CN107312770A - A kind of construction method in tumour BRCA1/2 genetic mutations library detected for high-flux sequence and its application - Google Patents

A kind of construction method in tumour BRCA1/2 genetic mutations library detected for high-flux sequence and its application Download PDF

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CN107312770A
CN107312770A CN201610263702.8A CN201610263702A CN107312770A CN 107312770 A CN107312770 A CN 107312770A CN 201610263702 A CN201610263702 A CN 201610263702A CN 107312770 A CN107312770 A CN 107312770A
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陈琰
郭飞飞
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Xiamen Spacegen Biotechnology Co Ltd
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Abstract

The invention discloses a kind of construction method in the tumour BRCA1/2 genetic mutations library detected for high-flux sequence, it is characterised in that：Cover the whole exon region mutation of mankind BRCA1/2 genes.The construction method of the present invention carries out single tube for the full exon sequence of BRCA1 and BRCA2 genes, it is rapidly completed the structure in library, whole library construction process only needs 2~3 hours, need only to manufal operation time 15~30 minutes, it can be solved at present with highly effective for needing to detect this difficult point to the full extron of gene on the basis of a small amount of clinical sample in clinically tumor disease with reference to high-flux sequence platform, and it is with low cost.

Description

A kind of structure in the tumour BRCA1/2 genetic mutations library detected for high-flux sequence Construction method and its application
Technical field
Present invention relates particularly to a kind of structure side in the tumour BRCA1/2 genetic mutations library detected for high-flux sequence Method and its application.
Background technology
Breast cancer, oophoroma are the most common malignant tumours for threatening women's health.The hair of country variant women with breast cancer Sick rate is dramatically different, with the U.S. and Northern Europe incidence of disease highest.Generation, the development of breast cancer are relevant with many factors, are by environment Factor and the coefficient result of inherent cause.BRCA1, BRCA2 are Breast Cancer Susceptibility Gene 1st, 2 abbreviation, i.e. breast cancer susceptibility gene 1/2.Wherein, the BRCA1 assignments of genes gene mapping are aobvious outside 23 in human chromosomal 17q21 Son is constituted；The BRCA2 assignments of genes gene mapping are made up of in human chromosomal 13q12.3 27 extrons.BRCA1, BRCA2 gene is all Tumor suppressor gene, important make is played in terms of DNA damage reparation, Cycle Regulation, gene transcriptional activation, chromatin are stable With.
There is BRCA1, BRCA2 in as shown by data, about 2% Female breast cancer patients and 10%-15% ovarian cancer patients Gene mutation.BRCA1, BRCA2 gene mutation state and familial breast cancer onset relation are very close, and gene mutation phenotype is past Toward the trend with induction breast cancer and oophoroma.Studies have shown that BRCA1 and BRCA2 mutation carriers are that occur mammary gland The people at highest risk of cancer, oophoroma, its lifelong breast cancer risk is dramatically increased：The probability that carriers of mutation suffers from breast cancer is 40-80%, the probability for suffering from oophoroma is 16-60% (it is respectively 12% and 1% that population, which suffers from cancer risk).BRCA2 genes Mutation also results in cancer of male breast (incidence 6%).
Reported according to breast cancer information centre (Breast Cancer Informationcore, BIC), BRCA1, BRCA2 base Because mutation has reached kind more than 3000, these mutation are distributed in whole code area, and most common disease cause mutation is frameshift mutation, nothing Justice mutation etc., without obvious mutantional hotspot.Big multimutation causes the formation of truncated protein, makes BRCA1, BRCA2 protein work( It can lose, so as to cause tumour.
High-frequency mutantional hotspot is not present in BRCA1, BRCA2 gene mutation, it means that, gene sequencing can not be detected only The site of the several fixations of BRCA1, BRCA2 gene, but to carry out full genome sequencing.This kit is complete to BRCA1/2 genes Extron carries out amplification library and prepared, and in carrying out sequencing reaction on high-flux sequence instrument, can rapidly detect BRCA1/2 genes 193 mutational sites (point mutation, small fragment is inserted, lacked, repeating) altogether of code area extron, specifying information is referring to table 1. By detecting BRCA1, BRCA2 gene mutation, it can reflect that breast cancer develops, can also be screened out breast cancer, oophoroma And the people at highest risk of other associated malignancies；It is tight by being carried out to the people at highest risk for carrying BRCA1, BRCA2 gene mutation Follow-up and monitoring, contribute to breast cancer discovery early, early diagnosis and and early treatment, preferably control curative effect so as to obtain Really.It is reported that, the women BRCA mutation carriers not yet suffered from breast cancer can pass through intervening measure (such as preventative breast Adenectomy takes TAM), examination or the two combine prevented and auxiliary direction personalized medicine.
Existing oncogene detection is main to use fluorescence quantitative PCR method, and detection flux is low, for BRCA1 and BRCA2 bases When being detected because of full extron, costly and sample size demand is big, causes clinical charge high and sample size deficiency.And industry is public at present The detection in Gene Mutation goldstandard recognized is Sanger PCR sequencing PCRs, and this kind of technical operation is complicated, and flux is relatively low, and sensitivity is low, has Certain missed detection risk, it is difficult to meet the detection demand of massive tumor patient.Therefore, a kind of accuracy rate height is developed, flux is big, pacify The complete full extron detection method of tumour BRCA1 and BRCA2 gene, is to effective supplement of existing tumour personalized medicines and complete It is kind.
High-flux sequence (High-Throughput Sequencing) also known as sequencing (Next Generation of future generation Sequencing, NGS), it can realize once parallel to hundreds of thousands to millions of target nucleic acid molecules progress sequencings, tool Have the characteristic of highoutput and high-res, provide not only abundant sequence variations information, and cause sequencing expense and Time greatly shortens, and is approved rapidly.Remarkable effect is played in many target researchs of cancer multi-path, this is highlighted brand-new The clinical importance of technological means.
The content of the invention
It is an object of the invention to overcome prior art defect, there is provided a kind of tumour detected for high-flux sequence The construction method in BRCA1/2 genetic mutations library.
Another object of the present invention is to provide the kit based on above-mentioned construction method.
The concrete technical scheme of the present invention is as follows：
A kind of construction method in the tumour BRCA1/2 genetic mutations library detected for high-flux sequence, covers the mankind The whole exon region mutation of BRCA1/2 genes, specifically includes following steps：
(1) the first basic amplimer group and the second basic amplimer group are designed for target gene BRCA1/2, this 5 ' ends of all forward primers and reverse primer of one basic amplimer group and the second basic amplimer group are equipped with extra 2~5 T, and 2~5 T close to 3 ' end first T there is PNA modifications, while the first basic amplimer group Tm values difference be no more than 1 DEG C, the Tm values of second basic amplimer group difference is no more than 1 DEG C；
(2) whether one asymmetric linking probe group of design and a universal primer for not forming complementation human genome or not, asymmetric Linking probe group contains multipair different asymmetric linking probe, and the asymmetric linking probe of each pair includes that itself ring-type can be formed Positive a probe and a reverse probe, the positive probe and reverse probe from 3 ' ends to 5 ' ends include successively one can with not The complementary series of 5 ' some continuous base pairings in end of symmetrical linking probe, one with the amplification sequence of the universal primer complementary pairing Row and a cohesive end recognition sequence corresponding with above-mentioned 2~5 T-phase, while each positive probe and reverse probe are respectively provided with phase The sequence label answered, the positive probe of the above-mentioned asymmetric linking probe of each pair and the Tm values difference of reverse probe are no more than 1 DEG C；
(3) template, above-mentioned first basic amplimer group are placed in into the first PCR containing RingCap-Taq enzymes to react Expanded in system, the first amplified production is obtained after purification；Template, above-mentioned second basic amplimer group are placed in one and contained Expanded in 2nd PCR reaction systems of RingCap-Taq enzymes, the second amplimer is obtained after purification；
(4) by the first amplified production, the second amplified production, asymmetric linking probe group, universal primer and above-mentioned RingCap-Taq enzymes are mixed into performing PCR, and the tumour BRCA1/2 genetic mutations text for high-flux sequence is obtained after purification Storehouse；
Above-mentioned RingCap-Taq enzymes are made up of Taq enzyme, DNA ligase and the end modified enzymes of DNA.
