CN104531862B - Detect the method and primer in the full exon sequence mutational site of mankind's BRCA1 and BRCA2 gene - Google Patents
Detect the method and primer in the full exon sequence mutational site of mankind's BRCA1 and BRCA2 gene Download PDFInfo
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Abstract
The present invention relates to technical field of gene detection, is to provide a kind of method and primer for detecting the full exon sequence mutational site of mankind's BRCA1 and BRCA2 gene.The specific primer includes the forward and reverse primer of whole 24 extrons of amplification covering BRCA1 genes, its base sequence such as SEQ ID NO：Shown in 001 062；With the amplification covering BRCA2 genes forward and reverse primer that all 27 extrons are dashed forward, its base sequence such as SEQ ID NO：Shown in 063 142.The present invention can be extended the whole full extron of BRCA1 and BRCA2 genes, be covered all mutational sites to be detected using the mutation of the Sanger PCR sequencing PCRs detection full extron of BRCA1 and BRCA2 genes；With very high specificity and accuracy；It is simple to operate, cost is low.The risk assessment of hereditary breast cancer will be greatly enhanced, effectively reduces the onset risk of breast cancer.
Description
Technical field
The present invention relates to technical field of gene detection, more particularly, to the detection full extron of mankind's BRCA1 and BRCA2 gene
The method and primer in series jump site.
Background technology
Breast cancer is one of most common malignant tumour of women, and the annual whole world has 1,300,000 breast cancer newly to send out patient, has more than
460000 patient dies from breast cancer.Breast cancer incidence is in the first place of female malignant in recent years in China, and with more than 3%
Speed cumulative year after year, the conservative estimation whole nation is annual to have women more than 40,000 to die from this disease.It is now recognized that most of hereditary breast cancers
It is due to caused by gene mutation, the wherein relation of tumor suppressor gene BRCA1 and BRCA2 and pathogenesis of breast carcinoma is more close,
Research confirms that the probability that breast cancer occurred before 40 years old for BRCA1 or BRCA2 gene mutation persons is up to 19%[1]。
Mutation of the BRCA1 or BRCA2 genes in different regions and race is not quite similar, and the two genes, which can encode, to be had
The albumen of multi-functional, its mutant phenotype often have the trend for inducing breast cancer and oophoroma.Current known BRCA1 and
BRCA2 and homologous recombination, DNA damage reparation, embryo growth, transcriptional control etc. are relevant[2].Wherein, damaged especially with both in DNA
Function in wound reparation, homologous recombination and transcriptional control is the most notable and important[3]。
BRCA1 is positioned at No. 17 chromosome q21 of the mankind.It is hereditary in a manner of autosomal dominant inheritance, and have very high outer
Aobvious rate[4].BRCA1 is about 100kb, containing 24 extrons.Its gene outcome is the phosphorylated protein that 1863 amino acid is formed
Matter, relative molecular weight about 200000.BRCA1 is the regulatory factor for regulating and controlling G/M phase key points, is that activation Chkl kinases institute is required
, and the induction G/M phases block and played an important role when the latter is to DNA damage.Meanwhile BRCA1 also controls Cdc25C and Cdc2/
The regulation of the expression of Cyclin B1 kinase proteins, phosphorylation and inner cellular localization, G/ in both protein on cells periodic processes
The M phases are smoothed out playing an important role[5].BRCA1 is positioned at centerbody in m period, and with the important set of centerbody
Into composition γ-tubulin interaction.P53 can also suppress BRCA1 expression in turn, reach stablize the work of itself whereby
With.The BRCA1 of wild type can also be apoptosis-induced and suppresses estrogen-dependent transcriptional pathway, and the path and breast epithelium are thin
Born of the same parents' hyperplasia is relevant, and inhibitory action weakens and caused a disease after gene mutation.In addition the BRCA1 of wild type can also adjust the work of prostaglandin
