CN102943120A - Method for detecting MTHFR (methylenetetrahydrofolate reductase) gene polymorphism - Google Patents

Abstract

The invention relates to application of pyrophosphoric acid sequencing technology in gene polymorphism detection, particularly a method for detecting MTHFR (methylenetetrahydrofolate reductase) gene polymorphism, an amplification primer of MTHFR gene, a sequencing primer for detecting MTHFR gene polymorphism and a kit containing the primers. The method provided by the invention is simple and quick to operate, has the advantages of favorable specificity, high sensitivity, high accuracy, high flux and low detection cost, and has wide application prospects.

Description

A kind of method that detects the mthfr gene polymorphism

Technical field

The invention belongs to biology field, relate to the tetra-sodium sequencing technologies in the application of gene pleiomorphism context of detection, be specifically related to a kind of method that detects the mthfr gene polymorphism, also relate to a kind of mthfr gene amplimer, detect the sequencing primer of mthfr gene polymorphism and contain their test kit.

Background technology

Single nucleotide polymorphism (Single Nucleotide Polymorphism, SNP) refers on genomic level by the caused dna sequence polymorphism of single nucleotide diversity.Generally speaking, SNP refers to that variation frequency is greater than 1% single nucleotide variations.General per 1000 bases just have a SNP in human genome, and the chances are 3 * 10 for the SNP total amount on the human genome ⁶Individual.At present, SNP becomes third generation genetic marker, and its each side such as early stage risk assessment, early diagnosis, prevention and treatment in disease has critical function and using value.

MTHFR(methylene radical four oxygen folic acid reductases) gene codified Methylene tetrahydrofolate reductase albumen, the Main Function of this enzyme are 5,10-CH2-THFA to be converted into the 5-methyltetrahydrofolate salt with biological function in the folic acid metabolism path.5-methyltetrahydrofolate salt can further enter the methyl transmission path, and the interprocedual ground connection that again methylates by homocysteine provides methyl and makes the homocysteine amount in the blood remain on a lower level for dna methylation and protein methylate.There is polymorphism in mthfr gene, can affect activity and the thermostability of Methylene tetrahydrofolate reductase.The polymorphism in mthfr gene 677 sites can cause that the thermolability of this enzyme increases, and enzymic activity descends, and causes homotype semicystinol concentration investigating rising in the blood.

The technical study of relevant SNP detection is very extensive both at home and abroad at present, and the main method that detects for SNP is as follows:

(1) SSCP technology

Being the single strand conformation polymorphism detection technique, is to make it to unwind and guarantee under the strand state through pyroprocessing under denaturing agent or the low ion concns through the purpose fragment of pcr amplification, then electrophoresis in certain density non-denaturing polyacrylamide gel.The single stranded DNA of equal length can be because of its order or single base difference, and formed space conformation will be different, and its mobility speed in gel is different, thereby demonstrates the difference of banding pattern.

(2) RFLP technology

It is the restriction fragment length polymorphism analytical technology, the party's ratio juris is that the variation of base can cause the disappearance of existing restriction enzyme site or the appearance of new restriction enzyme site, thereby cause Different Individual with same digestion with restriction enzyme the time, difference appears in dna fragmentation length.

(3) TaqMan probe technique

The TaqMan probe technique is to add a fluorescence labeling probe in conventional PCR reaction system, and 5 ' end of this probe and 3 ' end be mark report fluorophor and a cancellation fluorophor respectively.When probe was complete, the fluorescent signal of reporter group emission was absorbed by quenching group; During pcr amplification, if probe can with the complete annealing of template, the 5'-3' 5 prime excision enzyme activity of Taq enzyme is cut degraded with the probe enzyme so, make the report fluorophor separate with the cancellation fluorophor, thereby the fluorescence monitoring system can receive fluorescent signal.

(4) biochip technology

The principle of biochip technology is that the probe that will have specific base sequence is fixed on the special carrier, and testing gene is hybridized with the probe that fixes behind extraction, fluorescent mark, measures sequence to be measured according to intensity and the kind of fluorescence at last.

