CN107779507A - With the method for BstEII detection human oral cancer LINC00520 gene rs8008130 polymorphisms - Google Patents

Abstract

The invention discloses the method that human oral cancer LINC00520 gene rs8008130 polymorphisms are detected with BstEII, comprise the following steps：Extract the genomic DNA of sample；The forward primer and reverse primer of amplification people's LINC00520 gene pleiomorphism rs8008130 location proximate sequences are provided, using the human gene group DNA to be measured of extraction as template, enters performing PCR amplification, obtains amplified production；Digestion is carried out to the amplified production of acquisition using restriction enzyme, obtains corresponding digestion products；Digestion products are subjected to electrophoresis using 3% Ago-Gel, to judge LINC00520 gene pleiomorphisms rs8008130 each genotype.Method of the invention is simple, quick, safe and accurate, high sensitivity, is worth promoting in clinical and research, and can use for reference the parting for other Genetic polymorphisms.

Description

With BstEII detection human oral cancer LINC00520 gene rs8008130 polymorphisms Method

Technical field

The invention belongs to biological technical field, is related to one kind BstEII detection human oral cancer LINC00520 genes The method of rs8008130 polymorphisms.

Background technology

SNP (single nucleotide polymorphism, SNP), is primarily referred to as in genome water As the DNA sequence polymorphism caused by the variation of single nucleotide acid, including the form such as displacement/insertion of base and missing on flat. SNP is most common one kind in the heritable variation of the mankind, be widely used in simple and complex disease genetic linkage analysis/ Association analysis and the positioning of diseases predisposing gene, instruct tumor susceptibility gene to clone.The detection in more typical single base polymorphismses site Method includes PCR machine technology (PCR-RFLP), three-primer amplified allele Method (TP-PCR) and sequencing etc..Though these methods respectively have advantage, also respectively there is its weak point.

CAPs (cleaved amplification polymorphism sequence-tagged sites) CAPs skills Art is also known as PCR-RFLP, RFLP polymerase chain reaction (PCR-RFLP) technology.PCR-RFLP is one Kind is quick, easy, accurately detects the classical way of SNP genotype.Its principle is:Restriction enzyme is a kind of identification DNA Specific site (usual 4-6bp), and the enzyme cut in specific site.The specificity of restriction enzyme site means to specific etc. The complete digestion of position gene can produce same fragment sequence.And the displacement of base, insertion and missing can produce or eliminate one Individual specific restriction enzyme site, so as to change the size and number that fragment is produced after digestion.The difference of these endonuclease bamhi banding patterns is referred to as RFLP.If SNP is produced and is eliminated some restriction endonuclease sites, can be by PCR Product carries out digestion, electrophoresis is detected.The advantages of this method, is that quickly and easily endpoint is accurate.But its major defect It is the selection of restriction enzyme site, it is desirable to which pleomorphism site to be measured is related to a certain restriction enzyme site.The selection of PCR-RFLP enzymes has Limitation, not all SNP site can differentiate in this way, and part restriction endonuclease is expensive, tests into This height.Three-primer amplified allele method (TP-PCR) is a kind of recombinant DNA side for not needing restriction enzyme and ligase Method.There are 2 kinds of templates and 3 kinds of primers in reaction system, so as to produce a recombinant DNA molecules in same reaction system.This The major advantage of kind method is quick, the restriction enzyme sites independent of junction fragment.But its shortcoming is that amplification efficiency is low, amplification is special The opposite sex is poor, can not ensure the specificity and stability of PCR primer, genotyping result easily causes erroneous judgement.Taqman fluorescence probe method is A kind of new and effective accurate methods of genotyping, its advantage are detection sensitivity height, and parting is accurate.But this method needs costliness Instrument and longer step, cost it is higher.

Carcinoma of mouth (Oral cancer, OC) is department of stomatology common cancer, one of global malignant tumour of hair well, 90% Above is the squamous cell carcinoma in oral mucosa source.The new cases of the annual carcinoma of mouth in China just have nearly 4.56 ten thousand people.Due to mouth Chamber cancer morbidity is high, and prognosis is poor, mainly adds up Maxillary region, big for people's body and mind health effect, therefore preventing and controlling are not allowed to neglect Depending on.Because blood circulation is enriched in oral cavity, frequent plus tongue mechanical movement, Flora distribution is various in oral cavity, can be caused after canceration Oral cavity is infected, and Lymph Node Metastasis occurs in flora imbalance, severe patient.Long-chain non-coding RNA expression imbalance occurs with mouth neoplasm Between it is in close relations.