Property[6]。
The BRCA2 assignments of genes gene mapping are in No. 13 chromosome q12.Complete genome DNA is about 70kb, and wherein code area is contained
10987bp, and AT (about 64%) is rich in, its gene order is with BRCA1 without obvious relation.BRCA2 is made up of 27 extrons, its
In the 11st extron be about 4932bp, mRNA is about 10.2kb, and the BRCA2 albumen of coding contains 3418 amino acid.Normally
BRCA2 albumen is located in nucleus, participates in DNA reparation.It is similar with BRCA1 in the expression way of the amplification phase of cell cycle,
The transcription of the gene is can't detect in the cell of resting stage.BRCA2mRNA expression is obvious in the cell of fast breeding
Increase, and show cell cycle dependant, be low in early days in G0 phases and G1, peaked when demarcating the G1/S phases.These
As a result regulation important roles of the BRCA2 for cell growth is shown[7].Many studies have shown that：BRCA2 albumen and heredity
Breast cancer, oophoroma and anemia Fanconi's disease have substantial connection, and this may have because of itself and DNA double strand break repair
Close[8]。
At present, the high correlation of BRCA1, BRCA2 and Familial Occurrence breast cancer is very clear and definite.By right
The detection of the mutation of BRCA1 and BRCA2 genes, the occurrence and development of breast cancer can be predicted, can also be screened out breast cancer, oophoroma
And the people at highest risk of other associated malignancies, the early diagnosis beneficial to such disease are treated, and may be selected rationally effectively to control
Treatment scheme.
Current research is also shown that by detecting above two detection in Gene Mutation, breast cancer can be carried out preventative outer
Section performs the operation or chemoprophylaxis.Therefore, for have in one-level or second degree relative suffer from breast cancer or the individual of oophoroma have must
BRCA1 and BRCA2 detection in Gene Mutation is carried out, whether there is gene mutation carrying to understand.If testing result is positive, can be early
By operation, chemotherapy etc., some treatment means reduce pathogenesis of cancer risk.
The country has producer and carries out breast cancer correlation BRCA1 and BRCA2 gene mutation sequencing, but most of detections are pair
Local sequence in BRCA1 and BRCA2 genes is sequenced, and still whole coded sequences of BRCA1 and BRCA2 genes can not be entered
Row sequencing.
The content of the invention
Present invention solves the technical problem that one group is just to provide from individual test subjects peripheral blood with direct sequencing pair
The sequencing primer that BRCA1 and BRCA2 gene whole exon sequences are sequenced, to reach the individual pathogenesis of breast carcinoma risk of prediction
The factor, prevent the purpose of pathogenesis of breast carcinoma.
First purpose of the present invention is to provide the core of the full exon sequence sequencing relevant primer of BRCA1 and BRCA2 genes
Acid sequence, including 27 exons mutation sites of amplification covering BRCA1 genes 24 extrons of whole and BRCA2 genes whole
Forward and reverse primer, its base sequence are：
(1) primer in the whole 24 exons mutation sites of BRCA1 genes is detected：
Include the forward and reverse primer in the whole 24 exons mutation sites of amplification covering BRCA1 genes, its base sequence
For：
In the detection, the DNA pieces in the full exons mutation site of BRCA1 genes are covered first with above-mentioned forward and reverse primer pair
Duan Jinhang is expanded, and obtains amplified production, then amplified production is sequenced respectively using above-mentioned amplimer, obtains amplification production
The gene order of thing.
(2) primer in the whole 27 extron exons mutation sites of BRCA2 genes is detected：
Include the forward and reverse primer in the whole 27 extron exons mutation sites of amplification covering BRCA2 genes, its base
Sequence is：
In the detection, the DNA fragmentation in BRCA2 gene extrons mutational site is covered first with above-mentioned forward and reverse primer pair
Expanded, obtain amplified production, then amplified production is sequenced respectively using above-mentioned amplimer, obtain amplified production
Gene order.
