CN105200154B - The multi-PCR detection method of BRCA1 and BRCA2 gene mutation and kit - Google Patents
The multi-PCR detection method of BRCA1 and BRCA2 gene mutation and kit Download PDFInfo
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Abstract
The invention provides a kind of method detecting BRCA1 and BRCA2 sudden change and kit.The method of the present invention and kit employ two set PCR primer, first set PCR primer includes 189 pairs of PCR primer, divide two groups, first group includes primer 95 to (SEQ ID NO.5 SEQ ID NO.194), second group includes primer 94 to (SEQ ID NO.195 SEQ ID NO.382), 5 ' ends of each upstream primer add sequence SEQ ID NO.1, and 5 ' ends of each downstream primer add sequence SEQ ID NO.2；The upstream and downstream primer sequence of the second set PCR primer is SEQ ID NO.3 and SEQ ID NO.4.
Description
Technical field
The present invention relates to detection in Gene Mutation field, particularly relate to the detection of gene mutation multiplex PCR.
Background technology
BRCA1 and BRCA2 is two and has the gene that suppression malignant tumour occurs, at duplication, the something lost of regulation human body cell
Pass matter DNA injury repair, the normal growth aspect of cell plays an important role.The sudden change of BRCA1 and BRCA2 having been found that has number
Hundred kinds more than, some are relevant with HBC and oophoroma.Sum up the related cancer of BRCA1 with BRCA2 gene mutation
Lifetime risk shows, has BRCA1 gene mutation person to suffer from breast cancer and the risk of oophoroma is 50%-85% and 15%-respectively
45%, there is BRCA2 gene mutation person to suffer from breast cancer and the risk of oophoroma is 50%-85% and 10%-20% respectively.With commonly
Women compares, and these are all very high to suffer from cancer probability.
Owing to the sudden change of BRCA1 and BRCA2 gene has certain contact, institute to familial breast cancer, oophoroma
Diagnosis, prevention and treatment to associated cancer for the mutator to detect BRCA1 and BRCA2 has important clinical meaning.In addition,
Owing to the sudden change of BRCA1 and BRCA2 is numerous, detect these sudden changes very meaningful for the research of related gene, for example for
Polymorphism Analysis and family's evolutionary history research.
At present, commonly used BRCA detection method of gene mutation is mainly and limits small fragment length polymorphism analysis method
(RFLP method) and DNA direct sequencing.RFLP method is the method being combined PCR with Restriction Enzyme cut, below the method existence
Shortcoming: experimental implementation is loaded down with trivial details, detect cycle length, cost is high, there is the first round is digested false positive, the non-stopped pipe behaviour not exclusively causing
Make, be easy to pollute, be difficult to meet clinical detection requirement.DNA direct sequencing has the disadvantage in that the detection cycle is longer, cost
Stopped pipe high, non-operates, is difficult to avoid that cross pollution, flux be not high.In addition, the sensitivity of DNA direct Sequencing is relatively low, heterozygosis is dashed forward
The problems such as change, glue laminated are contracted, the existence of GC enrichment region make it difficult to obtain accurate data by once sequencing, it usually needs repeatedly
Repeating order-checking to be only possible to avoid false positive etc., therefore direct Sequencing method also difficulty is applicable to clinical detection.
Also disclosed patent is had to use PCR method to detect breast cancer susceptibility gene mutation, for example patent " detection breast cancer
Multiple PCR reagent kit of susceptibility gene mutation and preparation method thereof " (Authorization Notice No.: CN 101200766 B).But, this is special
Multiplex PCR used by Li is only limited on the fractional mutations site on BRCA1 and BRCA2 Gene Partial extron, and this is obvious
Can not cover hundreds of sudden change that BRCA1 and BRCA2 gene spreads on multiple extron, therefore this patent has relatively on using
Big limitation.
Therefore, this area needs to can be used for high sensitivity, high flux, low cost detection BRCA1 and BRCA2 gene mutation
Method and kit.