Carcinoma of mouth be gene and environment etc. it is multifactor collaboration or sequential effect under, and Neuro-endocrine-immunity regulate and control Under, sexual development is carried out to the stage has the specific disease of height.LINC00520 achieves in the research of tumor area in recent years Greater advance, but its research in carcinoma of mouth field carry out it is relatively fewer, therefore by the related LINC00520 sites of certain function Polymorphism combines with carcinoma of mouth can preferably inquire into its effect to Mediating Oral Carcinogenesis, disclose it and sent out with carcinoma of mouth Raw relevance and then discussion specific molecular mechanism therein.RNA (long intergenic non-between long-chain Noncoding gene Protein coding RNA 520, LINC00520) its encoding gene is located at human chromosomal 14q22.3.LINC00520 grows About 20kb, it is highly conserved long-chain non-coding RNA, is widely expressed in Various Tissues.LINC00520 is new hair in 2012 Existing gene, it has correlation with Spain children obesity.Full-length genome research in 2014 shows LINC00520 and Bangladesh A related gene phenotype of state adult human body weight.LncRNA also indicates in various cancers patient and the differential expression in healthy person LncRNA may be closely related with the occurrence and development of cancer.LINC00520 is in the galactophore epithelial cell converted by carcinogenic PI3K Middle increase, it expresses knockout of the increase dependent on mutation PIK3CA.Expression pattern analysis emphasizes LINC00520 in human breast cancer Rise, the priority enrichment especially in substrate sample breast cancer.Research shows that ShRNA mediations LINC00520 consumption prevents breast cancer The migration of cell, LINC00520 are adjusted by oncogene Src, PIK3CA and STAT3, potentially contribute to the molecular disease of breast cancer Studied because learning.The researchs such as Chen Mingzheng in 2015 show that LINC00520 is in low expression in cell line HK-1, and LINC00520 is non- Nasopharyngeal carcinoma group expression is higher than nasopharyngeal carcinoma group, the change of LINC00520 expressions in nasopharyngeal carcinoma and nasopharynx benign disease Prompt its differential expression relevant with the occurrence and development of nasopharyngeal carcinoma.

Three kinds of genotype in crowd be present in LINC00520 gene rs8008130 loci polymorphisms：AA type (human genomes Two allele bases of rs8008130 polymorphic sites are T), AC types (the two of human genome rs8008130 polymorphic sites Individual allele base is respectively A and C) and CC types (two allele bases of human genome rs8008130 polymorphic sites are equal For C) (the state-run medical library biology information technology center of NIH, http:// www.ncbi.nlm.nih.gov)。

People LINC00520 gene polymorphics rs8008130 identification is frequently with PCR-RFLP methods at present.But identification at present Expensive (the ginseng on part restriction endonuclease of restriction enzyme that people's LINC00520 gene polymorphics rs8008130 is used often Examine price and refer to table 1 below), experimental cost is high, it is difficult to which popularization uses in the lab.

Therefore, this area is present to simple to operate, and cost is low, the demand of the wide new detection SNP methods of use range.

The content of the invention

In order to overcome defect present in prior art, present invention offer one kind is simple to operate, cost is low, applied widely The general method that human oral cancer LINC00520 gene rs8008130 polymorphisms are detected with BstEII, before experimental cost is saved Put, by Created Restriction Site method (Created Restriction Site PCR, CRS-PCR), held using primer 3 ' wrong With technology, the identification of genotype is carried out after PCR primer carries out restriction enzyme digestion and electrophoresis, one kind is simple to operate, cost is low, suitable so as to provide With detection human oral cancer tumor susceptibility gene LINC00520 polymorphisms rs8008130 in extensive range method and detection kit.

Its technical scheme is as follows：

With the method for BstEII detection human oral cancer LINC00520 gene rs8008130 polymorphisms, comprise the following steps：

(a) genomic DNA of sample is extracted；

(b) provide amplification people's LINC00520 gene pleiomorphism rs8008130 location proximate sequences forward primer and reversely Primer, using the human gene group DNA to be measured of step (a) extraction as template, enter performing PCR amplification, obtain amplified production；

(c) digestion is carried out to the amplified production obtained in (b) using restriction enzyme, obtains corresponding digestion products；

(d) digestion products are subjected to electrophoresis using 3% Ago-Gel, to judge LINC00520 gene pleiomorphisms Rs8008130 each genotype.

Further, the design of forward primer described in step (b) and reverse primer is specially with synthesis：Base sequence information Obtained from NCBI dbSNP databases, checked on CHIP websites, determine LINC00520 polymorphic site nucleotide variation numbers According to.

Partial gene sequence containing LINC00520rs8008130 such as SEQ:ID:NO:Shown in 1；The 433rd of gene order Base M represents that A/C is polymorphic, as rs8008130 pleomorphism site.According to rs8008130 particular sequence and polymorphism The position of base is designed using Primer Premier 6.0, sets the parameter such as primer length and amplification purpose fragment length to carry out Design of primers, optimal upstream and downstream primer is selected according to G/C content and annealing temperature etc., then by the Blast in this primer and Pubmed Sequence alignment function (http://blast.ncbi.nlm.nih.gov/Blast.cgi) primer comparison is carried out, finally determine Upstream and downstream primer, forward primer sequence such as SEQ:ID:NO:Shown in 2；Reverse primer sequences such as SEQ:ID:NO:Shown in 3, wherein just Into primer sequence, 5th bit base reciprocal is G (correspondence position of amplified production is base mismatch G), therefore PCR primer after mispairing In polymorphic sequence nearby GGTGACC or GGTGACA be changed to by GTTGACC or GTTGACA (wherein the 7th base A/C be polymorphic Site, the 2nd bases G are base mismatch).Therefore C allele fragment can be identified G^GTGACC sequences in amplified production Restriction enzyme identifies (i.e. C allele fragment can be cut by restriction endonuclease).And then situation can be cut according to fragment to sentence Disconnected polymorphic gene type.

According to forward primer sequence and reverse primer sequences synthetic primer, the synthesis of primer can use generally in the art Method (such as solid-phase synthesis), also biotech firm can be entrusted to synthesize and detect forward and reverse primer.

Further, preparing PCR amplification system in step (b) in pcr amplification reaction is specially：2×Taq PCR Mix7.5μ L, the μ l of distilled water 5.9, the μ l of 0.3 μ l anti-sense primers of sense primer 0.3 and the μ l of template DNA 1.0,15.0 μ are produced after fully mixing LPCR amplification reaction systems；According to the first stage：94 DEG C of denaturation 5min, second stage includes 35 circulations, three steps altogether, first First 94 DEG C of denaturation 30s, 60.9 DEG C of annealing 45s, last 72 DEG C extend 45s, phase III：72 DEG C of extension 5min, final 4 DEG C of storages With standby, a length of 270bp pcr amplification product is produced.Product Sequence after amplification is such as SEQ:ID:NO:Shown in 4.