Second object of the present invention is the method for providing a kind of full exons mutation of detection BRCA1 and BRCA2 genes,
It comprises the following steps：
(1) sample DNA is extracted；
(2) SEQ ID NO are utilized：Whole 24 extrons of BRCA1 genes shown in 001-142 and BRCA2 genes whole 27
Forward and reverse amplimer corresponding to individual extron, the DNA in foregoing (1) is expanded, obtain covering BRCA1 and BRCA2 bases
Because of the amplified production in full exon sequence mutational site；
(3) SEQ ID NO are utilized：Whole 24 extrons of BRCA1 genes shown in 001-142 and BRCA2 genes whole 27
Forward and reverse amplimer corresponding to individual extron, forward and reverse sequencing is carried out to the amplified production in foregoing (2), obtains institute
State the gene order of amplified production；
By the gene order in (3) compared with wild type BRCA1 and BRCA2 gene extron subsequence, it is determined that mutation
Site whether there is.
Third object of the present invention is to provide a kind of detection BRCA1 and BRCA2 genes full exon sequence mutation position
The kit of point, the kit include sample DNA extraction agent；Absolute ethyl alcohol；Detection architecture PCR reaction solutions, sequencing system
Reaction solution, positive reference substance, negative controls and blank control product.Wherein detection architecture PCR reaction solutions include SEQ ID NO：
All 24 extrons and all forward and reverse amplifications corresponding to 27 extrons of BRCA2 genes of BRCA1 genes shown in 001-142
Primer, it is characterised in that such as SEQ ID NO：Sequence shown in 001-142, amplified production covering BRCA1 and BRCA2 genes are entirely outer
Aobvious subsequence.
Further, the detection architecture PCR reaction solutions also include 2XPCR Buffer；dNTPs；Roche DNA
Polymerase。
Further, the sequencing system also include sequencing refined solution, EDTA, absolute ethyl alcohol, 75% ethanol, HIDI and
Bigdye Terminator V3.1。
Further, the sequencing refined solution includes shrimp alkaline phosphotase and exonuclease 1.
The present invention devises the forward and reverse primer in the full exons mutation site of amplification covering BRCA1 and BRCA2 genes, and
Sequencing reaction can be completed simultaneously.Enter performing PCR amplification to censorship sample, using Sanger PCR sequencing PCRs, PCR primer is carried out positive and negative
To sequencing reaction amplification, denaturation, direct Sequencing can be accurately detected in the full extron of BRCA1 and BRCA2 genes respectively after purification
The catastrophe in mutational site.The reaction conditions such as concentration, annealing temperature by adjusting forward and reverse primer, can reach amplification efficiency
To optimal.
The beneficial effect that the present invention obtains：Using the prominent of the Sanger PCR sequencing PCRs detection full extron of BRCA1 and BRCA2 genes
Become, the whole full extron of BRCA1 and BRCA2 genes can be extended, cover all mutational sites to be detected；With very high spy
The opposite sex and accuracy；Using PCR method amplifying target genes and it is sequenced and detects its gene mutation, there is high sensitivity, operation is simple
Single, low cost and other advantages.The risk assessment of hereditary breast cancer will be greatly enhanced, effectively reduces the onset risk of breast cancer.
Brief description of the drawings
Fig. 1 is the testing result figure of sample 6 in the embodiment of the present invention；
Fig. 2 is the testing result figure of sample 17 in the embodiment of the present invention；
Fig. 3 is the testing result figure of sample 1 in the embodiment of the present invention.
Embodiment
Embodiments of the invention are described in detail below in conjunction with accompanying drawing.It should be noted that do not conflicting
In the case of, the feature in embodiment and embodiment in the application can be mutually combined.
Embodiment 1
Detect the primer sequence of BRCA1 and BRCA2 gene extrons mutation
(1) primer in BRCA1 gene extrons mutational site is detected
Including SEQ ID NO：Sequence shown in 001-062, for amplification covering BRCA1 gene extrons mutational site
Forward and reverse primer.