Content of the invention
The invention aims to offer can be used for high sensitivity, high flux, low cost detection BRCA1 and BRCA2 dash forward
The method becoming and kit.BRCA1 and BRCA2 gene coding region is captured by the present invention mainly by multiple PCR technique
Enrichment, the coding region sequence after then measuring enrichment by high throughput sequencing technologies, eventually through bioinformatic analysis high pass
Amount sequencing data, thus find catastrophe and the frequency thereof of code area.
Therefore, the invention provides a kind of method detecting BRCA1 and BRCA2 sudden change, described method includes
1) capture: carry out the first round specific multi-PRC reaction, use 189 to expand primer to sample DNA, institute
Stating 189 pairs of PCR primer divides two test tubes to react, and first test tube primer 95 is to (SEQ ID NO.5-SEQ ID
NO.194), (SEQ ID NO.195-SEQ ID NO.382), 5 ' ends of each upstream primer are added by second test tube primer 94
Add sequence SEQ ID NO.1:
" ACACTCTTTCCCTACACGACGCTCTTCCGATCTTAC ", 5 ' ends of each downstream primer add sequence SEQ
ID NO.2: " GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGTA "；
2) storehouse is built: carry out second and take turns PCR reaction, use PCR primer pair: upstream primer SEQ ID NO.3:
" AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGAC ", downstream primer SEQ ID NO.4:
" CAAGCAGAAGACGGCATACGAGATATCACGTTGTGACTGGAGTTCAGACGT " is to step 1) expansion that obtains
Volume increase thing expands；
3) checking order, to step 2) amplified production that obtains checks order；
4) analyze, to step 3) sequencing result that obtains is analyzed, and completes the abrupt climatic change of full code area.
Present invention also offers a kind of BRCA1 and BRCA2 gene mutation multiple PCR detection kit, described kit bag
Include: two set PCR primer and PCR amplification premix solution (for example, from KAPA2G Fast thermal starting multiplex amplification kit)；
Two described set PCR primer, first set PCR primer includes 189 pairs of PCR primer, divides two groups, and first group includes primer
95 pairs (SEQ ID NO.5-SEQ ID NO.194), second group includes primer 94 to (SEQ ID NO.195-SEQ ID
NO.382), 5 ' end interpolation SEQ ID NO.1 of each upstream primer:
" ACACTCTTTCCCTACACGACGCTCTTCCGATCTTAC " sequence, 5 ' ends of each downstream primer add SEQ
ID NO.2: " GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGTA " sequence；
The upstream primer sequence SEQ ID NO.3 of the second set PCR primer is:
" AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGAC ", downstream primer sequence SEQ ID
NO.4 is
“CAAGCAGAAGACGGCATACGAGATATCACGTTGTGACTGGAGTTCAGACGT”。
In the kit of the present invention, described first set is specific multiple PCR primer, and described second set PCR primer is
A pair Standard PCR primer, at its 5 ' end with sequencing sequence.
In a preferred embodiment, described kit also includes distilled water.
In a preferred embodiment, in described kit, the reaction system of the PCR reaction of first set primer is pressed
Following proportions: first set PCR primer 10 parts, PCR amplification premix solution (such as PCR expands premix solution) 12.5
Part, DNA profiling 3 parts, 4.5 parts of water.
In a preferred embodiment, the reaction system of the second set PCR reaction presses following proportions: second overlaps
PCR primer 2.5 parts, PCR amplification premix solution (such as PCR expands premix solution) 7.5 parts, first round PCR reclaim product 5
Part, 4.5 parts of water.
Compared to the prior art, detection method and the kit of the present invention has the advantage that
1) with low cost；
2) low to sample requirement amount to be detected, the extremely low DNA sample in blood plasma and serum can be analyzed；
3) blanket type design of primers, can realize full code area abrupt climatic change, not only can be analyzed known mutations, moreover it is possible to
Find new sudden change；
4) highly sensitive；
5) detect the time short, in conjunction with high throughput sequencing technologies, full code area abrupt climatic change in the short time, can be realized.
