CN105274213A - Method and kit for determination of human PLIN1 gene rs894160 site polymorphism - Google Patents

Abstract

The invention discloses a method and a kit for determination of human PLIN1 gene rs894160 site polymorphism. The method is as follows: providing to-be-tested human genomic DNA; providing upstream and downstream primers for amplification of sequences nearby human PLIN1 gene rs894160 site, wherein the upstream primer has a mismatched base C; taking the to-be-tested human genomic DNA as a template, using the upstream and downstream primers for PCR amplification reaction to obtain an amplification product containing CTCGAR or CCTCGARG fragment, wherein R is to be confirmed base A or G on the human PLIN1 gene rs894160 site; providing a restriction endonuclease; using the restriction endonuclease for enzyme digestion of the amplification product to obtain a corresponding enzyme digestion product; and according to the resulting enzyme digestion product, determining whether the to be confirmed base R on the human PLIN1 gene rs894160 site is A or G. The restriction endonuclease is restriction endonuclease which only be capable of cutting one of CTCGAG fragment or CTCGAA fragment, or restriction endonuclease which only be capable of cutting one of CCTCGAGG fragment or CCTCGAAG fragment. The determination method is fast and reliable, and can greatly reduce the determination cost.

Description

Measure method and the test kit of people PLIN1 gene rs894160 loci polymorphism

Technical field

The present invention relates to the method measuring single nucleotide polymorphism.

Background technology

SNP (single nucleotide polymorphism) refers to and comprises the forms such as the displacement of single base, insertion and disappearance by the mutant dna sequence that the change of single core thuja acid causes.Gene pleiomorphism is individual inherent hereditary feature, does not change with environment, neither the special or non-specific clinical manifestation of certain disease, and therefore its application does not exist Diagnosis and Treat effect.

Genetic polymorphism detection is widely used in genetic arts and medical field, is exemplified below: 1. study the genetics such as the origin of species and evolution phenomenon.According to genovariation situation, obtain the information of biomacromolecule, infer organic evolution history, illustrate spore relation.Be applied in archeology to determine the sibship of ancient human's heritable variation feature and different population.2. research polymorphic with population genetics etc. genetics research.Polymorphic also closely related with various characteristics of human body, comprise height, body weight, the colour of skin, Facial Features etc.3. may be used for prophylaxis in preventive medicine field.By the tolerance of researching human body to poisonous substance, find to be easy to, to the individuality of certain poisonous substance generation toxic action, avoid this individuality and toxicant exposure in time, protect this Susceptible population.Such as, carry out polymorphic detection to the crowd preparing to be engaged in the organic solvents such as Contact benzene, examination goes out easily to occur the individuality that hematotoxicity, neurotoxicity etc. are reacted, and makes it away from organic solvents such as benzene, protects this type of Susceptible population to encroach on from poisonous substance.4. may be used for medicament selection in pharmacology and dosage is determined.Can judge that diseased individuals is responsive to certain poisonous side effect of medicine by polymorphic detection, thus avoid using this kind of medicine to exempt its toxic action.By judging the individual level of response determination drug dose to effect of drugs, reduce drug dose and side effect thereof.

PLIN1 gene is positioned at karyomit(e) 15q26.1, and the perilipin albumen of coding plays an important role in fat metabolic process.PLIN1 gene rs894160 site is polymorphic, there are two kinds of allelotrope A and G in crowd, form the genotype of three kinds of PLIN1 genes: AA type (two allelotrope bases of human genome rs894160 polymorphic site are A), AG type (two allelotrope bases of human genome rs894160 polymorphic site are respectively A and G) and GG type (two allelotrope bases of human genome rs894160 polymorphic site are G).

Polymerase chain reaction-restriction fragment length polymorphic (PCR-RFLP) technology is a kind of quick, easy, accurate, low cost mensuration SNP (single nucleotide polymorphism) genotypic classical way.The method carries out amplified reaction by primer to template, forms sufficient amount and stable amplified production, by cutting and detect the fragment length of digestion products to the enzyme of amplified production, finally determines SNP.PLIN1 gene rs894160 polymorphic site takes PCR-RFLP technology to detect, and required restriction endonuclease is MnlI restriction endonuclease, and its price costly, needs to develop new detection technique.

Summary of the invention

The object of this invention is to provide the reliable means of the mensuration people PLIN1 gene rs894160 loci polymorphism of the diagnosis of a kind of non-diseases or therapeutic purpose.

According to a first aspect of the invention, provide a kind of method measuring people PLIN1 gene rs894160 loci polymorphism, comprising:

Human gene group DNA to be measured is provided;

There is provided upstream primer and the downstream primer of amplification people PLIN1 gene rs894160 location proximate sequence, wherein upstream primer has base mismatch C;

With described human gene group DNA to be measured for template, utilize upstream primer and downstream primer to carry out pcr amplification reaction to obtain the amplified production containing CTCGAR fragment or CCTCGARG fragment, wherein R is base A undetermined on people PLIN1 gene rs894160 site or G;

Restriction enzyme is provided;

Utilize restriction enzyme to carry out enzyme to gained amplified production and cut (reaction) to obtain corresponding digestion products; And

Determine that the base R undetermined on people PLIN1 gene rs894160 site is A or G according to gained digestion products,

Wherein, restriction enzyme is for can cut one of CTCGAG fragment and CTCGAA fragment, or the restriction enzyme of one of CCTCGAGG fragment and CCTCGAAG fragment.