(5) Sanger sequencing technologies

Sanger sequencing technologies principle is to utilize archaeal dna polymerase with single stranded DNA as template, to synthesize accurately DNA complementary strand.This endonuclease capable is with 2 ', 3 '---and dideoxyribonucleoside triphosphate is made substrate and it is aggregated to 3 ' end of newborn oligonucleotide chain, thereby stops its extension.Containing template, primer, dATP, dTTP, dGTP, in dCTP and a kind of ddNTP reaction tubes, archaeal dna polymerase synthesizes a series of take the new chain of ddNTP as the different lengths of 3 ' end, after electrophoresis and radioautograph, can promptly read dna sequence dna.Its testing process comprises plasmid extraction or PCR product purification, template quality evaluation, template is quantitative and dna sequencing reaction.

(6) tetra-sodium sequencing technologies

Along with the development of science and technology, the research that is applied as SNP and the clinical detection of tetra-sodium sequencing technologies provide new technology platform.The tetra-sodium sequencing technologies is a brand-new dna sequencing technology, and this technology is by the enzyme cascade chemiluminescence reaction in 4 kinds of enzymatic same reaction systems.The principle of this technology is:

The 1st step: sequencing primer is combined with strand PCR product template, then add enzyme mixture (comprising 4 kinds of enzymes, archaeal dna polymerase, ATP sulfurylase, luciferase and apyrase) and substrate mixture (comprising adenosine-5'-phosphinylidyne sulfuric acid (APS) and fluorescein);

The 2nd step: add dNTPs(in the reaction system and only add a kind of dNTP at every turn, comprise dATP, dTTP, dCTP or dGTP), if this dNTP can just with the template base pairing, then it can be under the effect of archaeal dna polymerase, add 3 ' end of sequencing primer to, discharge simultaneously the tetra-sodium (PPi) of equimolecular quantity.

The 3rd step: under the effect of ATP sulfurylase, the PPi of generation can with APS in conjunction with forming ATP; Under the catalysis of luciferase, the ATP of generation again can with fluorescein in conjunction with forming oxyluciferin, produce simultaneously visible light.Can obtain a special detected peaks by ccd video camera, the height of peak value is directly proportional with the nucleotide base number that synthesizes.

The 4th step: the ATP of unconjugated dNTPs and generation degrades under the effect of bisphosphatase in the reaction system.When degraded was finished, another kind of dNTP just can be added in the reaction system.

The 5th step: the interpolation of dNTPs is carried out according to order.Because ɑ-sulfo-deoxidation Triphosaden (dATP ɑ S) can effectively be used by archaeal dna polymerase and can not identified by luciferase, so substitute natural dATP with dATP ɑ S in this system.Along with program continue carry out, synthesize complementary DNA chain and can determine dna nucleotide sequence according to the peak value figure that obtains.

The tetra-sodium sequencing technologies is simple to operate fast, specificity is good, highly sensitive, accuracy is high, flux is high, testing cost is low, for single nucleotide polymorphism research and clinical detection provide desirable technology platform.

The invention provides a kind of method of utilizing the tetra-sodium sequencing technologies to carry out mthfr gene C677T polymorphic detection, and designed specificity amplification primer and tetra-sodium sequencing primer, this amplimer mthfr gene nucleic acid fragment that can from genomic dna, increase.Method of the present invention is utilized the tetra-sodium sequencing technologies, and the mthfr gene polymorphism in the biological specimen is detected.

Summary of the invention

A first aspect of the present invention provides a kind of method that detects the mthfr gene polymorphism, and it may further comprise the steps:

Use the amplification step of the amplimer amplification mthfr gene shown in SEQ ID NO.1 and/or the SEQ ID NO.2.Preferably, described primer is to can be by biotin labeling; Further preferably, the primer shown in the SEQ ID NO.1 is by biotin labeling.

Forward primer F:5'-TGACTGTCATCCCTATTGGCAGGTTACCCCAAAGGCCACCCC-3'(SEQ ID NO.1);

Reverse primer R:5'-GATCCCGGGGACGATGGGGCAAGTGATGCCCATG-3'(SEQ ID NO.2).

The method of detection mthfr gene polymorphism of the present invention, it can may further comprise the steps further:

(1) amplification step of the amplification of the amplimer shown in use SEQ ID NO.1 and/or SEQ ID NO.2 mthfr gene;

(2) amplified production that step (1) is obtained becomes the step of single stranded DNA template;

(3) step that the single stranded DNA template of using the sequencing primer shown in the SEQ ID NO.3 that step (2) is obtained is carried out the tetra-sodium order-checking.

