CN105779636A - PCR primer used for amplifying human breast cancer susceptibility gene BRCA1 and BRCA2 coding sequence and application thereof - Google Patents
PCR primer used for amplifying human breast cancer susceptibility gene BRCA1 and BRCA2 coding sequence and application thereof Download PDFInfo
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Abstract
The invention relates to a PCR primer for a human breast cancer susceptibility gene BRCA1 and BRCA2 coding sequence based on a NGS technology and application thereof. The PCR primer for the human breast cancer susceptibility gene BRCA1 and BRCA2 coding sequence comprises at least one capturing primer pair, and sequences of a forward primer and a reverse primer of each capturing primer pair respectively comprise a specific sequence and a linker sequence connected with a terminal 5' of the specific sequence. The PCR primer used for amplifying the human breast cancer susceptibility gene BRCA1 and BRCA2 coding sequence based on the NGS technology and application thereof have the advantages that experiment operations related in the whole method are simple, only a PCR reagent and a primer combination are related, cost is low, and a ditag sequencing linker sequence is introduced for distinguishing different samples, so that high throughput sample sequencing detection can be realized, and gene detection support can be effectively provided for risk assessment on human breast cancer and other BRCA susceptible gene-related hereditary tumours or targeted medication specific to BRCA mutation.
Description
Technical field
The present invention relates to field of medical molecular biology, especially relate to a kind of PCR primer for expanding human breast carcinoma tumor susceptibility gene BRCA1 and BRCA2 coded sequence and application.
Background technology
Breast carcinoma is the modal cancer of women.Show that the annual breast carcinoma new cases about 220,000 of China account for the 15% of whole world breast carcinoma new cases (1,500,000) according to " 2012 China's tumor registration annual report " data.In all malignant tumor of women, breast cancer incidence, up to 16.8%, has 43 people's morbidities in about every 100,000 people, and age of onset to present rejuvenation, sickness rate in rising trend.Sickness rate rose rapidly when 25 years old, peaked when 50 years old.Therefore, the high incidence of breast carcinoma serious threat China women's health.
Familial Occurrence breast carcinoma accounts for the 10%～30% of all pathogenesis of breast carcinomas.In the breast cancer susceptibility gene having been found that up to now, BRCA1 and BRCA2 is the most direct tumor susceptibility gene, and the breast carcinoma that the two gene mutation causes accounts for the 5%～10% of familial breast cancer.BRCA1 gene mapping, in 17q21, comprises 24 exons, encodes 1863 aminoacid；BRCA2 gene mapping, in 13q12-13, comprises 27 exons, and encoding proteins is containing 3418 aminoacid.The multiple important cells functions such as BRCA1 and BRCA2 cell growth inhibiting, participation cell cycle regulating, DNA damage reparation and apoptosis, play a very important role in maintaining Genome stability.If undergoing mutation of BRCA1/2 gene, then the tumorigenic function of suppression that it has will be impacted.BRCA1/2 gene or ovarian cancer susceptible gene.The BRCA1 mutation carriers risk suffered from breast cancer with ovarian cancer throughout one's life is 50%～85% and 15%～45% respectively according to statistics；BRCA2 mutation carriers suffers from breast cancer and the risk of ovarian cancer is 50%～85% and 10%～20% respectively.BRCA1/2 sudden change is also relevant and also relevant with prostate cancer risk with cancer of pancreas, malignant melanoma.Therefore, " BRCA1andBRCA2:CancerRiskandGeneticTesting " guide that National Cancer Institute is issued is pointed out: first the patient suffering from breast carcinoma or ovarian cancer in family is carried out gene test, if it find that this patient is with harmful BRCA1 or BRCA2 gene mutation, so other member in family can carry out gene test again, seeing whether also to have there is detrimental mutation, this kind of way has significantly high clinical value.
BRCA1 coding region has had been found that hundreds of kind is suddenlyd change, and the sudden change of BRCA2 is up to thousands of kinds.For BRCA1, BRCA2 technique of gene detection, traditional Sanger order-checking is main detection means.From detection sensitivity, Sanger order-checking generally can only detect the variation of more than 20% mutation rate, and for the whole coded sequence detection of BRCA1, BRCA2, flux ratio is relatively low, the cycle is longer in detection, and single pattern detection expense is also higher.Along with new-generation sequencing (NGS) technology maturation and order-checking cost lasting reduction, NGS replace Sanger sequencing technologies carry out the trend that breast carcinoma BRCA gene test is future market.