Further, the digestion system of progress digestion is specially in step (c)：The μ l of PCR reaction products 5, restriction enzyme 0.5 μ l, the Buffer1.0 μ l and μ l of nuclease free pure water 8.5 amount to 15 μ l digestion systems, 60 DEG C of digestion 3h in water-bath, Produce digestion products.

Further, step (d) agarose gel electrophoresis, determine that gene pleiomorphism is specially：By digestion products with 3% fine jade Sepharose is under conditions of 4-10V/cm, electrophoresis 20-40min, to uviol lamp under can distinguish after obvious band and mirror of taking pictures It is fixed.For different genotype, rs8008130 sites different genotype shows different bands, wild-type genotype CC, a length of The band of 250bp, 20bp two；Heterozygous genotypes AC, a length of 270bp, 250bp and the bands of 20bp tri-；Heterozygous genotypes AA, 270bp One band.

Compared with prior art, beneficial effects of the present invention：

The present invention uses Created Restriction Site method (Created Restriction Site PCR, CRS-PCR), using drawing Thing base mispairing Technology design detection human oral cancer LINC00520 polymorphisms rs8008130.Because this method applies primer 3 ' end mispairing technologies, PCR primer can carry out the appraisal of genotype after carrying out restriction enzyme digestion and electrophoresis, therefore have in practice There is very big flexibility, and detection method is simple and easy, is a kind of preferable side for carrying out single base mutation loci gene type identification Method.It is provided by the present invention to detect the letter of human oral cancer tumor susceptibility gene LINC00520 polymorphism rs8008130 methods of genotyping Single, quick, safe and accurate, high sensitivity, it is worth promoting in clinical and research, and can uses for reference for other Genetic polymorphisms Parting.

Brief description of the drawings

Fig. 1 is the electrophoresis pattern in LINC00520rs8008130 sites；

Fig. 2 is LINC00520rs8008130 sites CC genotype Sequencing chromatogram (backward sequencing)；

Fig. 3 is LINC00520rs8008130 sites AC genotype Sequencing chromatogram (backward sequencing)；

Fig. 4 is LINC00520rs8008130 sites AA genotype Sequencing chromatogram (backward sequencing).

Embodiment

Technical scheme is further illustrated with reference to the accompanying drawings and examples.

The method of the present invention comprises the following steps：

(a) genomic DNA of sample is extracted；

(b) provide amplification people's LINC00520 gene pleiomorphism rs8008130 location proximate sequences forward primer and reversely Primer, using the human gene group DNA to be measured of step (a) extraction as template, enter performing PCR amplification, obtain amplified production；

(c) digestion is carried out to the amplified production obtained in (b) using restriction enzyme, obtains corresponding digestion products；

(d) digestion products are subjected to electrophoresis using 3% Ago-Gel, to judge LINC00520 gene pleiomorphisms Rs8008130 each genotype.

Wherein, it is AA genotype to have a band person after electrophoresis, and two band persons are CC genotype, and three band persons are AC genotype.The technologies such as the amplification that is related in the above method, extracting genome, digestion can use the routine operation side of this area Method.

Wherein, its 3 ' end of the forward primer is adjacent with polymorphic rs8008130 bases, and the 5th bit base reciprocal is that G (expands The correspondence position for increasing production thing is base mismatch G), to form G^GTGACC structures with polymorphic C allele in amplified production (the 2nd bases G is base mismatch, and the 7th base C is polymorphic allele).

Sequence near SNP can be changed into and can be determined in advance by the base mismatch on primer by the method for the present invention Restriction enzyme identification sequence.Therefore, the needs present in traditional PCR-RFLP methods can be overcome using expensive The defects of restriction endonuclease, and then greatly reduce detection SNP cost.Specifically：Due to inverse the 5th in the forward primer sequence Bit base is T (correspondence position of amplified production is base mismatch G), thus after mispairing in PCR primer it is polymorphic nearby sequence by GTTGACC or GTTGACA is changed to GGTGACC or GGTGACA, and (wherein the 7th base A/C is polymorphic site, the 2nd bases G For base mismatch).Therefore C allele fragment can be identified the restriction enzyme identification of G^GTGACC sequences in amplified production (i.e. C allele fragment can be cut by restriction endonuclease)；And then situation can be cut according to fragment to judge polymorphic gene type.More Specifically, understand that A/C is polymorphic in gene original series to be identified by first three restriction endonuclease in table 1 through sequence analysis.Such as pass through alkali 5th bit base T before pleomorphism site is changed into G by base error-prone PCR, then it is that C allele includes after PCR is expanded that this is polymorphic GGTGACC sequences can be identified the restriction enzyme BstEII identifications of G^GTNACC sequences, and A allele can not be cut.

The invention provides a kind of detection oral cavity cancer susceptibility gene LINC00520 gene pleiomorphism rs8008130 sites Method, due to identifying that the restriction enzyme BstEII of specific site is applied widely, price is relatively inexpensive, and then substantially reduces Detection SNP cost.Found through sequence analysis, 4 kinds of enzymes in the usable table 1 of the polymorphic methods of the A/C are detected, wherein preceding 3 Kind enzyme is expensive, and digestion effect is undesirable.Using Created Restriction Site method (Created Restriction Site PCR, CRS-PCR) to 5th bit base reciprocal changes into G by T in positive primer sequence after, polymorphic sequence life nearby in PCR primer Into GGTGACC sequences, so as to be identified by restriction enzyme BstEII.Due to identifying the restriction enzyme of specific site BstEII is applied widely, and price is relatively inexpensive, and then greatly reduces detection SNP cost.Therefore, simple behaviour is considered The property made and economic and practical, restriction enzyme BstEII are only selections.