Sequencing primer P:5'-GAAAAGCTGCGTGATGATGAAATCG-3'(SEQ ID NO.3)

In a preferred embodiment of the present invention, the method for described detection mthfr gene polymorphism, wherein said amplification condition is: 95 ℃ of denaturation 15min; 94 ℃ of sex change 30s, 60 ℃ of annealing 30s, 72 ℃ are extended 30s, totally 45 circulations; 72 ℃ are extended 10min.

In another preferred embodiment of the present invention, the method of described detection mthfr gene polymorphism, wherein, step (2) is that the amplified production that step (1) obtains is added binding buffer solution and the coated magnetic bead of Streptavidin, at room temperature hatch certain hour, preferred 15 seconds; PCR product after magnetic bead is combined washs certain hour (preferably washing 5 seconds), sex change liquid (QIAGEN in ethanolic soln (being preferably 70% ethanolic soln), article No. 979007) hatches certain hour (preferred 5 seconds), elutriant (QIAGEN in, article No. 979008) hatches certain hour (preferred 10 seconds) in, obtain the single stranded DNA template;

Preferably, the method for claim 2, wherein, step (3) is further comprising the steps of,

The single stranded DNA template that step (2) is obtained changes the annealing buffer (QIAGEN that contains the sequencing primer shown in the SEQ ID NO.3 over to, article No. 979009) in, (for example 80 ℃) hatch certain hour (for example 2 minutes) under suitable temperature condition, be cooled to room temperature, obtain the strand sequencing template;

Preferably, when carrying out the tetra-sodium order-checking in the step (3), carry out tetra-sodium sequencing reaction: TAGACTCTGCACGTA according to following base allocation order.

In a preferred embodiment of the present invention, the method for detection mthfr gene polymorphism of the present invention is for detection of the polymorphism of mthfr gene C677T; Preferably, described mthfr gene derives from people's whole blood or human oral epithelial cells.

A second aspect of the present invention provides a kind of primer for mthfr gene amplification pair, and its sequence is shown in SEQ ID NO.1 and SEQ ID NO.2, and preferably, described primer is to can be by biotin labeling.

A third aspect of the present invention provides a kind of sequencing primer for detection of the mthfr gene polymorphism, and its sequence is shown in SEQ ID NO.3.

Sequencing primer shown in amplimer shown in SEQ ID NO.1 and the SEQ ID NO.2, the SEQ ID NO.3 all can be according to the conventional synthetic method preparation of this area.It is synthetic by Invitrogen company to use above-mentioned primer in the embodiments of the invention.

A fourth aspect of the present invention provides a kind of cdna amplification kit, wherein contains the described primer of second aspect present invention pair.

A fifth aspect of the present invention provides a kind of detection kit, it contains the described primer of second aspect present invention pair, with the described sequencing primer of third aspect present invention, with optional other required reagent of sequencing reaction, preferred other reagent are the required reagent of tetra-sodium sequencing reaction, the for example used enzyme mixture (comprising 4 kinds of enzymes, archaeal dna polymerase, ATP sulfurylase, luciferase and apyrase) of tetra-sodium sequencing reaction, substrate mixture (comprising adenosine-5'-phosphinylidyne sulfuric acid (APS) and fluorescein) etc.

A sixth aspect of the present invention provide the described primer of second aspect present invention to or the purposes of the described sequencing primer of the third aspect or the described detection kit of the described cdna amplification kit of fourth aspect or the 5th aspect, the purposes in detecting the mthfr gene polymorphism; Be preferred for detecting the polymorphism of mthfr gene C677T; Preferably, described mthfr gene derives from people's whole blood or human oral epithelial cells.