According to embodiments of the invention, on gained amplified production, two bases G A of being separated by between base mismatch C and the R of upstream primer.Surprisingly, so arrange base mismatch endonuclease reaction will be made to carry out extremely smooth, there will not be enzyme to cut the phenomenons such as insufficient.Further, so arrange upstream primer base mismatch and PCR reaction is well on, improve sensitivity and the specific degree of PCR, this may be relevant with making the secondary structural change of extension increasing sequence after base mismatch.

In an alternate embodiment of the present invention where, on gained amplified production, upstream primer terminal bases is adjacent with R.

In a preferred embodiment of the invention, on gained amplified production, upstream primer terminal bases is not adjacent with R.Such as, upstream primer end can be ... the structures such as CG.Be difficult to the imagination, the upstream primer with this design can promote endonuclease reaction unexpectedly further greatly, and this may have certain to associate with amplification bonding force.

In a preferred embodiment of the invention, restriction enzyme can adopt the BsoBI or its isoschizomers that only can cut CTCGAG fragment.This kind of BsoBI enzyme is cheap, greatly can reduce cost of determination.

According to embodiments of the invention, gained digestion products comprises three kinds of clip types: have total fragment length do not cut off amplified production, cut off after have compared with the amplified production of long segment and after cutting off, there is amplified production (usually can not effectively observe) compared with short-movie section.The clip types comprised by observation (judgement) digestion products determines that the base undetermined on people PLIN1 gene rs894160 site is A or G.Such as, when adopting BsoBI enzyme, judge compared with the observations of these two fragments of long segment according to total fragment with after cutting off: if observe digestion products only have a kind of there is total fragment length do not cut off amplified production, then base undetermined is A; Only have one to have the cut-out product of comparatively long segment (being less than total fragment length) if observe digestion products, then base undetermined is G; If observe said two devices, then base undetermined not only comprises A but also comprise G.

In one embodiment of the invention, the clip types that judgement (or observation) digestion products comprises is schemed to contrast to carry out with reference after being taken pictures by gel electrophoresis associating ultraviolet lamp.In this case, preferably reference figure is that amplified production obtains and only has first with reference to picture position and second with reference to picture position after the gel electrophoresis standard setting time after ultraviolet lamp is taken pictures, wherein first with reference to picture position for be the amplified production of the total fragment length do not cut off, second with reference to picture position then for be cut off after there is amplified production compared with long segment.Such as, the present invention adopt with reference to figure be by 118bp and 87bp synthesized two base fragments simultaneously after gel electrophoresis same time ultraviolet lamp take pictures and form.Compared with the compound reference figure of conventional DNAladder, owing to have employed, only there are two specific reference figure with reference to picture position, this method of the present invention intuitively can be observed rapidly or judge the clip types that digestion products comprises, and avoids erroneous judgement to greatest extent simultaneously.After endonuclease reaction, preferably carry out electrophoresis by after concentrated for digestion products 1 ~ 2 times of concentration, increase brightness of image, improve judgment accuracy.

In a preferred embodiment of the invention, make total fragment length of amplified production between 100 to 300bp by designing the length of upstream primer and downstream primer, and the fragment length of upstream primer is at least 35bp, preferred length 40 ~ 60bp (amplified reaction especially smoothly fully between this dominant area), makes enzyme fall Partial Fragment length earnestly and accounts for the per-cent of total fragment length more than 20%.This design of the present invention has the electrophoresis ultraviolet photo of the amplified production compared with long segment position after can making to have not cutting off amplified production and cutting off of total fragment length is distinguished significantly.But if total fragment is long, then electrophoretic mobility shift is by not obvious; Too short, image can thicken a slice, cannot accurately distinguish.The fragment length scope of upstream primer ensure that pcr amplification reaction can carry out smoothly, the amplification that there will be when avoiding base mismatch not foot phenomenon.

In a preferred embodiment of the invention, by controlling the scope of annealing temperature in pcr amplification program between 55 ~ 70 DEG C, preferable temperature 60 ~ 69 DEG C (this temperature can improve the specificity of amplified reaction), make the PCR primer purity of design higher.This design of the present invention can make pcr amplification product band clear, occurs, be easy to electrophoresis identification without assorted band.But if temperature is too low, then few the and assorted band of specific band is too much, is difficult to after electrophoresis distinguish and judges; If temperature is too high, then affects pcr amplification efficiency and make specific amplified production very few, affect observing effect.

