CN105779636B

CN105779636B - PCR primer for amplifying human breast cancer susceptibility gene BRCA1 and BRCA2 coding sequence and application

Info

Publication number: CN105779636B
Application number: CN201610334650.9A
Authority: CN
Inventors: 张巨永; 陈绍宇; 黎美燕
Original assignee: Lbp Medicine Science & Technology Co ltd
Current assignee: Lbp Medicine Science & Technology Co ltd
Priority date: 2016-05-18
Filing date: 2016-05-18
Publication date: 2020-05-19
Anticipated expiration: 2036-05-18
Also published as: CN105779636A

Abstract

The invention relates to a PCR primer of human breast cancer susceptibility genes BRCA1 and BRCA2 coding sequences based on NGS technology and application thereof. The PCR primers of the human breast cancer susceptibility genes BRCA1 and BRCA2 coding sequences comprise at least one pair of capture primer pairs, and the sequences of the forward primer and the reverse primer of each pair of capture primer pairs comprise a specific sequence and a joint sequence connected with the 5' end of the specific sequence. The PCR primers for amplifying the coding sequences of the human breast cancer susceptibility genes BRCA1 and BRCA2 based on the NGS technology and the application thereof have the advantages that the experiment operation related to the whole method is simple, only PCR reagents and primer combinations are related, the cost is low, meanwhile, double-label sequencing joint sequences are introduced for distinguishing different samples, the high-throughput sample sequencing detection can be realized, and the gene detection support can be effectively provided for the risk assessment of the hereditary tumors related to the human breast cancer and other BRCA susceptibility genes or the targeted medication of BRCA mutation.

Description

PCR primer for amplifying human breast cancer susceptibility gene BRCA1 and BRCA2 coding sequence and application

Technical Field

The invention relates to the field of medical molecular biology, in particular to a PCR primer for amplifying coding sequences of human breast cancer susceptibility genes BRCA1 and BRCA2 and application thereof.

Background

Breast cancer is the most common cancer in women. According to the data of '2012 Chinese tumor registration annual report', about 22 ten thousand new breast cancer cases in China each year account for 15% of new breast cancer cases (150 ten thousand) all over the world. In all malignant tumors in women, the incidence rate of breast cancer is as high as 16.8%, 43 people occur in about every 10 ten thousand people, and the incidence age is younger and the incidence rate is in an increasing trend. The incidence of disease increases rapidly by age 25 and peaks at age 50. Therefore, the high incidence of breast cancer has seriously threatened the health of women in China.

Familial hereditary breast cancer accounts for 10% -30% of all breast cancer cases. Among the breast cancer susceptibility genes discovered so far, BRCA1 and BRCA2 are the most direct susceptibility genes, and the breast cancer caused by the mutations of the two genes accounts for 5% -10% of the familial breast cancer. The BRCA1 gene is located at 17q21, comprises 24 exons and encodes 1863 amino acids; the BRCA2 gene is located at 13q12-13, contains 27 exons, and codes protein with 3418 amino acids. BRCA1 and BRCA2 inhibit cell growth, participate in cell cycle regulation, DNA damage repair and apoptosis, and play an important role in maintaining genome stability. If the BRCA1/2 gene is mutated, its function of inhibiting tumorigenesis is affected. The BRCA1/2 gene is also an ovarian cancer susceptibility gene. According to statistics, the risks of breast cancer and ovarian cancer of BRCA1 gene mutation carriers in life are respectively 50-85% and 15-45%; the risk of breast cancer and ovarian cancer of BRCA2 gene mutation carriers is 50-85% and 10-20%, respectively. The BRCA1/2 mutation is also associated with pancreatic cancer, malignant melanoma, and with a risk of prostate cancer in men. Thus, the national Cancer institute published guidelines "BRCA 1 andBCRCA 2: Cancer Risk and Genetic Testing" states that: genetic testing of a patient suffering from breast or ovarian cancer in a family is performed and if the patient is found to have a deleterious mutation in the BRCA1 or BRCA2 genes, then the other members of the family can be genetically tested to see if the deleterious mutation is also present, which is of great clinical value.