Summary of the invention
Based on this, it is necessary to provide a kind of PCR primer and application for expanding human breast carcinoma tumor susceptibility gene BRCA1 and BRCA2 coded sequence based on NGS technology.
A kind of PCR primer for expanding human breast carcinoma tumor susceptibility gene BRCA1 and BRCA2 coded sequence, primer pair is caught including at least one pair of, the forward primer catching primer pair described in every pair all includes specific sequence and holds, with the 5 ' of described specific sequence, the joint sequence being connected with the sequence of reverse primer, catching forward primer and the described specific sequence respectively SEQIDNO.m and SEQIDNO. (m+112) in reverse primer of primer pair described in every pair, wherein m is 1,2,3 ... or 112.
A kind of PCR primer for expanding human breast carcinoma tumor susceptibility gene BRCA1 and BRCA2 coded sequence, including sequencing primer pair, the forward primer of described sequencing primer pair includes at least one in the sequence such as primer shown in SEQIDNO.227～SEQIDNO.234, and the reverse primer of described sequencing primer pair includes at least one in the sequence such as primer shown in SEQIDNO.235～SEQIDNO.246.
A kind of detection kit, including the PCR primer for expanding human breast carcinoma tumor susceptibility gene BRCA1 and BRCA2 coded sequence described in any of the above-described item.
A kind of method catching human breast carcinoma tumor susceptibility gene BRCA1 and BRCA2 coded sequence, comprises the steps:
With the human gene group DNA of extraction for template, catch primer pair with at least one in above-mentioned PCR primer and carry out substance pcr amplification for primer, or it is combined as primer with at least one multi-primers in above-mentioned PCR primer and carries out multiplexed PCR amplification, obtain the PCR primer library containing human breast carcinoma tumor susceptibility gene BRCA1 and BRCA2 complete encoding sequence.
Adopt the PCR primer library that the above-mentioned method catching human breast carcinoma tumor susceptibility gene BRCA1 and BRCA2 coded sequence obtains.
A kind of method of sequencing library building human breast carcinoma tumor susceptibility gene BRCA1 and BRCA2 coded sequence, comprises the steps:
Using the above-mentioned method catching human breast carcinoma tumor susceptibility gene BRCA1 and BRCA2 coded sequence, amplification obtains the PCR primer library of human breast carcinoma tumor susceptibility gene BRCA1 and BRCA2 coded sequence；
With described PCR primer library for template, at least one sequencing primer corresponding in such as above-mentioned PCR primer carries out secondary PCR amplification to for primer, it is thus achieved that the sequencing library of human breast carcinoma tumor susceptibility gene BRCA1 and BRCA2 coded sequence.
The method adopting the sequencing library of above-mentioned structure human breast carcinoma tumor susceptibility gene BRCA1 and BRCA2 coded sequence builds the sequencing library obtained.
Above-mentioned PCR primer and application for expanding human breast carcinoma tumor susceptibility gene BRCA1 and BRCA2 coded sequence based on NGS technology, experimental implementation involved by whole method is simple, pertain only to PCR reagent and combination of primers, cost are low, it is simultaneously introduced double; two label sequencing joint sequence for distinguishing different samples, the order-checking detection of high flux sample can be realized, effectively human breast carcinoma and other BRCA tumor susceptibility gene correlated inheritance tumor risk can be assessed or provide gene test support for the BRCA targeting medication suddenlyd change.