In the detection, the DNA fragmentation in BRCA1 gene extrons mutational site is covered first with above-mentioned forward and reverse primer pair
Expanded, obtain amplified production, then amplified production is sequenced respectively using above-mentioned amplimer, obtain amplified production
Gene order.
(2) primer in BRCA2 gene extrons mutational site is detected
Including SEQ ID NO：Sequence shown in 063-142, for amplification covering BRCA2 gene extrons mutational site
Forward and reverse primer.
In the detection, the DNA fragmentation in BRCA2 gene extrons mutational site is covered first with above-mentioned forward and reverse primer pair
Expanded, obtain amplified production, then amplified production is sequenced respectively using above-mentioned amplimer, obtain amplified production
Gene order.
Embodiment 2
Detect the kit in BRCA1 and BRCA2 gene extrons mutational site
Including：Tissue DNA extraction agent box (such as extracts kit using Kai Jie companies)；Absolute ethyl alcohol；Detect body
It is PCR reaction solutions, sequencing system reaction solution, positive reference substance, negative controls and blank control product.Wherein detection architecture PCR
Reaction solution includes：2x PCR Buffer；2mM dNTPs；Roche DNA Polymerase(1U/μl)；SEQ ID NO：001-
The forward and reverse primer of the full extron of covering BRCA1 and BRCA2 genes shown in 142.
Sequencing system includes：Sequencing refined solution, EDTA (1.25mmol), absolute ethyl alcohol, 75% ethanol, HIDI (are highly gone
Formamide), SEQ ID NO：The forward and reverse amplification of the full extron of covering BRCA1 and BRCA2 genes shown in 001-142
Each 3.2 μm of primer, and Bigdye Terminator V3.1 (buying from Applied Biosystems companies of the U.S.), wherein
Sequencing refined solution includes shrimp alkaline phosphotase (SAP) 0.6U and exonuclease 1 (EXONI) 1.2U.
Embodiment 3
The method for detecting BRCA1 genes and BRCA2 gene mutation sites
(1) genomic DNA in blood is extracted：
1) 500 μ L blood are extracted and add 1000 μ l erythrocyte cracked liquids, reverse tear is even, and room temperature places 5 min, during which runs again
Mix several times.3000rpm centrifuges 5min, sucks and asks, leaves leukocyte cell pellet, add 200 μ l buffer solution GA, vibrate to thorough
Mix.
2) 20 μ l Proteinase K Solutions are added, are mixed.
3) resistance of 200 μ l buffer solutions is added, fully reverse to mix, 70 DEG C of placement 10min, solution strains limpid, brief centrifugation
To remove the globule of cap wall.
4) 200 μ l absolute ethyl alcohols are added, fully vibrate tear even 15 seconds, now it is possible that flocculent deposit, brief centrifugation
To remove the globule of cap wall.
5) previous step resulting solution and flocculent deposit all add in an adsorption column CB3 to (adsorption column is put into collecting pipe
In), 12,000rpm (13,400X g) are centrifuged 30 seconds, outwell waste liquid, adsorption column CB3 is put back in collecting pipe.
6) 500 μ l buffer solutions GD (please first checking whether that oneself adds absolute ethyl alcohol before use) are added into adsorption column CB3,
12,000rpm (13,400Xg) centrifuge 30 seconds, outwell waste liquid, adsorption column CB3 is put into collecting pipe.
7) 700 μ l rinsing liquids PW (please first checked whether before use and added absolute ethyl alcohol) are added into adsorption column CB3,
12,000rpm (13,400Xg) centrifuge 30 seconds, outwell waste liquid, adsorption column CB3 is put into collecting pipe.
8) 500 μ l rinsing liquids PW are added into adsorption column CB3,12,000rpm (13,400Xg) centrifuge 30 seconds, outwelled useless
Liquid.