Several hundred mutations have been found in the coding region of BRCA1, whereas up to thousands of mutations in BRCA 2. Aiming at BRCA1 and BRCA2 gene detection technologies, the traditional Sanger sequencing is a main detection means. In terms of detection sensitivity, Sanger sequencing can only detect variation with mutation rate of more than 20%, and for detection of all coding sequences of BRCA1 and BRCA2, the detection flux is low, the period is long, and the detection cost of a single sample is also high. With the maturity of the New Generation Sequencing (NGS) technology and the continuous reduction of sequencing cost, the trend of NGS to replace Sanger sequencing technology for breast cancer BRCA gene detection is a future market trend.

Disclosure of Invention

Based on the above, there is a need to provide a PCR primer for amplifying coding sequences of human breast cancer susceptibility genes BRCA1 and BRCA2 based on NGS technology and application thereof.

A PCR primer for amplifying coding sequences of human breast cancer susceptibility genes BRCA1 and BRCA2, comprising at least one pair of capture primer pairs, the sequences of the forward and reverse primers of each pair comprising a specific sequence and an adaptor sequence attached to the 5' end of the specific sequence, the specific sequences in the forward and reverse primers of each pair being SEQ ID No. m and SEQ ID No. (m +112), respectively, wherein m is 1, 2, 3 … … or 112.

A PCR primer for amplifying coding sequences of human breast cancer susceptibility genes BRCA1 and BRCA2 comprises a sequencing primer pair, wherein a forward primer of the sequencing primer pair comprises at least one primer with a sequence shown as SEQ ID NO. 227-SEQ ID NO.234, and a reverse primer of the sequencing primer pair comprises at least one primer with a sequence shown as SEQ ID NO. 235-SEQ ID NO. 246.

A detection kit, comprising any one of the PCR primers for amplifying the coding sequences of human breast cancer susceptibility genes BRCA1 and BRCA 2.

A method for capturing coding sequences of human breast cancer susceptibility genes BRCA1 and BRCA2 comprises the following steps:

and (3) performing single PCR amplification by taking the extracted human genome DNA as a template and at least one capture primer pair in the PCR primers as primers, or performing multiplex PCR amplification by taking at least one multiplex primer combination in the PCR primers as primers to obtain a PCR product library containing the full-length coding sequence of the human breast cancer susceptibility genes BRCA1 and BRCA 2.

The PCR product library obtained by the method for capturing the coding sequences of the human breast cancer susceptibility genes BRCA1 and BRCA 2.

A method for constructing a sequencing library of coding sequences of human breast cancer susceptibility genes BRCA1 and BRCA2, comprising the following steps:

amplifying to obtain a PCR product library of the coding sequences of the human breast cancer susceptibility genes BRCA1 and BRCA2 by using the method for capturing the coding sequences of the human breast cancer susceptibility genes BRCA1 and BRCA 2;

and (3) performing secondary PCR amplification by taking the PCR product library as a template and at least one sequencing primer pair corresponding to the PCR primers as primers to obtain a sequencing library of the coding sequences of the human breast cancer susceptibility genes BRCA1 and BRCA 2.

The sequencing library is constructed by adopting the method for constructing the sequencing library of the coding sequences of the human breast cancer susceptibility genes BRCA1 and BRCA 2.

The PCR primers for amplifying the coding sequences of the human breast cancer susceptibility genes BRCA1 and BRCA2 based on the NGS technology and the application thereof have the advantages that the experiment operation related to the whole method is simple, only PCR reagents and primer combinations are related, the cost is low, meanwhile, the double-label sequencing adaptor sequence is introduced to distinguish different samples, the high-throughput sample sequencing detection can be realized, and the gene detection support can be effectively provided for the risk assessment of the hereditary tumor related to the human breast cancer and other BRCA susceptibility genes or the targeted medication of BRCA mutation.