The kit of the present invention can be used for high sensitivity, high flux, low cost detection BRCA sudden change, assists clinician real
The individualized treatment of existing tumour patient, reduces Operative risk and patient burden.
It should be appreciated that aforementioned description substantially and follow-up detailed description are exemplary illustration and explanation, should not
It is used as the restriction of the claimed content to the present invention.
Brief description
With reference to the accompanying drawing enclosed, as follows by by embodiment of the present invention of the more purpose of the present invention, function and advantage
Description is illustrated, wherein:
Fig. 1: the second takes turns PCR reacts through 2% agarose electrophoresis qualification result.
Detailed description of the invention
By with reference to one exemplary embodiment, the purpose of the present invention and function and for realizing the side of these purposes and function
Method will be illustrated.But, the present invention is not limited to one exemplary embodiment disclosed below；Can be come by multi-form
It is realized.The essence of specification is only the detail helping the various equivalent modifications Integrated Understanding present invention.
The invention provides a kind of method detecting BRCA1 and BRCA2 sudden change, although in some cases, to these bases
The detection of cause has important Auxiliary Significance for diagnosis, prevention and the treatment of associated cancer, but the sudden change of these genes is not must
So cause the generation of described disease, be therefore not related to diagnosis or the treatment method of disease to the detection of these genes.In addition, this
The method of the detection sudden change of invention can also have other purposes many, for example, carry out Polymorphism Analysis and family's evolutionary history grinds
Study carefully.The method of the present invention can be also used for the sample research of nonspecific object, for example, detect mixing blood product.
In the present invention, sequence SEQ ID NO.5-SEQ ID NO.382 matches formation two-by-two and 189 draws first set PCR
Thing, i.e. SEQ ID NO.5 and SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.8......SEQ ID NO.381 and
SEQ ID NO.382 matches respectively and forms 189 primers pair.
In the present invention, described 189 pairs of first set PCR primer divide two groups, and first group includes primer 95 to (SEQ ID
NO.5-SEQ ID NO.194), second group include primer 94 to (SEQ ID NO.195-SEQ ID NO.382), this be through
Inventor is by computer simulation and carries out testing the result being adjusted, and so makes the intersection avoiding between primer anti-
Should so that improve efficiency and the quality of PCR.
In the present invention, 5 ' ends of each upstream primer add sequence SEQ ID NO.1
" ACACTCTTTCCCTACACGACGCTCTTCCGATCTTAC ", 5 ' ends of each downstream primer add sequence SEQ ID NO.2
" GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGTA ", the consensus sequence that upstream and downstream primer is added is second to take turns PCR
The binding site of reaction primer.
In the present invention, the upstream primer sequence of the second set PCR primer is: SEQ ID NO.3
" AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGAC ",
Downstream primer sequence is SEQ ID NO.4
“CAAGCAGAAGACGGCATACGAGATATCACGTTGTGACTGGAGTTCAGACGT ", its middle and lower reaches underscore partial sequence
It is barcode sequence, be used for distinguishing multiple sample.
In the present invention, PCR amplification premix solution, i.e. contains the PCR such as Taq archaeal dna polymerase, dNTPs, buffer solution and expands
Increasing the solution of required component (except template and primer), such as PCR expands premix solution.
In one embodiment of the invention, the application process of the multiple PCR detection kit of the present invention is as follows:
1) DNA (for example conventionally) of detected sample is extracted as template；
2) carrying out first round multiplexed PCR amplification reaction with first set primer, first group and second group of primer are to respectively two
Individual test tube is carried out；
3) pcr amplification product is purified recovery by magnetic bead；
4) carry out second as template with the second set primer with the product that first round PCR reclaims and take turns PCR reaction；
5) pcr amplification product is purified recovery by magnetic bead；
6) electroresis appraisal；
7) collect product and carry out upper machine order-checking (such as second generation high-flux sequence)；
8) identify whether the gene of BRCA1 and BRCA2 there occurs sudden change by bioinformatic analysis.