In a preferred embodiment of the invention, by controlling to extend the scope of time between 10 ~ 60s in pcr amplification program, preferred time 15 ~ 30s (this time can improve the efficiency of amplified reaction and the specificity of amplified production), make the PCR reaction times of design shorter, amplified production purity is higher.This design of the present invention can make pcr amplification product band clear, occurs, be easy to electrophoresis identification, and the PCR time is shorter without assorted band.But if the time is too short, then specific band can not increase completely and make amplified production less, be difficult to after electrophoresis distinguish judgement; If overlong time, then increase the occurrence probability of non-specific band, occur that assorted band affects observing effect.

In the present invention, PCR reacted constituent can comprise DNA profiling, upstream and downstream primer, TaqDNApolymerase (archaeal dna polymerase or also can be called " Taq enzyme "), damping fluid, Mg ²⁺deng.In a preferred embodiment of the invention, TaqDNApolymerase (archaeal dna polymerase) and TaqAntibody thereof can be used in pcr amplification reaction.TaqAntibody is the monoclonal antibody of TaqDNApolymerase, it suppresses DNA polymerase activity after being combined with TaqDNApolymerase, both avidity is very high, even if the activity of TaqDNApolymerase still can be closed at 65 DEG C, the non-specific amplification that therefore can effectively suppress the non-specific annealing of primer and primer dimer to cause, has high amplification sensitivity and specificity.ChampagneTaqAntibody only needs to get final product complete deactivation at 95 DEG C of heating 30s, and release TaqDNApolymerase is active, ensure that subsequent PCR amplification reaction can be carried out smoothly.

The Tris-HCl damping fluid that pH value is 8.1 ~ 8.7 can be added in PCR reaction, adjust PCR solution ph when 72 DEG C of extensions between 6.8 ~ 7.8, thus make Taq enzyme play activity better in slight alkali environment.In addition, gelatin (0.01%) can also be added in PCR solution to reduce the adsorption of PCR pipe to Taq enzyme, stabilized enzyme activity and provide protection, promote PCR reaction.

In addition, Mg in PCR solution ²⁺when concentration is between 1.5 ~ 2.0mmol/L, can well control PCR react productive rate and specificity.

The final concentration that PCR reacts primer is generally about 0.1 ~ 1 μM, and the too high meeting of concentration causes non-specific amplification, and too low then amplified production very little.

In addition, can also add solubility promoter methyl-sulphoxide (DMSO) and ammonium sulfate to reduce base mispairing level in PCR reaction, the amplification efficiency of GC template is rich in raising.This with the secondary structure eliminating primer and template, may reduce DNA melting temperature(Tm) and makes DNA sex change completely relevant.

According to a further aspect in the invention, provide a kind of test kit for measuring people PLIN1 gene rs894160 loci polymorphism, it comprises:

For upstream primer and the downstream primer of pcr amplification people PLIN1 gene rs894160 location proximate sequence, wherein upstream primer has base mismatch C to obtain the amplified production containing CTCGAR fragment or CCTCGARG fragment, and wherein R is base A undetermined on people PLIN1 gene rs894160 site or G; And

The restriction enzyme of one of CTCGAG fragment and CTCGAA fragment can be identified, or identify the restriction enzyme of one of CCTCGAGG fragment and CCTCGAAG fragment.

Can also comprise other composition in mentioned reagent box, such as PCR reacts Mix and (comprises 4 kinds of dNTP mixtures, Mg ²⁺, TaqDNApolymerase, TaqAntibody, damping fluid, DMSO and ammonium sulfate) and for implement measure specification sheets etc.

It will be understood by those skilled in the art that unless there are obvious conflict, the correlated characteristic of a first aspect of the present invention and second aspect can combine mutually.

Mensuration means of the present invention are not only fast and reliable, and cost of determination reduces greatly.

Accompanying drawing explanation

Fig. 1 describes the digestion products electrophoresis ultraviolet photo that method according to the present invention obtains.Wherein Marker is: standard reference position; Swimming lane 1:GG genotype; Swimming lane 2:AG genotype; Swimming lane 3:AA genotype.

Embodiment

Describe the present invention below.It will be appreciated by those skilled in the art that following detailed description only for illustration of and non-limiting the present invention.

(1) acquisition of DNA to be measured

Gather different crowd peripheral blood, after extracting genomic dna, carry out the mensuration of PLIN1 gene rs894160 loci polymorphism below.

For the selection of DNA to be measured, the not special restriction of the present invention.Both can be obtain in vitro sample from the body fluid of human body or tissue, the genome of degradation treatment in advance can also be through.For convenience of implementation, usually preferably in vitro sample is extracted from blood.Wherein said tissue comprises the tissue containing all or at least PLIN1 gene in human body, and with whether express this PLIN1 gene and have nothing to do.The method extracting DNA from body fluid and/or tissue well known to a person skilled in the art, and can with reference to conventional molecular biology manual, and such as " molecular cloning " the 2nd edition carries out.

(2) design of primers and synthesis

Search Gene database and the snp database of NCBI website, obtain PLIN1 gene complete sequence and rs894160 polymorphic site information respectively.

