CN107828868A - With the method for HindIII identification human breast carcinoma BCAR4 gene rs13334967 polymorphisms - Google Patents
With the method for HindIII identification human breast carcinoma BCAR4 gene rs13334967 polymorphisms Download PDFInfo
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Abstract
The invention discloses the method that human breast carcinoma BCAR4 gene rs13334967 polymorphisms are identified with HindIII, comprise the following steps：Extract the genomic DNA of sample；The forward primer and reverse primer of amplification human breast carcinoma BCAR4 gene pleiomorphism rs13334967 location proximate sequences are provided, using the human gene group DNA to be measured as template, pcr amplification reaction is carried out, obtains amplified production；Digestion is carried out to the amplified production using restriction enzyme, obtains corresponding digestion products；Digestion products are subjected to electrophoresis using 3% Ago-Gel, to judge each genotype in BCAR4 gene pleiomorphism rs13334967 sites.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
Technical field
The invention belongs to biological technical field, is related to one kind HindIII identification human breast carcinoma BCAR4 genes
The method of rs13334967 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 the detection in the more typical single base polymorphismses sites of tumor susceptibility gene clone
Method includes PCR machine technology (PCR-RFLP), three-primer amplified allele
The methods of method (TP-PCR) and sequencing.Though these methods respectively have advantage, also respectively there is its weak point.
PCR-RFLP is a kind of quick, easy, the accurate classical way for detecting SNP genotype.Its principle is:It is restricted
Restriction endonuclease is that one kind identifies DNA specific sites (usual 4-6bp), and the enzyme cut in specific site.Restriction enzyme site
Specificity means that the complete digestion to specific allele can produce same fragment sequence.And the displacement of base, insertion and
Missing can produce or eliminate a specific restriction enzyme site, so as to change the size and number that fragment is produced after digestion.These enzymes
The difference of section section banding pattern is referred to as RFLP.If SNP produces and eliminated some restriction enzyme
Site, then can be by being detected to PCR primer progress digestion, electrophoresis.The advantages of this method, is that quickly and easily terminal is sentenced
It is disconnected accurate.But its major defect 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, and not all SNP site can differentiate in this way, and in part
Enzyme cutting is expensive, and experimental cost is high.Three-primer amplified allele method (TP-PCR) is that one kind does not need restriction enzyme
With the recombinant DNA method of ligase.There are 2 kinds of templates and 3 kinds of primers in reaction system, so as to be produced in same reaction system
One recombinant DNA molecules.The major advantage of this method is quick, the restriction enzyme sites independent of junction fragment.But its shortcoming
It is that amplification efficiency is low, specific amplification is poor, can not ensure the specificity and stability of PCR primer, and genotyping result easily causes erroneous judgement.
Taqman fluorescence probe method is a kind of new and effective accurate methods of genotyping, and its advantage is detection sensitivity height, and parting is accurate
Really.But this method needs expensive instrument and longer step, cost higher.
BCAR4 (Breast cancer anti-estrogen resistance 4) is a kind of long-chain non-coding RNA, its
Encoding gene is located at human chromosomal 16p13.13.BCAR4 initially resists the sieve of estrogen drug resistant gene in breast cancer cell
Choose and be found abnormal expression.Later research is shown：CCL21 discharges SNIP inhibitor --- the acetylation group that p300 is relied on
Albumen, BCAR4 is attached on transcripton SNIP1 and PNUTS, activate non-classical Hedgehog/GLI2 transcription pathways to promote
Cell invasion.In mouse model, BCAR4 expression is related to advanced breast cancer, and BCAR4 target therapeutic agent LNAs energy
Enough obvious transfers for suppressing breast cancer.It is another to there is research to show：High-level BCAR4 mRNA and the shorter no transfer of breast cancer patients
Survival rate is related to overall survival.BCAR is a kind of powerful transformed gene, can cause estrogen independent growths, antiestrogenic
Resistance, makes tumour be formed in vivo.Increasing research finds lncRNA SNP (single
Nucleotide polymorphism, SNP) it is related to breast cancer susceptibility.BCAR4 has been found
The lncRNA of pass, and gene mutation is possible to influence lncRNA adjustment effect, promote cell occur canceration and tumour occur,
Development.
