by BsrFI - Google Patents

Abstract

The invention discloses a kit for identifying polymorphic rs2274976 of a human MTHFR gene by BsrFI. 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 basic group 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 an ACCGGT structure with polymorphic G alleles. The forward 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 selected from BsrFI, BsaWI and AgeI 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 BsrFI identifier's mthfr gene polymorphum 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 polymorphum 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 SNP.More specifically, the present invention relates to the method for identifier's mthfr gene polymorphum rs2274976.

Background technology

SNP (SNP) is meant the mutant dna sequence that the change of single Nucleotide causes, comprises the forms such as displacement, insertion and disappearance of single base.SNP has been widely used in the location of genetic linkage analysis, association analysis and the diseases predisposing gene of simple and complex disease at present, 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 one type of 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, produce segmental size and number thereby change enzyme cutting back.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 detects through 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 costs an arm and a leg.

5; 10-MTHFR (5; 10-methylenetetrahydrofolate reductase MTHFR) is 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.Discover that at present this proteic genetic flaw of coding and arteriosclerotic vascular obstruction disease 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 possibly 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 in snp database, is numbered.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; The costing an arm and a leg of this restriction endonuclease (can be with reference to the table 1 of back) about the reference price of part restriction endonuclease; Influenced the expense of experiment, be difficult in the laboratory, popularize and use.And because the vice proper 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 polymorphum rs2274976 that use range is wide, and it may further comprise the steps:

A) human gene group DNA to be measured is provided;

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

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

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

Can near the sequence the SNP be changed into through the base mismatch on the primer through method of the present invention can be by the sequence of predetermined restriction enzyme identification; 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; After the mispairing in the PCR product polymorphic near sequence change to ACCGGT or ACCAGT by AGCGGT or AGCAGT (wherein second base be base mismatch; The 4th base is the A/G polymorphic site), so G allelotrope fragment can be identified the restriction enzyme of CCGG sequence or discern the restriction enzyme identification (being that G allelotrope fragment can be cut by restriction endonuclease) of ACCGGT sequence in the amplified production.After the same mispairing in the PCR product polymorphic near sequence change to CCAGT or CCGGT by GCGGT or GCAGT (wherein first base be a base mismatch; 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, can know that A/G is polymorphic in the gene original series can be by preceding four kinds of restriction endonucleases identification in the table 1 through sequential analysis.As the polymorphic site front second bit base G being replaced with C through base mispairing PCR; Then this polymorphicly contains the restriction endonuclease identification (like MspI, HpaII) that the CCGG sequence can be identified the CCGG sequence when the G allelotrope behind pcr amplification, contains the restriction endonuclease identification (like 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 discern the restriction enzyme of same DNA sequence) identification equally, and A allelotrope can not cut.Through 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 discern (like 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.

Several kinds of restriction endonuclease recognition sequences of table 1 NEB 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 influence of primer to sensitivity, specific degree and the amplification efficiency of pcr amplification mainly considered in the design of forward and reverse primer.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 in amplified production, to form ACCGGT with polymorphic G allelotrope; The 4th bases G is polymorphic allelotrope) structure; Form CCAGT (first base C is a 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 is near (being positioned at the polymorphic site upper reaches because of forward primer of being determined by reverse primer; Its position is roughly definite); Usually amplified production length is between the 100-200bp, so promptly 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 pairing 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 uv lamp, taking pictures.

In another preferred embodiment of the present invention, said restriction enzyme is selected from MspI, HpaII, BsrFI, BsrI, BsaWI, AgeI and their isoschizomers, and more preferably said 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 does not receive special restriction, as long as can obtain amplified production.Significant parameter-the 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, ultimate demand is confirmed with experiment.The PCR time of extending confirms 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 implementation of the present invention, the pcr amplification condition that is adopted 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 present invention does not have special restriction.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.Implement the genomic dna that preferably extracts usually for ease from blood.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 from body fluid and/or tissue, extracting DNA 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; The test kit of a kind of identifier's of being used for mthfr gene polymorphum rs2274976 also is provided in the present invention; Comprise: the forward primer and the reverse primer of sequence near the amplification people mthfr gene polymorphum rs2274976 site, wherein, said forward primer is adjacent with the polymorphic base of rs2274976 at its 3 ' terminal bit base last; Penultimate is base mismatch C; So that in amplified production, form the ACCGGT structure with polymorphic G 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 the restriction enzyme that is selected from BsrFI, BsaWI, AgeI; And said test kit also comprises and is used to the specification sheets implementing to identify.