In a preferred embodiment of the invention, the first amplimer group includes BA2-001-F, BA2-001- R、BA1-001-F、BA1-001-R、BA2-002-F、BA2-002-R、BA1-002-F、BA1-002-R、BA2-003-F、BA2- 003-R、BA2-004-F、BA2-004-R、BA1-003-F、BA1-003-R、BA1-004-F、BA1-004-R、BA1-005-F、 BA1-005-R、BA1-006-F、BA1-006-R、BA1-007-F、BA1-007-R、BA1-008-F、BA1-008-R、BA1- 009-F、BA1-009-R、BA1-010-F、BA1-010-R、BA1-011-F、BA1-011-R、BA1-012-F、BA1-012-R、 BA2-005-F、BA2-005-R、BA2-006-F、BA2-006-R、BA2-007-F、BA2-007-R、BA2-008-F、BA2- 008-R、BA2-009-F、BA2-009-R、BA1-013-F、BA1-013-R、BA2-010-F、BA2-010-R、BA2-011-F、 BA2-011-R、BA2-012-F、BA2-012-R、BA2-013-F、BA2-013-R、BA2-014-F、BA2-014-R、BA2- 015-F、BA2-015-R、BA2-016-F、BA2-016-R、BA2-017-F、BA2-017-R、BA2-018-F、BA2-018-R、 BA2-019-F、BA2-019-R、BA2-020-F、BA2-020-R、BA2-021-F、BA2-021-R、BA2-022-F、BA2- 022-R、BA2-023-F、BA2-023-R、BA2-024-F、BA2-024-R、BA2-025-F、BA2-025-R、BA2-026-F、 BA2-026-R、BA2-027-F、BA2-027-R、BA2-028-F、BA2-028-R、BA2-029-F、BA2-029-R、BA2- 030-F、BA2-030-R、BA2-031-F、BA2-031-R、BA2-032-F、BA2-032-R、BA2-033-F、BA2-033-R、 BA2-034-F、BA2-034-R、BA2-035-F、BA2-035-R、BA2-036-F、BA2-036-R、BA2-037-F、BA2- 037-R、BA2-038-F、BA2-038-R、BA2-039-F、BA2-039-R、BA2-040-F、BA2-040-R、BA2-041-F、 BA2-041-R、BA2-042-F、BA2-042-R、BA2-043-F、BA2-043-R、BA2-044-F、BA2-044-R、BA2- 045-F、BA2-045-R、BA2-046-F、BA2-046-R、BA2-047-F、BA2-047-R、BA2-048-F、BA2-048-R、 BA2-049-F、BA2-049-R、BA2-050-F、BA2-050-R、BA2-051-F、BA2-051-R、 BA2-052-F、BA2- 052-R、BA2-053-F、BA2-053-R、BA2-054-F、BA2-054-R、BA2-055-F、BA2-055-R、BA2-056-F、 BA2-056-R、BA2-057-F、BA2-057-R、BA2-058-F、BA2-058-R、BA2-059-F、BA2-059-R、BA1- 014-F、BA1-014-R、BA1-015-F、BA1-015-R、BA1-016-F、BA1-016-R、BA1-017-F、BA1-017-R、 BA1-018-F、BA1-018-R、BA1-019-F、BA1-019-R、BA1-020-F、BA1-020-R、BA1-021-F、BA1- 021-R、BA1-022-F、BA1-022-R、BA1-023-F、BA1-023-R、BA1-024-F、BA1-024-R、BA1-025-F、 BA1-025-R、BA1-026-F、BA1-026-R、BA1-027-F、BA1-027-R、BA1-028-F、BA1-028-R、BA1- 029-F、BA1-029-R、BA1-030-F、BA1-030-R、BA1-031-F、BA1-031-R、BA1-032-F、BA1-032-R、 BA1-033-F、BA1-033-R、BA1-034-F、BA1-034-R、BA1-035-F、BA1-035-R、BA1-036-F、BA1- 036-R、BA2-060-F、BA2-060-R、BA2-061-F、BA2-061-R、BA1-037-F、BA1-037-R、2-BA2-062- F, 2-BA2-062-R, 2-BA1-038-F and 2-BA1-038-R, its sequence is successively such as the institutes of SEQ 01~SEQ of ID ID 200 Show.
In a preferred embodiment of the invention, the second amplimer group includes 2-BA2-063-F, 2-BA2- 063-R、2-BA2-064-F、2-BA2-064-R、2-BA2-065-F、2-BA2-065-R、2-BA2-066-F、2-BA2-066- R、2-BA2-067-F、2-BA2-067-R、2-BA2-068-F、2-BA2-068-R、2-BA2-069-F、2-BA2-069-R、2- BA2-070-F、2-BA2-070-R、2-BA1-039-F、2-BA1-039-R、2-BA1-040-F、2-BA1-040-R、2-BA1- 041-F、2-BA1-041-R、2-BA1-042-F、2-BA1-042-R、2-BA1-043-F、2-BA1-043-R、2-BA1-044- F、2-BA1-044-R、2-BA1-045-F、2-BA1-045-R、2-BA1-046-F、2-BA1-046-R、2-BA2-071-F、2- BA2-071-R、2-BA2-072-F、2-BA2-072-R、2-BA2-073-F、2-BA2-073-R、2-BA2-074-F、2-BA2- 074-R、2-BA2-075-F、2-BA2-075-R、2-BA1-047-F、2-BA1-047-R、2-BA2-076-F、2-BA2-076- R、2-BA1-048-F、2-BA1-048-R、2-BA2-077-F、2-BA2-077-R、2-BA2-078-F、2-BA2-078-R、2- BA2-079-F、2-BA2-079-R、2-BA2-080-F、2-BA2-080-R、2-BA2-081-F、2-BA2-081-R、2-BA2- 082-F、2-BA2-082-R、2-BA2-083-F、2-BA2-083-R、2-BA2-084-F、2-BA2-084-R、2-BA2-085- F、2-BA2-085-R、2-BA2-086-F、2-BA2-086-R、2-BA2-087-F、2-BA2-087-R、2-BA2-088-F、2- BA2-088-R、2-BA2-089-F、2-BA2-089-R、2-BA2-090-F、2-BA2-090-R、2-BA2-091-F、2-BA2- 091-R、2-BA2-092-F、2-BA2-092-R、2-BA2-093-F、2-BA2-093-R、2-BA2-094-F、2-BA2-094- R、2-BA2-095-F、2-BA2-095-R、2-BA2-096-F、2-BA2-096-R、2-BA2-097-F、2-BA2-097-R、2- BA2-098-F、2-BA2-098-R、2-BA2-099-F、2-BA2-099-R、2-BA2-100-F、2-BA2-100-R、2-BA2- 101-F、2-BA2-101-R、2-BA2-102-F、2-BA2-102-R、2-BA2-103-F、2-BA2-103-R、2-BA2-104- F、2-BA2-104-R、2-BA2-105-F、2-BA2-105-R、2-BA2-106-F、2-BA2-106-R、2-BA2-107-F、 2- BA2-107-R、2-BA2-108-F、2-BA2-108-R、2-BA2-109-F、2-BA2-109-R、2-BA2-110-F、2-BA2- 110-R、2-BA2-111-F、2-BA2-111-R、2-BA2-112-F、2-BA2-112-R、2-BA2-113-F、2-BA2-113- R、2-BA2-114-F、2-BA2-114-R、2-BA2-115-F、2-BA2-115-R、2-BA2-116-F、2-BA2-116-R、2- BA2-117-F、2-BA2-117-R、2-BA2-118-F、2-BA2-118-R、2-BA2-119-F、2-BA2-119-R、2-BA2- 120-F、2-BA2-120-R、2-BA2-121-F、2-BA2-121-R、2-BA1-049-F、2-BA1-049-R、2-BA1-050- F、2-BA1-050-R、2-BA1-051-F、2-BA1-051-R、2-BA1-052-F、2-BA1-052-R、2-BA1-053-F、2- BA1-053-R、2-BA1-054-F、2-BA1-054-R、2-BA1-055-F、2-BA1-055-R、2-BA1-056-F、2-BA1- 056-R、2-BA1-057-F、2-BA1-057-R、2-BA1-058-F、2-BA1-058-R、2-BA1-059-F、2-BA1-059- R、2-BA1-060-F、2-BA1-060-R、2-BA1-061-F、2-BA1-061-R、2-BA1-062-F、2-BA1-062-R、2- BA1-063-F、2-BA1-063-R、2-BA1-064-F、2-BA1-064-R、2-BA1-065-F、2-BA1-065-R、2-BA1- 066-F、2-BA1-066-R、2-BA1-067-F、2-BA1-067-R、2-BA1-068-F、2-BA1-068-R、2-BA1-069- F、2-BA1-069-R、2-BA1-070-F、2-BA1-070-R、2-BA1-071-F、2-BA1-071-R、2-BA1-072-F、2- BA1-072-R, 2-BA1-073-F, 2-BA1-073-R, 2-BA1-074-F and 2-BA1-074-R, its sequence is successively such as SEQ ID Shown in 201~SEQ ID 390.
In a preferred embodiment of the invention, the asymmetric linking probe group includes Ion-BC1-FF, Ion- BC1-FR、Ion-BC1-RF、Ion-BC1-RR、Ion-BC2-FF、Ion-BC2-FR、Ion-BC2-RF、Ion-BC2-RR、Ion- BC3-FF、Ion-BC3-FR、Ion-BC3-RF、Ion-BC3-RR、Ion-BC4-FF、Ion-BC4-FR、Ion-BC4-RF、Ion- BC4-RR、Ion-BC5-FF、Ion-BC5-FR、Ion-BC5-RF、Ion-BC5-RR、Ion-BC6-FF、Ion-BC6-FR、Ion- BC6-RF、Ion-BC6-RR、Ion-BC7-FF、Ion-BC7-FR、Ion-BC7-RF、Ion-BC7-RR、Ion-BC8-FF、Ion- BC8-FR、Ion-BC8-RF、Ion-BC8-RR、Ion-BC9-FF、Ion-BC9-FR、Ion-BC9-RF、Ion-BC9-RR、Ion- BC10-FF、Ion-BC10-FR、Ion-BC10-RF、Ion-BC10-RR、Ion-BC11-FF、Ion-BC11-FR、Ion-BC11- RF、Ion-BC11-RR、Ion-BC12-FF、Ion-BC12-FR、Ion-BC12-RF、Ion-BC12-RR、Ion-BC13-FF、 Ion-BC13-FR、Ion-BC13-RF、Ion-BC13-RR、Ion-BC14-FF、Ion-BC14-FR、Ion-BC14-RF、Ion- BC14-RR、Ion-BC15-FF、Ion-BC15-FR、Ion-BC15-RF、Ion-BC15-RR、Ion-BC16-FF、Ion-BC16- FR, Ion-BC16-RF and Ion-BC16-RR, its sequence successively as shown in SEQ 391~SEQ of ID ID 454, above-mentioned BC1~ 16 represent eight kinds of different sequence labels respectively.