Wherein, described multiplexed PCR amplification reaction condition is preferably as follows:
First round PCR reacts amplification system:
First round PCR reacts amplification condition:
First round PCR amplification afterproduct purifies:
1) PCR primer adds isopyknic recovery buffer solution, and (Tris-Hcl weighs 121.1gTris-Hcl and is placed in 1L burning
In Bei, add in the deionized water getting over 800mL, be sufficiently stirred for.Add the Hcl of 42mL, be settled to 1L.)
With 10ul magnetic bead (Beckman-B46053), concussion mixes 30s；
2) mixture is positioned over 30s on magnetic frame；
3) sucking-off supernatant, dries 30s, leaves magnetic frame；
4) adding washing lotion W1 (70% ethanol), concussion mixes 30s；
5) mixture is positioned over 30s on magnetic frame；
6) sucking-off supernatant, dries 30s, leaves magnetic frame；
7) washing lotion W2 (H is added2O), concussion mixes 30s；
8) mixture is positioned over 30s on magnetic frame；
9) sucking-off supernatant, dries 2min, leaves magnetic frame；
10) (ATE, 48.4gTris-Hcl are dissolved in 150mL deionized water, heat 45 DEG C of stirrings extremely to add 20ul eluent
Dissolving, 14.61g EDTA adds above-mentioned solution, continues stirring.Add 11.42m LAC, add while stirring, after adding AC, become clear
Clearly, EDTA thoroughly dissolves.Surveying PH is 8.6, adds deionized water and is settled to 200mL), concussion mixes 30s；
11) mixture is positioned over 30s on magnetic frame；
12) supernatant is collected standby.
Second takes turns PCR amplification system:
Second take turns PCR reaction amplification condition:
Second takes turns PCR amplification afterproduct purification step purifies with the amplification afterproduct of first round PCR above.
Embodiment
First, the preparation of multiple PCR reagent kit of the present invention and assembling
1. the design of two-wheeled PCR primer and preparation
The first round 189 to PCR primer,
First round PCR primer, divides two groups, and first group includes that primer 95 is right, and second group includes that primer 94 is right: often organizes and takes
1ul (single primer concentration 1uM), mixes；
Second takes turns PCR primer (overall primer concentration 0.1uM): take 1.25ul；
All primers are all through automatic dna synthesizer synthesis.
2. the assembling of kit
PCR expands premix solution 1 (KAPA, No:KK5801): 12.5ul；
PCR expands premix solution 2 (KAPA, No:KK2601): 7.5ul；
First round PCR primer first group: 5ul, first round PCR primer second group: 5ul
Second takes turns PCR primer test tube: 1.25ul.
2nd, the application experiment of multiple PCR reagent kit of the present invention
1. detect sample
The sample source being detected is in the blood of certain volunteer.
2. detection method
DNA extracts: conventionally extract the DNA of detected sample as template.
PCR expands:
The first round PCR reaction amplification system first round described above PCR reaction amplification system.
The first round PCR reaction amplification condition first round described above PCR reaction amplification condition.
The first round PCR amplification afterproduct purification step first round described above PCR amplification afterproduct purifies.
Second takes turns PCR amplification system described above second takes turns PCR reaction amplification system.
Second takes turns PCR reaction amplification condition described above second takes turns PCR reaction amplification condition.
Second takes turns the PCR amplification afterproduct purification step first round described above PCR amplification afterproduct purifies.
Measuring A pipe 260/280 is 1.79, corresponding DNA concentration 5.02ng/ul.
Measuring B pipe 260/280 is 1.81, corresponding DNA concentration 6.87ng/ul.
Electroresis appraisal result is shown in Fig. 1.
3. testing result
Machine in the result of two-wheeled PCR is checked order, and carries out bioinformatic analysis.
1) first quality evaluation is carried out to sequencing data.
2) the sequencing data amount after assessment is added up, as shown in the table.
