CN107760771A - A kind of breast cancer susceptibility gene BRCA1 and BRCA2 full genomes capture primer, kit and its method - Google Patents
A kind of breast cancer susceptibility gene BRCA1 and BRCA2 full genomes capture primer, kit and its method Download PDFInfo
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Abstract
The invention provides a kind of nucleotide sequence, kit and its application for the gene copy number augmentation detections of HER 2, includes amplification BRCA1 (Gene ID:672) 8 pairs of pcr amplification primer things of full length gene；Expand BRCA2 (Gene ID:675) 8 pairs of pcr amplification primer things of full length gene, the DNA for expanding capture can be used for follow-up high-flux sequence to analyze.Capture technique and multiple PCR technique are targetted compared to probe, this method is simple to operate, cost is low, sequencing data homogeneity is good, less sequencing data amount can reach more than 99% coverage, help to provide technique of gene detection support for the related hereditary tumor risk assessment of breast cancer susceptibility gene or medication and the foundation of database.
Description
Technical field
The invention belongs to technical field of gene detection, and in particular to a kind of full bases of breast cancer susceptibility gene BRCA1 and BRCA2
Because of capture primer, kit and its application.
Background technology
BRCA1 and BRCA2 belong to tumor suppressor gene, are breast cancer and the susceptible two related genes of oophoroma, mainly join
With DNA damage reparation.BRCA1 and BRCA2 is relevant with homologous recombination, DNA damage reparation, embryo growth, transcriptional control.BRCA1
The reason for BRCA2 gene mutations being most of autosomal dominant Familial Occurrence breast cancer, ovarian tumors, especially
BRCA2 gene mutations and male breast carcinoma relation are more close.The mammary gland of BRCA mutation carriers suffers from cancer risk factor throughout one's life
Significantly rise, and easily fall ill in one's early years.Breast cancer is the most common malignant tumour of women, and the whole world in 2007 has 1,300,000 breast cancer new
Patient is sent out, the patient more than 460,000 dies from breast cancer.Chinese breast cancer incidence in recent years is in the first place of female malignant, and
With more than 3% speed cumulative year after year, estimating that the whole nation is annual has women more than 40,000 to die from this disease.Its pathogenesis is mainly summarized as
Genetic predisposition, endocrinopathy, pass through lactation virus spread particle etc..About 5% breast cancer is caused by gene mutation, its
Middle tumor suppressor gene BRCA1 and BRCA2 and pathogenesis of breast carcinoma relation are more close, and breast occurred before 40 years old for BRCA1 mutation persons
Gland cancer probability is up to 19%.
The high correlation of BRCA1, BRCA2 and Familial Occurrence breast cancer is unquestionable.Pass through the inspection to BRCA1
Survey, the occurrence and development of breast cancer can be reflected, can also be screened out the high-risk people of breast cancer, oophoroma and other associated malignancies
Group, the early diagnosis beneficial to such disease are treated., can be with early detection breast cancer and other several evils by being detected to BRCA2
Property tumour, such as prostate cancer, oophoroma, and may be selected rationally effective therapeutic scheme.
At present, the detection for BRCA1 and BRCA2 is concentrated on extron, but equally there are some researches show on introne
The variation of nucleotide sequence, RNA shearing can be also influenceed, so as to influence the function of gene.For BRCA1 and BRCA2 capture skill
Art mainly includes multiplex PCR and target capture technology, the design complicated compared to multiplex PCR capture technique and Preference, targeting
The high cost of capture technique, this patent use long-range PCR, can quickly and easily, low cost completion BRCA1 and
The capture of BRCA2 full genomes.With reference to follow-up high throughput sequencing technologies, less sequencing data amount can reach more than 99%
Coverage, help to propose the related hereditary tumor risk assessment of breast cancer susceptibility gene or medication and the foundation of database
Supported for technique of gene detection.
The content of the invention
The technical problems to be solved by the invention are to provide a kind of breast cancer susceptibility gene BRCA1 and BRCA2 full genomes and caught
Obtain primer, kit and its application.
In order to solve the above technical problems, the technical solution adopted by the present invention is as follows：
A kind of primer that can expand breast cancer susceptibility gene BRCA1 and BRCA2 full genome, including following nucleotide sequence：
(1) primer pair of BRCA1 full length genes, its nucleotide sequence SEQ ID NO. are expanded：Shown in 1~16；
(2) primer pair of BRCA2 full length genes, its nucleotide sequence SEQ ID NO. are expanded：Shown in 17~32；
Above-mentioned primer pair is used in conjunction with one-time detection.
A kind of guarantor of above-mentioned breast cancer susceptibility gene BRCA1 and BRCA2 full genomes primer and its complementary series in the present invention
Within the scope of shield.
The present invention another technical solution be：A kind of breast cancer susceptibility gene BRCA1 and BRCA2 full genome capture agent
Box, include above-mentioned primer SEQ ID NO.：1~16, SEQ ID NO.：The buffer solution of the amplifications of PCR shown in 17~32, enzyme, dNTP.