Drawings

FIG. 1 is a schematic diagram of a multi-sample multiplex PCR loading plate;

FIG. 2 is a schematic representation of the design of full-coverage PCR primers for the coding exon sequences of the BRCA1 and BRCA2 genes;

FIG. 3 is an electrophoretogram of 7 sets of multiplex PCR amplification products, G1-G7 are products, N is a negative control, and the products are concentrated between 200-300 bp;

FIG. 4 is a schematic representation of the uniformity results of 112 amplicons obtained after multiplex PCR product sequencing of exon sequences encoding BRCA1 and BRCA2 genes from a sample;

FIG. 5 is a second round of PCR product electrophoresis with sequencing adapters in the region of interest and product sizes between 300-425 bp;

FIG. 6 is a schematic of a mutation analysis procedure involving conversion of raw sequencing data into Fastq format with elimination of linkers, low quality sequences and primers; the processed sequences were aligned to the human reference genome by alignment tool bwa; removing the repetitive sequence; three variation detection tools with higher accuracy are used for searching variation sites, and each tool uses different algorithms; comparing normal SNP sites in the filtering result of the thousand-person genome database; annotating and analyzing the remaining mutation sites to determine whether the mutation sites are harmful; and reading the analysis result and evaluating the morbidity risk rate of the subject.

Detailed Description

To facilitate an understanding of the invention, the invention will now be described more fully with reference to the accompanying drawings. Preferred embodiments of the present invention are shown in the drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

This example provides PCR primers for amplifying the coding sequences of the human breast cancer susceptibility genes BRCA1 and BRCA 2. The PCR primers include at least one pair of capture primers designed to cover the specific sequences of the amplicons for the coding regions of human BRCA1 and BRCA 2. As shown in fig. 2, the sequences of the forward primer and the reverse primer of each pair of capture primer pairs each include the specific sequence and a linker sequence connected to the 5' end of the specific sequence, wherein the specific sequences in the forward primer and the reverse primer of each pair of capture primer pairs are SEQ ID No.1 and SEQ ID No.113, SEQ ID No.2 and SEQ ID No.114, SEQ ID No.3 and SEQ ID No.115 … …, or SEQ ID No.112 and SEQ ID No.224, respectively (i.e. the corresponding relationship between the specific sequences in the forward primer and the reverse primer of each pair of capture primer pairs is SEQ ID No. m and SEQ ID No. (m +112), wherein m is 1, 2, 3 … …, or 112), as shown in table 1 below:

TABLE 1

Preferably, in this embodiment, the linker sequence of the forward primer in the capture primer pair is GACGCTCTTCCGATCTCTG (shown as SEQ ID No. 225), the linker sequence of the reverse primer in the capture primer pair is TGTGCTCTTCCGATCTGAC (shown as SEQ ID No. 226), the specific sequences shown as SEQ ID No.1 and SEQ ID No.113, and the sequences of the forward primer and the reverse primer in the corresponding capture primer pair are GACGCTCTTCCGATCTCTGCTAATGTGTTAAAGTTCATTGGAACA and TGTGCTCTTCCGATCTGACAGTTCTTCAGTTAAGAAAATCAGCA, respectively, the same applies.

Further preferably, in this embodiment, the PCR primers comprise a plurality of pairs of capture primers. The multiple pairs of capture primer pairs are combined to form multiple primer combinations, and each multiple primer combination comprises multiple pairs of capture primer pairs. The multiplex primer combination of this example includes at least one of 7 sets of combinations as shown in table 2 below:

TABLE 2

Further, in this embodiment, the PCR primers further comprise a sequencing primer pair with a characteristic sequence. The forward primer of the sequencing primer pair comprises at least one of primers with sequences shown as SEQ ID NO. 227-SEQ ID NO. 234. The reverse primer of the sequencing primer pair comprises at least one of primers with sequences shown as SEQ ID No. 235-SEQ ID No.246, and is specifically shown as the following table 3.

TABLE 3

The PCR primers described in this example can be used alone, in pairs or in combination of multiple primers to form a PCR primer reagent for use in a detection kit, and for example, this example also provides a detection kit comprising the above PCR primers for amplifying the coding sequences of human breast cancer susceptibility genes BRCA1 and BRCA 2.