3) reference sequences comparison and depth data statistics
Data (clean after using Burrows-Wheeler Aligner (BWA) software that previous step is generated Quality Control
Data) comparing with full-length genome, BWA is a comparison software that analysis is accurate, analysis efficiency is high.Comparison generates after completing
Sequence Alignment Map (SAM) document result.Then picard software is used to remove the repetitive sequence (PCR in SAM
During produce unnecessary information), finally, we utilize samtools software that the destination file of SAM is changed into BAM file,
BAM form is exactly the compression result of SAM form, and the explanation of SAM file format refers to http: //
samtools.sourceforge.net/SAM1.pdf。
| Project | Value |
| Sequencing sequence average length (Average read length) | 139 |
| Order-checking base average mass values (Average base quality) | 32 |
| Averagely build storehouse length (Average lab size) | 193.8 |
| Repetitive rate (Duplication rate) (%) | Nothing |
| Comparison rate (Align rate) (%) | 82.56 |
| The target area size (Total target base) of capture | 15915 |
| The size (Covered target base) in actual sequencing result coverage goal region | 15662 |
| Coverage rate (Coverage rate) (%) | 98.41 |
| Total valid data amount (Total effective base) (Mb) | 84.51 |
| The valid data amount (Effective base on target) (Mb) of target area | 14.89 |
| Capture rate (Capture rate) (%) | 17.62 |
| The mean depth (Target average depth) of target area | 935.54 |
| The ratio (Target 4X rate) (%) shared by region more than 4 layers for the target area degree of depth | 95.97 |
| The ratio (Target 10X rate) (%) shared by region more than 10 layers for the target area degree of depth | 95.27 |
| (the ratio Target 20X rate shared by region more than 20 layers for the target area degree of depth) (%) | 92.57 |
| Capture region title (Panel name) | BRCA1/2 |
Comparison rate: the sequence-repetitive sequence/clean data of human genome in comparison
Coverage rate: the target area size of the size/capture in actual sequencing result coverage goal region
Total valid data amount: the sequence-repetitive sequence of human genome in comparison
Capture rate: the valid data amount of target area/
4) in this example, the sudden change of discovery is distributed:
| Extron same sense mutation (exonic_synonymous) | 9 |
| Extron nonsense mutation (exonic_nonsynonymous) | 12 |
| Exon 1 sudden change sum (exonic) | 21 |
| Introne region mutation sum (intronic) | 4 |
| Variable brief introduction region mutation sum (splicing) | 1 |
| 5 ' UTR region mutations sum (5-UTR) | 1 |
Although already in connection with preferred embodiment, invention has been described, it is to be understood that protection scope of the present invention is simultaneously
It is not limited to embodiment as described herein.In conjunction with explanation and the practice of the present invention disclosing here, other of the present invention are implemented
Example is all easy to for those skilled in the art to expect and understand.Illustrate and embodiment is to be considered only as exemplary, this
Bright true scope and purport are all defined in the claims.
Claims (6)
1. a BRCA1 and BRCA2 gene mutation multiple PCR detection kit, described kit includes: two set PCR primer；
Two described set PCR primer, first set PCR primer includes 189 pairs of PCR primer, divides two groups, and first group includes that primer 95 is right
SEQ ID NO.5-SEQ ID NO.194, second group include primer 94 to SEQ ID NO.195-SEQ ID NO.382, each
5 ' ends of upstream primer add sequence SEQ ID NO.1, and 5 ' ends of each downstream primer add sequence SEQ ID NO.2；
The upstream and downstream primer sequence of the second set PCR primer is SEQ ID NO.3 and SEQ ID NO.4 respectively.
2. the kit of claim 1, described kit also includes PCR amplification premix solution.
3. the kit of claim 2, described kit includes from KAPA 2G Fast thermal starting multiplex amplification kit
PCR expands premix solution.
4. the kit of claim 1, described kit also includes water.
5. the kit of Claims 2 or 3, in described kit, the reaction system of the PCR reaction of first set primer is pressed as follows
Proportions: first set PCR primer 10 parts, PCR amplification premix solution 12.5 parts, DNA profiling 3 parts, 4.5 parts of water.
6. the kit of claim 1, in described kit, the reaction system of the second set PCR reaction is by following proportions:
PCR primer 2.5 parts, PCR amplification premix solution 7.5 parts, first round PCR reclaim product 5 parts, 4.5 parts of water.