In the specific embodiment of the present invention, the method for described detection mthfr gene polymorphism, it may further comprise the steps:

A) adopt ordinary method to extract genomic dna, obtain extracting genome DNA liquid;

B) pcr amplification reaction

In the pcr amplification reaction system, add the extracting genome DNA liquid 2uL that obtains in the step a) and carry out pcr amplification, amplification system comprises: Auele Specific Primer is synthetic to SEQ ID No.1 and SEQID No.2(Invitrogen company) reaction density the time 0.2uM, 2 * PyroMark PCRMaster Mix 12.5uL, 10 * CoralLoad Concentrate 2.5uL(Pyromark PCRKit, QIAGEN, article No. 978703), residue adds water and complements to 25uL.The pcr amplification condition is: 95 ℃ of denaturation 15min, and 45 circulations (72 ℃ are extended 30s for 94 ℃ of sex change 30s, 60 ℃ of annealing 30s), 72 ℃ are extended 10min.

C) strand sequencing template preparation

The amplified production (15uL) that obtains in the step b) is added binding buffer solution (40uL, available from QIAGEN, article No. 979006) and the coated magnetic bead of Streptavidin (3uL is available from GE Healthcare, article No. 17-5113-01), at room temperature hatch 15 seconds; PCR product after magnetic bead is combined washs 5 seconds, sex change liquid (90mL in 70% ethanolic soln (110mL), available from QIAGEN, article No. 979007) hatch in 5 seconds, (110mL is available from QIAGEN for elutriant, article No. 979008) hatched in 10 seconds, above-mentioned amplified production becomes the single stranded DNA template.Again described single stranded DNA template is changed over to and contain the specific nucleotide sequencing primer (sequencing primer shown in the SEQID NO.3,0.4uM) annealing buffer (40uL, available from QIAGEN, article No. 979009) in, under 80 ℃, hatched 2 minutes, and be cooled to room temperature and obtain the strand sequencing template.

D) determine that the base allocation order is: TAGACTCTGCACGTA.

E) tetra-sodium sequencing reaction

Be ready to the above-mentioned used reagent of the used enzyme mixture of tetra-sodium sequencing reaction, substrate mixture and dNTPs(and all come from test kit-Pyromark Gold Q96Reagents, QIAGEN company, article No. 972804), add the reagent cabin, single stranded DNA template after the resulting separation and purification in the step c) is put into the tetra-sodium sequenator, carry out the tetra-sodium sequencing reaction according to the base allocation order that designs in the step d).

F) Analysis deterrmination of sequencing result

Instrument is analyzed sequencing data automatically, according to sequencing result Analysis deterrmination mthfr gene 677 loci polymorphism types.

The beneficial effect of the invention

The present invention utilizes pcr amplification and tetra-sodium sequencing technologies, and the mthfr gene polymorphism in the biological specimen is detected.The present invention has designed specificity amplification primer and tetra-sodium sequencing primer, this amplimer is amplification mthfr gene nucleic acid fragment from genomic dna specifically, and the tetra-sodium sequencing primer of using the present invention's design can make more Simple fast of mthfr gene polymorphic detection.Method of the present invention is simple to operate fast, and specificity is good, highly sensitive, accuracy is high, flux is high, testing cost is low, has broad application prospects.

Description of drawings

Fig. 1-1, Fig. 1-2 and Fig. 1 the-the 3rd, the capillary electrophoresis detection figure of mthfr gene plasmid amplification product among the embodiment 1,

Wherein Fig. 1-1 is wild plasmid amplified production detected result; Fig. 1-2 is mutant plasmid amplification product detected result; Fig. 1-3 is that wild-type and mutant equal proportion mixing plasmid are the amplified production detected result of template.

Fig. 1-4, Fig. 1-5 and Fig. 1-the 6th, the tetra-sodium sequencer map of mthfr gene plasmid amplification product among the embodiment 1,

Wherein Fig. 1-4 is wild plasmid tetra-sodium sequencer map; Fig. 1-5 is mutant plasmid tetra-sodium sequencer map; Fig. 1-6 is the tetra-sodium sequencer map that wild-type and mutant equal proportion are mixed plasmid.

Fig. 2-1, Fig. 2-2 and Fig. 2-the 3rd, the tetra-sodium sequencer map that among the embodiment 2 whole blood sample is detected.

Fig. 3-1, Fig. 3-2 and Fig. 3-the 3rd, the tetra-sodium sequencer map that among the embodiment 3 the mouth epithelial cells sample is detected.