Accompanying drawing explanation
Fig. 1 is model schematic diagram on multiple sample multiplex PCR；
Fig. 2 is the schematic diagram designing all standing PCR primer for BRCA1 and BRCA2 gene code exon sequence；
Fig. 3 is 7 groups of multiplexed PCR amplification product electrophoretograms, and G1-G7 is product, and N is negative control, and product concentrates between 200-300bp；
Fig. 4 is after BRCA1 and the BRCA2 gene code exon sequence to a sample carries out multiple PCR products order-checking, the homogeneity result schematic diagram of 112 obtained amplicons；
Fig. 5 second takes turns PCR primer electrophoretogram, and sequence measuring joints on the band of purpose region, product size is between 300-425bp；
Fig. 6 is mutation analysis schematic flow sheet, including converting thereof into Fastq form and remove joint, low quality sequence and primer obtaining raw sequencing data；Treated sequence passes through comparison instrument bwa comparison on mankind's reference gene group；Remove repetitive sequence；Using the variation detection instrument searching variant sites that three accuracys rate are higher, each instrument uses algorithms of different；Normal SNP site in comparison thousand human genome filtering based on database result；Residual variation site is annotated and is analyzed judge its whether harmful variation；Interpretive analysis result also assesses the steps such as subject onset risk rate.
Detailed description of the invention
For the ease of understanding the present invention, below with reference to relevant drawings, the present invention is described more fully.Accompanying drawing gives presently preferred embodiments of the present invention.But, the present invention can realize in many different forms, however it is not limited to embodiment described herein.On the contrary, the purpose providing these embodiments is to make the understanding to the disclosure more thorough comprehensively.
Present embodiments provide a kind of PCR primer for expanding human breast carcinoma tumor susceptibility gene BRCA1 and BRCA2 coded sequence.This PCR primer include at least one pair of for people BRCA1 and BRCA2 coding region design all standing amplicon specific sequence catch primer pair.nullAs shown in Figure 2，The sequence of every pair of forward primer and reverse primer catching primer pair all includes this specific sequence and holds, with the 5 ' of this specific sequence, the joint sequence being connected，Wherein，Every pair of forward primer catching primer pair and described specific sequence respectively SEQIDNO.1 and the SEQIDNO.113 in reverse primer、SEQIDNO.2 and SEQIDNO.114、SEQIDNO.3 and SEQIDNO.115 ... or SEQIDNO.112 and SEQIDNO.224 (corresponding relation namely catching the forward primer of primer pair and the described specific sequence in reverse primer described in every pair is SEQIDNO.m and SEQIDNO. (m+112)，Wherein m is 1、2、3 ... or 112)，Shown in table 1 specific as follows:
Table 1
nullPreferably，In the present embodiment，This catches the joint sequence of forward primer in primer pair is GACGCTCTTCCGATCTCTG (as shown in SEQIDNO.225)，This catches the joint sequence of reverse primer in primer pair is TGTGCTCTTCCGATCTGAC (as shown in SEQIDNO.226)，Specific sequence as shown in SEQIDNO.1 and SEQIDNO.113，Sequence respectively GACGCTCTTCCGATCTCTGCTAATGTGTTAAAGTTCATTGGAACA and TGTGCTCTTCCGATCTGACAGTTCTTCAGTTAAGAAAATCAGCA of the forward primer catching primer pair of its correspondence and reverse primer，Other are in like manner.
It is further preferred that in the present embodiment, this PCR primer includes multipair catching primer pair.This is multipair catches combination between primer pair and constitutes multi-primers combination, and often the combination of group multi-primers includes multipair catching primer pair.The multi-primers combination of the present embodiment includes at least one in the combination of as shown in table 2 below 7 group:
Table 2
Further, in the present embodiment, this PCR primer also includes the sequencing primer pair with characteristic sequence.The forward primer of sequencing primer pair includes at least one in the sequence such as primer shown in SEQIDNO.227～SEQIDNO.234.The reverse primer of sequencing primer pair includes at least one in the sequence such as primer shown in SEQIDNO.235～SEQIDNO.246, shown in table 3 specific as follows.
Table 3
Each PCR primer described in the present embodiment can individually, in pairs or constitute PCR primer reagent according to multi-primers combination and be applied in detection kit, as the present embodiment additionally provides a kind of detection kit, it contains the above-mentioned PCR primer for expanding human breast carcinoma tumor susceptibility gene BRCA1 and BRCA2 coded sequence.