Rs894160 polymorphic site R front and rear part base sequence (base sequence represents with 5' → 3', capital and small letter same meaning) following (SEQIDNO:1):

Carry out upstream primer and downstream primer design according to gene order, wherein, upstream primer introduces base mismatch.On final gained amplified production, two bases G A of being separated by between base mismatch C and the R of upstream primer.

In the present invention, upstream primer has base mismatch C, and length is 35 ~ 60bp.In one of them embodiment, primer is as follows, upstream primer: 5'GTAATAAGGAGTCTCTGTTTGTGGGGCTCCCT cgA3'(SEQIDNO:2), downstream primer: 5'AAAAATTCAATGAACCAGAGTGTCC3'(SEQIDNO:3), wherein upstream primer underscore base is base mismatch.

(3) pcr amplification product is prepared

Formulate pcr amplification program and condition according to upstream primer and downstream primer feature and PCR corresponding reagent, prepare pcr amplification product.In one of them embodiment, get genomic dna (50 ~ 80ng), upstream and downstream primer (final concentration is 0.1 ~ 1 μM), 2 × PCRMix7.5 μ L (comprise 4 kinds of dNTP mixtures, Mg ²⁺, TaqDNApolymerase, TaqAntibody, damping fluid, DMSO and ammonium sulfate) and distilled water jointly form 15 μ L reaction systems, adjustment pH value of solution be about 7.3.PCR reaction conditions is: 95 DEG C of denaturation 30s, and DEG C annealing 30s → 72 DEG C, 95 DEG C of sex change 30s → 60 extend 20s totally 30 circulations, and 72 DEG C extend 7min.Stable amplified production is obtained after PCR reaction.

(4) endonuclease reaction

The present invention can adopt BsoBI, XhoI, PaeR7I, AvaI, SmlI, PspXI restriction endonuclease, and the polymorphic qualification for PLIN1 gene rs894160 site provides the restriction endonuclease that plurality of optional is selected, and can select suitable restriction endonuclease during experiment according to market value.Current part restriction endonuclease less expensive, as shown in table 1.

Table several endonuclease recognition sequence of 1NEB company and price thereof

Note: Y is C or T, R be A or G, V be A or C or G, B be C or G or T, N is A or T or C or G

In one of them embodiment, get PCR primer 10 μ L, add 5U restriction endonuclease BsoBI, 2 μ L10 × enzyme cutting buffering liquids and 7.5 μ L distilled waters and form 20 μ L reaction systems, in 37 DEG C of water-baths, enzyme cuts 4 ~ 12h, obtain digestion products, digestion products is observed through 1 times of concentrated rear electrophoresis.

(5) electrophoresis test

In one of them embodiment, if PCR primer can not be cut after BsoBI enzyme is cut, be still 118bp, if be cut open, occur that (cut generation sticky end due to BsoBI enzyme makes its complementary strand bases number different to 87bp with 31bp, therefore after cutting, fragment length is as the criterion with strand base number in gene order and calculates), be wherein difficult in 31bp fragment electrophoretic figure differentiate.

By digestion products in 2 ~ 4% sepharoses under 3 ~ 8V/cm condition, electrophoresis 30 ~ 80min, mensuration of taking pictures under ultraviolet lamp.Restriction enzyme digestion and electrophoresis figure is shown in Fig. 1, and the presence or absence according to 118bp and 87bp fragment judges genotype: AA type is 118bp fragment, and AG type has 118bp, 87bp fragment, and GG type is 87bp fragment.

Here implements some embodiments of the present invention.

Embodiment 1 is extracted human peripheral leucocytes DNA and is measured rs894160 polymorphism

1 materials and methods

1.1 main agents and instrument

Reagent: 2 × PCRMix (comprises 4 kinds of dNTP mixtures, Mg ²⁺, TaqDNApolymerase, TaqAntibody, damping fluid, DMSO and ammonium sulfate), restriction enzyme BsoBI (NEB company), agarose (Biowest company), primer is synthesized by Shanghai Sangon company.

Instrument: 9600 type PCR instrument (PE company), miniature electrophoresis chamber (PharmaciaBiotech, EPS1000), GelDoc2000 gel imaging instrument (Bio-RAD company).

1.2 extract DNA as testing gene group DNA profiling from peripheral blood leucocyte

EDTA-K ₂anticoagulant tube gathers human peripheral 1mL, white corpuscle separation is carried out with reference to leukocytic separation method in " practical flow cytometry icones ", white corpuscle genomic dna is extracted with reference to NaCl salting-out method in " molecular cloning ", and as human gene group DNA's template to be measured.

1.3 sequences are searched and design of primers

PLIN1 gene order and rs894160 polymorphic site information is searched to design primer in NCBI website, specific as follows:

Upstream primer: 5'GTAATAAGGAGTCTCTGTTTGTGGGGCTCCCT cgA3'(SEQIDNO:2);

Downstream primer: 5'AAAAATTCAATGAACCAGAGTGTCC3'(SEQIDNO:3)

1.4PCR amplification

Get genomic dna (50 ~ 80ng), upstream and downstream primer (final concentration is 0.1 ~ 1 μM), 2 × PCRMix7.5 μ L (comprise 4 kinds of dNTP mixtures, Mg ²⁺, TaqDNApolymerase, TaqAntibody, damping fluid, DMSO and ammonium sulfate), sterilizing distilled water supplies 15 μ l reaction systems, adjustment pH value of solution be about 7.3.