Embodiment

Below in conjunction with embodiment embodiment of the present invention are described in detail, but it will be understood to those of skill in the art that the following example only is used for explanation the present invention, and should not be considered as limiting scope of the present invention.Unreceipted actual conditions person among the embodiment carries out according to the condition of normal condition or manufacturers's suggestion.The unreceipted person of production firm of agents useful for same or instrument, being can be by the conventional products of commercial acquisition.

The mensuration of embodiment 1MTHFR gene plasmid sequence

The structure of step a) plasmid

Plasmid construction work is finished by TAKARA company, and used carrier is pMD18-T carrier (available from TAKARA, article No. D101A).This plasmid insertion sequence is that mthfr gene (NCBIReference Sequence:NC_000001.10) is from the sequence fragment in 9601-10000 site.Wherein wild plasmid contains mthfr gene 677C sequence, and the mutant plasmid contains mthfr gene 677T sequence.

The step b) plasmid extraction

Adopt QIAGEN Plasmid Mini Kit(available from QIAGEN, article No. 12123) carry out the extraction of plasmid DNA, concrete operation step is with reference to shop instruction.

Step c) gene fragment amplification

Adopt PyroMark PCR Kit(available from QIAGEN, article No. 978703)

PCR System 9700(AB) carries out the amplification of gene fragment on.Amplification system is as follows: specificity amplification primer F(SEQ ID No.1) and R(SEQ ID No.2) working concentration be 0.2uM, 2 * PyroMark PCR Master Mix 12.5uL, 10 * CoralLoadConcentrate 2.5uL, getting above-mentioned steps b) the plasmid 2uL that extracts is as template, adds ddH ₂O complements to cumulative volume 25uL.

Amplification program is as follows: 95 ℃ of denaturation 15min, and 45 circulations (72 ℃ are extended 30s for 94 ℃ of sex change 30s, 60 ℃ of annealing 30s), 72 ℃ are extended 10min.

The base sequence of described specificity amplification primer is:

Forward primer F:5'-TGACTGTCATCCCTATTGGCAGGTTACCCCAAAGGCCACCCC-3'(SEQ ID No.1);

Reverse primer R:5'-GATCCCGGGGACGATGGGGCAAGTGATGCCCATG-3'(SEQ ID No.2), wherein SEQ ID No.1 is biotin labeled primer, and biotin labeling is in the 5' of primer end.Primer shown in above-mentioned SEQ ID No.1 and the SEQ ID No.2 entrusts Invitrogen company synthetic.

Obtain pcr amplification product through above-mentioned amplification, the pcr amplification product that obtains is used QIAxcel System(QIAGEN) and QIAxcel DNA High Resolution Kit(available from QIAGEN, article No. 929002) carry out capillary electrophoresis and detect, the amplified production electrophoresis detection result of wild plasmid as Figure 1-1; The amplified production electrophoresis detection result of mutant plasmid is shown in Fig. 1-2; Wild-type and mutant equal proportion mix plasmid be template amplified production electrophoresis detection result as Figure 1-3.From Fig. 1-1, Fig. 1-2 and Fig. 1-3 can find out (wherein A B position shown in the C be respectively purpose band position), wild plasmid, mutant plasmid and wild-type and mutant equal proportion are mixed in three amplification systems that plasmid is template all can obtain the single purpose band, and specific amplification products satisfies tetra-sodium order-checking requirement nothing but.

The preparation of step d) single-stranded template

The single-stranded template preparation is used PyroMark Q96 Vaccum Station(available from QIAGEN) finish, concrete operations are carried out to specifications, the recommendation consumption of the consumption by specification of all reagent, be about to step c) amplification gained pcr amplification product (15uL), (40uL is available from QIAGEN to add binding buffer solution, article No. 979006) and the coated magnetic bead (3uL of Streptavidin, available from GE Healthcare, article No. 17-5113-01), at room temperature hatched 15 seconds; Pcr amplification product is combined with the magnetic bead that Streptavidin is coated with by the vitamin H of mark, PCR product after magnetic bead is combined washs 5 seconds, sex change liquid (90mL in 70% ethanolic soln (110mL), available from QIAGEN, article No. 979007) hatches 5 seconds, elutriant (110mL in, available from QIAGEN, article No. 979008) hatched in 10 seconds, above-mentioned amplified production becomes the single stranded DNA template.Again described single stranded DNA template is changed in the annealing buffer that contains sequencing primer (working concentration is 0.4uM) (40uL, available from QIAGEN, article No. 979009), under 80 ℃, hatched 2 minutes, be cooled to room temperature and obtain the single stranded DNA template.