In the present embodiment, this detection kit also can contain pcr amplification related reagent further, such as the pcr amplification related reagent etc. containing PCR buffer, dNTP solution, DMSO and archaeal dna polymerase.Wherein, PCR buffer is 10 × buffer, its pH value is 7.5～8.0, solvent is water, solute includes Tris-HCl (pH8.0,25 DEG C) that concentration is 500～1000mM, concentration is the NaCl of 500～1000mM, concentration is the dithiothreitol, DTT of 10～50mM, 1～10mg/mLBSA, 150～200mMMgCl2And the ATP of 80～120mM；DNTP solution is concentration is the dNTPs mixed liquor of 10mM；DMSO is mass percentage concentration is the dimethyl sulfoxide of 100%；Archaeal dna polymerase is thermal starting TagDNA polymerase, archaeal dna polymerase is saved in preservation solution, the pH value preserving solution is 8.0～8.5.0, and NonidetP40 that component includes Tris-HCl (pH8.0) that concentration is 10～30mM, concentration is the KCl of 0.05M～0.20M, concentration is the EDTA of 0.05mM～0.20mM, concentration expressed in percentage by volume is 0.1%～0.5% Tween20, concentration expressed in percentage by volume are 0.1%～0.5%, concentration are the dithiothreitol, DTT of 0.05mM～0.15mM and volumetric concentration is the glycerol of 40%～60%.
The present embodiment additionally provides a kind of method catching human breast carcinoma tumor susceptibility gene BRCA1 and BRCA2 coded sequence and the PCR primer library using the method to obtain.The method comprises the steps:
With the human gene group DNA of extraction for template, catch primer pair with at least one in above-mentioned PCR primer and carry out substance pcr amplification for primer, or combine with at least one multi-primers in above-mentioned PCR primer and carry out multiplexed PCR amplification, obtain the PCR primer library containing human breast carcinoma tumor susceptibility gene BRCA1 and BRCA2 complete encoding sequence.
Wherein, human gene group DNA respectively organizes from human peripheral, human body and/or uses mouth desquamated cells the method that purification column extracts and/or magnetic bead extracts to extract human gene group DNA.The related reagent used in pcr amplification process is the pcr amplification related reagent in above-mentioned detection kit.
nullPreferably，The method catching human breast carcinoma tumor susceptibility gene BRCA1 and BRCA2 coded sequence is to combine with at least one multi-primers in above-mentioned PCR primer to carry out multiplexed PCR amplification，The annealing temperature of its pcr amplification is 56～60 DEG C，Condition is first 90～100 DEG C and processes 1.5～2.5min，Process 15～30 seconds at 90～100 DEG C again、Process at processing 85～95 seconds and 68～75 DEG C at 56～60 DEG C and within 55～65 seconds, carry out 30～40 circulations，Then preserve after processing 3～5 minutes at 68～75 DEG C，Preferred PCR condition is 95 DEG C and processes 2min，Process 20 seconds at 95 DEG C again、Process at processing 90 seconds and 72 DEG C under 56～60 DEG C (such as 58 DEG C) and within 60 seconds, carry out 35 circulations，Then 16 DEG C of preservations after processing 4 minutes at 72 DEG C.The step in method separating-purifying PCR primer library by purification column purification and/or magnetic beads for purifying is also included after obtaining PCR primer library.
Additionally, the present embodiment additionally provides a kind of method of sequencing library building human breast carcinoma tumor susceptibility gene BRCA1 and BRCA2 coded sequence and uses the method to build the sequencing library obtained.The method of this structure sequencing library comprises the steps:
Using the above-mentioned method catching human breast carcinoma tumor susceptibility gene BRCA1 and BRCA2 coded sequence, amplification obtains the PCR primer library of human breast carcinoma tumor susceptibility gene BRCA1 and BRCA2 coded sequence；
With PCR primer library for template, carry out secondary PCR amplification with at least one sequencing primer in above-mentioned PCR primer to for primer, it is thus achieved that the sequencing library of human breast carcinoma tumor susceptibility gene BRCA1 and BRCA2 coded sequence.
In the present embodiment, the condition of this secondary PCR amplification is first 90～100 DEG C and processes 1.5～2.5min, 12～18 circulations within 25～35 seconds, are carried out again to process at processing 25～35 seconds and 68～75 DEG C at processing 8～12 seconds, 60～70 DEG C at 90～100 DEG C, then preserve after processing 3～5 minutes at 68～75 DEG C, preferably first 95 DEG C process 2min, 15 circulations within 30 seconds, are carried out again, then 16 DEG C of preservations after 72 DEG C of process 4 minutes to process at processing 30 seconds and 72 DEG C at processing 10 seconds, 65 DEG C at 95 DEG C.