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 said test kit, like this can be conveniently to the detection of amplified production.Further, preferred said restriction enzyme is BsrFI.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 to specification sheets of implementing to identify 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

Through embodiment of the present invention technical scheme of the present invention is described in detail below.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 notion 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 said 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.

Be described in detail in the face of each key step down

(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, confirm 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, confirm the 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; After the mispairing in the PCR product polymorphic near sequence change to ACCGGT or ACCAGT by AGCGGT or AGCAGT (wherein the 4th base is that A/G is polymorphic), so G allelotrope fragment can be identified the restriction enzyme of CCGG sequence or discern the restriction enzyme identification (being that G allelotrope fragment can be cut by restriction endonuclease) of ACCGGT sequence in the amplified production.In the time should polymorphicly being A allelotrope, 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 behind the pcr amplification discerns.Judge polymorphic genotype according to fragment incision situation;

According to forward primer sequence and reverse primer sequence synthesized primer thing, synthetic said 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.With synthetic forward, reverse primer dilution is that 10 μ mol/L concentration are subsequent use;

(2) preparation pcr amplification product

Pcr amplification uses the reaction system of 15-100 μ l to be: 2 * PCR MIX consumption is 7.5-50 μ l, the said human gene group DNA to be detected of 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 promptly 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 processed;

With the pcr amplification reaction system after 94-95 ℃ of preparatory 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, promptly 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 tgcUnderscore partly is forward, the pairing sequence of reverse primer in the 133bp sequence 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: the pcr amplification product of 2-15 μ l, identification CCGG sequence or restriction enzyme 1-10U and the 10 * enzyme cutting buffering liquid discerning the ACCGGT sequence or discern the CCAGT sequence account for 1/10 of TV; The sterilization distilled water is supplemented to 10-30 μ l, and mixing gets enzyme and cuts system; Enzyme was cut system in 37 ℃ of water-bath 4-16 hours, promptly get enzyme and cut product;

The restriction enzyme of said identification CCGG sequence is restriction endonuclease MspI, HpaII and isoschizomers thereof; The restriction enzyme of said identification ACCGGT sequence is restriction endonuclease BsrFI, BsaWI, AgeI and isoschizomers thereof; The restriction enzyme of said 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 pairing sequence of reverse primer in the fragment 114bp sequence, and 2bp represents at the place 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, confirm gene pleiomorphism:

Enzyme is cut product in 3% sepharose under the 3-8V/cm condition, electrophoresis 40-80 minute, the evaluation of under uv lamp, taking pictures; Wherein, for the different gene type, 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 133bp and 114bp segmental having or not: 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 sequencing result is identical with the result that the use prior art detects gained.

Be some embodiment of 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 appearance (PE company), miniature electrophoresis chamber (Pharmacia Biotech, EPS1000), Gel Doc 2000 gel imaging appearance (Bio-RAD company).

The order-checking of PCR product is given birth to worker's biotechnology ltd by Shanghai and is accomplished.

1.2 sequence is searched and design of primers

Search mthfr gene sequence and SNP information in the NCBI website, confirm 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 Blood DNA System of TIANGEN company poba gene group DNA extraction system (staple 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; 10, centrifugal 20 seconds of 000r/min abandons supernatant;

In centrifuge tube, add the damping fluid mixed solution of 150 μ l, 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;

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

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, promptly got Whole Blood Genomic DNA.

1.4PCR amplification and enzyme are cut evaluation

Human gene group DNA 100ng (preparation method is as stated), 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 preparatory 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 appearance behind the electrophoresis, the result is 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 uv lamp, taking pictures; Wherein, 133bp segment (being as the criterion with upstream sequence, as follows) occurs after the amplification of MTHFR site, 133bp, 114bp (downstream sequence is compared upstream sequence owing to the MspI enzyme is cut two strand bases of generation sticky end minimizing), three kinds of segments (wherein can not see in the 19bp electrophorogram) of 19bp (downstream sequence is compared upstream sequence and produced two strand bases of sticky end increase because the MspI enzyme is cut) after the MspI enzyme is cut, can occur.

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 adopted 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 is adopted is HpaII (a NEB company).

The result:

The amplified production sequence that is obtained is (159bp, SEQ ID NO:8) as follows:

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 following:

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 following:

ctttgccctg?tggattgac 19

Above-mentioned enzyme is cut product under 3% sepharose 6V/cm condition, electrophoresis 50 minutes, the evaluation of under uv lamp, taking pictures; Wherein, The 159bp segment appears after the MTHFR site amplification, through enzyme cut, 159bp appears in electrophoresis, 140bp, three kinds of segments of 19bp, genotype basis for estimation 159 and 140bp be segmental to 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, the method below just adopting is extracted 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 (tttgccctgt ggattgacc), and reverse primer is SEQ NO:12 (tctcgcattc tgggtggg), and annealing temperature is 58 ℃.Restriction enzyme is MspI (a 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 Blood 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;

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

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;

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

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, promptly got genomic dna in the peripheral blood blood clot.