Three kinds of genotype in crowd be present in BCAR4 gene rs13334967 loci polymorphisms：AA type (human genomes
Two allele bases of rs13334967 polymorphic sites are A), AT types be (human genome rs13334967 polymorphic sites
Two allele bases are respectively A and T) and TT types (two allele alkali of human genome rs13334967 polymorphic sites
Base is T) (the state-run medical library biology information technology center of NIH, http://
www.ncbi.nlm.nih.gov)。
Human breast carcinoma BCAR4 gene polymorphics rs13334967 identification should use PCR-RFLP methods in theory.But mesh
The preceding restriction enzyme used without identification human breast carcinoma BCAR4rs13334967 gene pleiomorphisms, is caused to SNP experiments
It is certain to hinder.
Therefore, this area is present 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 breast carcinoma BCAR4 gene rs13334967 polymorphisms are identified with HindIII, saving the premise of experimental cost
Under, by Created Restriction Site method (Created Restriction Site PCR, CRS-PCR), mispairing is held using primer 3 '
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, applicable so as to provide
The method and detection kit of detection human breast carcinoma BCAR4rs13334967 polymorphisms in extensive range.
Its technical scheme is as follows：
With the method for HindIII identification human breast carcinoma BCAR4 gene rs13334967 polymorphisms, comprise the following steps：
(a) genomic DNA of sample is extracted；
(b) forward primer of amplification people's BCAR4 gene pleiomorphism rs13334967 location proximate sequences is provided and reversely drawn
Thing, using the human gene group DNA to be measured as template, pcr amplification reaction is carried out, obtains amplified production.
(c) digestion is carried out to the amplified production using restriction enzyme, obtains corresponding digestion products；
(d) digestion products are subjected to electrophoresis using 3% Ago-Gel, to judge BCAR4 gene pleiomorphisms
Each genotype in rs13334967 sites.
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 BCAR4 polymorphic site nucleotide variation data
Partial gene sequence containing BCAR4rs13334967 such as SEQ:ID:NO:Shown in 1；507th alkali of gene order
Base R represents that A/G is polymorphic, as rs8012083 pleomorphism site.It is more that 501st base W of gene order represents A/T
The pleomorphism site of state, as rs13334967.Used according to the position of rs13334967 particular sequence and polymorphic base
The upstream and downstream primer that Primer Premier 6.0 are designed, forward primer sequence such as SEQ:ID:NO:Shown in 2；Reverse primer sequence
Row such as SEQ:ID:NO:Shown in 3, the 2nd bit base reciprocal is A (the amplified production correspondence positions of formation wherein in forward primer sequence
For base mismatch A), thus after mispairing in PCR primer it is polymorphic nearby sequence by ATGCTA or ATGCTT be changed to AAGCTA or
AAGCTT (wherein the 2nd base A is base mismatch, and the 6th base A/T is polymorphic site).Therefore T equipotential bases in amplified production
The restriction enzyme Hind III that A^AGCTT sequences can be identified because of fragment identifies that (i.e. T allele fragment can be by inscribe digestion
Open.And then situation can be cut according to fragment to judge 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, 62.1 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 163bp pcr amplification product is produced.Product Sequence such as SEQ after amplification: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, 37 DEG C of digestion 1- in water-bath
16h, produce digestion products.Consider economic and practical and simple operations, select Hind III to carry out digestion identification.
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, rs13334967 sites different genotype shows different bands, wild homozygous genotype
AA, a length of bands of 163bp mono-；It is mutated heterozygous genotypes AT, a length of 163bp, 140bp and the bands of 23bp tri-；It is mutated heterozygous genes
The band of type TT, 140bp, 23bp two.