In the present embodiment, after obtaining sequencing library, also include the step using the method separation purification sequencing library of magnetic beads for purifying and/or purification column purification.
Above-mentioned PCR primer and application for expanding human breast carcinoma tumor susceptibility gene BRCA1 and BRCA2 coded sequence based on NGS technology, experimental implementation involved by whole method is simple, pertain only to PCR reagent and combination of primers, cost are low, it is simultaneously introduced double; two label sequencing joint sequence for distinguishing different samples, the order-checking detection of high flux sample can be realized, effectively human breast carcinoma and other BRCA tumor susceptibility gene correlated inheritance tumor risk can be assessed or provide gene test support for the BRCA targeting medication suddenlyd change.
Below in conjunction with specific embodiment, the human breast carcinoma tumor susceptibility gene BRCA1 of the present invention and PCR primer and the application of BRCA2 coded sequence are explained.
45 parts of buccal saliva specimens of the following example human peripheral sample to 45 parts of unknown BRCA1 and BRCA2 abrupt climatic change result and correspondence, have carried out PCR and have caught and build storehouse.It addition, catch build storehouse to carrying out identical PCR through 2 strain breast cancer cell lines of Sanger order-checking and 4 parts of BRCA carriers of mutation, specifically include following steps:
1, sample extraction
The tissue blood cell DNA using sky root respectively extracts test kit and new hundred base saliva of buccal cavity magnetic beads extract test kit, extracts DNA from 45 parts of buccal saliva specimens (5ml) of 2 strain breast cancer cells, 45 parts of human peripheral samples (200 μ l) and correspondence.Obtain the eluted product (DNA of extraction) of 50 μ about l, quality through Nanodrop Detection and Extraction DNA, result OD260/OD230=1.8～2.5, OD260/OD280=1.8～2.5, illustrate that DNA extraction is qualified, can be used for the template in next step pcr amplification.
2, first round multiplex PCR catches the coded sequence of human breast carcinoma tumor susceptibility gene BRCA1 and BRCA2
Using 96 orifice plates as shown in Figure 1 that sample is carried out PCR reaction, wherein each sample carries out 7 hole multi-PRC reactions, and the primer used by 7 holes is the combination of corresponding 7 shown in table 2 group multi-primers respectively.Separately arranging a negative control without template, the primer that the primer used by negative control uses with sample the first hole multi-PRC reaction is the same.Each sample uses 1 row's reacting hole on 96 orifice plates, and labelling also records each 96 orifice plates and hole site corresponding to sample, as shown in Figure 1.
PCR reaction system is 20 μ l, and used PCR reaction reagent is the above-mentioned pcr amplification related reagent distributed rationally.This PCR reaction system concrete is as shown in table 4 below:
Table 4
| Component | Volume 8--> |
| 10 × buffer | 2μl |
| DNTPs solution | 0.4μl |
| DMSO | 1μl |
| Template DNA (5～20ng) | 1μl |
| Thermal starting Taq archaeal dna polymerase | 0.2μl |
| Multi-primers combination (10 μMs) | 2μl |
| Water (HPLC rank) | 13.4 |
| Altogether | 20μl |
PCR parameter as shown in table 5 below is used to carry out pcr amplification:
Table 5
Pcr amplification runs on the ABI-2720PCR instrument of ABI company.After PCR completes, taking 5 μ lPCR products and carry out electrophoresis detection on the agarose gel of 2%, result is shown in Fig. 3.
3, multiplex PCR catches mixing and the purification of product
PCR primer obtained in the previous step is blended in the EP pipe of a 1.5ml, and every Guan Jun takes 10 μ l, respectively marker samples information, and shakes mixing.The Ampure magnetic bead using 1:1 volume ratio is purified, and obtains the DNA of 50 μ l.