PCR reaction conditions is: 95 DEG C of denaturation 30s, and DEG C annealing 30s → 72 DEG C, 95 DEG C of sex change 30s → 60 extend 20s totally 30 circulations, and 72 DEG C extend 7min.Stable amplified production is obtained after PCR reaction.

1.5 enzymes cut qualification

After pcr amplification, get PCR primer 10 μ l, add 5U restriction endonuclease BsoBI and 2 μ l10 × enzyme cutting buffering liquid and sterilizing distilled water and form 20 μ l reaction systems, after enzyme cuts 4h in 37 DEG C of water-baths, obtain digestion products.

After above-mentioned digestion products is concentrated through 1 times, under 3% sepharose 5V/cm condition, electrophoresis 40min, qualification Polymorphic type of taking pictures under ultraviolet lamp.

2 results

2.1PCR amplification

Sequence (it is positioned at 466 ~ 583 places of SEQIDNO:1 base sequence, altogether 118bp) after amplification, the amplified production sequence obtained following (SEQIDNO:4):

In amplification after product sequence, underscore part is respectively the sequence corresponding to upstream, downstream primer, and R represents the polymorphic i.e. SNP site rs894160 of A/G.

2.2 enzymes cut result

Product Sequence after restriction endonuclease BsoBI enzyme is cut is as follows respectively:

When this polymorphic site contains G allelotrope, PCR primer enzyme can be formed after cutting and cut fragment 31bp (this fragment downstream sequence forms sticky end because enzyme is cut and increases by 4 bases than upstream sequence) sequence (SEQIDNO:5):

gtaataaggagtctctgtttgtggggctccc31

When this polymorphic site contains G allelotrope, PCR primer enzyme can be formed after cutting and cut fragment 87bp (this fragment downstream sequence forms sticky end because enzyme is cut and reduces by 4 bases than upstream sequence) sequence (SEQIDNO:6):

Take pictures under ultraviolet lamp after digestion products electrophoresis qualification, result is shown in Figure 1.Wherein, there is 118bp segment after the amplification of rs894160 site.There will be 118bp, 87bp and 31bp tri-kinds of segments (wherein 31bp not easily differentiates in electrophorogram) after BsoBI enzyme is cut.

After BsoBI enzyme is cut, carry out genotype judgement: as shown in Figure 1, AA type is 118bp fragment, and AG type has 118bp and 87bp two kinds of fragments, and GG type is 87bp fragment.

The polymorphic result in 2.3PLIN1 gene rs894160 site

Detect 89 routine personnel altogether, detected result finds that GG type 55 example, AG type 22 example and AA type 12 example appear in rs894160 site.

Embodiment 2 human peripheral whole blood sample measures rs894160 polymorphism

Main agents is except restriction endonuclease is XhoI, and all the other are with embodiment 1; Instrument is with embodiment 1.Peripheral blood genomic dna is extracted with reference to phenol-chloroform extraction process in " molecule clone technology ", and as human gene group DNA's template to be measured.

Sequence searches same embodiment 1, and design primer sequence is as follows:

Upstream primer: 5'TGTAATAAGGAGTCTCTGTTTGTGGGGCTCCCT cg3'(SEQIDNO:7);

Downstream primer: 5'GAAAAATTCAATGAACCAGAGTGTCC3'(SEQIDNO:8)

Get genomic dna (50 ~ 80ng), upstream and downstream primer (final concentration is 0.1 ~ 1 μM), 2 × PCRMix10 μ L (comprise 4 kinds of dNTP mixtures, Mg ²⁺, TaqDNApolymerase, TaqAntibody, damping fluid, DMSO and ammonium sulfate), sterilizing distilled water supplies 20 μ l reaction systems, adjustment pH value of solution be about 7.3.

PCR reaction conditions is: 95 DEG C of denaturation 30s, and DEG C annealing 30s → 72 DEG C, 95 DEG C of sex change 30s → 60 extend 20s totally 30 circulations, and 72 DEG C extend 10min.

Enzyme cuts qualification: get PCR primer 10 μ l, and add 5U restriction endonuclease XhoI, 2 μ l10 × enzyme cutting buffering liquids and sterilizing distilled water and form 20 μ l reaction systems, in 37 DEG C of water-baths, enzyme cuts 4h, obtains digestion products.After digestion products is concentrated through 1 times, under 2.5% sepharose 5V/cm condition, electrophoresis 40min, qualification of taking pictures under ultraviolet lamp.

Result:

Sequence (it is positioned at 465 ~ 584 places of SEQIDNO:1 base sequence, altogether 120bp) after amplification, the amplified production sequence obtained following (SEQIDNO:9):

In amplification after product sequence underscore part be upstream, sequence corresponding to downstream primer, R represents the polymorphic i.e. SNP site rs894160 of A/G.