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 BCAR4 polymorphisms rs13334967.Mispairing skill is held because this method applies primer 3 '
Art, PCR primer can carry out the appraisal of genotype after carrying out restriction enzyme digestion and electrophoresis, therefore have very big spirit in practice
Activity, and detection method is simple and easy, is a kind of better method for carrying out single base mutation loci gene type identification.The present invention is built
Vertical BCAR4 polymorphism rs13334967 methods of genotyping is simple, quick, safe and accurate, high sensitivity, is worth in clinic
Promoted with research, and the parting for other Genetic polymorphisms can be used for reference.
Brief description of the drawings
Fig. 1 is the electrophoresis pattern in BCAR4rs13334967 sites；
Fig. 2 is that BCAR4rs13334967 sites AA genotype Sequencing chromatogram is (reverse；
Fig. 3 is BCAR4rs13334967 sites AT genotype Sequencing chromatogram (reverse)；
Fig. 4 is that BCAR4rs13334967 sites TT genotype Sequencing chromatogram is (reverse.
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 human breast carcinoma BCAR4 gene pleiomorphism rs13334967 location proximate sequences forward primer and
Reverse primer, using the human gene group DNA to be measured as template, pcr amplification reaction is carried out, obtains amplified production.
(c) digestion is carried out to the amplified production using restriction enzyme, obtains corresponding digestion products；
(d) digestion products are subjected to electrophoresis using 3% Ago-Gel, to judge BCAR4 gene pleiomorphisms
Each genotype in rs13334967 sites.
Wherein, its 3 ' end of the forward primer 1st bit base reciprocal is close with polymorphic rs13334967 bases, inverse the
2 bit bases are A (correspondence position of amplified production is base mismatch A), to be formed in amplified production with polymorphic T allele
AAGCTT structures (the 2nd base A is base mismatch, and the 6th base T is polymorphic site).
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, can overcome restriction endonuclease present in traditional PCR-RFLP methods select by
The defects of limiting, or needing to use expensive restriction endonuclease.Specifically：Due to the 2nd bit base reciprocal in the forward primer sequence
For A (correspondence position of amplified production is base mismatch A), thus after mispairing in PCR primer polymorphic sequence nearby by ATGCTA or
ATGCTT is changed to AAGCTA or AAGCTT (wherein the 2nd base A is base mismatch, and the 6th base A/T is polymorphic site).
Therefore T allele fragment can be identified restriction enzyme identification (the i.e. T allele piece of AAGCTT sequences in amplified production
Section can be cut by restriction endonuclease)；And then situation can be cut according to fragment to judge polymorphic gene type.More specifically, through sequence point
Analysis understands that the polymorphic no restriction endonucleases of A/T can identify cutting in gene original series.Such as by base mispairing PCR by polymorphism
The 4th bit base T changes into A before site, then it is that T allele can be known after PCR is expanded comprising AAGCTT sequences that this is polymorphic
The restriction enzyme Hind III of other AAGCTT sequences is identified, and A allele can not be cut.
Found through sequence analysis, detect the A/T polymorphisms, without can be with the restriction endonuclease of Direct Recognition corresponding site.Adopting
With Created Restriction Site method (Created Restriction Site PCR, CRS-PCR) to inverse the 2nd in positive primer sequence
After bit base changes into A by T, the polymorphic generation AAGCTT sequences of sequence nearby in PCR primer, so as to by restriction enzyme
Hind III is identified.Due to identifying that the restriction enzyme Hind III of specific site is applied widely, price is relatively inexpensive, not only solves
Having determined does not have the problem of restriction endonuclease identifies the SNP, and the cost for detecting SNP is more cheap.Therefore, simple operations are considered
Property and economic and practical, restriction enzyme Hind III are only selections.
Restriction enzyme Hind III price is from NEB companies (http://www.neb-china.com), it is restricted interior
The selection of enzyme cutting 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.In restricted
The recognition sequences of enzyme cutting Hind III are A^AGCTT, and its price is 205 yuan/5000U, cheap, economical and practical.