4, secondary PCR amplification builds sequencing library
After PCR primer dilute with water obtained in the previous step 100 times, for the second template taking turns PCR reaction, second takes turns PCR uses the above-mentioned sequencing primer with label joint to expanding, and gained PCR primer is sequencing library.Used PCR reaction reagent is the above-mentioned pcr amplification related reagent distributed rationally.
PCR reaction system is 20 μ l, as shown in table 6 below:
Table 6
The PCR parameter shown in table 7 below is used to carry out pcr amplification:
Table 7
PCR reaction runs on the ABI-2720PCR instrument of ABI company.After PCR completes, taking 5 μ lPCR products and carry out electrophoresis detection on the agarose gel of 2%, result is referring to Fig. 5.
5, sequencing library purification, quantitatively and mixing
The 20 μ l sequencing libraries from different samples, use the Ampure magnetic bead of 1:1 volume ratio to be purified, obtain the sequencing library after the purification of 20 μ l.Taking each sequencing library 1 μ l after purification, use Qubit to carry out quantitatively, and record the concentration of each sequencing library, result is in Table 7.According to the concentration surveyed, from each sequencing library, take the DNA of 100ng, be blended in the PE pipe of a 1.5ml, and shake mixing.
Table 7: sequencing library concentration
Sample 91-92 is cell line sample, and sample 93-96 is positive sample.
6, machine order-checking on mixing sequencing library
Using illumina sequenator (NextSeq500) and sequencing reagent (NextSeq500MidOutputKit, 300cycles), with PE150, double; two index programs check order.
7, interpretation of result
The information of the sequence label characteristic sequence according to the sequencing primer centering in sequencing result, by sequencing data and each sample one_to_one corresponding.Then, use the known conventional alignment programs in order-checking field, such as BLAST and SOAP, the sequencing sequence of each sample and human genome are compared, and target area (i.e. the whole coded sequence of BRCA1 and BRCA2) coverage rate and the assessment of multiple PCR primer homogeneity is carried out according to sequencing sequence genomic in comparison, result is as shown in Figure 4.96 sample mean order-checking degree of depth are between 2000X 3000X, average criterion areal coverage is 99.5% (99.0%-99.9%), it is 98.5% (97.2%-99.9%) that the order-checking degree of depth is not less than the coverage rate of 50X (reaching good mutation analysis data statistics requirement), it is 95% (93.1%-97.0%) that the order-checking degree of depth is not less than on average the check order coverage rate of the degree of depth of 0.1x, referring to table 8.The multi-primers combination that these target area coverage data statistics show designed by this example and optimize is very effective.It addition, carry out mutation analysis for the sequencing data of BRCA1 and BRCA2 coded sequence in comparison to human genome, used mutation analysis software is samtools, GATK, varscans, referring to Fig. 6.The testing result obtained for 2 strain cell lines is completely the same with known result, other 30 parts of whole blood samples and corresponding buccal saliva specimens analyze obtained sudden change result, also it is all correct through Sanger order-checking, the sudden change result obtained with buccal saliva specimens further from the peripheral blood whole blood of same person is consistent, referring to the mutation rate that the NGS of table 9 mutation rate checked order and Sanger check order, it was shown that the inventive method and test kit may be used for human breast carcinoma tumor susceptibility gene BRCA1 and BRCA2 whole coding sequence abrupt climatic change.
Table 8: sample order-checking deep statistical
Table 9: positive sample Sanger testing result and NGS testing result
Sample 91-92 is cell line sample, and sample 93-96 is known positive sample, and sample 41-42 is saliva and the blood sample of same person respectively.
Found that: the result that the method for the employing present invention is obtained is consistent with the sudden change result of 2 strain cell lines of known BRCA1 and BRCA2 abrupt information and 4 parts of BRCA carriers of mutation；45 parts of buccal saliva specimens for 45 parts of human peripheral samples of unknown mutation information and correspondence, through BRCA2 sudden change (the same sample that illumina order-checking finds, peripheral blood and buccal saliva specimens detect simultaneously), sudden change result is also 100% accurately through Sanger checking.
Each technical characteristic of embodiment described above can combine arbitrarily, for making description succinct, the all possible combination of each technical characteristic in above-described embodiment is not all described, but, as long as the combination of these technical characteristics is absent from contradiction, all it is considered to be the scope that this specification is recorded.