In this embodiment, the detection kit further comprises reagents related to PCR amplification, such as reagents related to PCR amplification comprising PCR buffer, dNTP solution, DMSO, and DNA polymerase. Wherein the PCR buffer solution is 10 times the buffer solution, the pH value is 7.5-8.0, the solvent is water, the solute comprises 500-1000 mM Tris-HCl (pH 8.0,25 ℃), 500-1000 mM NaCl, 10-50 mM dithiothreitol, 1-10 mg/mL BSA, 150-200 mM MgCl₂And 80-120 mM ATP; the dNTP solution is a dNTPs mixture with the concentration of 10 mM; DMSO is dimethyl sulfoxide with the mass percentage concentration of 100%; the DNA polymerase is hot start Tag DNA polymerase, the DNA polymerase is stored in a storage solution, the pH value of the storage solution is 8.0-8.5.0, and the components comprise Tris-HCl (pH 8.0) with the concentration of 10-30 mM, KCl with the concentration of 0.05-0.20M, EDTA with the concentration of 0.05-0.20 mM, Tween20 with the volume percentage concentration of 0.1-0.5%, Nonidet P40 with the volume percentage concentration of 0.1-0.5%, dithiothreitol with the concentration of 0.05-0.15 mM and glycerol with the volume concentration of 40-60%.

The embodiment also provides a method for capturing the coding sequences of human breast cancer susceptibility genes BRCA1 and BRCA2 and a PCR product library obtained by using the method. The method comprises the following steps:

and (3) performing single PCR amplification by taking the extracted human genome DNA as a template and at least one capture primer pair in the PCR primers as primers, or performing multiple PCR amplification by taking at least one multiple primer combination in the PCR primers to obtain a PCR product library containing the full-length coding sequence of the human breast cancer susceptibility genes BRCA1 and BRCA 2.

The human genomic DNA can be extracted from human peripheral blood, human tissues and/or oral exfoliated cells by using a purification column extraction method and/or a magnetic bead extraction method. The related reagent used in the PCR amplification process is the PCR amplification related reagent in the detection kit.

Preferably, the method for capturing the coding sequences of the human breast cancer susceptibility genes BRCA1 and BRCA2 is to perform multiplex PCR amplification by using at least one multiplex primer combination in the PCR primers, wherein the annealing temperature of the PCR amplification is 56-60 ℃, the conditions are that the PCR amplification is firstly performed for 1.5-2.5 min at 90-100 ℃, then performed for 30-40 cycles at 90-100 ℃ for 15-30 seconds, 56-60 ℃ for 85-95 seconds and 68-75 ℃ for 55-65 seconds, and then performed for 3-5 minutes at 68-75 ℃, and then stored, the preferred PCR conditions are that the PCR amplification is performed for 2min at 95 ℃, then performed for 20 seconds at 95 ℃, 56-60 ℃ (such as 58 ℃) for 90 seconds and 72 ℃ for 35 cycles, and then performed for 35 cycles at 72 ℃ for 4 minutes and then stored at 16 ℃. After obtaining the PCR product library, the method also comprises a step of separating and purifying the PCR product library by a purification column purification and/or magnetic bead purification method.

In addition, the embodiment also provides a method for constructing a sequencing library of coding sequences of human breast cancer susceptibility genes BRCA1 and BRCA2 and a sequencing library constructed by using the method. The method for constructing the sequencing library comprises the following steps:

and (3) performing secondary PCR amplification by taking the PCR product library as a template and at least one sequencing primer pair in the PCR primers as primers to obtain a sequencing library of the coding sequences of the human breast cancer susceptibility genes BRCA1 and BRCA 2.