In embodiments of the invention, the design of forward primer and reverse primer is carried out using Primer6.0 softwares, if
The principle of meter considers the influence of sensitivity, specificity and amplification efficiency that primer pair PCR is expanded.Mutually it is unworthy of according to base
To principle design primer, primer length typically between 15~30 bases, it is long or it is short can cause poor specificity, it is long also
Its elongating temperature can be caused to be more than 74 DEG C, be unsuitable for Taq DNA polymerase and reacted.Primer G/C content 40%~60% it
Between, for Tm values 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 should not have complementary series between primer, otherwise primer itself, which can be folded into hairpin structure, makes primer renaturation itself.Primer 5 '
End and middle △ G values should be of a relatively high, and 3 ' end △ G values are relatively low.The single-stranded of amplified production can not form secondary structure.Primer
There should be specificity, after design of primers completion, BLAST detections should be carried out to it, to ensure itself and other genes without mutual
Benefit property.
Final forward primer selected from following nucleotide sequence by forming：GAGTGCTGAAAGCCTCATTACAAAG.
Reverse primer is by selected from following nucleotides row:CAGGAAATAGACAGAAGCCACAAGA.
The main sensitivity for considering primer pair PCR amplifications of design, specificity and the amplification effect of forward primer and reverse primer
The influence of rate.Primer generally is designed according to base pair complementarity principle, the sequence of primer and template wants close complementary.Primer length
For 15-30bp, it is too short or it is long cause poor specificity, it is long also to cause its elongating temperature to be more than 74 DEG C and be unfavorable for PCR
Reaction.Forward primer contains base A for 2nd reciprocal in its 3 ' end, to be formed in amplified production with polymorphic T allele
AAGCTT structures, so as to be identified by the restriction endonucleases of Hind III.Amplified production total length is that 163bp. amplified productions are as follows：
Underscore part is respectively forward primer and reverse primer, and the 28th bit base W represents the polymorphic i.e. SNP sites of A/T
rs13334967.24th bit base is the base A (being base T originally) after mispairing.The amplified production of a length of 163bp is through restricted
Restriction endonuclease Hind III can produce 163bp after cutting, 140bp, 23bp totally three kinds of clip types.Genotype result of determination：It is homozygous wild
Frequency of genotypes AA, a length of bands of 163bp mono-；It is mutated heterozygous genotypes AT, a length of 163bp, 140bp and the bands of 23bp tri-；It is mutated miscellaneous
Close the band of genotype TT, 140bp, 23bp two.
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 the 3U water-baths of restriction endonuclease Hind III.Digestion products carry out agarose using 3% agarose
Gel electrophoresis, wild homozygous genotype is judged according to different bands under ultra violet lamp, be mutated heterozygous genotypes and dash forward
Become homozygous genotype.
In specific embodiments of the present invention, present invention detection human breast carcinoma BCAR4 gene polymorphics rs13334967's
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 Hind III to pcr amplification product, obtains digestion products；
(4) digestion products progress agarose enters row agarose gel electrophoresis, according to different bands under ultra violet lamp
Judge wild homozygous genotype, be mutated 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, determines that BCAR4 is more
State site nucleotide variation data
Partial gene sequence containing BCAR4rs13334967 is as follows：
501st base W of gene order represents that A/T is polymorphic, as rs13334967 pleomorphism site.According to
Rs13334967 particular sequence and the position of polymorphic base are most had up and down using what Primer Premier 6.0 were designed
It is as follows to swim primer；
Forward primer sequence：5’-GAGTGCTGAAAGCCTCATTACAAAG-3’
Reverse primer sequences：5’-CAGGAAATAGACAGAAGCCACAAGA-3’.
The 2nd bit base reciprocal is that (the amplified production correspondence position of formation is base mismatch to A wherein in forward primer sequence
A), polymorphic sequence nearby is changed to AAGCTA or AAGCTT (wherein the by ATGCTA or ATGCTT in PCR primer therefore after mispairing
2 base A are base mismatch, and the 6th base A/T is polymorphic site).Therefore T allele fragment can be known in amplified production
The restriction enzyme Hind III of other A^AGCTT sequences identifies that (i.e. T allele fragment can be cut by restriction endonuclease.And then can be with
Situation is cut according to fragment to judge 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, 62.1 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 163bp are produced for final 4 DEG C of storages
Thing.Product Sequence after amplification is：
Underscore part is respectively forward primer and reverse primer, and the 28th bit base W represents the polymorphic i.e. SNP sites of A/T
rs13334967.24th bit base is the base A (being base T originally) after mispairing.