Embodiment described above only have expressed the several embodiments of the present invention, and it describes comparatively concrete and detailed, but can not therefore be construed as limiting the scope of the patent.It should be pointed out that, for the person of ordinary skill of the art, without departing from the inventive concept of the premise, it is also possible to making some deformation and improvement, these broadly fall into protection scope of the present invention.Therefore, the protection domain of patent of the present invention should be as the criterion with claims.
Claims (13)
1. the PCR primer being used for expanding human breast carcinoma tumor susceptibility gene BRCA1 and BRCA2 coded sequence, it is characterized in that, primer pair is caught including at least one pair of, the forward primer catching primer pair described in every pair all includes specific sequence and holds, with the 5 ' of described specific sequence, the joint sequence being connected with the sequence of reverse primer, catching forward primer and the described specific sequence respectively SEQIDNO.m and SEQIDNO. (m+112) in reverse primer of primer pair described in every pair, wherein m is 1,2,3 ... or 112.
2. the PCR primer for expanding human breast carcinoma tumor susceptibility gene BRCA1 and BRCA2 coded sequence as claimed in claim 1, it is characterized in that, joint sequence in the described forward primer catching primer pair is such as shown in SEQIDNO.225, described in catch the joint sequence in the reverse primer of primer pair such as shown in SEQIDNO.226.
3. the PCR primer for expanding human breast carcinoma tumor susceptibility gene BRCA1 and BRCA2 coded sequence as claimed in claim 1 or 2, it is characterized in that, primer pair is caught described in multipair, catch combination between primer pair described in multipair and constitute multi-primers combination, described PCR primer includes at least one in the combination of following 7 groups of multi-primerses: first group includes sequence and be: SEQIDNO.13 and 125, 25 and 137, 77 and 189, 18 and 130, 9 and 121, 91 and 203, 97 and 209, 3 and 115, 108 and 220, 64 and 176, 103 and 215, 93 and 205, 68 and 180, 98 and 210, 59 and 171 and 104 and 216 catch primer pair；
Second group includes sequence and is: SEQIDNO.37 and 149,111 and 223,10 and 122,26 and 138,20 and 132,86 and 198,7 and 119,61 and 173,74 and 186,24 and 136,39 and 151,42 and 154,44 and 156,109 and 221,105 and 217 and 2 and 114 catch primer pair；
3rd group includes sequence and is: SEQIDNO.95 and 207,112 and 224,83 and 195,19 and 131,28 and 140,41 and 153,53 and 165,17 and 129,30 and 142,71 and 183,22 and 134,6 and 118,11 and 123,106 and 218,60 and 172 and 94 and 206 catch primer pair；
4th group includes sequence and is: SEQIDNO.36 and 148,14 and 126,57 and 169,5 and 117,21 and 133,16 and 128,87 and 199,27 and 139,23 and 135,45 and 157,40 and 152,92 and 204,110 and 222,89 and 201,65 and 177 and 34 and 146 catch primer pair；
5th group includes sequence and is: SEQIDNO.50 and 162,69 and 181,29 and 141,51 and 163,32 and 144,63 and 175,75 and 187,81 and 193,66 and 178,55 and 167,79 and 191,84 and 196,72 and 184,1 and 113,43 and 155 and 47 and 159 catch primer pair；
6th group includes sequence and is: SEQIDNO.49 and 161,12 and 124,76 and 188,38 and 150,56 and 168,52 and 164,62 and 174,102 and 214,90 and 202,31 and 143,15 and 127,107 and 219,8 and 120,35 and 147,99 and 211 and 46 and 158 catch primer pair；
7th group includes sequence and is: SEQIDNO.82 and 194,70 and 182,88 and 200,85 and 197,78 and 190,4 and 116,58 and 170,80 and 192,101 and 213,48 and 160,54 and 166,96 and 208,100 and 212,33 and 145,73 and 185 and 67 and 179 catch primer pair.
4. the PCR primer for expanding human breast carcinoma tumor susceptibility gene BRCA1 and BRCA2 coded sequence as claimed in claim 1 or 2, it is characterized in that, also include sequencing primer pair, the forward primer of described sequencing primer pair includes at least one in the sequence such as primer shown in SEQIDNO.227～SEQIDNO.234, and the reverse primer of described sequencing primer pair includes at least one in the sequence such as primer shown in SEQIDNO.235～SEQIDNO.246.