The price of the restriction enzyme provided in table 1 is from NEB companies (http://www.neb-china.com), limitation The selection of property restriction endonuclease obtains (http from WatCut restriction endonuclease analysis softwares://watcut.uwaterloo.ca/ template.phpAct=snp_new), in this, as the reference of restriction enzyme enzyme recognition site and price.

1 four kinds of restriction endonuclease recognition sequences of table and its price

In an embodiment of the present invention, the design of forward primer and reverse primer is carried out using Primer6.0 softwares, design Principle considers the influence of sensitivity, specificity and amplification efficiency that primer pair PCR is expanded.According to base pair complementarity Principle designs primer, primer length typically between 15~30 bases, it is long or it is short can cause poor specificity, it is long also to lead Cause its elongating temperature to be more than 74 DEG C, be unsuitable for Taq DNA polymerase and reacted.Primer G/C content is between 40%~60%, Tm For value preferably close to 72 DEG C, G/C content is too high or too low to be all unfavorable for initiation reaction.Base wants random distribution, primer itself and primer Between should not have complementary series, otherwise primer itself, which can be folded into hairpin structure, makes primer renaturation itself.Primer 5 ' is held with Between △ G values should be of a relatively high, and 3 ' end △ G values it is relatively low.The single-stranded of amplified production can not form secondary structure.Primer should have Specificity, after design of primers is completed, BLAST detections should be carried out to it, to ensure it with other genes without complementarity.

On this basis, the sense primer finally chosen is 5 '-TTTGTAGATG CATCACTCTG GTGAC-3 ', downstream Primer is 5 '-AATCTTTGAA TCCACCTATG ACC-3 '.The fragment 270bp in the face that thus primer amplifies, amplified production It is as follows：

Underscore part is respectively forward primer and reverse primer, and the 26th bit base M represents the polymorphic i.e. SNP sites of A/C rs8008130.21st bit base is the bases G (being base T originally) after mispairing.The amplified production of a length of 270bp is through restricted 270bp can be produced after restriction endonuclease BstEII digestions, 250bp, 20bp totally three kinds of clip types.Genotype result of determination：Wild base Because of type CC, the band of a length of 250bp, 20bp two；Heterozygous genotypes AC, a length of 270bp, 250bp and the bands of 20bp tri-；Heterozygous genes Type AA, a length of bands of 270bp mono-.

Gel electrophoresis is divided into agarose gel electrophoresis and polyacrylamide gel electrophoresis, and agarose (Agarose) is a kind of Linear polysaccharide polymer, it is to be extracted from Red seaweeds product agar.Cooled down after agarose solution is heated to boiling point Solidification will form good electrophoretic medium, and its density is determined by the concentration of agarose, the preparation electricity available for DNA fragmentation Swimming.Polyacrylamide gel mainly has two ways：First, the non denatured polyacrylamide for separating and purifying double chain DNA fragment Amine gel, second, for separation and the denaturing polyacrylamide gel of purification of single stranded DNA fragmentation.But consider practicality, just Profit and economy, it can yet be regarded as a kind of optimal selection using agarose gel electrophoresis.In the present invention, purpose amplified production makes With digestion 1-16h in restriction endonuclease BstEII 3U water-baths.Digestion products carry out agar using 3% agarose Sugared gel electrophoresis, wild homozygous genotype, heterozygous genotypes and mutation are judged according to different bands under ultra violet lamp Homozygous genotype.

In specific embodiments of the present invention, present invention detection people LINC00520 gene polymorphic rs8008130 sites Comprise the following steps that：

(l) human gene group DNA's template to be measured is extracted, human gene group DNA's template is the people that human body any part obtains Genomic templates.

(2) PCR amplifying genom DNAs, performing PCR amplification is entered to the templet gene group DNA of extraction, obtained containing polymorphic sequence nearby The PCR primer of row.

(3) endonuclease reaction is carried out with restriction endonuclease BstEII to pcr amplification product, obtains digestion products；

(4) digestion products enter row agarose gel electrophoresis using agarose, according to different bands under ultra violet lamp Judge wild homozygous genotype, heterozygous genotypes and mutant homozygous genotype.

Above-mentioned each key step is described in detail below：

(1) design of primers and synthesis

Base sequence information obtains from NCBI dbSNP databases, is checked on CHIP websites, it is determined that LINC00520 polymorphic site nucleotide variation data.

The partial gene sequence of the rs8008130 containing LINC00520 is as follows：

Gene sequence 433rd base M of row represents that A/C is polymorphic, as rs8008130 pleomorphism site.According to rs8008130 specific sequence The position of row and polymorphic base is designed using Primer Premier 6.0, sets primer length and amplification purpose fragment length Carry out design of primers etc. parameter, optimal upstream and downstream primer selected according to G/C content and annealing temperature etc., then by this primer with Blast sequence alignment functions (http in Pubmed://blast.ncbi.nlm.nih.gov/Blast.cgi) carry out primer Compare, the upstream and downstream primer finally determined is：

Forward primer sequence：5’-TTTGTAGATGCATCACTCTGGTGAC-3’

Reverse primer sequences：5’-AATCTTTGAATCCACCTATGACC-3’

The 5th bit base reciprocal is G (correspondence position of amplified production is base mismatch G) wherein in forward primer sequence, because Polymorphic sequence nearby is changed to GGTGACC or GGTGACA the (the wherein the 7th by GTTGACC or GTTGACA in PCR primer after this mispairing Individual base A/C is polymorphic site, and the 2nd bases G is base mismatch).Therefore C allele fragment can be identified in amplified production The restriction enzyme identification of G^GTGACC sequences (i.e. C allele fragment can be cut by restriction endonuclease).And then can be according to piece Section incision situation judges polymorphic gene type.