(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 37 DEG C of digestion 1-16h, produce digestion products in water-bath.Consider economical real
With property and simple operations, Hind III is selected to 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, rs13334967 sites different genotype show different band (see Fig. 1), wild homozygous genotype AA, a length of 163bp
One band；It is mutated heterozygous genotypes AT, a length of 163bp, 140bp and the bands of 23bp tri- (23bp is with invisible)；It is mutated heterozygosis base
Because of type TT, the band of 140bp, 23bp two (23bp is with invisible).Each genotype further identifies through being sequenced, sequencing result
(see Fig. 2-Fig. 4) display is identical with the result that prior art measures.
The human peripheral whole blood sample of embodiment 1. determines human breast carcinoma BCAR4rs13334967 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), Hind III (Thermo), agarose (SIGMA) etc..
1.2 design of primers
In NCBI dbSNP databases, BCAR4rs13334967 nucleotide sequence is searched, in primer-design software
In Primer Premier 6.0, the parameter such as primer length and amplification purpose fragment length is set to carry out design of primers, according to GC
Content and annealing temperature etc. select optimal upstream and downstream primer, and the 2nd bit base reciprocal in reverse primer sequences is set into A and (formed
Amplified production correspondence position be base mismatch A), therefore after mispairing in PCR primer polymorphic sequence nearby by ATGCTA or
ATGCTT is changed to AAGCTA or AAGCTT (wherein the 2nd base A is base mismatch, and the 6th base A/T is polymorphic site).
Therefore T allele fragment can be identified the restriction enzyme Hind III of A^AGCTT sequences and identify (i.e. T etc. in amplified production
Position genetic fragment can be cut by restriction endonuclease).Again by the Blast sequence alignment functions (http in this primer and Pubmed://
Blast.ncbi.nlm.nih.gov/Blast.cgi primer comparison) is carried out, the upstream and downstream primer finally determined is
Forward primer sequence：5’-GAGTGCTGAAAGCCTCATTACAAAG-3’
Reverse primer sequences：5’-CAGGAAATAGACAGAAGCCACAAGA-3’.
The selection of 1.3 restriction enzymes
According to the base sequence in five sites before and after rs13334967 in dbSNP, with the online restriction enzymes of WatCut
Analysis software, on-line search obtain the information of the restriction enzyme in recognizable mutational site, consider its digestion specificity
And the restriction enzyme Hind III that economic and practical Sexual behavior mode is optimal.
1.4 extract the genomic DNA of sample to be tested from 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
2.1 PCR are expanded
(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.
The PCR reaction conditions in (3) 3 sites：First stage is the pre-degeneration stage, 94 DEG C/5min；Second stage bag
Include three steps totally 35 circulation, set gradually for 94 DEG C/35s, 62.1 DEG C of annealing time 45s, 72 DEG C/30s；Three phases
72℃/5min。
2.2 endonuclease reaction
Digestion system is μ l of pcr amplification product 5, the μ l of restriction enzyme 0.5, Buffer 1.0 μ l, finally uses distilled water
Cumulative volume is supplemented to 15 μ l.37 DEG C of water-bath 1-16 hours in water-bath are placed in after mixing.
The judgement of 2.3 genotype
The rs13334967 loci gene types of table 1 judge
The human breast carcinoma tissue sample of embodiment 2. determines human breast carcinoma BCAR4rs13334967 polymorphisms
The step of with embodiment 1, is essentially identical, simply adopts and extracts DNA works from breast cancer tissue's sample in the following method
For DNA to be measured.
Breast cancer tissue is cut off, phenol-chloroform method extracts breast cancer tissue's genomic DNA as DNA to be measured.
1) breast cancer tissue's block is thawed, the tissue that blood stains, clip 0.1g or so are washed away with physiological saline is milled, and is added
1ml aqua sterilisa, overturn and mix, 10000 leave the heart 10 minutes, abandon supernatant.Ttom of pipe should have precipitation.It is repeated twice
2) 200ul DNA lysates are added, 5ul Proteinase K mixes, and 55 degree 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.