In a preferred embodiment of the invention, the universal primer is C-primer, its sequence such as SEQ ID Shown in 455.
A kind of kit based on above-mentioned construction method, including：
One the oneth DNA is enriched with reaction component, including the first amplimer group；
One the 2nd DNA is enriched with reaction component, including the second amplimer group；
One RingCap-Taq enzymes, are made up of Taq enzyme, DNA ligase and the end modified enzymes of DNA；
The reaction component of one Barcode1~16, including asymmetric linking probe group and universal primer, specifically include and are connected in one 16 reacting holes risen, each reacting hole is respectively provided with the asymmetric linking probe and universal primer of corresponding sequence label；
One positive quality control product；
With a negative quality-control product.The beneficial effects of the invention are as follows：
1st, construction method of the invention carries out single tube for the full exon sequence of BRCA1 and BRCA2 genes, is rapidly completed text The structure in storehouse, whole library construction process only needs 2~3 hours, needs only to manufal operation time 15~30 minutes, with reference to height Flux microarray dataset can with it is highly effective solve at present in clinically tumor disease on the basis of a small amount of clinical sample Need to detect this difficult point to the full extron of gene, and it is with low cost.
2nd, the library sequence prepared by construction method of the invention by current high-flux sequence system identification and can be examined Survey, so as to realize that library construction carries out the application of the detection of nucleotide sequence, the detection of nucleic acids can be applied to current a variety of high fluxs Microarray dataset, genetic chip platform, hybridization check platform.
3rd, construction method of the invention is for the sample from formaldehyde fixation FFPE and the sample institute from peripheral blood The DNA of acquisition is still applicable, and the detection method based on it is still with the amplification same with fresh tissue sample DNA and detection energy Power.
Brief description of the drawings
Fig. 1 is that the nucleic acid library that embodiments of the invention are built carries out high-flux sequence total data figure.
Fig. 2 is the detection homogeneity result figure that embodiments of the invention detect BRCA1/2 gene mutations.
Fig. 3 is that embodiments of the invention detect one of detection mutation result of BRCA1/2 gene mutations.
Fig. 4 is the two of the detection mutation result that the embodiment of the present invention detects BRCA1/2 gene mutations.
Embodiment
Technical scheme is further detailed and described below by way of embodiment combination accompanying drawing.
HotStar-Taq enzymes, T4DNA ligases, the end modified enzymes of DNA, RingCap buffer in following embodiments, MgCl2The precious biotech firm of DaLian, China is purchased from dNTPs.
Embodiment 1
A kind of construction method in the tumour BRCA1/2 genetic mutations library detected for high-flux sequence, covers the mankind The whole exon region mutation of BRCA1/2 genes, the information such as table 1 below of specific exons mutation：
Table 1
Specifically include following steps：
A kind of construction method in the tumour BRCA1/2 genetic mutations library detected for high-flux sequence, covers the mankind The whole exon region mutation of BRCA1/2 genes, specifically includes following steps：
(1) the first basic amplimer group and the second basic amplimer group are designed for target gene BRCA1/2, this 5 ' ends of all forward primers and reverse primer of one basic amplimer group and the second basic amplimer group are equipped with extra 2~5 T, and 2~5 T close to 3 ' end first T there is PNA modifications, while the first basic amplimer group Tm values difference be no more than 1 DEG C, the Tm values of second basic amplimer group difference is no more than 1 DEG C；Specifically, described first expands Increasing primer sets includes BA2-001-F, BA2-001-R, BA1-001-F, BA1-001-R, BA2-002-F, BA2-002-R, BA1- 002-F、BA1-002-R、BA2-003-F、BA2-003-R、BA2-004-F、BA2-004-R、BA1-003-F、BA1-003-R、 BA1-004-F、BA1-004-R、BA1-005-F、BA1-005-R、BA1-006-F、BA1-006-R、BA1-007-F、BA1- 007-R、BA1-008-F、BA1-008-R、BA1-009-F、BA1-009-R、BA1-010-F、BA1-010-R、BA1-011-F、 BA1-011-R、BA1-012-F、BA1-012-R、BA2-005-F、BA2-005-R、BA2-006-F、BA2-006-R、BA2- 007-F、BA2-007-R、BA2-008-F、BA2-008-R、 BA2-009-F、BA2-009-R、BA1-013-F、BA1-013-R、 BA2-010-F、BA2-010-R、BA2-011-F、BA2-011-R、BA2-012-F、BA2-012-R、BA2-013-F、BA2- 013-R、BA2-014-F、BA2-014-R、BA2-015-F、BA2-015-R、BA2-016-F、BA2-016-R、BA2-017-F、 BA2-017-R、BA2-018-F、BA2-018-R、BA2-019-F、BA2-019-R、BA2-020-F、BA2-020-R、BA2- 021-F、BA2-021-R、BA2-022-F、BA2-022-R、BA2-023-F、BA2-023-R、BA2-024-F、BA2-024-R、 BA2-025-F、BA2-025-R、BA2-026-F、BA2-026-R、BA2-027-F、BA2-027-R、BA2-028-F、BA2- 028-R、BA2-029-F、BA2-029-R、BA2-030-F、BA2-030-R、BA2-031-F、BA2-031-R、BA2-032-F、 BA2-032-R、BA2-033-F、BA2-033-R、BA2-034-F、BA2-034-R、BA2-035-F、BA2-035-R、BA2- 036-F、BA2-036-R、BA2-037-F、BA2-037-R、BA2-038-F、BA2-038-R、BA2-039-F、BA2-039-R、 BA2-040-F、BA2-040-R、BA2-041-F、BA2-041-R、BA2-042-F、BA2-042-R、BA2-043-F、BA2- 043-R、BA2-044-F、BA2-044-R、BA2-045-F、BA2-045-R、BA2-046-F、BA2-046-R、BA2-047-F、 BA2-047-R、BA2-048-F、BA2-048-R、BA2-049-F、BA2-049-R、BA2-050-F、BA2-050-R、BA2- 051-F、BA2-051-R、BA2-052-F、BA2-052-R、BA2-053-F、BA2-053-R、BA2-054-F、BA2-054-R、 BA2-055-F、BA2-055-R、BA2-056-F、BA2-056-R、BA2-057-F、BA2-057-R、BA2-058-F、BA2- 058-R、BA2-059-F、BA2-059-R、BA1-014-F、BA1-014-R、BA1-015-F、BA1-015-R、BA1-016-F、 BA1-016-R、BA1-017-F、BA1-017-R、BA1-018-F、BA1-018-R、BA1-019-F、BA1-019-R、BA1- 020-F、BA1-020-R、BA1-021-F、BA1-021-R、BA1-022-F、BA1-022-R、BA1-023-F、BA1-023-R、 BA1-024-F、BA1-024-R、BA1-025-F、BA1-025-R、BA1-026-F、BA1-026-R、BA1-027-F、BA1- 027-R、BA1-028-F、BA1-028-R、BA1-029-F、BA1-029-R、BA1-030-F、BA1-030-R、BA1-031-F、 BA1-031-R、BA1-032-F、BA1-032-R、BA1-033-F、BA1-033-R、BA1-034-F、BA1-034-R、BA1- 035-F、BA1-035-R、BA1-036-F、BA1-036-R、BA2-060-F、BA2-060-R、BA2-061-F、BA2-061-R、 BA1-037-F, BA1-037-R, 2-BA2-062-F, 2-BA2-062-R, 2-BA1-038-F and 2-BA1-038-R, its sequence according to It is secondary as shown in SEQ 01~SEQ of ID ID 200；The second amplimer group include 2-BA2-063-F, 2-BA2-063-R, 2-BA2-064-F、2-BA2-064-R、2-BA2-065-F、2-BA2-065-R、2-BA2-066-F、2-BA2-066-R、2- BA2-067-F、2-BA2-067-R、2-BA2-068-F、2-BA2-068-R、2-BA2-069-F、2-BA2-069-R、2-BA2- 