5. the PCR primer being used for expanding human breast carcinoma tumor susceptibility gene BRCA1 and BRCA2 coded sequence, it is characterized in that, including sequencing primer pair, the forward primer of described sequencing primer pair includes at least one in the sequence such as primer shown in SEQIDNO.227～SEQIDNO.234, and the reverse primer of described sequencing primer pair includes at least one in the sequence such as primer shown in SEQIDNO.235～SEQIDNO.246.
6. a detection kit, it is characterised in that include the PCR primer for expanding human breast carcinoma tumor susceptibility gene BRCA1 and BRCA2 coded sequence any one of Claims 1 to 4 or as described in claim 5.
7. the method catching human breast carcinoma tumor susceptibility gene BRCA1 and BRCA2 coded sequence, it is characterised in that comprise the steps:
With the human gene group DNA of extraction for template, catch primer pair with at least one in PCR primer as claimed in claim 1 or 2 and carry out substance pcr amplification for primer, or it is combined as primer with at least one multi-primers in PCR primer as claimed in claim 3 and carries out multiplexed PCR amplification, obtain the PCR primer library containing human breast carcinoma tumor susceptibility gene BRCA1 and BRCA2 complete encoding sequence.
8. the method catching human breast carcinoma tumor susceptibility gene BRCA1 and BRCA2 coded sequence as claimed in claim 7, it is characterized in that, be respectively organize from human peripheral, human body and/or mouth desquamated cells uses the method that purification column extracts and/or magnetic bead extracts to extract human gene group DNA.
9. the method catching human breast carcinoma tumor susceptibility gene BRCA1 and BRCA2 coded sequence as according to any one of claim 7～8, it is characterized in that, it is combine with at least one multi-primers in PCR primer as claimed in claim 3 to carry out multiplexed PCR amplification, the annealing temperature of its pcr amplification is 56～60 DEG C, condition is first 90～100 DEG C and processes 1.5～2.5min, process at processing 85～95 seconds and 68～75 DEG C at processing 15～30 seconds, 56～60 DEG C at 90～100 DEG C again and within 55～65 seconds, carry out 30～40 circulations, preservation after then processing 3～5 minutes at 68～75 DEG C.
10. the method catching human breast carcinoma tumor susceptibility gene BRCA1 and BRCA2 coded sequence as according to any one of claim 7～8, it is characterised in that also include the step by PCR primer library described in the method separating-purifying of purification column purification and/or magnetic beads for purifying.
11. the method for the sequencing library building human breast carcinoma tumor susceptibility gene BRCA1 and BRCA2 coded sequence, it is characterised in that comprise the steps:
Using the method catching human breast carcinoma tumor susceptibility gene BRCA1 and BRCA2 coded sequence as according to any one of claim 7～10, amplification obtains the PCR primer library of human breast carcinoma tumor susceptibility gene BRCA1 and BRCA2 coded sequence；
With described PCR primer library for template, at least one sequencing primer corresponding in the PCR primer as described in claim 4 or 5 carries out secondary PCR amplification to for primer, it is thus achieved that the sequencing library of human breast carcinoma tumor susceptibility gene BRCA1 and BRCA2 coded sequence.
12. the method building the sequencing library of human breast carcinoma tumor susceptibility gene BRCA1 and BRCA2 coded sequence as claimed in claim 11, it is characterized in that, the condition of described secondary PCR amplification is first 90～100 DEG C and processes 1.5～2.5min, 12～18 circulations within 25～35 seconds, are carried out again, preservation after then processing 3～5 minutes at 68～75 DEG C to process at processing 25～35 seconds and 68～75 DEG C at processing 8～12 seconds, 60～70 DEG C at 90～100 DEG C.
13. the method for the sequencing library building human breast carcinoma tumor susceptibility gene BRCA1 and BRCA2 coded sequence as described in claim 11 or 12, it is characterised in that also include the step of sequencing library described in the method separation purification of use magnetic beads for purifying and/or purification column purification.
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