A kind of method of breast cancer susceptibility gene BRCA1 and BRCA2 full genomes capture, comprises the following steps：
(1) human peripheral genomic DNA is extracted；
(2) using the human peripheral genomic DNA of extraction as the primer in template, claim 1, PCR amplification buffers,
Enzyme, dNTP, capture 8 pcr amplification reactions of BRCA1 full genomes and 8 amplified reactions of capture BRCA2 full genomes；
(3) electrophoresis detection is carried out using 1% Ago-Gel, judges whether PCR amplifications succeed.
Preferably, in the step (2), 8 pcr amplification reactions and the capture full bases of BRCA2 of BRCA1 full genomes are captured
8 amplified reactions of cause, the amplification system of each amplified reaction are：The μ L of cumulative volume 20, include 5 × PrimeSTAR GXL
The μ L of 4 μ L, dNTP Mixture (2.5mM each) of Buffer, 1.6 μ L, PrimeSTAR GXL DNA Polymerase 1, on
Swim primer (SEQ ID NO.：1,3,5,7,9,11,13,15,17,19,21,23,25,27,29) 0.4 μM, anti-sense primer (SEQ
ID NO.：2,4,6,8,10,12,14,16,18,20,22,24,26,28,30) 0.4 μM, template DNA 50-100ng, deionization
Water supplies volume to 20 μ L.
As again with preferred, in the step (2), the amplification program in PCR reactions is：95 DEG C, 2min；95 DEG C, 45s,
68 DEG C, 10min, 25 circulations；16 DEG C, terminating reaction.
Beneficial effect：
, can be quickly simple the invention provides a kind of breast cancer susceptibility gene BRCA1 and BRCA2 full genome capture agent boxes
The capture of single completion BRCA1 and BRCA2 full genomes.The DNA captured can be directly used for follow-up flux sequencing.Compared to target
To capture technique and multiple PCR technique, this method is simple to operate, and cost is low, and sequencing data homogeneity is good, less sequencing data
Amount can reach more than 99% coverage, contribute to the related hereditary tumor risk assessment of breast cancer susceptibility gene or use
The foundation of medicine and database provides technique of gene detection and supported.
Brief description of the drawings
Fig. 1 is BRCA1 and BRCA2 amplification diagrams；
BRCA1：1-8；BRCA2：9-16.
Embodiment
According to following embodiments, the present invention may be better understood.It is however, as it will be easily appreciated by one skilled in the art that real
Apply the content described by example and be merely to illustrate the present invention, without should be also without limitation on sheet described in detail in claims
Invention.
Embodiment 1：
Step 1：DNA is extracted
200 μ L peripheral bloods are taken, it is (new purchased from Hangzhou to carry out DNA extractions according to Whole Blood Genomic DNA extracts kit specification
Prompt bio tech ltd), extraction DNA is used for subsequent experimental or deposits in -20 DEG C.
Step 2：PCR is expanded and detection
(1) BRCA1 and BRCA2 primer pairs, its nucleotide sequence SEQ ID NO. are expanded：Shown in 1-32；
(2) in the connecting legs of PCR 8, it is formulated as follows reaction system：
The μ L of cumulative volume 20, include 5 × PrimeSTAR GXL Buffer 4 μ L, dNTP Mixture (2.5mM each)
1.6 μ L, PrimeSTAR GXL DNA Polymerase 1 μ L, sense primer (SEQ ID NO.：1,3,5,7,9,11,13,
15,17,19,21,23,25,27,29) 0.4 μM, anti-sense primer (SEQ ID NO.：2,4,6,8,10,12,14,16,18,20,
22,24,26,28,30) 0.4 μM, template DNA 50-100ng, deionized water supplies volume to 20 μ L.Amplification in PCR reactions
Program is：95 DEG C, 2min；95 DEG C, 45s, 68 DEG C, 10min, 25 circulations；16 DEG C, terminating reaction.
Step 3：Interpretation of result
After reaction terminates, analyzed using agarose gel electrophoresis, judge whether PCR amplifications succeed.
Embodiment 2：
Step 1：DNA is extracted
200 μ L peripheral bloods are taken, it is (new purchased from Hangzhou to carry out DNA extractions according to Whole Blood Genomic DNA extracts kit specification
Prompt bio tech ltd), extraction DNA is used for subsequent experimental or deposits in -20 DEG C.
Step 2：PCR is expanded and detection
(1) BRCA1 and BRCA2 primer pairs, its nucleotide sequence SEQ ID NO. are expanded：Shown in 1-32；
(2) in the connecting legs of PCR 8, it is formulated as follows reaction system：
The μ L of cumulative volume 20, include 5 × PrimeSTAR GXL Buffer 4 μ L, dNTP Mixture (2.5mM each)
1.6 μ L, PrimeSTAR GXL DNA Polymerase 1 μ L, sense primer (SEQ ID NO.：1,3,5,7,9,11,13,
15,17,19,21,23,25,27,29) 0.4 μM, anti-sense primer (SEQ ID NO.：2,4,6,8,10,12,14,16,18,20,
22,24,26,28,30) 0.4 μM, template DNA 50-100ng, deionized water supplies volume to 20 μ L.Amplification in PCR reactions
Program is：95 DEG C, 2min；95 DEG C, 45s, 68 DEG C, 10min, 25 circulations；16 DEG C, terminating reaction.