070-F、2-BA2-070-R、2-BA1-039-F、2-BA1-039-R、2-BA1-040-F、2-BA1-040-R、2-BA1-041- F、2-BA1-041-R、2-BA1-042-F、2-BA1-042-R、2-BA1-043-F、2-BA1-043-R、2-BA1-044-F、2- BA1-044-R、2-BA1-045-F、2-BA1-045-R、2-BA1-046-F、2-BA1-046-R、2-BA2-071-F、2-BA2- 071-R、2-BA2-072-F、2-BA2-072-R、2-BA2-073-F、 2-BA2-073-R、2-BA2-074-F、2-BA2-074- R、2-BA2-075-F、2-BA2-075-R、2-BA1-047-F、2-BA1-047-R、2-BA2-076-F、2-BA2-076-R、2- BA1-048-F、2-BA1-048-R、2-BA2-077-F、2-BA2-077-R、2-BA2-078-F、2-BA2-078-R、2-BA2- 079-F、2-BA2-079-R、2-BA2-080-F、2-BA2-080-R、2-BA2-081-F、2-BA2-081-R、2-BA2-082- F、2-BA2-082-R、2-BA2-083-F、2-BA2-083-R、2-BA2-084-F、2-BA2-084-R、2-BA2-085-F、2- BA2-085-R、2-BA2-086-F、2-BA2-086-R、2-BA2-087-F、2-BA2-087-R、2-BA2-088-F、2-BA2- 088-R、2-BA2-089-F、2-BA2-089-R、2-BA2-090-F、2-BA2-090-R、2-BA2-091-F、2-BA2-091- R、2-BA2-092-F、2-BA2-092-R、2-BA2-093-F、2-BA2-093-R、2-BA2-094-F、2-BA2-094-R、2- BA2-095-F、2-BA2-095-R、2-BA2-096-F、2-BA2-096-R、2-BA2-097-F、2-BA2-097-R、2-BA2- 098-F、2-BA2-098-R、2-BA2-099-F、2-BA2-099-R、2-BA2-100-F、2-BA2-100-R、2-BA2-101- F、2-BA2-101-R、2-BA2-102-F、2-BA2-102-R、2-BA2-103-F、2-BA2-103-R、2-BA2-104-F、2- BA2-104-R、2-BA2-105-F、2-BA2-105-R、2-BA2-106-F、2-BA2-106-R、2-BA2-107-F、2-BA2- 107-R、2-BA2-108-F、2-BA2-108-R、2-BA2-109-F、2-BA2-109-R、2-BA2-110-F、2-BA2-110- R、2-BA2-111-F、2-BA2-111-R、2-BA2-112-F、2-BA2-112-R、2-BA2-113-F、2-BA2-113-R、2- BA2-114-F、2-BA2-114-R、2-BA2-115-F、2-BA2-115-R、2-BA2-116-F、2-BA2-116-R、2-BA2- 117-F、2-BA2-117-R、2-BA2-118-F、2-BA2-118-R、2-BA2-119-F、2-BA2-119-R、2-BA2-120- F、2-BA2-120-R、2-BA2-121-F、2-BA2-121-R、2-BA1-049-F、2-BA1-049-R、2-BA1-050-F、2- BA1-050-R、2-BA1-051-F、2-BA1-051-R、2-BA1-052-F、2-BA1-052-R、2-BA1-053-F、2-BA1- 053-R、2-BA1-054-F、2-BA1-054-R、2-BA1-055-F、2-BA1-055-R、2-BA1-056-F、2-BA1-056- R、2-BA1-057-F、2-BA1-057-R、2-BA1-058-F、2-BA1-058-R、2-BA1-059-F、2-BA1-059-R、2- BA1-060-F、2-BA1-060-R、2-BA1-061-F、2-BA1-061-R、2-BA1-062-F、2-BA1-062-R、2-BA1- 063-F、2-BA1-063-R、2-BA1-064-F、2-BA1-064-R、2-BA1-065-F、2-BA1-065-R、2-BA1-066- F、2-BA1-066-R、2-BA1-067-F、2-BA1-067-R、2-BA1-068-F、2-BA1-068-R、2-BA1-069-F、2- BA1-069-R、2-BA1-070-F、2-BA1-070-R、2-BA1-071-F、2-BA1-071-R、2-BA1-072-F、2-BA1- 072-R, 2-BA1-073-F, 2-BA1-073-R, 2-BA1-074-F and 2-BA1-074-R, its sequence is successively such as SEQ ID 201 Shown in~SEQ ID 390；
(2) whether one asymmetric linking probe group of design and a universal primer for not forming complementation human genome or not, asymmetric Linking probe group contains multipair different asymmetric linking probe, and the asymmetric linking probe of each pair includes that itself ring-type can be formed Positive a probe and a reverse probe, the positive probe and reverse probe from 3 ' ends to 5 ' ends include successively one can with not The complementary series of 5 ' some continuous base pairings in end of symmetrical linking probe, one with the amplification of the universal primer complementary pairing Sequence and a cohesive end recognition sequence corresponding with above-mentioned 2~5 T-phase, while each positive probe and reverse probe are respectively provided with Corresponding sequence label, the positive probe of the above-mentioned asymmetric linking probe of each pair and the Tm values difference of reverse probe are no more than 1 DEG C； Specifically, the asymmetric linking probe group include Ion-BC1-FF, Ion-BC1-FR, Ion-BC1-RF, Ion-BC1-RR, Ion-BC2-FF、Ion-BC2-FR、Ion-BC2-RF、Ion-BC2-RR、Ion-BC3-FF、Ion-BC3-FR、Ion-BC3-RF、 Ion-BC3-RR、Ion-BC4-FF、Ion-BC4-FR、Ion-BC4-RF、Ion-BC4-RR、Ion-BC5-FF、Ion-BC5-FR、 Ion-BC5-RF、Ion-BC5-RR、Ion-BC6-FF、Ion-BC6-FR、Ion-BC6-RF、Ion-BC6-RR、Ion-BC7-FF、 Ion-BC7-FR、Ion-BC7-RF、Ion-BC7-RR、Ion-BC8-FF、Ion-BC8-FR、Ion-BC8-RF、Ion-BC8-RR、 Ion-BC9-FF、Ion-BC9-FR、Ion-BC9-RF、Ion-BC9-RR、Ion-BC10-FF、Ion-BC10-FR、Ion-BC10- RF、Ion-BC10-RR、Ion-BC11-FF、Ion-BC11-FR、Ion-BC11-RF、Ion-BC11-RR、Ion-BC12-FF、 Ion-BC12-FR、Ion-BC12-RF、Ion-BC12-RR、Ion-BC13-FF、Ion-BC13-FR、Ion-BC13-RF、Ion- BC13-RR、Ion-BC14-FF、Ion-BC14-FR、Ion-BC14-RF、Ion-BC14-RR、Ion-BC15-FF、Ion-BC15- FR, Ion-BC15-RF, Ion-BC15-RR, Ion-BC16-FF, Ion-BC16-FR, Ion-BC16-RF and Ion-BC16-RR, Its sequence is successively as shown in SEQ 391~SEQ of ID ID 454, and above-mentioned BC1~16 represent 16 kinds of different label sequences respectively Row；The universal primer is C-primer, and its sequence is as shown in SEQ ID 455；
(3) template, above-mentioned first basic amplimer group are placed in into the first PCR containing RingCap-Taq enzymes to react Expanded in system, the first amplified production is obtained after purification；Template, above-mentioned second basic amplimer group are placed in one and contained Expanded in 2nd PCR reaction systems of RingCap-Taq enzymes, the second amplimer is obtained after purification；
(4) by the first amplified production, the second amplified production, asymmetric linking probe group, universal primer and above-mentioned RingCap-Taq enzymes are mixed into performing PCR, and the tumour BRCA1/2 genetic mutations text for high-flux sequence is obtained after purification Storehouse；
Above-mentioned RingCap-Taq enzymes are made up of Taq enzyme, DNA ligase and the end modified enzymes of DNA；
(5) machine testing on library is carried out by Ion torrent PGM high-flux sequences instrument, obtains target sequence information, Data message is carried out by VC softwares and compares analysis, sample mutation status are obtained.
Above-mentioned template from the sample scope of application include the fresh pathological tissue of surgery excision, formaldehyde fixes paraffin bag Bury pathological anatomy, paraffin section, whole blood, blood plasma, serum, the sample such as pleural effusion.
Fresh pathological tissue, takes mung bean size, and about 1g weights extract base using Qiagen company organizations DNA extraction kit Because of a group DNA, specific steps press kit operating instruction.
Wax stone sample is cut into 5~8 μm of sections, takes 5, or 5~8 μm of sections being made take 5, de- by dimethylbenzene After wax, genomic DNA is extracted using Qiagen companies FFPE DNA extraction kit, specific steps are said by kit operation It is bright.
Whole blood, blood plasma, serum and pleural effusion sample, base is extracted using Qiagen company organizations DNA extraction kit Because of a group DNA, specific steps press kit operating instruction.The μ l of whole blood 200 are extracted every time, and blood plasma, serum and pleural effusion are many In 800 μ l.Carried DNA is dissolved in Tris-HCl (10mmol/L, pH 8.0), and quality is extracted through UV spectrophotometer measuring, and Concentration is determined, adjust DNA concentration with Tris-HCl solution (10mmol/L, pH 8.0) is used as PCR to 100ng/ μ l or 2ng/ μ l The template of amplification.