Product Sequence after XhoI enzyme is cut is as follows respectively:

When this polymorphic site contains G allelotrope, PCR primer enzyme can be formed after cutting and cut fragment 32bp (this fragment downstream sequence forms sticky end because enzyme is cut and increases by 4 bases than upstream sequence) sequence (SEQIDNO:10):

tgtaataaggagtctctgtttgtggggctccc32

When this polymorphic site contains G allelotrope, PCR primer enzyme can be formed after cutting and cut fragment 88bp (this fragment downstream sequence forms sticky end because enzyme is cut and reduces by 4 bases than upstream sequence) sequence (SEQIDNO:11):

To take pictures under ultraviolet lamp after digestion products electrophoresis qualification, after the amplification of rs894160 site, occur 120bp segment.Cut rear electrophoresis through XhoI enzyme and there will be 120bp, 88bp and 32bp tri-kinds of segments (wherein 32bp not easily differentiates in electrophorogram).

Cut rear electrophoresis through XhoI enzyme and carry out genotype judgement: AA type is 120bp fragment, and AG type has 120bp and 88bp two kinds of fragments, GG type is 88bp fragment.

Polymorphic result: detect 96 routine personnel altogether, detected result finds that GG type 60 example, AG type 22 example and AA type 14 example appear in rs894160 site.

Embodiment 3 human peripheral blood clot measures rs894160 polymorphism

Blood clot genomic dna is extracted with reference to phenol-chloroform extraction process in " molecule clone technology ".

It is SEQNO:12 that PCR reacts the upstream primer adopted, and downstream primer is SEQNO:13.

Upstream primer: 5'GTGTAATAAGGAGTCTCTGTTTGTGGGGCTCCCT cgA3'(SEQIDNO:12);

Downstream primer: 5'TGAAAAATTCAATGAACCAGAGTGTCC3'(SEQIDNO:13)

Pcr amplification is except annealing temperature is 61 DEG C, and remaining reaction condition is with embodiment 1; Enzyme cuts qualification except restriction endonuclease adopts PaeR7I enzyme, and all the other conditions are with embodiment 1.

Result:

Sequence (it is positioned at 464 ~ 585 places of SEQIDNO:1 base sequence, altogether 122bp) after amplification, the amplified production sequence obtained following (SEQIDNO:14):

In amplification after product sequence underscore part be upstream, sequence corresponding to downstream primer, R represents the polymorphic i.e. SNP site rs894160 of A/G.

Product Sequence after PaeR7I enzyme is cut is as follows respectively:

When this polymorphic site contains G allelotrope, PCR primer enzyme can be formed after cutting and cut fragment 33bp (this fragment downstream sequence forms sticky end because enzyme is cut and increases by 4 bases than upstream sequence) sequence (SEQIDNO:15):

gtgtaataaggagtctctgtttgtggggctccc33

When this polymorphic site contains G allelotrope, PCR primer enzyme can be formed after cutting and cut fragment 89bp (this fragment downstream sequence forms sticky end because enzyme is cut and reduces by 4 bases than upstream sequence) sequence (SEQIDNO:16):

To take pictures under ultraviolet lamp after digestion products electrophoresis qualification, after the amplification of rs894160 site, occur 122bp segment.Cut rear electrophoresis through PaeR7I enzyme and there will be 122bp, 89bp and 33bp tri-kinds of segments (wherein 33bp not easily differentiates in electrophorogram).

Cut rear electrophoresis through PaeR7I enzyme and carry out genotype judgement: AA type is 122bp fragment, and AG type has 122bp and 89bp two kinds of fragments, GG type is 89bp fragment.

Polymorphic result: detect 80 routine personnel altogether, detected result finds that GG type 45 example, AG type 23 example and AA type 12 example appear in rs894160 site.

Embodiment 4 human oral mucosa cells measures rs894160 polymorphism

Main agents is except restriction endonuclease is AvaI, and all the other are with embodiment 1; Instrument is with embodiment 1.Adopt minim DNA to extract test kit and extracting genome DNA is carried out to Oral Mucosal Cells.

It is SEQNO:17 that PCR reacts the upstream primer adopted, and downstream primer is SEQNO:18.

Upstream primer: 5'GCTGTGTAATAAGGAGTCTCTGTTTGTGGGGCTCCCT cgA3'(SEQIDNO:17);

Downstream primer: 5'GTGAAAAATTCAATGAACCAGAGTGTCC3'(SEQIDNO:18)

Pcr amplification is except annealing temperature is 59 DEG C, and remaining reaction condition is with embodiment 1; Enzyme cuts qualification except restriction endonuclease adopts AvaI enzyme, and all the other conditions are with embodiment 1.

Result:

Sequence (it is positioned at 461 ~ 586 places of SEQIDNO:1 base sequence, altogether 126bp) after amplification, the amplified production sequence obtained following (SEQIDNO:19):

In amplification after product sequence underscore part be upstream, sequence corresponding to downstream primer, R represents the polymorphic i.e. SNP site rs894160 of A/G.