9) adsorption column CB3 is put back in collecting pipe, 12,000rpm (13,400X g) are centrifuged 2 minutes, outwell waste liquid.It will inhale
Attached column CB3 is placed in room temperature and placed several minutes, thoroughly to dry rinsing liquid remaining in sorbing material.
10) adsorption column CB3 is transferred in a clean centrifuge tube, 100 μ l is vacantly added dropwise to the middle part of adsorbed film
Elution buffer TE, room temperature place 2-5min, and 12,000rpm (13,400X g) are centrifuged 2 minutes, and solution is collected into centrifuge tube
In, obtain poba gene group DNA solution.
(2) reagent configures:
Detection architecture PCR reaction solutions are formulated as follows:
| Reagent name | Dosage |
| 2X PCR Buffer | 10.0μl |
| dNTP | 4.0μl |
| Forward primer (10 μm) | 0.5μl |
| Reverse primer (10 μm) | 0.5μl |
| Roche DNA Polymerase | 0.5μl |
| ddH20 | 3.5μl |
| Sample DNA templates | 1.0μl |
| Amount to | 20.0μl |
Wherein, the amplimer base sequence of each gene overlay area such as SEQ ID NO：Shown in 001-142.
Each each X μ l of code area detection architecture PCR reaction solutions are configured by detection people's number, per the μ l of person-portion 19 packing：
X=19 μ l reaction solutions X (+1 part of blank control of n parts+1 part of sample+1 part of positive control negative control)
N is detection number of samples.
(3) it is loaded：The poba gene group DNA solution obtained in 1 μ l steps (1) is added to detection architecture PCR reaction solutions
In；For positive control experiment, directly add 1 μ l positive reference substances；For negative control experiment, directly plus 1 μ l feminine genders are right
According to product；For blank control experiment, add 1 μ l physiological saline.
(4) Touch-down PCR are expanded：Detection is carried out on Standard PCR instrument, can include ABI verity with instrument
PCR instrument (Applied Biosystems companies of the U.S.) etc..Reaction condition is as follows：95 DEG C of pre-degenerations 10 minutes；95 DEG C 30 seconds, 64
DEG C 90 seconds, 72 DEG C 30 seconds, totally 15 circulations；95 DEG C 30 seconds, 55 DEG C 60 seconds, 72 DEG C 30 seconds, totally 25 circulation；72 DEG C 10 minutes；4
DEG C preserve.
(5) product purification is sequenced:
Take 9 μ l PCR primers that refined solution is sequenced with 2 μ l.Purified according to following procedure, obtain purified product:
| The stage of reaction | Reaction condition |
| Purifying | 37℃50min |
| Denaturation | 95℃5min |
| Preserve | 4℃ |
(6) Sanger is sequenced：
By 1 μ l purified products respectively with SEQ ID NO：Forward and reverse amplimer shown in 001-142 is according to such as lower body
System is mixed：
| Reagent name | Dosage |
| Forwards/reverse primer | 1μl |
| Bigdye Terminator V3.1 | 1μl |
| ddH20 | 2μl |
| Purified product | 1μl |
| Amount to | 6μl |
Sequencing reaction program:
95 DEG C of pre-degenerations 4 minutes；95 DEG C 15 seconds, 50 DEG C 20 seconds, 60 DEG C 2 minutes, totally 25 circulation；4 DEG C of preservations.
(7) product purification is sequenced:
2 μ l 125mmol EDTA is added into the product for completing sequencing reaction, stands 5min；Add the anhydrous second of 15ml
Alcohol, it is vortexed and mixes；3700rpm centrifuges 30min；Centrifugation 15sec is inverted, adds 50ml70% ethanol, is vortexed and mixes；3700rpm
Centrifuge 15min；Centrifugation 15sec is inverted, is placed on 95 DEG C of metal baths；Denaturation 5 minutes is carried out after adding 10 μ l HIDI.It is denatured journey
After sequence terminates, upper sequenator ABI3730 sequencings.