12000 leave the heart 10 minutes.
4) supernatant is taken, is careful not to wash intermediate medium and lower floor's liquid.Isometric chloroform is added, overturns and mixes.
12000 leave the heart 10 minutes.
5) supernatant is taken, is careful not to wash intermediate medium and lower floor's liquid.Add 1/10th sodium acetate and 2.5
Absolute ethyl alcohol again, overturn and mix.
6) 12000 the heart is left 10 minutes.Abandon supernatant.
7) ethanol of 1ml 70% is added, its white precipitate is suspended, overturns and mixes for several times.12000 leave the heart 10 minutes.
8) supernatant is abandoned, then room temperature, which is uncapped, stands minute and make the volatilization of its ethanol clean.
9) aqua sterilisa dissolving DNA is added, it is general to add the dissolving of 50ul aqua sterilisas
10) -20 DEG C of preservations, produce breast cancer tissue's genomic DNA
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, rs13334967 sites different genotype shows different bands (see figure
1), wild homozygous genotype AA, a length of bands of 163bp mono-；It is mutated heterozygous genotypes AT, a length of 163bp, 140bp and 23bp tri-
Band；It is mutated the band of heterozygous genotypes TT, 140bp, 23bp two.Each genotype further identifies through being sequenced, sequencing result
(see Fig. 2-Fig. 4) display is identical with the result that prior art measures.
From above-described embodiment as can be seen that the present invention is directed to using CRS-PCR identification BCAR4rs13334967 polymorphisms,
Normal PCR-RFLP methods are improved, primer 3 ' is applied and holds mispairing technology, overcome the choosing of normal PCR-RFLP methods restriction endonuclease
It is small to select leeway, expensive, the shortcomings of experimental cost is high, the detection side for the BCAR4rs13334967 polymorphisms that this research is established
Method, restriction endonuclease price is very cheap, while PCR primer purity is higher, and digestion result is easy to differentiate.The detection method passes through sequencing
Checking, 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 BCAR4 polymorphisms using primer base mispairing Technology design
rs13334967.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 the BCAR4 polymorphisms designed
Rs13334967 upstream and downstream primer, forward primer sequence：5 '-GAGTGCTGAAAGCCTCATTACAAAG-3 ', reverse primer
Sequence：5’-CAGGAAATAGACAGAAGCCACAAGA-3’.And restriction enzyme Hind III use.In a word, it is of the invention
The BCAR4 polymorphism rs13334967 methods of genotyping of foundation is simple, quick, safe and accurate, high sensitivity, is worth facing
Promoted in bed and 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>Zhengzhou University
<120>With the method for HindIII identification human breast carcinoma BCAR4 gene rs13334967 polymorphisms
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1001
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 1
agacaagaag agactataaa ttgctgaccc acccacgctg agacccatgc gtatttgatt 60
tttttttttt ttttttttga gacggagttt cgctcttttt gcccaggccg gagtgcaatg 120
gtgcaatctc ggctcaccgc gacctctgcc tcccgggttc aagcatttct cctggctcag 180
cctcccaagt agctgggatt acaggcgcct gccaccccgt ctggcgaatt ttttgtattt 240
ttagtagaga caggtttcac cacgttggcc tggattgtct cgatctcctg acctcgtgat 300
ccacccgcct cggcctccca aagtgctggg attacaggcg tgagccactg cacccggccg 360
catatttgat ttctatgttt atctacaggt caagtacaga ttgcccaagt tcaagagaaa 420
gacttgattg ttcttcctct ctttcctttt cacatgcaac acgtggattg agtgagtgct 480
gaaagcctca ttacaaagct wccctatcct tctccatttt ttcttttcac ctctcccctc 540
ctgcccactt tttcccttta aatattgaag ccctttaaat cctctttgga aaaagtgctg 600
actccagatc ctcttgtggc ttctgtctat ttcctgggca tgtcctcaat gttagctaaa 660
ataaacctct acagtgattg agaccttctg agacttctgg tttacaagtg aaatttcatg 720
aagctatctc aagacatcca aaaagactgc ttctgaaaat gaatcctcac gcttgggagg 780
ccgaggcagg tggatcgctt gagcccagga gttcaagact agcctgggga acgtggcaaa 840