Product Sequence after AvaI enzyme is cut is as follows respectively:

When this polymorphic site contains G allelotrope, PCR primer enzyme can be formed after cutting and cut fragment 36bp sequence (this fragment downstream sequence forms sticky end because enzyme is cut and increases by 4 bases than upstream sequence) (SEQIDNO:20):

gctgtgtaataaggagtctctgtttgtggggctccc36

When this polymorphic site contains G allelotrope, PCR primer enzyme can be formed after cutting and cut fragment 90bp sequence (this fragment downstream sequence forms sticky end because enzyme is cut and reduces by 4 bases than upstream sequence) (SEQIDNO:21):

To take pictures under ultraviolet lamp after digestion products electrophoresis qualification, after the amplification of rs894160 site, occur 126bp segment.Cut rear electrophoresis through AvaI enzyme and there will be 126bp, 90bp and 36bp tri-kinds of segments (wherein 36bp not easily differentiates in electrophorogram).

Cut rear electrophoresis through AvaI enzyme and carry out genotype judgement: AA type is 126bp fragment, and AG type has 126bp and 90bp two kinds of fragments, GG type is 90bp fragment.

Polymorphic result: detect 124 routine personnel altogether, detected result finds that GG type 67 example, AG type 28 example and AA type 29 example appear in rs894160 site.

It is polymorphic that embodiment 5 human lung tissue sample measures rs894160

Main agents is except restriction endonuclease is PspXI, and all the other are with embodiment 1; Instrument is with embodiment 1.Phenol-chloroform extraction process is adopted to carry out lung tissue block genome DNA extraction.

It is SEQNO:22 that PCR reacts the upstream primer adopted, and downstream primer is SEQNO:23.

Upstream primer: 5'AGCTGTGTAATAAGGAGTCTCTGTTTGTGGGGCTCCCT cgA3'(SEQIDNO:22);

Downstream primer: 5'AGTGAAAAATTCAATGAACCAGAGTGTCC3'(SEQIDNO:23)

Pcr amplification is except annealing temperature is 61 DEG C, and remaining reaction condition is with embodiment 1; Enzyme cuts qualification except restriction endonuclease adopts PspXI enzyme, and remaining reaction condition is with embodiment 1.

Result:

Sequence (it is positioned at 460 ~ 587 places of SEQIDNO:1 base sequence, altogether 128bp) after amplification, the amplified production sequence obtained following (SEQIDNO:24):

In amplification after product sequence underscore part be upstream, sequence corresponding to downstream primer, R represents the polymorphic i.e. SNP site rs894160 of A/G.

Product Sequence after PspXI enzyme is cut is as follows respectively:

When this polymorphic site contains G allelotrope, PCR primer enzyme can be formed after cutting and cut fragment 37bp sequence (this fragment downstream sequence forms sticky end because enzyme is cut and increases by 4 bases than upstream sequence) (SEQIDNO:25):

agctgtgtaataaggagtctctgtttgtggggctccc37

When this polymorphic site contains G allelotrope, PCR primer enzyme can be formed after cutting and cut fragment 91bp sequence (this fragment downstream sequence forms sticky end because enzyme is cut and reduces by 4 bases than upstream sequence) (SEQIDNO:26):

To take pictures under ultraviolet lamp after digestion products electrophoresis qualification, after the amplification of rs894160 site, occur 128bp segment.Cut rear electrophoresis through PspXI enzyme and there will be 128bp, 91bp and 37bp tri-kinds of segments (wherein 37bp not easily differentiates in electrophorogram).

Cut rear electrophoresis through PspXI enzyme and carry out genotype judgement: AA type is 128bp fragment, and AG type has 128bp and 91bp two kinds of fragments, GG type is 91bp fragment.

Polymorphic result: detect 118 routine personnel altogether, detected result finds that GG type 67 example, AG type 22 example and AA type 29 example appear in rs894160 site.

Embodiment 6 human hair sample measures rs894160 polymorphism

Adopt minim DNA to extract test kit and extracting genome DNA is carried out to hair swatch.

It is SEQNO:27 that PCR reacts the upstream primer adopted, and downstream primer is SEQNO:28.

Upstream primer: 5'CCCCAAAAGCTGTGTAATAAGGAGTCTCTGTTTGTGGGGCTCCCT cgA3'(SEQIDNO:27);

Downstream primer: 5'TTTTAAGAGTGAAAAATTCAATGAACCAGAGTGTCC3'(SEQIDNO:28)

Pcr amplification is except annealing temperature is 62 DEG C, and remaining reaction condition is with embodiment 1.

Enzyme cuts qualification: get PCR primer 10 μ l, and add 5U restriction endonuclease SmlI, 2 μ l10 × enzyme cutting buffering liquids and sterilizing distilled water and form 20 μ l reaction systems, in 55 DEG C of water-baths, enzyme cuts 4h, obtains digestion products.After digestion products is concentrated through 1 times, under 2.5% sepharose 5V/cm condition, electrophoresis 40min, qualification of taking pictures under ultraviolet lamp.