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| CN105586427B (en) * | 2016-03-10 | 2020-06-19 | 厦门艾德生物医药科技股份有限公司 | Primers, kit and method for detecting human BRCA1 and BRCA2 gene mutation |
| CN105754998A (en) * | 2016-04-01 | 2016-07-13 | 山东美加赛培生物科技有限公司 | Primer and kit for amplifying related genes of breast cancer and ovarian cancer and application thereof |
| CN107312770A (en) * | 2016-04-26 | 2017-11-03 | 厦门飞朔生物技术有限公司 | A kind of construction method in tumour BRCA1/2 genetic mutations library detected for high-flux sequence and its application |
| CN105779636B (en) * | 2016-05-18 | 2020-05-19 | 广州安必平医药科技股份有限公司 | PCR primer for amplifying human breast cancer susceptibility gene BRCA1 and BRCA2 coding sequence and application |
| CN107523609A (en) * | 2016-06-22 | 2017-12-29 | 海门中科基因生物科技有限公司 | Hereditary breast cancer and oophoroma BRCA1/2 gene hot mutant site detection kits |
| CN106191269A (en) * | 2016-07-22 | 2016-12-07 | 上海产业技术研究院 | Universal BRCA1 gene multiplex PCR builds storehouse test kit |
| CN106191271A (en) * | 2016-07-22 | 2016-12-07 | 上海产业技术研究院 | Tailored version BRCA2 gene multiplex PCR builds storehouse test kit |
| CN106191268A (en) * | 2016-07-22 | 2016-12-07 | 上海产业技术研究院 | Tailored version BRCA1 gene multiplex PCR builds storehouse test kit |
| CN106191272A (en) * | 2016-07-22 | 2016-12-07 | 上海产业技术研究院 | Universal BRCA2 gene multiplex PCR builds storehouse test kit |
| CN106701936A (en) * | 2016-12-20 | 2017-05-24 | 湖南圣湘生物科技有限公司 | Breast cancer susceptibility gene BRCA1 and BRCA2 detection kit and method |
| CN107955835B (en) * | 2017-05-27 | 2021-05-14 | 广州市达瑞生物技术股份有限公司 | Primer pool and detection method for detecting BRCA1/2 gene mutation |
| CN107400714B (en) * | 2017-08-21 | 2020-12-29 | 广州永诺生物科技有限公司 | Multiple PCR primer group and kit for detecting drug-related genes for colorectal cancer |
| CN110714071B (en) * | 2018-07-12 | 2023-06-20 | 深圳华大智造科技股份有限公司 | High-throughput detection kit for human BRCA1/2 gene mutation |
| CN111349699A (en) * | 2018-12-24 | 2020-06-30 | 深圳华大智造科技有限公司 | Kit and method for detecting BRCA gene mutation from cervical secretions |
| CN110079868A (en) * | 2019-03-20 | 2019-08-02 | 上海思路迪生物医学科技有限公司 | BRCA1/2 genetic mutation detects library constructing method and kit |
| CN112458195B (en) * | 2020-12-28 | 2023-03-24 | 广州迈景基因医学科技有限公司 | Multiplex PCR primer set, kit and method for detecting sexually transmitted pathogens based on high-throughput sequencing |
| CN113403380A (en) * | 2021-06-11 | 2021-09-17 | 中国科学院北京基因组研究所(国家生物信息中心) | Complex disease related SNP site primer composition and application |
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| CN102628082B (en) * | 2012-04-10 | 2014-09-17 | 张影频 | Method for qualitatively and quantitatively detecting nucleic acid based on high-flux sequencing technology |
| CN104531862B (en) * | 2014-12-19 | 2017-12-29 | 钱学庆 | Detect the method and primer in the full exon sequence mutational site of mankind's BRCA1 and BRCA2 gene |
| CN104694663B (en) * | 2015-04-13 | 2015-12-02 | 玉峰惠仁生物医药科技（北京）有限公司 | BRCA gene susceptible SNP site detection composition |
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