Kit based on above-mentioned construction method includes：
One the oneth DNA is enriched with reaction component, including the first amplimer group, and the first amplimer group includes BRCA- Primer-Mix1 to BRCA-primer-Mix10, the first DNA per person-portion is enriched with the BRCA-primer-Mix1 of reaction component Formula to BRCA-primer-Mix10 is as shown in the table：
BRCA-primer-Mix1 preparation formula tables：
Sequence number Title Concentration (μM) Volume (μ L)
1 BA2-001-F 50 0.06
2 BA2-001-R 50 0.07
3 BA1-001-F 50 0.07
4 BA1-001-R 50 0.08
5 BA2-002-F 50 0.08
6 BA2-002-R 50 0.08
7 BA1-002-F 50 0.08
8 BA1-002-R 50 0.08
9 BA2-003-F 50 0.08
10 BA2-003-R 50 0.08
11 BA2-004-F 50 0.08
12 BA2-004-R 50 0.08
13 BA1-003-F 50 0.07
14 BA1-003-R 50 0.08
15 BA1-004-F 50 0.08
16 BA1-004-R 50 0.08
17 BA1-005-F 50 0.08
18 BA1-005-R 50 0.08
19 BA1-006-F 50 0.08
20 BA1-006-R 50 0.08
It is total 1.49
BRCA-primer-Mix2 preparation formula tables：
BRCA-primer-Mix3 preparation formula tables：
Sequence number Title Concentration (μM) Volume (μ L)
1 BA2-009-F 50 0.08
2 BA2-009-R 50 0.06
3 BA1-013-F 50 0.09
4 BA1-013-R 50 0.06
5 BA2-010-F 50 0.06
6 BA2-010-R 50 0.05
7 BA2-011-F 50 0.06
8 BA2-011-R 50 0.08
9 BA2-012-F 50 0.08
10 BA2-012-R 50 0.07
11 BA2-013-F 50 0.07
12 BA2-013-R 50 0.05
13 BA2-014-F 50 0.06
14 BA2-014-R 50 0.06
15 BA2-015-F 50 0.09
16 BA2-015-R 50 0.08
17 BA2-016-F 50 0.08
18 BA2-016-R 50 0.08
19 BA2-017-F 50 0.06
20 BA2-017-R 50 0.08
It is total 1.40
BRCA-primer-Mix4 preparation formula tables：
Sequence number Title Concentration (μM) Volume (μ L)
1 BA2-018-F 50 0.07
2 BA2-018-R 50 0.06
3 BA2-019-F 50 0.08
4 BA2-019-R 50 0.06
5 BA2-020-F 50 0.05
6 BA2-020-R 50 0.05
7 BA2-021-F 50 0.05
8 BA2-021-R 50 0.08
9 BA2-022-F 50 0.08
10 BA2-022-R 50 0.07
11 BA2-023-F 50 0.07
12 BA2-023-R 50 0.06
13 BA2-024-F 50 0.06
14 BA2-024-R 50 0.06
15 BA2-025-F 50 0.09
16 BA2-025-R 50 0.07
17 BA2-026-F 50 0.09
18 BA2-026-R 50 0.08
19 BA2-027-F 50 0.06
20 BA2-027-R 50 0.08
It is total 1.37
BRCA-primer-Mix5 preparation formula tables：
BRCA-primer-Mix6 preparation formula tables：
Sequence number Title Concentration (μM) Volume (μ L)
1 BA2-038-F 50 0.05
2 BA2-038-R 50 0.09
3 BA2-039-F 50 0.08
4 BA2-039-R 50 0.06
5 BA2-040-F 50 0.07
6 BA2-040-R 50 0.05
7 BA2-041-F 50 0.09
8 BA2-041-R 50 0.08
9 BA2-042-F 50 0.06
10 BA2-042-R 50 0.07
11 BA2-043-F 50 0.06
12 BA2-043-R 50 0.06
13 BA2-044-F 50 0.06
14 BA2-044-R 50 0.06
15 BA2-045-F 50 0.09
16 BA2-045-R 50 0.08
17 BA2-046-F 50 0.07
18 BA2-046-R 50 0.07
19 BA2-047-F 50 0.08
20 BA2-047-R 50 0.06
It is total 1.39
BRCA-primer-Mix7 preparation formula tables：
BRCA-primer-Mix8 preparation formula tables：
Sequence number Title Concentration (μM) Volume (μ L)
1 BA2-058-F 50 0.06
2 BA2-058-R 50 0.08
3 BA2-059-F 50 0.09
4 BA2-059-R 50 0.06
5 BA1-014-F 50 0.07
6 BA1-014-R 50 0.08
7 BA1-015-F 50 0.07
8 BA1-015-R 50 0.08
9 BA1-016-F 50 0.07
10 BA1-016-R 50 0.08
11 BA1-017-F 50 0.09
12 BA1-017-R 50 0.06
13 BA1-018-F 50 0.09
14 BA1-018-R 50 0.08
15 BA1-019-F 50 0.09
16 BA1-019-R 50 0.08
17 BA1-020-F 50 0.07
18 BA1-020-R 50 0.07
19 BA1-021-F 50 0.07
20 BA1-021-R 50 0.07
It is total 1.51
BRCA-primer-Mix9 preparation formula tables：
BRCA-primer-Mix10 preparation formula tables：
Sequence number Title Concentration (μM) Volume (μ L)
1 BA1-032-F 50 0.09
2 BA1-032-R 50 0.08
3 BA1-033-F 50 0.08
4 BA1-033-R 50 0.08
5 BA1-034-F 50 0.08
6 BA1-034-R 50 0.07
7 BA1-035-F 50 0.06
8 BA1-035-R 50 0.07
9 BA1-036-F 50 0.07
10 BA1-036-R 50 0.08
11 BA2-060-F 50 0.08
12 BA2-060-R 50 0.06
13 BA2-061-F 50 0.08
14 BA2-061-R 50 0.08
15 BA1-037-F 50 0.09
16 BA1-037-R 50 0.06
17 2-BA2-062-F 50 0.08
18 2-BA2-062-R 50 0.06
19 2-BA1-038-F 50 0.06
20 2-BA1-038-R 50 0.06
It is total 1.47
One the 2nd DNA is enriched with reaction component, including the second amplimer group, and the second amplimer group includes BRCA- Primer-Mix11 to BRCA-primer-Mix20, the 2nd DNA per person-portion is enriched with the BRCA-primer- of reaction component Mix11 to BRCA-primer-Mix20 formula is as shown in the table：
BRCA-primer-Mix11 preparation formula tables
BRCA-primer-Mix12 preparation formula tables
Sequence number Title Concentration (μM) Volume (μ L)
1 2-BA1-041-F 50 0.08
2 2-BA1-041-R 50 0.08
3 2-BA1-042-F 50 0.08
4 2-BA1-042-R 50 0.08
5 2-BA1-043-F 50 0.08
6 2-BA1-043-R 50 0.07
7 2-BA1-044-F 50 0.06
8 2-BA1-044-R 50 0.07
9 2-BA1-045-F 50 0.07
10 2-BA1-045-R 50 0.08
11 2-BA1-046-F 50 0.06
12 2-BA1-046-R 50 0.06
13 2-BA2-071-F 50 0.08
14 2-BA2-071-R 50 0.08
15 2-BA2-072-F 50 0.09
16 2-BA2-072-R 50 0.06
17 2-BA2-073-F 50 0.08
18 2-BA2-073-R 50 0.06
19 2-BA2-074-F 50 0.06
20 2-BA2-074-R 50 0.07
It is total 1.45
BRCA-primer-Mix13 preparation formula tables
BRCA-primer-Mix14 preparation formula tables
Sequence number Title Concentration (μM) Volume (μ L)
1 2-BA2-083-F 50 0.08
2 2-BA2-083-R 50 0.08
3 2-BA2-084-F 50 0.09
4 2-BA2-084-R 50 0.09
5 2-BA2-085-F 50 0.09
6 2-BA2-085-R 50 0.07
7 2-BA2-086-F 50 0.09
8 2-BA2-086-R 50 0.07
9 2-BA2-087-F 50 0.07
10 2-BA2-087-R 50 0.06
11 2-BA2-088-F 50 0.06
12 2-BA2-088-R 50 0.06
13 2-BA2-089-F 50 0.08
14 2-BA2-089-R 50 0.08
15 2-BA2-090-F 50 0.08
16 2-BA2-090-R 50 0.06
17 2-BA2-091-F 50 0.08
18 2-BA2-091-R 50 0.06
19 2-BA2-092-F 50 0.06
20 2-BA2-092-R 50 0.07
It is total 1.48
BRCA-primer-Mix15 preparation formula tables
Sequence number Title Concentration (μM) Volume (μ L)
1 2-BA2-093-F 50 0.08
2 2-BA2-093-R 50 0.08
3 2-BA2-094-F 50 0.09
4 2-BA2-094-R 50 0.09
5 2-BA2-095-F 50 0.09
6 2-BA2-095-R 50 0.07
7 2-BA2-096-F 50 0.09
8 2-BA2-096-R 50 0.07
9 2-BA2-097-F 50 0.07
10 2-BA2-097-R 50 0.06
11 2-BA2-098-F 50 0.06
12 2-BA2-098-R 50 0.06
13 2-BA2-099-F 50 0.08
14 2-BA2-099-R 50 0.08
15 2-BA2-100-F 50 0.08
16 2-BA2-100-R 50 0.06
17 2-BA2-101-F 50 0.08
18 2-BA2-101-R 50 0.06
19 2-BA2-102-F 50 0.06
20 2-BA2-102-R 50 0.07
It is total 1.48
BRCA-primer-Mix16 preparation formula tables
BRCA-primer-Mix17 preparation formula tables
Sequence number Title Concentration (μM) Volume (μ L)
1 2-BA2-113-F 50 0.09
2 2-BA2-113-R 50 0.08
3 2-BA2-114-F 50 0.06
4 2-BA2-114-R 50 0.07
5 2-BA2-115-F 50 0.08
6 2-BA2-115-R 50 0.08
7 2-BA2-116-F 50 0.09
8 2-BA2-116-R 50 0.06
9 2-BA2-117-F 50 0.08
10 2-BA2-117-R 50 0.06
11 2-BA2-118-F 50 0.06
12 2-BA2-118-R 50 0.07
13 2-BA2-119-F 50 0.07
14 2-BA2-119-R 50 0.07
15 2-BA2-120-F 50 0.07
16 2-BA2-120-R 50 0.07
17 2-BA2-121-F 50 0.07
18 2-BA2-121-R 50 0.07
19 2-BA1-049-F 50 0.06
20 2-BA1-049-R 50 0.08
It is total 1.44
BRCA-primer-Mix18 preparation formula tables
BRCA-primer-Mix19 preparation formula tables
Sequence number Title Concentration (μM) Volume (μ L)
1 2-BA1-060-F 50 0.09
2 2-BA1-060-R 50 0.08
3 2-BA1-061-F 50 0.06
4 2-BA1-061-R 50 0.08
5 2-BA1-062-F 50 0.08
6 2-BA1-062-R 50 0.08
7 2-BA1-063-F 50 0.09
8 2-BA1-063-R 50 0.06
9 2-BA1-064-F 50 0.08
10 2-BA1-064-R 50 0.07
11 2-BA1-065-F 50 0.07
12 2-BA1-065-R 50 0.07
13 2-BA1-066-F 50 0.08
14 2-BA1-066-R 50 0.06
15 2-BA1-067-F 50 0.08
16 2-BA1-067-R 50 0.07
17 2-BA1-068-F 50 0.08
18 2-BA1-068-R 50 0.08
19 2-BA1-069-F 50 0.06
20 2-BA1-069-R 50 0.08
It is total 1.50
BRCA-primer-Mix20 preparation formula tables
Sequence number Title Concentration (μM) Volume (μ L)
1 2-BA1-070-F 50 0.09
2 2-BA1-070-R 50 0.08
3 2-BA1-071-F 50 0.06
4 2-BA1-071-R 50 0.08
5 2-BA1-072-F 50 0.08