The base sequence of wherein said sequencing primer is: P:5'-GAAAAGCTGCGTGATGATGAAATCG-3'(SEQ ID No.3), entrust Invitrogen company synthetic.

Step e) base allocation order

Open PYROMARK ID software, click Simple Entries, the primer catalogue of this experiment of preserving in the primer-design software is introduced, program can generate the tetra-sodium sequence chart (Pyrogram) of a Dispensation Order and expection automatically.Determine that the base allocation order is TAGACTCTGCACGTA.

Step f) tetra-sodium sequencing reaction

Sequencing reaction is at PYROMARK Q96ID(QIAGEN) carry out under the SNP pattern of instrument.The used reagent that checks order all comes from test kit-Pyromark Gold Q96Reagents, available from QIAGEN company, and article No. 972804.The enzyme mixture that the tetra-sodium sequencing reaction is used, substrate mixture and dNTPs add the reagent cabin, and resulting single stranded DNA template in the step d) is put into the tetra-sodium sequenator, carry out the tetra-sodium sequencing reaction according to the base allocation order that designs in the step e).Primer strand extends along with the adding of different dNTPs, follows the carrying out of enzymatic reaction, and ccd video camera detects the fluorescent signal that sends, and tetra-sodium order-checking collection of illustrative plates is seen Fig. 1-4, Fig. 1-5 and Fig. 1-6.

The Analysis deterrmination of step g) sequencing result

The tetra-sodium sequencing result of wild plasmid is shown in Fig. 1-4, and its sequence that records is that GCTCCC(is reverse), instrument judges that according to peak figure its mthfr gene 677 loci gene types are CC, and is consistent with the expection sequencing result of this wild plasmid;

The tetra-sodium sequencing result of mutant plasmid is shown in Fig. 1-5, and its sequence that records is that ACTCCC(is reverse), instrument judges that according to peak figure its mthfr gene 677 loci gene types are TT, and is consistent with the expection sequencing result of this mutant plasmid;

Wild-type and mutant equal proportion are mixed the tetra-sodium sequencing result of plasmid shown in Fig. 1-6, its sequence that records is reverse for [A/G] CTCCC(), instrument judges that according to peak figure its mthfr gene 677 loci gene types are CT, and is consistent with this expection sequencing result that mixes plasmid.

The detection of embodiment 2 whole blood samples

Adopt DNeasy Blood﹠amp; Tissue Kit(is available from QIAGEN, article No. 69504) carry out the extracting genome DNA of whole blood sample (whole blood sample 1,2 and 3 is all from the sample of clinical collection), concrete operation step obtains DNA extraction liquid with reference to shop instruction.

The Analysis deterrmination of the amplification of mthfr gene fragment, the preparation of single-stranded template, base allocation order, tetra-sodium sequencing reaction and sequencing result is with each step among the embodiment 1.

The tetra-sodium sequencing result of sample 1 is shown in Fig. 2-1, and recording sequence is that GCTCCC(is reverse), instrument judges that according to peak figure its mthfr gene 677 loci gene types are the pure and mild wild-type of CC();

The tetra-sodium sequencing result of sample 2 is shown in Fig. 2-2, and recording sequence is that ACTCCC(is reverse), instrument judges that according to peak figure its mthfr gene 677 loci gene types are the pure and mild mutant of TT();

The tetra-sodium sequencing result of sample 3 is shown in Fig. 2-3, and it is reverse for [A/G] CTCCC(to record sequence), instrument judges that according to peak figure its mthfr gene 677 loci gene types are CT(heterozygous mutant type).

The detection of embodiment 3 mouth epithelial cells samples

With GDNA Normalized Buccal Cell Kit(is available from Invitrogen, article No. CS11020) carries out mouth epithelial cells sample (mouth epithelial cells sample sample 4,5 and 6 is all from the sample of clinical collection) extracting genome DNA, concrete operations obtain DNA extraction liquid with reference to shop instruction.