According to forward primer sequence and reverse primer sequences synthetic primer, the synthesis of primer can use generally in the art Method (such as solid-phase synthesis), also biotech firm can be entrusted to synthesize and detect forward and reverse primer.

(2) PCR amplification system is prepared：2 × Taq PCR Mix7.5 μ l, the μ l of distilled water 5.9, the μ l downstreams of sense primer 0.3 The μ l of the primer 0.3 and μ l of template DNA 1.0,15.0 μ lPCR amplification reaction systems are produced after fully mixing；According to the first stage：94 DEG C denaturation 5min, second stage altogether include 35 circulation three steps, 94 DEG C first denaturation 30s, 60.9 DEG C annealing 45s, finally 72 DEG C of extension 45s, phase III：72 DEG C of extension 5min, with standby, the PCR amplifications for producing a length of 270bp are produced for final 4 DEG C of storages Thing.Product Sequence after amplification is：

Underscore part is respectively forward primer and reversely drawn Thing, the 26th bit base M represent the polymorphic i.e. SNP site rs8008130 of A/C.21st bit base is that the bases G after mispairing (was alkali originally Base T).

(3) digestion system：The μ l of PCR reaction products 5, μ l, Buffer1.0 the μ l of restriction enzyme 0.5 and nuclease free The μ l of pure water 8.5 amount to 15 μ l digestion systems, and 60 DEG C of digestion 3h, produce digestion products in water-bath.Consider economical and practical Property and simple operations final choice BstEII carry out digestion identification.

(4) agarose gel electrophoresis, gene pleiomorphism is determined：By digestion products with 3% Ago-Gel in 4-10V/ Under conditions of cm, electrophoresis 20-40min, to uviol lamp under can distinguish after obvious band and identification of taking pictures.For different genes Type, rs8008130 sites different genotype show different band (see Fig. 1), wild-type genotype CC, a length of 250bp, 20bp Two bands；Heterozygous genotypes AC, a length of 270bp, 250bp and the bands of 20bp tri-；The band of heterozygous genotypes AA, 270bp mono-.Each base Because type is further identified through being sequenced, sequencing result (see Fig. 2-4) display is identical with the result that prior art measures.

Here is to implement some embodiments of the present invention：

The human oral cancer peripheral blood whole blood sample of embodiment 1. determines LINC00520rs8008130 polymorphisms

1 materials and methods

1.1 key instruments and reagent

Instrument：BCD-228CH refrigerators (newly fly electrical equipment), HH-2 digital displays thermostat water bath (magnificent peak instrument), SmartGe gels Imager (Beijing match intelligence is started an undertaking), GT9612 grads PCRs instrument (hundred Tykes biology), WD900SL23-2 model micro-wave oven (Glanzs Electrical equipment), DG-300C types electrophoresis apparatus (the prosperous biology of ancient cooking vessel state) etc..

Reagent：NEP004-1DNA extracts kits (ancient cooking vessel state prosperous biology), 50bp DNA Ladder (Lay humorously thing), 2 × Taq PCR Mix (Lay humorously thing), BstEII (NEB), agarose (SIGMA) etc..

1.2 design of primers

In NCBI dbSNP databases, LINC00520rs8008130 nucleotide sequence is searched, it is soft in design of primers Designed in part Primer Premier 6.0, set the parameter such as primer length and amplification purpose fragment length to carry out design of primers, Optimal upstream and downstream primer is selected according to G/C content and annealing temperature etc., then by the Blast sequence alignments in this primer and Pubmed Function (http://blast.ncbi.nlm.nih.gov/Blast.cgi) primer comparison is carried out, the upstream and downstream finally determined is drawn Thing is：

Forward primer sequence：5’-TTTGTAGATGCATCACTCTGGTGAC-3’

Reverse primer sequences：5’-AATCTTTGAATCCACCTATGACC-3’

The 5th bit base reciprocal is G (correspondence position of amplified production is base mismatch G) wherein in forward primer sequence, because Polymorphic sequence nearby is changed to GGTGACC or GGTGACA the (the wherein the 7th by GTTGACC or GTTGACA in PCR primer after this mispairing Individual base A/C is polymorphic site, and the 2nd bases G is base mismatch).Therefore C allele fragment can be identified in amplified production The restriction enzyme identification of G^GTGACC sequences (i.e. C allele fragment can be cut by restriction endonuclease).And then can be according to piece Section incision situation judges polymorphic gene type.

According to forward primer sequence and reverse primer sequences synthetic primer, the synthesis of primer can use generally in the art Method (such as solid-phase synthesis), also biotech firm can be entrusted to synthesize and detect forward and reverse primer.

The selection of 1.3 restriction enzymes

According to the base sequence in rs8008130 sites in dbSNP, with the online restriction endonuclease analysis softwares of WatCut, On-line search obtains the information of the restriction enzyme in recognizable mutational site, and it is specific and economic and practical to consider its digestion The optimal restriction enzyme BstEII of Sexual behavior mode.