6 2-BA1-072-R 50 0.08
7 2-BA1-073-F 50 0.09
8 2-BA1-073-R 50 0.06
9 2-BA1-074-F 50 0.08
10 2-BA1-074-R 50 0.07
11 TE - 0.73
It is total 1.50
One RingCap-Taq enzymes, are made up of, ratio 0.8~1.2 Taq enzyme, DNA ligase and the end modified enzymes of DNA: 0.8~1.2:0.8~1.2, preferred proportion 1:1:1；
The reaction component of one Barcode1~16, including asymmetric linking probe group and universal primer, specifically include and are connected in one 16 reacting holes risen, each reacting hole is respectively provided with the asymmetric linking probe and universal primer of corresponding sequence label, The formula of the reaction component of Barcode1~16 per person-portion is as shown in the table：
One negative quality-control product, seedless sour water；
With a positive quality control product (Positive mutants sequent synthesis plasmid), concentration is 2ng/ μ L.
Mentioned reagent box is tested with foregoing construction method, with BRCA1 and BRCA2 genes NM_000059: exon3:c.71_96del:Detection tumour BRCA1 and the BRCA2 gene that the present invention is analyzed exemplified by p.L24fs mutation are entirely outer aobvious The method of son mutation.Experiment 3 plants of cell line and mutant plasmid sequence, (band is c.71_96del by respectively BRCA-M1:p.L24fs Mutation), H460 (BRCA1/2 genes wild type), 293T (BRCA1/2 genes wild type), (BRCA1/2 genes are wild by SW480 Type)；The whole blood sample of 100 parts of breast cancer household heredity factors, 20 parts of clinical breast cancer samples (including flesh tissue, paraffin section, Pleural effusion, whole blood)：
The template amount (testing sample, positive quality control product and negative quality-control product) of the first and second PCR reaction systems is 5 μ L, RingCap-Taq enzyme are (specifically by HotStar-Taq enzymes, T4DNA ligases and the end modified enzymes of DNA with 1:1:1 ratio Composition, material concentration：5U/ μ L/ are every kind of) addition be 0.25 μ L, remaining component is as shown in the table：
First PCR reaction systems
2nd PCR reaction systems
PCR amplification programs set such as following table：
The purifying of the first and second amplified productions obtained by above-mentioned first and second PCR reaction systems is specific as follows：
Take out Agencourt AMPure XP reagents and be placed in room temperature, while magnetic bead is broken up, while preparing fresh 70% Ethanol (the μ L nuclease-free waters of 230 μ L absolute ethyl alcohols+100), it is necessary to be Fresh.
First round purification step
(1) Agencourt of 12.5 μ L (0.5x sample volumes) is separately added into each μ L products of example reaction pipe 25 AMPure XP reagents, draw piping and druming 5 times up and down, mix DNA is resuspended completely；
(2) it is incubated at room temperature 5 minutes；
(3) it is placed on above magnetic frame, is incubated 5 minutes, until solution clarification；
(4) carefully Aspirate supernatant is placed in new centrifuge tube, should not disturb magnetic bead；Note：Contain amplification in supernatant Product should not be abandoned.
Second wheel purification step
(1) the Agencourt AMPure of 30 μ L (1.2x sample volumes) are added into the μ L of supernatant 25 of above-mentioned absorption XP reagents, draw piping and druming 5 times up and down, mix DNA is resuspended completely；
(2) it is incubated at room temperature 5 minutes；
(3) it is placed on above magnetic frame, is incubated 3 minutes, until solution clarification, carefully siphons away and discard supernatant, no Disturb magnetic bead；Note：It should not be abandoned containing amplification library on magnetic bead.
(4) 70% ethanol of 150 μ l Fresh is added, did not had Magnetic bead sample, centrifuge tube both forward and reverse directions movement 5 It is secondary, then it is incubated 2 minutes on magnetic frame, removes supernatant；
(5) repeat the above steps 4, second of washing of progress；
(6) ensure that ethanol drop is all siphoned away from hole, plate be positioned on magnetic frame, air at room temperature is dried 5 minutes, It is careful not to over-drying.
(7) sample cell is taken away from magnetic frame, 25 μ L TE (PH8.0) buffer solutions is added in every hole and fully infiltrate magnetic Pearl.Fully vibration is mixed, and liquid is collected into ttom of pipe by quick centrifugation.(it can also select to be drawn on the liquid of more than half with rifle It is lower to blow and beat at least 5 times to mix)；Note：Supernatant, which contains amplification library, to be abandoned.
Sample cell is placed on magnetic frame 2 minutes.Contain amplified production in supernatant.Take out 20 μ L of supernatant.
The first amplified production and the second amplified production obtained by above-mentioned purifying prepare the tumour for high-flux sequence BRCA1/2 genetic mutations library PCR reaction template amount for 5 μ L, RingCap-Taq enzymes (specifically by HotStar-Taq enzymes, T4DNA ligases and the end modified enzyme compositions of DNA, material concentration：5U/ μ L/ are every kind of) addition be 0.25 μ L, remaining component It is as shown in the table：
Sequence number Material Material concentration Consumption (μ L)
1 RingCap buffer 10× 2.5
2 MgCl2 25mM 4
3 dNTPs 10mM 2
4 The reaction component of Barcode1~16 50μM 2
5 H2O Purified water 9.5
Cumulative volume 20
PCR amplification programs set such as following table：
Purified, be made for high-flux sequence by the first amplified production of purifying and the second amplified production method respectively Tumour BRCA1/2 genetic mutations library.
The detection in the above-mentioned tumour BRCA1/2 genetic mutations library for high-flux sequence is specific as follows：
As shown in figure 1, the library sequence prepared by the construction method of the present invention can be by current high-flux sequence system identification And detected, so as to realize that library construction carries out the application of the detection of nucleotide sequence, the detection of nucleic acids can be applied to many at present Plant high-flux sequence platform, genetic chip platform, hybridization check platform.And as shown in Figures 2 to 4, construction method of the invention The DNA obtained for fixing the sample of FFPE and the sample from peripheral blood from formaldehyde is still applicable, based on it Detection method still has the amplification same with fresh tissue sample DNA and detectability.
(BRCA1/2 genes are wild by foregoing 100 parts of whole blood samples and cell line H460 (BRCA1/2 genes wild type), 293T Raw type), SW480 (BRCA1/2 genes wild type), (band is c.71_96del for mutant plasmid:P.L24fs is mutated) body through the present invention System's detection, only mutant plasmid DNA detects mutant nucleotide sequence, and other samples further demonstrate this method without mutant nucleotide sequence Specificity.
Sensitivity analysis：By mutant plasmids DNA from the continuous 10 times of gradient dilutions of 100ng/ μ L, respectively 100ng/ μ L, 10ng/ μ L, 1ng/ μ L, 100pg/ μ L, 10pg/ μ L, 1pg/ μ L.5 μ L DNA are added per secondary response.As a result the text of the present invention is shown The sensitivity of base construction method is high, and 50 copy DNA genomes can be detected.