The result:

The amplified production sequence that is obtained is (192bp, SEQ ID NO:13) as follows:

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 following:

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 following:

tttgccctgt?ggattgac 18

Above-mentioned enzyme is cut product under 3% sepharose 5V/cm condition, electrophoresis 60 minutes, the evaluation of under uv lamp, taking pictures; Wherein, The 192bp segment appears after the MTHFR site amplification, through enzyme cut, 192bp appears in electrophoresis, 174bp, three kinds of segments of 18bp, genotype basis for estimation 192 and 174bp be segmental to 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, the method below just adopting is extracted 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 saline water 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) and mixing; Add 50 μ l mass concentrations again and be 10% sodium lauryl sulphate, 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;

The absolute ethyl alcohol that in the 4th centrifuge tube, adds-20 ℃ of precoolings of 2.5 times of volumes of clear liquid in the pipe, 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 exsiccant DNA throw out;

Add 100 μ l sterilization distilled water dissolving exsiccant DNA throw out ,-20 ℃ of preservations promptly get the lung tissue genomic dna, and are subsequent use.

The result:

The gained enzyme is cut product under 3% sepharose 4V/cm condition, electrophoresis 70 minutes, the evaluation of under uv lamp, taking pictures; Wherein, The 133bp segment appears after the MTHFR site amplification, through enzyme cut, 133bp appears in electrophoresis, 114bp, three kinds of segments of 19bp, genotype basis for estimation 133bp and 114bp be segmental to 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 sequencing result is identical with the result that the use prior art detects gained.

Can find out from the foregoing description, the present invention is directed to the shortcoming of using the polymorphic rs2274976 of PCR-RFLP identification of M THFR, promptly must use identification former polymorphic near the restriction endonuclease of sequence, the restriction endonuclease choice is little, factor such as cost an arm and a leg.The present invention overcomes this shortcoming through the mispairing primer that design comprises restriction enzyme site.This method can be thought the application specific IC 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 makes a kind of mutant of primer 3 ' end and single base mutation behind pcr amplification, form a restriction enzyme site, and 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, have very big handiness, and detection method is simple, cost is low; Enzyme is cut the result and is prone to differentiate, and is a kind of better method of carrying out genotype identification.The present invention designs this polymorphic sequence of new primer amplification again through improving experimental technique, makes near sequence change this gene polymorphic in the amplified production, can use other restriction endonucleases and identify, detects thereby can select more cheap restriction endonuclease to accomplish.

The present invention is described in detail; It is to be noted that above-mentioned only is embodiments of the invention, is not that the present invention is done any pro forma restriction; Those skilled in the art are not in breaking away from technical scheme scope of the present invention; When above-mentioned disclosed technology contents capable of using modifies the technique effect that acquisition is equal to, be the content that does not break away from technical scheme of the present invention in every case, and then still belong in the scope of technical scheme of the present invention.

Claims (5)

1. test kit that is used for identifier's mthfr gene polymorphum rs2274976; Comprise: the forward primer and the reverse primer of sequence near the amplification people mthfr gene polymorphum rs2274976 site; Wherein, Said forward primer is adjacent with the polymorphic base of rs2274976 at its 3 ' terminal bit base last, and penultimate is base mismatch C, so that in amplified production, form the ACCGGT structure with polymorphic G allelotrope; Described forward primer is SEQ ID NO:1 or SEQ NO:11, and reverse primer is SEQ ID NO:2, SEQ IDNO:7 or SEQ NO:12; Described test kit also comprises the restriction enzyme that is selected from BsrFI, BsaWI, AgeI; And said test kit also comprises and is used to the specification sheets implementing to identify.

2. according to the test kit of claim 1, wherein restriction enzyme is BsrFI.

3. according to the test kit of claim 1, wherein said forward primer is SEQ ID NO:1, and reverse primer is SEQ ID NO:2.

4. according to the test kit of claim 1, wherein said forward primer is SEQ ID NO:1, and reverse primer is SEQID NO:7.

5. according to the test kit of claim 1, wherein said forward primer is SEQ ID NO:11, and reverse primer is SEQ ID NO:12.

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