1.4 extract the genomic DNA of sample to be tested from oral cancer patient's whole blood

In strict accordance with centrifugation column type DNA extraction kit operating procedure extraction genomic DNA.

L) after 300 μ l haemocytes are added in 1.5ml centrifuge tubes, 900 μ l cell pyrolysis liquids is added and are mixed evenly, in ice After upper placement 10min, 12000rpm centrifuges 1min in centrifuge, abandons supernatant, adds 900 μ l cell pyrolysis liquids again, use rifle Blow afloat after precipitating and mixing, repeat the above steps；

2) 600 μ l solution B solutions are added into sediment, after gently blowing afloat precipitation with liquid-transfering gun, add 10 μ l Proteinase K mixes, after placing 10min in 70 DEG C of water-baths, 12000rpm centrifugations 5min；

3) supernatant in centrifuge tube is transferred in the new centrifuge tube compiled number, then 500 μ l is added into new centrifuge tube Absolute ethyl alcohol, during which it is possible that flocky precipitate, the floccule are DNA；

4) liquid of mixing is transferred in centrifugal column (if once turning endless, graded is transferred to), is stored at room temperature 2min, then 12000rpm centrifuges 1min, abandons waste liquid；

5) the solution C rinsing liquids that 700 μ l have added respective volume absolute ethyl alcohol are added, are stored at room temperature 2min, 12000rpm centrifuges 1min, abandons waste liquid；

6) the solution D rinsing liquids that 700 μ l have added respective volume absolute ethyl alcohol are added, are stored at room temperature 2min, 12000rpm centrifuges 1min, abandons waste liquid；

7) 500 μ l solution D rinsing liquids are added, 12000rpm centrifugation 1min, abandon waste liquid；

8) 2min is centrifuged again, centrifugal column is placed in the new centrifuge tube compiled number, opening is put into 37 DEG C of insulating boxs 10min is until without obvious ethanol flavor；

9) 65 DEG C of the μ l of solution E 100 are had been preheated with the addition of silicon substrate plasma membrane center, room temperature places 5min, 12000rpm centrifuges 1min, repeats the above steps once, and the liquid after centrifugation is the genomic DNA extracted eventually；

10) draw the DNA that extract of 2 μ l with NanoPhotometer Pearl micro-spectrophotometers determine its concentration and Purity.

2 results

2.1PCR amplification

(1) according to the concentration of extraction genomic DNA, the DNA of research object is diluted, makes final concentration of 20 μ g/ μ l.

(2) PCR amplification system is μ l of 2 × Taq PCR Mix 7.5, sense primer and each 0.3 μ l of anti-sense primer, template DNA1.0 μ l, cumulative volume finally is supplemented to 15 μ l with distilled water.

(3) the PCR reaction conditions in rs8008130 sites：First stage is the pre-degeneration stage, 94 DEG C/5min；Second Stage include three steps totally 35 circulation, set gradually for 94 DEG C/35s, 60.9 DEG C of annealing time 45s, 72 DEG C/30s；3rd Individual 72 DEG C/5min of stage.

2.2 endonuclease reaction

Digestion system is μ l of pcr amplification product 5, the μ l of upper restriction enzyme 0.5, Buffer 1.0 μ l, is finally steamed with double Water supplements cumulative volume to 15 μ l.60 DEG C of water-baths 3 hours are placed in water-bath after mixing.

The judgement of 2.3 genotype

The rs8008130 loci gene types of table 2 judge

The human oral cancer tissue specimen of embodiment 2. determines LINC00520rs8008130 polymorphisms

The step of with embodiment 1, is essentially identical, simply adopts and extracts DNA works from carcinoma of mouth tissue specimen in the following method For DNA to be measured.

The oral cavity cancerous tissue for the pathological diagnosis that operation is cut is obtained, the gene of oral cavity cancerous tissue is extracted using phenol-chloroform method Group DNA is as follows as DNA to be measured, extraction step：

1) carcinoma of mouth tissue block is thawed, blood stains is washed away with physiological saline, clip 0.1g tissue is milled, and is added 1ml aqua sterilisa, overturn and mix, 10000rpm centrifugation 10min, abandon supernatant.Ttom of pipe should have precipitation, and above step repeats two It is secondary.

2) 200 μ l DNA lysates are added, 5 μ l Proteinase K is mixed, and 55 DEG C of water-baths are digested overnight.

3) isometric phenol chloroform mixed liquor (1 is added after the completion of digesting:1), acutely concussion, makes it become milk coffee color. 12000rpm centrifuges 10min.

4) avoid touching intermediate medium and lower floor's liquid when taking supernatant.Mixing is overturned after adding isometric chloroform. 12000rm centrifuges 10min.

5) supernatant is taken, pays attention to avoiding touching intermediate medium and lower floor's liquid.Add 1/10 sodium acetate and 2.5 times Absolute ethyl alcohol, overturn and mix, 12000rpm centrifugation 10min, abandon supernatant.

6) ethanol of 1ml 70% is added, its white precipitate is suspended, overturns and mixes for several times, 12000rpm centrifugation 10min, is abandoned Supernatant, being stored at room temperature 5-10min makes ethanol volatilization clean.

7) 50 μ l aqua sterilisa dissolving DNAs are added, produce carcinoma of mouth tissue gene group DNA, -20 DEG C of preservations.