Step 3：Product purification
(1) 16 pcr amplification products will be obtained in step (2), respectively takes 10 μ L to be mixed, obtain 160 μ L PCR amplifications
Product.
(2) added more than in 160 μ L PCR primers 160 μ L Agencourt AMPure XP Reagent (Beckman,
CAT#：A63880), purification process is carried out according to reagent operation specification, is finally eluted using 100 μ L deionized waters.
Step 4：Library construction
(1) 50ng purified products are taken, using TruePrep DNA Library Prep Kit V2for
(promise is only praised, CAT#：TD501-01) carry out building storehouse operation.
(2) using Agencourt AMPure XP Reagent sortings 350bp library fragments.
Step 5：Upper machine sequencing
It is sequenced using MiSeq (Illumina), sequencing pattern is PE150.
Step 6：Data analysis
Sequencing result is analyzed using bioinformatics software.
It is described above, it is preferred embodiments of the present invention, and the limitation that non-invention is any formal or substantial, should
Point out, for those skilled in the art, under the premise of the scope of the present invention is not departed from, it can also be modified
And improvement, these are improved and supplement should also be considered as protection scope of the present invention.
Sequence table
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Claims (5)
1. a kind of primer for expanding breast cancer susceptibility gene BRCA1 and BRCA2 full genome, it is characterised in that including following nucleic acid
Sequence：
(1) primer pair of BRCA1 full length genes, its nucleotide sequence SEQ ID NO. are expanded：Shown in 1~16；
(2) primer pair of BRCA2 full length genes, its nucleotide sequence SEQ ID NO. are expanded：Shown in 17~32；
Above-mentioned primer is used in conjunction with one-time detection.
2. a kind of breast cancer susceptibility gene BRCA1 and BRCA2 full genome capture agent boxes, it is characterised in that comprising claim 1
The primer of described amplification breast cancer susceptibility gene BRCA1 and BRCA2 full genome, the buffer solution of PCR amplifications, enzyme, dNTP.
3. a kind of breast cancer susceptibility gene BRCA1 and the method for BRCA2 full genomes capture, it is characterised in that comprise the following steps：
(1) human peripheral genomic DNA is extracted；
(2) using the human peripheral genomic DNA of extraction as the primer in template, claim 1, PCR amplification buffers, enzyme,
DNTP, capture 8 pcr amplification reactions of BRCA1 full genomes and 8 amplified reactions of capture BRCA2 full genomes；
(3) electrophoresis detection is carried out using 1% Ago-Gel, judges whether PCR amplifications succeed.
4. breast cancer susceptibility gene BRCA1 according to claim 3 and the method for BRCA2 full genomes capture, its feature exist
In in the step (2), 8 amplifications for capturing 8 pcr amplification reactions and capture BRCA2 full genomes of BRCA1 full genomes are anti-
Should, the amplification system of each amplified reaction is：The μ L of cumulative volume 20, include 5 × PrimeSTAR GXL Buffer 4 μ L, dNTP
1.6 μ L, PrimeSTAR GXL DNA Polymerase of Mixture (2.5mM each) 1 μ L, sense primer (SEQ ID
NO.：1,3,5,7,9,11,13,15,17,19,21,23,25,27,29) 0.4 μM, anti-sense primer (SEQ ID NO.：2,4,6,
8,10,12,14,16,18,20,22,24,26,28,30) 0.4 μM, template DNA 50-100ng, deionized water supplies volume extremely
20μL。
5. breast cancer susceptibility gene BRCA1 according to claim 3 and the method for BRCA2 full genomes capture, its feature exist
In in the step (2), the amplification program in PCR reactions is：95 DEG C, 2min；95 DEG C, 45s, 68 DEG C, 10min, 25 are followed
Ring；16 DEG C, terminating reaction.
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| CN201711138801.4A CN107760771A (en) | 2017-11-16 | 2017-11-16 | A kind of breast cancer susceptibility gene BRCA1 and BRCA2 full genomes capture primer, kit and its method |
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| US11421238B2 (en) | 2018-02-20 | 2022-08-23 | Longas Technologies Pty Ltd | Method for introducing mutations |
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| US11421238B2 (en) | 2018-02-20 | 2022-08-23 | Longas Technologies Pty Ltd | Method for introducing mutations |
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| CN109402257B (en) * | 2018-11-03 | 2023-06-23 | 杭州链康医学检验实验室有限公司 | Primer, method and kit for detecting mutation sites of human BRCA1and BRCA2 gene full-coding sequences |
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