Selective capability analysis：Fixed each PCR reactions STb gene consumption, 100ng/ reactions and 10ng/ reactions.First will be prominent The DNA and wild-type cell system DNA concentration for becoming plasmid are adjusted to 20ng/ μ L and 2ng/ μ L.So each reaction plus 5 μ L templates As 100ng/ reacts and 10ng/ reactions.DNA profiling is simulated in configuration as follows.
A:50% is 100ng/ μ L mutant cells DNA.
B:30% takes 40 μ L 100ng/ μ L wild-type cell DNA is mixed into after the μ L of A liquid 60, and concussion is mixed.
C:20% takes 60 μ L 100ng/ μ L wild-type cell DNA is mixed into after the μ L of A liquid 40, and concussion is mixed.
D:15% takes 50 μ L 100ng/ μ L wild-type cell DNA is mixed into after the μ L of B liquid 50, and concussion is mixed.
E:10% takes 50 μ L 100ng/ μ L wild-type cell DNA is mixed into after the μ L of C liquid 50, and concussion is mixed.
F:5% takes 50 μ L 100ng/ μ L wild-type cell DNA is mixed into after the μ L of E liquid 50, and concussion is mixed.
G:1% takes 80 μ L 100ng/ μ L wild-type cell DNA is mixed into after the μ L of F liquid 20, and concussion is mixed.
H:0.5% takes 50 μ L 100ng/ μ L wild-type cell DNA is mixed into after the μ L of G liquid 50, and concussion is mixed.
I:0.1% takes 80 μ L 100ng/ μ L wild-type cell DNA is mixed into after the μ L of H liquid 20, and concussion is mixed.
As a result the selective enumeration method ability of the full exons mutation detection method of tumour BRCA1/2 genes for showing the present invention is The mutant DNA of 50 copies can be detected in 10ng STb genes, detectability is 1%.
Replica test：Each reaction is separately added into mutant plasmid DNA10ng, 1ng and 100pg, is repeated 10 times progress high Flux sequencing detection, 10 times result is consistent, coincidence rate 100%.
Collect the breast cancer sample of surgery excision, whole blood sample sample totally 50, wherein 20 tissue samples, 30 whole bloods. Wherein male 28, women 22.Age is 36-71 Sui, and average age is 55 years old.
For tumour BRCA1/2 genes, testing result of the present invention and DNA sequencing result are completely the same：Have 4 in 50 samples BRCA1 gene mutations occur for example, and 2 occur BRCA1 gene mutations, and 44 normal lung tissues' controls are wild type.
From the foregoing, it will be observed that tumour BRCA1 of the present invention and the reaction of BRCA2 construction of gene library can just detect tumour simultaneously The full exons mutation site of BRCA1 and BRCA2 genes, the library construction time only needs 3 hours, therefore the present invention is time saving and energy saving, accurate True property is high, can meet the quick diagnosis of mutation.Moreover, the coincidence rate of high-flux sequence method and traditional sequencing methods result is 100%, the sensitivity of high-flux sequence method and selective enumeration method ability are higher than in traditional sequencing methods, 10ng sample DNAs containing 1% Mutant DNA can be detected.
The foregoing is only a preferred embodiment of the present invention, therefore can not limit the scope that the present invention is implemented according to this, i.e., The equivalent changes and modifications made according to the scope of the claims of the present invention and description, all should still belong in the range of the present invention covers.

Claims (6)

1. a kind of construction method in the tumour BRCA1/2 genetic mutations library detected for high-flux sequence, it is characterised in that：Cover The whole exon region mutation of mankind BRCA1/2 genes are covered, following steps are specifically included：
(1) the first basic amplimer group and the second basic amplimer group, first base are designed for target gene BRCA1/2 5 ' ends of all forward primers and reverse primer of this amplimer group and the second basic amplimer group are equipped with extra 2~ 5 T, and first T close to 3 ' ends of 2~5 T has PNA modifications, while the Tm values of the first basic amplimer group Difference is no more than 1 DEG C, and the Tm values difference of the second basic amplimer group is no more than 1 DEG C；
(2) one asymmetric linking probe group of design and a universal primer for not forming complementation human genome or not, asymmetric connection Probe groups contain multipair different asymmetric linking probe, and the asymmetric linking probe of each pair includes that itself the one of ring-type can be formed Positive probe and a reverse probe, the positive probe and reverse probe from 3 ' end to 5 ' end successively include one can with it is asymmetric The complementary series of 5 ' some continuous base pairings in end of linking probe, one with the extension increasing sequence of the universal primer complementary pairing and One cohesive end recognition sequence corresponding with above-mentioned 2~5 T-phase, while each positive probe and reverse probe are respectively provided with accordingly Sequence label, the positive probe of the above-mentioned asymmetric linking probe of each pair and the Tm values difference of reverse probe are no more than 1 DEG C；
(3) template, above-mentioned first basic amplimer group are placed in a first PCR reaction systems containing RingCap-Taq enzymes It is middle to be expanded, the first amplified production is obtained after purification；Template, above-mentioned second basic amplimer group are placed in one and contained Expanded in 2nd PCR reaction systems of RingCap-Taq enzymes, the second amplimer is obtained after purification；
(4) by the first amplified production, the second amplified production, asymmetric linking probe group, universal primer and above-mentioned RingCap-Taq Enzyme is mixed into performing PCR, and the tumour BRCA1/2 genetic mutations library for high-flux sequence is obtained after purification；
Above-mentioned RingCap-Taq enzymes are made up of Taq enzyme, DNA ligase and the end modified enzymes of DNA.
2. a kind of structure in tumour BRCA1/2 genetic mutations library detected for high-flux sequence as claimed in claim 1 Method, it is characterised in that：The first amplimer group includes BA2-001-F, BA2-001-R, BA1-001-F, BA1-001- R、BA2-002-F、BA2-002-R、BA1-002-F、BA1-002-R、BA2-003-F、BA2-003-R、BA2-004-F、BA2- 004-R、BA1-003-F、BA1-003-R、BA1-004-F、BA1-004-R、BA1-005-F、BA1-005-R、BA1-006-F、 BA1-006-R、BA1-007-F、BA1-007-R、BA1-008-F、BA1-008-R、BA1-009-F、BA1-009-R、BA1- 010-F、BA1-010-R、BA1-011-F、BA1-011-R、BA1-012-F、BA1-012-R、BA2-005-F、BA2-005-R、 BA2-006-F、BA2-006-R、BA2-007-F、BA2-007-R、BA2-008-F、BA2-008-R、BA2-009-F、BA2- 009-R、BA1-013-F、BA1-013-R、BA2-010-F、BA2-010-R、BA2-011-F、BA2-011-R、BA2-012-F、 BA2-012-R、BA2-013-F、BA2-013-R、BA2-014-F、BA2-014-R、BA2-015-F、BA2-015-R、BA2- 016-F、BA2-016-R、BA2-017-F、BA2-017-R、BA2-018-F、BA2-018-R、BA2-019-F、BA2-019-R、 BA2-020-F、BA2-020-R、BA2-021-F、BA2-021-R、BA2-022-F、BA2-022-R、BA2-023-F、BA2- 023-R、BA2-024-F、BA2-024-R、BA2-025-F、BA2-025-R、BA2-026-F、BA2-026-R、BA2-027-F、 BA2-027-R、BA2-028-F、BA2-028-R、BA2-029-F、BA2-029-R、BA2-030-F、BA2-030-R、BA2- 031-F、BA2-031-R、BA2-032-F、BA2-032-R、BA2-033-F、BA2-033-R、BA2-034-F、BA2-034-R、 BA2-035-F、BA2-035-R、BA2-036-F、BA2-036-R、BA2-037-F、BA2-037-R、BA2-038-F、BA2- 038-R、BA2-039-F、BA2-039-R、BA2-040-F、BA2-040-R、BA2-041-F、BA2-041-R、BA2-042-F、 BA2-042-R、BA2-043-F、BA2-043-R、BA2-044-F、BA2-044-R、BA2-045-F、BA2-045-R、BA2- 046-F、BA2-046-R、BA2-047-F、BA2-047-R、BA2-048-F、BA2-048-R、BA2-049-F、BA2-049-R、 BA2-050-F、BA2-050-R、BA2-051-F、BA2-051-R、BA2-052-F、BA2-052-R、BA2-053-F、BA2- 053-R、BA2-054-F、BA2-054-R、BA2-055-F、BA2-055-R、BA2-056-F、BA2-056-R、BA2-057-F、 BA2-057-R、BA2-058-F、BA2-058-R、BA2-059-F、BA2-059-R、BA1-014-F、BA1-014-R、BA1- 015-F、BA1-015-R、BA1-016-F、BA1-016-R、BA1-017-F、BA1-017-R、BA1-018-F、BA1-018-R、 BA1-019-F、BA1-019-R、BA1-020-F、BA1-020-R、BA1-021-F、BA1-021-R、BA1-022-F、BA1- 022-R、BA1-023-F、BA1-023-R、BA1-024-F、BA1-024-R、BA1-025-F、BA1-025-R、BA1-026-F、 BA1-026-R、BA1-027-F、BA1-027-R、BA1-028-F、BA1-028-R、BA1-029-F、BA1-029-R、BA1- 030-F、BA1-030-R、BA1-031-F、BA1-031-R、BA1-032-F、BA1-032-R、BA1-033-F、BA1-033-R、 BA1-034-F、BA1-034-R、BA1-035-F、BA1-035-R、BA1-036-F、BA1-036-R、BA2-060-F、BA2- 060-R、BA2-061-F、BA2-061-R、BA1-037-F、BA1-037-R、2-BA2-062-F、2-BA2-062-R、2-BA1- 038-F and 2-BA1-038-R, its sequence is successively as shown in SEQ 01~SEQ of ID ID 200.