The Analysis deterrmination of the amplification of mthfr gene fragment, the preparation of single-stranded template, base allocation order, tetra-sodium sequencing reaction and sequencing result is with each step among the embodiment 1.

The tetra-sodium sequencing result of sample 4 is shown in Fig. 3-1, and recording sequence is that GCTCCC(is reverse), instrument judges that according to peak figure its mthfr gene 677 loci gene types are the pure and mild wild-type of CC();

The tetra-sodium sequencing result of sample 5 is shown in Fig. 3-2, and recording sequence is that ACTCCC(is reverse), instrument judges that according to peak figure its mthfr gene 677 loci gene types are the pure and mild mutant of TT();

The tetra-sodium sequencing result of sample 6 is shown in Fig. 3-3, and it is reverse for [A/G] CTCCC(to record sequence), instrument judges that according to peak figure its mthfr gene 677 loci gene types are CT(heterozygous mutant type).

Claims (10)

1. method that detects the mthfr gene polymorphism, it may further comprise the steps:

Use the amplification step of the amplimer amplification mthfr gene shown in SEQ ID NO.1 and/or the SEQ ID NO.2, preferably, described primer is to can be by biotin labeling.

2. method that detects the mthfr gene polymorphism, it may further comprise the steps:

(1) amplification step of the amplification of the amplimer shown in use SEQ ID NO.1 and/or SEQ ID NO.2 mthfr gene;

(2) amplified production that step (1) is obtained becomes the step of single stranded DNA template;

(3) step that the single stranded DNA template of using the sequencing primer shown in the SEQ ID NO.3 that step (2) is obtained is carried out the tetra-sodium order-checking.

3. claim 1 or 2 method, wherein said amplification condition is: 95 ℃ of denaturation 15min; 94 ℃ of sex change 30s, 60 ℃ of annealing 30s, 72 ℃ are extended 30s, totally 45 circulations; 72 ℃ are extended 10min.

4. claim 1 or 2 method, the method is for detection of the polymorphism of mthfr gene C677T; Preferably, described mthfr gene derives from people's whole blood or human oral epithelial cells.

5. the method for claim 2, wherein, step (2) is that the amplified production that step (1) obtains is added binding buffer solution and the coated magnetic bead of Streptavidin, at room temperature hatches certain hour, preferred 15 seconds; PCR product after magnetic bead is combined washs to hatch in certain hour (preferably washing 5 seconds), the sex change liquid in ethanolic soln (being preferably 70% ethanolic soln) hatches certain hour (preferred 10 seconds) in certain hour (preferred 5 seconds), the elutriant, obtain the single stranded DNA template;

Preferably, the method for claim 2, wherein, step (3) is further comprising the steps of,

The single stranded DNA template that step (2) is obtained changes in the annealing buffer that contains the sequencing primer shown in the SEQ ID NO.3, (for example 80 ℃) hatch certain hour (for example 2 minutes) under suitable temperature condition, be cooled to room temperature, obtain the strand sequencing template;

Preferably, when carrying out the tetra-sodium order-checking in the step (3), carry out tetra-sodium sequencing reaction: TAGACTCTGCACGTA according to following base allocation order.

6. be used for the primer pair of mthfr gene amplification, its sequence is shown in SEQ ID NO.1 and SEQID NO.2; Preferably, described primer is to can be by biotin labeling; Further preferably, the primer shown in the SEQ ID NO.1 is by biotin labeling.

7. for detection of the sequencing primer of mthfr gene polymorphism, its sequence is shown in SEQ IDNO.3.

8. cdna amplification kit wherein contains the primer pair of claim 7 and optional other required reagent of amplification.

9. detection kit, it contains the primer pair of claim 7, and sequencing primer claimed in claim 8, and optional other required reagent of sequencing reaction, and preferably other reagent are the required reagent of tetra-sodium sequencing reaction.

The primer of claim 6 to or the purposes of the detection kit of the cdna amplification kit of the sequencing primer of claim 7 or claim 8 or claim 9, the purposes in detecting the mthfr gene polymorphism; Be preferred for detecting the polymorphism of mthfr gene C677T; Preferably, described mthfr gene derives from people's whole blood or human oral epithelial cells.

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