In this embodiment, the conditions for the secondary PCR amplification are first 90 to 100 ℃ for 1.5 to 2.5min, then 90 to 100 ℃ for 8 to 12 seconds, 60 to 70 ℃ for 25 to 35 seconds, and 68 to 75 ℃ for 25 to 35 seconds for 12 to 18 cycles, then 68 to 75 ℃ for 3 to 5 minutes, and then the mixture is stored, preferably 95 ℃ for 2min, then 95 ℃ for 10 seconds, 65 ℃ for 30 seconds, and 72 ℃ for 30 seconds for 15 cycles, then 72 ℃ for 4 minutes, and then 16 ℃ for 16 ℃.

In this embodiment, the method further comprises a step of separating and purifying the sequencing library by using magnetic bead purification and/or purification column purification after obtaining the sequencing library.

The PCR primers of the coding sequences of the human breast cancer susceptibility genes BRCA1 and BRCA2 and the application thereof are described in the following by combining specific embodiments.

The following examples were performed on 45 human peripheral blood samples and corresponding 45 saliva samples from unknown BRCA1 and BRCA2 mutation tests by PCR capture pooling. In addition, the same PCR capture and library construction is carried out on 2 breast cancer cell lines which are sequenced by Sanger and 4 BRCA mutation carriers, and the method specifically comprises the following steps:

1. sample extraction

DNA was extracted from 2 breast cancer cells, 45 human peripheral blood samples (200. mu.l) and corresponding 45 oral saliva samples (5ml) using a tissue blood cell DNA extraction kit from Tiangen and a New Baiji oral saliva magnetic bead extraction kit, respectively. About 50 mu l of eluted product (extracted DNA) is obtained, and the quality of the extracted DNA is detected by Nanodrop, so that the result shows that the OD260/OD230 is 1.8-2.5, and the OD260/OD280 is 1.8-2.5, which indicates that the DNA is qualified for extraction and can be used as a template in the next PCR amplification.

2. First round of multiple PCR captures coding sequences of human breast cancer susceptibility genes BRCA1 and BRCA2

The samples were subjected to PCR reactions using 96-well plates as shown in FIG. 1, in which 7-well multiplex PCR reactions were performed for each sample, and the primers used for the 7 wells corresponded to 7 sets of multiplex primer combinations as shown in Table 2, respectively. A negative control without template addition was set, using the same primers as those used in the multiplex PCR reaction in the first well of the sample. Each sample used 1 row of reaction wells on a 96-well plate, and the corresponding well position for each 96-well plate and sample was marked and recorded, as shown in fig. 1.

The PCR reaction system is 20 mul, and the used PCR reaction reagent is the PCR amplification related reagent with the optimized configuration. The PCR reaction system is shown in the following Table 4:

TABLE 4

Components	Volume of
		10 Xbuffer	2μl
dNTPs solution	0.4μl
		DMSO	1μl
Template DNA (5 ng to 20ng)	1μl
		Hot start Taq DNA polymerase	0.2μl
Multiple primer combination (10. mu.M)	2μl
		Water (HPLC grade)	13.4
In all	20μl

PCR amplification was performed using the PCR parameters shown in table 5 below:

TABLE 5

PCR amplification was performed on an ABI-2720PCR instrument from ABI. After completion of PCR, 5. mu.l of the PCR product was electrophoretically detected on a 2% agarose gel, and the results are shown in FIG. 3.

3. Mixing and purification of multiple PCR Capture products

Mixing the PCR product obtained in the last step into a 1.5ml EP tube, taking 10 mul of each tube, marking sample information respectively, and shaking and mixing uniformly. Purification was performed using Ampure magnetic beads at a 1:1 volume ratio to obtain 50. mu.l of DNA.

4. Secondary PCR amplification construction sequencing library

And (3) diluting the PCR product obtained in the last step by 100 times with water, and then using the diluted PCR product as a template for a second round of PCR reaction, wherein the second round of PCR is amplified by using the sequencing primer pair with the tag joint, and the obtained PCR product is a sequencing library. The PCR reaction reagent is the PCR amplification related reagent with the optimized configuration.