Result:

Sequence (it is positioned at 453 ~ 594 places of SEQIDNO:1 base sequence, altogether 142bp) after amplification, the amplified production sequence obtained following (SEQIDNO:29):

In amplification after product sequence underscore part be upstream, sequence corresponding to downstream primer, R represents the polymorphic i.e. SNP site rs894160 of A/G.

Product Sequence after SmlI enzyme is cut is as follows respectively:

When this polymorphic site contains G allelotrope, PCR primer enzyme can be formed after cutting and cut fragment 44bp sequence (this fragment downstream sequence forms sticky end because enzyme is cut and increases by 4 bases than upstream sequence) (SEQIDNO:30):

ccccaaaagctgtgtaataaggagtctctgtttgtggggctccc44

When this polymorphic site contains G allelotrope, PCR primer enzyme can be formed after cutting and cut fragment 98bp sequence (this fragment downstream sequence forms sticky end because enzyme is cut and reduces by 4 bases than upstream sequence) (SEQIDNO:31):

To take pictures under ultraviolet lamp after digestion products electrophoresis qualification, after the amplification of rs894160 site, occur 142bp segment.Cut rear electrophoresis through SmlI enzyme and there will be 142bp, 98bp and 44bp tri-kinds of segments (wherein 44bp not easily differentiates in electrophorogram).

Cut rear electrophoresis through SmlI enzyme and carry out genotype judgement: AA type is 142bp fragment, and AG type has 142bp and 98bp two kinds of fragments, GG type is 98bp fragment.

Polymorphic result: detect 140 routine personnel altogether, detected result finds that GG type 68 example, AG type 33 example and AA type 39 example appear in rs894160 site.

Claims (10)

1. measure a method for people PLIN1 gene rs894160 loci polymorphism, comprising:

Human gene group DNA to be measured is provided;

There is provided upstream primer and the downstream primer of amplification people PLIN1 gene rs894160 location proximate sequence, wherein upstream primer has base mismatch C;

With described human gene group DNA to be measured for template, utilize upstream primer and downstream primer to carry out pcr amplification reaction to obtain the amplified production containing CTCGAR fragment or CCTCGARG fragment, wherein R is base A undetermined on people PLIN1 gene rs894160 site or G;

Restriction enzyme is provided;

Utilize restriction enzyme to carry out enzyme to gained amplified production to cut to obtain corresponding digestion products;

And determine that the base R undetermined on people PLIN1 gene rs894160 site is A or G according to gained digestion products,

Wherein, restriction enzyme is for can cut one of CTCGAG fragment and CTCGAA fragment, or the restriction enzyme of one of CCTCGAGG fragment and CCTCGAAG fragment.

2. method according to claim 1, wherein on gained amplified production, two bases G A of being separated by between base mismatch C and the R of upstream primer.

3. method according to claim 2, wherein on gained amplified production, upstream primer terminal bases is adjacent with R or non-conterminous.

4. method according to claim 1, wherein restriction enzyme is BsoBI, XhoI, PaeR7I, AvaI, SmlI or its isoschizomers that can cut CTCGAG fragment.

5. method according to claim 1, wherein restriction enzyme is PspXI or its isoschizomers that can cut CCTCGAGG fragment.

6. method according to claim 1, wherein gained digestion products comprises three kinds of clip types: have total fragment length do not cut off amplified production, cut off after have compared with the amplified production of long segment and after cutting off, there is amplified production compared with short-movie section, by judging that the clip types that digestion products comprises determines that the base undetermined on people PLIN1 gene locus rs894160 is A or G.

7. method according to claim 6, wherein judge clip types that digestion products comprises taken pictures by gel electrophoresis associating ultraviolet lamp after with reference to scheming to contrast to carry out.

8. method according to claim 7, wherein reference figure is that amplified production obtains and only has first with reference to picture position and second with reference to picture position after the gel electrophoresis standard setting time after ultraviolet lamp is taken pictures, wherein first with reference to picture position for be the amplified production of the total fragment length do not cut off, second with reference to picture position then for be cut off after there is amplified production compared with long segment.

9. method according to claim 1, wherein make total fragment length of amplified production between 100 to 300bp by designing the length of upstream primer and downstream primer, and the fragment length of upstream primer is at least 35bp, makes enzyme fall Partial Fragment earnestly and account for the per-cent of the length of total fragment more than 20%.

10., for measuring a test kit for people PLIN1 gene rs894160 loci polymorphism, it comprises:

For upstream primer and the downstream primer of pcr amplification people PLIN1 gene rs894160 location proximate sequence, wherein upstream primer has base mismatch C to obtain the amplified production containing CTCGAR fragment or CCTCGARG fragment, and wherein R is base A undetermined on people PLIN1 gene rs894160 site or G; And

The restriction enzyme of one of CTCGAG fragment and CTCGAA fragment can be identified, or identify the restriction enzyme of one of CCTCGAGG fragment and CCTCGAAG fragment.