(8) result judges：By sequencing result and BRCA1 wild-type reference sequences (NCBI Reference Sequence:
) and BRCA2 wild-type reference sequences (NCBI Reference Sequence NC_000017.11:NC_000013.11) introduce
Row is compared, and result is reported according to actual catastrophe.
Embodiment 4
Clinical samples are detected using kit for detecting nucleic acid of the present invention.
Inspection women anti-freezing blood specimen 20 is fetched and delivered, 20 samples are by the methods described of embodiment 3 extraction sample genome
DNA, reagent preparation simultaneously detect.Every part of sample adds 1 μ l in detection architecture PCR reaction solutions and does the positive simultaneously, negative, blank pair
According to.Each sample is repeated 2 times, and after 2 sequencings, compares the situation of mutation, and the 3rd will be carried out for the skimble-scamble sample of result
Secondary sequencing.Finally, judged according to sequencing result for prognosis.
Testing result such as following table：
The positive sequencing result of sample 6 causes frameshift mutation as shown in figure 1, be c.2901insCT mutated for BRCA1 genes,
It is excessive risk to prompt hereditary breast cancer morbidity.
The positive sequencing result of sample 17 is carried as shown in Fig. 2 be c.2699_2704delTAAATG mutated for BRCA2 genes
Show that hereditary breast cancer morbidity is excessive risk.
The positive sequencing result of sample 1 is as shown in figure 3, be wild type, it is low-risk to prompt hereditary breast cancer morbidity.
The sequencing result of remaining sample is wild type, it is low-risk to prompt hereditary breast cancer morbidity with sample 1.
The preferred embodiments of the present invention are the foregoing is only, are not intended to limit the invention, for the skill of this area
For art personnel, the present invention can have various modifications and variations.Within the spirit and principles of the invention, that is made any repaiies
Change, equivalent substitution, improvement etc., should be included in the scope of the protection.
【Bibliography】
1.Casey,G.The BRCA1and BRCA2breast cancer genes.Curr Opin Oncol 9,88-
93(1997).
2.Garvin,A.M.,Attenhofer-Haner,M.&Scott,R.J.BRCA1and BRCA2mutation
analysis in 86early onset breast/ovarian cancer patients.J Med Genet 34,990-5
(1997).
3.Friedman,L.S.et al.Confirmation of BRCA1by analysis of germline
mutations linked to breast and ovarian cancer in ten families.Nat Genet 8,
399-404(1994).
4.Miki,Y.et al.A strong candidate for the breast and ovarian cancer
susceptibility gene BRCA1.Science 266,66-71(1994).
5.Brown,M.A.et al.Regulation of BRCA1.Nature 372,733(1994).
6.Futreal,P.A.et al.BRCA1mutations in primary breast and ovarian
carcinomas.Science 266,120-2(1994).
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gene BRCA2.Nature 378,789-92(1995).
。
Claims (1)
- A kind of 1. kit for detecting the full exon sequence mutational site of mankind's BRCA1 and BRCA2 gene, it is characterised in that：Institute Stating kit includes：Sample DNA extraction agent, absolute ethyl alcohol, detection architecture PCR reaction solutions, system reaction solution is sequenced, it is positive right According to product, negative controls and blank control product；Wherein detection architecture PCR reaction solutions include：71 pairs of amplimers, 2xPCR Buffer；dNTPs；Roche DNA Polymerase；Sequencing system includes：Refined solution is sequenced；EDTA；Absolute ethyl alcohol；75% Ethanol；Height deionized formamide and Bigdye Terminator V3.1；Wherein described sequencing refined solution includes shrimp alkalescence phosphorus Sour enzyme and exonuclease；Wherein described 71 pairs of amplimers are：The forward and reverse primer in the whole 24 exons mutation sites of amplification covering BRCA1 genes, its base sequence such as SEQ ID NO：Shown in 001-062；With the forward and reverse primer in the whole 27 exons mutation sites of amplification covering BRCA2 genes, its base Sequence such as SEQ ID NO：Shown in 063-142.
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