As a result：By digestion products with 3% agarose gel electrophoresis 20-40min, to uviol lamp under can distinguish obvious bar Band and identification of taking pictures.For different genotype, rs8008130 sites different genotype shows different band (see Fig. 1), CC wild-type genotypes, the band of a length of 250bp, 20bp two；AC heterozygous genotypes, a length of 270bp, 250bp and the bands of 20bp tri-；AA Heterozygous genotypes, the bands of 270bp mono-.Each genotype further identifies that sequencing result (see Fig. 2-4) display is with showing through being sequenced The result for having technology to measure is identical.

The invention is intended to provide a kind of detection human oral cancer tumor susceptibility gene simple to operate, cost is low, applied widely LINC00520 polymorphisms rs8008130 method.From above-described embodiment as can be seen that the present invention using CRS-PCR for being identified LINC00520 polymorphism rs8008130 sites, are improved to normal PCR-RFLP methods, apply primer 3 ' and hold mispairing technology, It is low to overcome normal PCR-RFLP method inscribe enzyme selectivities, expensive, the shortcomings of experimental cost is high, what this research was established The detection method of LINC00520rs8008130 polymorphisms, restriction endonuclease price is very cheap, while PCR primer purity is higher, enzyme Result is cut to be easy to differentiate.The detection method is by sequence verification, and its result is accurately and reliably.

SNP detection techniques are very ripe, and the detection method in more typical single base polymorphismses site is drawn including sequencing, three Thing amplified allele method (TP-PCR), PCR machine technology (PCR-RFLP) etc. Method.Though these methods respectively have advantage, also respectively there is its weak point.Sequencing can directly measure all prominent in DNA sequence dna Become the base alternative case in site, but this method needs expensive instrument and longer step, cost higher；TP-PCR methods are accurate True property is low, as a result easily causes erroneous judgement；PCR-RFLP method comparative maturities, but require pleomorphism site to be measured and a certain restriction enzyme site Correlation, the selection of enzyme have limitation, and often cost is higher.

Therefore, in view of restriction enzyme used in the present invention is cheap, and primer synthesis and each reagent compared with For economy, CRS-PCR is simple to operate in addition, reproducible, therefore is most economical efficient gene type detection method.

The present invention is improved to normal PCR-RFLP technologies, using Created Restriction Site method (Created Restriction Site PCR, CRS-PCR), detect LINC00520 polymorphisms using primer base mispairing Technology design rs8008130.Mispairing technology, PCR primer is held to carry out base after carrying out restriction enzyme digestion and electrophoresis because this method applies primer 3 ' Because of the appraisal of type, therefore there is very big flexibility in practice, and detection method is simple and easy, is a kind of progress The better method of single base mutation loci gene type identification.The key problem in technology point of this invention is that the LINC00520 designed is more State property rs8008130 upstream and downstream primer,

Forward primer sequence：5’-TTTGTAGATGCATCACTCTGGTGAC-3’

Reverse primer sequences：5’-AATCTTTGAATCCACCTATGACC-3’

And restriction enzyme BstEII use.It is provided by the present invention to detect human oral cancer tumor susceptibility gene LINC00520 polymorphism rs8008130 methods of genotyping is simple, quick, safe and accurate, high sensitivity, be worth clinical and Promoted in research, and the parting for other Genetic polymorphisms can be used for reference.

Best mode for carrying out the invention is the foregoing is only, any one skilled in the art drapes over one's shoulders in the present invention In the technical scope of dew, the simple change of technical scheme can be become apparent to or equivalence replacement each falls within the protection of the present invention In the range of.

Sequence table

<110>Affiliated hospital of Zhengzhou University first

<120>With the method for BstEII detection human oral cancer LINC00520 gene rs8008130 polymorphisms