3. a kind of structure in tumour BRCA1/2 genetic mutations library detected for high-flux sequence as claimed in claim 1 Method, it is characterised in that：The second amplimer group includes 2-BA2-063-F, 2-BA2-063-R, 2-BA2-064-F, 2- BA2-064-R、2-BA2-065-F、2-BA2-065-R、2-BA2-066-F、2-BA2-066-R、2-BA2-067-F、2-BA2- 067-R、2-BA2-068-F、2-BA2-068-R、2-BA2-069-F、2-BA2-069-R、2-BA2-070-F、2-BA2-070- R、2-BA1-039-F、2-BA1-039-R、2-BA1-040-F、2-BA1-040-R、2-BA1-041-F、2-BA1-041-R、2- BA1-042-F、2-BA1-042-R、2-BA1-043-F、2-BA1-043-R、2-BA1-044-F、2-BA1-044-R、2-BA1- 045-F、2-BA1-045-R、2-BA1-046-F、2-BA1-046-R、2-BA2-071-F、2-BA2-071-R、2-BA2-072- F、2-BA2-072-R、2-BA2-073-F、2-BA2-073-R、2-BA2-074-F、2-BA2-074-R、2-BA2-075-F、2- BA2-075-R、2-BA1-047-F、2-BA1-047-R、2-BA2-076-F、2-BA2-076-R、2-BA1-048-F、2-BA1- 048-R、2-BA2-077-F、2-BA2-077-R、2-BA2-078-F、2-BA2-078-R、2-BA2-079-F、2-BA2-079- R、2-BA2-080-F、2-BA2-080-R、2-BA2-081-F、2-BA2-081-R、2-BA2-082-F、2-BA2-082-R、2- BA2-083-F、2-BA2-083-R、2-BA2-084-F、2-BA2-084-R、2-BA2-085-F、2-BA2-085-R、2-BA2- 086-F、2-BA2-086-R、2-BA2-087-F、2-BA2-087-R、2-BA2-088-F、2-BA2-088-R、2-BA2-089- F、2-BA2-089-R、2-BA2-090-F、2-BA2-090-R、2-BA2-091-F、2-BA2-091-R、2-BA2-092-F、2- BA2-092-R、2-BA2-093-F、2-BA2-093-R、2-BA2-094-F、2-BA2-094-R、2-BA2-095-F、2-BA2- 095-R、2-BA2-096-F、2-BA2-096-R、2-BA2-097-F、2-BA2-097-R、2-BA2-098-F、2-BA2-098- R、2-BA2-099-F、2-BA2-099-R、2-BA2-100-F、2-BA2-100-R、2-BA2-101-F、2-BA2-101-R、2- BA2-102-F、2-BA2-102-R、2-BA2-103-F、2-BA2-103-R、2-BA2-104-F、2-BA2-104-R、2-BA2- 105-F、2-BA2-105-R、2-BA2-106-F、2-BA2-106-R、2-BA2-107-F、2-BA2-107-R、2-BA2-108- F、2-BA2-108-R、2-BA2-109-F、2-BA2-109-R、2-BA2-110-F、2-BA2-110-R、2-BA2-111-F、2- BA2-111-R、2-BA2-112-F、2-BA2-112-R、2-BA2-113-F、2-BA2-113-R、2-BA2-114-F、2-BA2- 114-R、2-BA2-115-F、2-BA2-115-R、2-BA2-116-F、2-BA2-116-R、2-BA2-117-F、2-BA2-117- R、2-BA2-118-F、2-BA2-118-R、2-BA2-119-F、2-BA2-119-R、2-BA2-120-F、2-BA2-120-R、2- BA2-121-F、2-BA2-121-R、2-BA1-049-F、2-BA1-049-R、2-BA1-050-F、2-BA1-050-R、2-BA1- 051-F、2-BA1-051-R、2-BA1-052-F、2-BA1-052-R、2-BA1-053-F、2-BA1-053-R、2-BA1-054- F、2-BA1-054-R、2-BA1-055-F、2-BA1-055-R、2-BA1-056-F、2-BA1-056-R、2-BA1-057-F、2- BA1-057-R、2-BA1-058-F、2-BA1-058-R、2-BA1-059-F、2-BA1-059-R、2-BA1-060-F、2-BA1- 060-R、2-BA1-061-F、2-BA1-061-R、2-BA1-062-F、2-BA1-062-R、2-BA1-063-F、2-BA1-063- R、2-BA1-064-F、2-BA1-064-R、2-BA1-065-F、2-BA1-065-R、2-BA1-066-F、2-BA1-066-R、2- BA1-067-F、2-BA1-067-R、2-BA1-068-F、2-BA1-068-R、2-BA1-069-F、2-BA1-069-R、2-BA1- 070-F、2-BA1-070-R、2-BA1-071-F、2-BA1-071-R、2-BA1-072-F、2-BA1-072-R、2-BA1-073- F, 2-BA1-073-R, 2-BA1-074-F and 2-BA1-074-R, its sequence is successively such as the institutes of SEQ 201~SEQ of ID ID 390 Show.
4. a kind of structure in tumour BRCA1/2 genetic mutations library detected for high-flux sequence as claimed in claim 1 Method, it is characterised in that：The asymmetric linking probe group includes Ion-BC1-FF, Ion-BC1-FR, Ion-BC1-RF, Ion- BC1-RR、Ion-BC2-FF、Ion-BC2-FR、Ion-BC2-RF、Ion-BC2-RR、Ion-BC3-FF、Ion-BC3-FR、Ion- BC3-RF、Ion-BC3-RR、Ion-BC4-FF、Ion-BC4-FR、Ion-BC4-RF、Ion-BC4-RR、Ion-BC5-FF、Ion- BC5-FR、Ion-BC5-RF、Ion-BC5-RR、Ion-BC6-FF、Ion-BC6-FR、Ion-BC6-RF、Ion-BC6-RR、Ion- BC7-FF、Ion-BC7-FR、Ion-BC7-RF、Ion-BC7-RR、Ion-BC8-FF、Ion-BC8-FR、Ion-BC8-RF、Ion- BC8-RR、Ion-BC9-FF、Ion-BC9-FR、Ion-BC9-RF、Ion-BC9-RR、Ion-BC10-FF、Ion-BC10-FR、 Ion-BC10-RF、Ion-BC10-RR、Ion-BC11-FF、Ion-BC11-FR、Ion-BC11-RF、Ion-BC11-RR、Ion- BC12-FF、Ion-BC12-FR、Ion-BC12-RF、Ion-BC12-RR、Ion-BC13-FF、Ion-BC13-FR、Ion-BC13- RF、Ion-BC13-RR、Ion-BC14-FF、Ion-BC14-FR、Ion-BC14-RF、Ion-BC14-RR、Ion-BC15-FF、 Ion-BC15-FR, Ion-BC15-RF, Ion-BC15-RR, Ion-BC16-FF, Ion-BC16-FR, Ion-BC16-RF and Ion- BC16-RR, its sequence successively as shown in SEQ 391~SEQ of ID ID 454, above-mentioned BC1~16 represent respectively eight kinds it is different Sequence label.
5. a kind of structure in tumour BRCA1/2 genetic mutations library detected for high-flux sequence as claimed in claim 1 Method, it is characterised in that：The universal primer is C-primer, and its sequence is as shown in SEQ ID 455.
6. a kind of kit of the construction method based on described in any claim in claim 1 to 5, it is characterised in that：Bag Include：
One the oneth DNA is enriched with reaction component, including the first amplimer group；
One the 2nd DNA is enriched with reaction component, including the second amplimer group；
One RingCap-Taq enzymes, are made up of Taq enzyme, DNA ligase and the end modified enzymes of DNA；
The reaction component of one Barcode1~16, including asymmetric linking probe group and universal primer, specifically include what is connected together 16 reacting holes, each reacting hole is respectively provided with the asymmetric linking probe and universal primer of corresponding sequence label；
One positive quality control product；
With a negative quality-control product.
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CN108179177A (en) * 2017-12-29 2018-06-19 博迪泰（厦门）生物科技有限公司 A kind of kit and detection method of quick detection nucleic acid mutation
CN108624686A (en) * 2018-03-30 2018-10-09 南京世和基因生物技术有限公司 A kind of probe library, detection method and the kit of detection BRCA1/2 mutation
CN109295230A (en) * 2018-10-24 2019-02-01 福建翊善生物科技有限公司 A method of the polygene combined abrupt climatic change based on ctDNA assesses tumour dynamic change
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CN108624686B (en) * 2018-03-30 2019-06-04 南京世和基因生物技术有限公司 A kind of probe library, detection method and the kit of detection BRCA1/2 mutation
CN110714071A (en) * 2018-07-12 2020-01-21 深圳华大智造科技有限公司 High-throughput detection kit for human BRCA1/2 gene mutation
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
CN109295230A (en) * 2018-10-24 2019-02-01 福建翊善生物科技有限公司 A method of the polygene combined abrupt climatic change based on ctDNA assesses tumour dynamic change
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