accctgtctc tacaaaaaat aaataaataa attaaataaa atacaaaaaa ttagccacgc 900
atggtggcac gtgcctgtag taccagatac cctgggagct gaggcttcag taagccttga 960
ttgtgccact gcactccagc ctgggagata gaaaggcccc a 1001
<210> 2
<211> 25
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 2
gagtgctgaa agcctcatta caaag 25
<210> 3
<211> 25
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 3
caggaaatag acagaagcca caaga 25
<210> 4
<211> 162
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 4
gagtgctgaa agcctcatta caagctwccc tatccttctc cattttttct tttcacctct 60
cccctcctgc ccactttttc cctttaaata ttgaagccct ttaaatcctc tttggaaaaa 120
gtgctgactc cagatcctct tgtggcttct gtctatttcc tg 162
Claims (5)
1. with HindIII identify human breast carcinoma BCAR4 gene rs13334967 polymorphisms method, it is characterised in that including with
Lower step：
(a) genomic DNA of sample is extracted；
(b) provide amplification human breast carcinoma BCAR4 gene pleiomorphism rs13334967 location proximate sequences forward primer and reversely
Primer, using the human gene group DNA to be measured as template, pcr amplification reaction is carried out, obtains amplified production；
(c) digestion is carried out to the amplified production using restriction enzyme, obtains corresponding digestion products；
(d) digestion products are subjected to electrophoresis using 3% Ago-Gel, to judge BCAR4 gene pleiomorphisms rs13334967
Each genotype in site.
2. the side according to claim 1 that human breast carcinoma BCAR4 gene rs13334967 polymorphisms are identified with HindIII
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 BCAR4 polymorphic site nucleotide variation data；
Partial gene sequence containing BCAR4rs13334967 such as SEQ:ID:NO:Shown in 1；507th base R generation of gene order
Table A/G is polymorphic, as rs8012083 pleomorphism site；It is polymorphic that 501st base W of gene order represents A/T, i.e.,
For rs13334967 pleomorphism site；Primer is used according to the position of rs13334967 particular sequence and polymorphic base
The upstream and downstream primer that Premier 6.0 is designed, forward primer sequence such as SEQ:ID:NO:Shown in 2；Reverse primer sequences are such as
SEQ:ID:NO:Shown in 3, the 2nd bit base reciprocal is A wherein in forward primer sequence, therefore polymorphic attached in PCR primer after mispairing
Nearly sequence is changed to AAGCTA or AAGCTT by ATGCTA or ATGCTT；Therefore T allele fragment can be known in amplified production
The restriction enzyme Hind III of other A^AGCTT sequences identifies, and then, situation is cut according to fragment to judge polymorphic gene type；
According to forward primer sequence and reverse primer sequences synthetic primer, the synthesis of primer uses solid-phase synthesis, or commission
Biotech firm synthesizes and detects forward and reverse primer.
3. the side according to claim 1 that human breast carcinoma BCAR4 gene rs13334967 polymorphisms are identified with HindIII
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, 62.1 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 163bp pcr amplification product is produced；Product Sequence such as SEQ after amplification:ID:NO:Shown in 4.
4. the side according to claim 1 that human breast carcinoma BCAR4 gene rs13334967 polymorphisms are identified with HindIII
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, 37 DEG C of digestion 1- in water-bath
16h, produce digestion products.
5. the side according to claim 1 that human breast carcinoma BCAR4 gene rs13334967 polymorphisms are identified with HindIII
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, rs13334967 sites different genotype shows different bands, wild homozygous genotype
AA, a length of bands of 163bp mono-；It is mutated heterozygous genotypes AT, a length of 163bp, 140bp and the bands of 23bp tri-；It is mutated heterozygous genes
The band of type TT, 140bp, 23bp two.
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