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1398

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 1

ctgctctcct caaaccaagg gctcattgct gtttcctgat cccttgtcca gtgtccagcc 60

tggtttttca tcttgtttct cacacatggc tctttctccc ccaactctgg cttggatccc 120

cccaccccac gggtcttctt ccagggagac tgcgtacctc ctacagcttc agctcttaag 180

tgggacccta actgggaatc tatattcatt tcctaaggct gctgtaacag agtaccacaa 240

actgggaggc tcacaacaac agaaatgttt tctctacagt tctgaatgct agaagtctga 300

aatcaaggtg ttggccagct atggtccctc tgagggctct gggggaagat ctttccttgc 360

ctaaaattct agcttctgct ggttgcctgc gattcttggc actctggttt gtagatgcat 420

cactctgttg acmaaaagag tcaaactctg taaaatggta gaagagatgt attctgagcc 480

aaatacaaga gacaaatggc tcataacaca gccctcaaga gatcctgaga acatgtgtcc 540

aaggtggtca aggtacagct tggttttata cattttaggg agacatgaga catcaaatac 600

acttaagata tacattggtt ttgtccagaa aggggggaca aatggaagct tccaggtcat 660

aggtggattc aaagattttc tgattgatga ttggttgaaa gagttaagtt attgtttaaa 720

gacttaggaa tgtctgggtt aagatgatgg gttgcgagac caaggttttg tcatgcaagt 780

aaggcctcca ggtagcaggc ttcagggaga gtagatggta aatgcttctt atcagatgta 840

taagtttgat aagtccattt ccaagtaaga tcactttctg aggtgccagg tacatatcca 900

ttttgggggg accttattca acccagtaca ctgctgtagg ttatattacc actttccttt 960

tagacatttt ttgtttgttt gttttttgtg atggagtctc actctgttgc ccaggctgga 1020

gtgcagtggt gtgatcttgg ctcactgcaa gctccgcctc ccgggttcac gccattctcc 1080

tgcctcagcc tcctgagtag ctgggactac aggcacccac caccatgcct ggttaattaa 1140

tttttttttt tgtattttta gtagggacag ggtttcactg tgttagccaa gatggtttcg 1200

atctcctgac ctcatgatcc tcctgcctcg gcctcccaaa gtgctgggat tacaggcttt 1260

aaggcatttc tttgttcatg ttccgcagat gttttcaact caatatgtca aataataacc 1320

ttattttcac accaatcctc taagcagaac ctgctccttc tgaagggatt aatggagtca 1380

ctttccttcc agtaacag 1398

<210> 2

<211> 25

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 2

tttgtagatg catcactctg gtgac 25

<210> 3

<211> 23

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 3

aatctttgaa tccacctatg acc 23

<210> 4

<211> 270

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 4

tttgtagatg catcactctg gtgacmaaaa gagtcaaact ctgtaaaatg gtagaagaga 60

tgtattctga gccaaataca agagacaaat ggctcataac acagccctca agagatcctg 120

agaacatgtg tccaaggtgg tcaaggtaca gcttggtttt atacatttta gggagacatg 180

agacatcaaa tacacttaag atatacattg gttttgtcca gaaagggggg acaaatggaa 240

gcttccaggt cataggtgga ttcaaagatt 270

Claims (5)

1. the method for human oral cancer LINC00520 gene rs8008130 polymorphisms is detected with BstEII, it is characterised in that including Following steps：

(a) genomic DNA of sample is extracted；

(b) forward primer of amplification people's LINC00520 gene pleiomorphism rs8008130 location proximate sequences is provided and reversely drawn Thing, using the human gene group DNA to be measured of step (a) extraction as template, enter performing PCR amplification, obtain amplified production；

(c) digestion is carried out to the amplified production obtained in (b) using restriction enzyme, obtains corresponding digestion products；

(d) digestion products are subjected to electrophoresis using 3% Ago-Gel, to judge LINC00520 gene pleiomorphisms Rs8008130 each genotype.

2. the side according to claim 1 that human oral cancer LINC00520 gene rs8008130 polymorphisms are detected with BstEII Method, it is characterised in that the design of forward primer described in step (b) and reverse primer is specially with synthesis：Base sequence information Obtained from NCBI dbSNP databases, checked on CHIP websites, determine LINC00520 polymorphic site nucleotide variation numbers According to；

Partial gene sequence containing LINC00520rs8008130 such as SEQ:ID:NO:Shown in 1；433rd base of gene order M represents that A/C is polymorphic, as rs8008130 pleomorphism site；According to rs8008130 particular sequence and polymorphic base Position designed using Primer Premier 6.0, set primer length and amplification purpose fragment length to carry out design of primers, Optimal upstream and downstream primer is selected according to G/C content and annealing temperature, then by the Blast sequence alignment work(in this primer and Pubmed Primer comparison, the upstream and downstream primer finally determined, forward primer sequence such as SEQ can be carried out:ID:NO:Shown in 2；Reverse primer sequence Row such as SEQ:ID:NO:Shown in 3, the 5th bit base reciprocal is G wherein in forward primer sequence, therefore more in PCR primer after mispairing Nearby sequence is changed to GGTGACC or GGTGACA to state by GTTGACC or GTTGACA；Therefore C allele pieces in amplified production Section can be identified the restriction enzyme identification of G^GTGACC sequences；And then situation is cut to judge polymorphic gene according to fragment Type；

According to forward primer sequence and reverse primer sequences synthetic primer, the synthesis of primer uses solid-phase synthesis or student on commission Thing company synthesizes and detects forward and reverse primer.

3. the side according to claim 1 that human oral cancer LINC00520 gene rs8008130 polymorphisms are detected with BstEII Method, it is characterised in that preparing PCR amplification system in step (b) in pcr amplification reaction is specially：2×Taq PCR Mix7.5μ L, the μ l of distilled water 5.9, the μ l of 0.3 μ l anti-sense primers of sense primer 0.3 and the μ l of template DNA 1.0,15.0 μ are produced after fully mixing LPCR amplification reaction systems；According to the first stage：94 DEG C of denaturation 5min, second stage includes 35 circulations, three steps altogether, first First 94 DEG C of denaturation 30s, 60.9 DEG C of annealing 45s, last 72 DEG C extend 45s, phase III：72 DEG C of extension 5min, final 4 DEG C of storages With standby, a length of 270bp pcr amplification product is produced；Product Sequence after amplification is such as SEQ:ID:NO:Shown in 4.

4. the side according to claim 1 that human oral cancer LINC00520 gene rs8008130 polymorphisms are detected with BstEII Method, it is characterised in that the digestion system of progress digestion is specially in step (c)：The μ l of PCR reaction products 5, restriction enzyme 0.5 μ l, the Buffer1.0 μ l and μ l of nuclease free pure water 8.5 amount to 15 μ l digestion systems, 60 DEG C of digestion 3h in water-bath, Produce digestion products.

5. the side according to claim 1 that human oral cancer LINC00520 gene rs8008130 polymorphisms are detected with BstEII Method, it is characterised in that step (d) agarose gel electrophoresis, determine that gene pleiomorphism is specially：By digestion products with 3% fine jade Sepharose is under conditions of 4-10V/cm, electrophoresis 20-40min, to uviol lamp under can distinguish after obvious band and mirror of taking pictures It is fixed；For different genotype, rs8008130 sites different genotype shows different bands, wild-type genotype CC, a length of The band of 250bp, 20bp two；Heterozygous genotypes AC, a length of 270bp, 250bp and the bands of 20bp tri-；Heterozygous genotypes AA, 270bp One band.