The PCR reaction system was 20. mu.l, as shown in Table 6 below:

TABLE 6

PCR amplification was performed using the PCR parameters shown in table 7 below:

TABLE 7

The PCR reaction was run on an ABI-2720PCR machine from ABI. After completion of PCR, 5. mu.l of the PCR product was electrophoretically detected on a 2% agarose gel, and the results are shown in FIG. 5.

5. Sequencing library purification, quantification and mixing

Mu.l of the sequencing library from different samples were purified using Ampure magnetic beads in a 1:1 volume ratio to give 20. mu.l of purified sequencing library. Mu.l of each purified sequencing library was taken, quantified using a Qubit, and the concentration of each sequencing library was recorded, the results are shown in Table 7. Based on the concentrations determined, 100ng of DNA was taken from each sequencing library, mixed in a 1.5ml PE tube and vortexed.

Table 7: sequencing library concentration

Samples 91-92 were cell line samples and samples 93-96 were positive samples.

6. On-machine sequencing of a Mixed sequencing library

Sequencing was performed using an illumina sequencer (NextSeq500) and sequencing reagents (NextSeq500 Mid Output Kit, 300cycles) with PE150, a double index program.

7. Analysis of results

And according to the information of the tag sequence characteristic sequences in the sequencing primer pair in the sequencing result, the sequencing data corresponds to each sample one by one. Then, the sequenced sequences of each sample were aligned to the human genome using commonly used alignment programs known in the sequencing art, such as BLAST and SOAP, and region of interest (i.e., the full coding sequence of BRCA1 and BRCA 2) coverage and multiplex PCR primer homogeneity evaluations were performed on the sequenced sequences of the aligned genomes, with the results shown in fig. 4. The average sequencing depth of 96 samples is between 2000X and 3000X, the average target area coverage rate is 99.5 percent (99.0 percent to 99.9 percent), the coverage rate of the sequencing depth is not less than 50X (meeting the statistical requirement of better mutation analysis data) is 98.5 percent (97.2 percent to 99.9 percent), the coverage rate of the sequencing depth is not less than 0.1X average sequencing depth is 95 percent (93.1 percent to 97.0 percent), and the reference is shown in Table 8. These statistics of target region coverage data indicate that the multiplex primer combinations designed and optimized in this example are very effective. In addition, mutation analysis was performed on the sequencing data aligned to the BRCA1 and BRCA2 coding sequences in the human genome using mutation analysis software samtools, GATK, varscan, see fig. 6. The detection results obtained by 2 cell lines are completely consistent with the known results, the mutation results obtained by analyzing other 30 whole blood samples and corresponding oral cavity saliva samples are all correct through Sanger sequencing, and the mutation results obtained by analyzing the whole blood and the oral cavity saliva samples from the same person are consistent, see the mutation rate of NGS sequencing and the mutation rate of Sanger sequencing in Table 9, which indicates that the method and the kit can be used for the mutation detection of the full coding sequence of the human breast cancer susceptibility genes BRCA1 and BRCA 2.

Table 8: sample sequencing depth statistics

Table 9: positive sample Sanger test results and NGS test results

Samples 91-92 are cell line samples, samples 93-96 are known positive samples, and samples 41-42 are saliva and blood samples of the same person, respectively.

As a result, it was found that: the results obtained by the method of the invention are consistent with the mutation results of 2 cell lines and 4 BRCA mutation carriers with known BRCA1 and BRCA2 mutation information; for 45 human peripheral blood samples with unknown mutation information and 45 corresponding oral saliva samples, a BRCA2 mutation (the same sample is detected in the peripheral blood and oral saliva samples simultaneously) is discovered through illumina sequencing, and the mutation result is 100% accurate through Sanger verification.

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims

1. A PCR primer for amplifying coding sequences of human breast cancer susceptibility genes BRCA1 and BRCA2, comprising 112 pairs of capture primer pairs, wherein the sequences of the forward primer and the reverse primer of each pair are both composed of a specific sequence and a linker sequence connected with the 5' end of the specific sequence, the specific sequences in the forward primer and the reverse primer of each pair are SEQ ID No. m and SEQ ID No. (m +112), wherein m is 1, 2, 3 … … 112;

the sequence of an adaptor in a forward primer of the capture primer pair is shown as SEQ ID NO.225, and the sequence of an adaptor in a reverse primer of the capture primer pair is shown as SEQ ID NO. 226;

the primers also comprise a sequencing primer pair, wherein a forward primer of the sequencing primer pair is composed of primers with sequences shown as SEQ ID NO. 227-SEQ ID NO.234, and a reverse primer of the sequencing primer pair is composed of primers with sequences shown as SEQ ID NO. 235-SEQ ID NO. 246.

2. The PCR primers for amplifying coding sequences of human breast cancer susceptibility genes BRCA1 and BRCA2 of claim 1, wherein said primers comprise 112 pairs of said capture primers, and wherein said pairs of capture primers comprise a multiplex primer combination consisting of 7 sets of multiplex primer combinations: the first group consists of the sequences: capture primer pairs of SEQ ID nos. 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;

the second group consists of the sequences: capture primer pairs of SEQ ID nos. 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;

the third group consists of sequences: capture primer pairs of SEQ ID nos. 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;

the fourth group is composed of the sequences: capture primer pairs of SEQ ID nos. 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;

the fifth group consists of the sequences: capture primer pairs of SEQ ID nos. 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;

the sixth group consists of the sequences: capture primer pairs of SEQ ID nos. 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;

the seventh group consists of the sequences: the capture primer pairs of SEQ ID nos. 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.

3. A test kit comprising the PCR primers of claim 1 or 2 for amplifying the coding sequences of BRCA1 and BRCA2 of human breast cancer susceptibility genes.

4. A method for capturing the coding sequences of human breast cancer susceptibility genes BRCA1 and BRCA2 for non-diagnostic purposes, comprising the steps of:

carrying out PCR amplification by using the extracted human genome DNA as a template and the capture primer pair in the PCR primers as claimed in claim 1 or 2 as primers to obtain a PCR product library containing the full-length coding sequence of human breast cancer susceptibility genes BRCA1 and BRCA 2.

5. The method of claim 4, wherein the human genomic DNA is extracted from human peripheral blood, human tissues and/or exfoliated cells using a purification column and/or magnetic bead extraction.

6. The method for capturing the coding sequences of human breast cancer susceptibility genes BRCA1 and BRCA2 of any of claims 4 to 5, wherein the PCR amplification is performed by the PCR primers of claim 2, wherein the annealing temperature of the PCR amplification is 56 to 60 ℃, the annealing temperature is 90 to 100 ℃ for 1.5 to 2.5min, then 90 to 100 ℃ for 15 to 30 seconds, 56 to 60 ℃ for 85 to 95 seconds, and 68 to 75 ℃ for 55 to 65 seconds for 30 to 40 cycles, and then 68 to 75 ℃ for 3 to 5 minutes before storage.

7. The method for capturing the coding sequences of human breast cancer susceptibility genes BRCA1 and BRCA2 of any of claims 4 to 5, further comprising the step of isolating and purifying the PCR product library by purification column purification and/or magnetic bead purification.

8. A method for constructing a sequencing library of human breast cancer susceptibility genes BRCA1 and BRCA2 coding sequences for non-diagnostic purposes, comprising the steps of:

amplifying a PCR product library of coding sequences of human breast cancer susceptibility genes BRCA1 and BRCA2 using the method of capturing coding sequences of human breast cancer susceptibility genes BRCA1 and BRCA2 of any one of claims 4-7;

performing secondary PCR amplification by using the PCR product library as a template and the sequencing primer pair in the PCR primers as claimed in claim 1 or 2 as primers to obtain a sequencing library of the coding sequences of human breast cancer susceptibility genes BRCA1 and BRCA 2.

9. The method for constructing the sequencing library of the coding sequences of the human breast cancer susceptibility genes BRCA1 and BRCA2 as claimed in claim 8, wherein the conditions of the secondary PCR amplification are that the treatment is carried out at 90-100 ℃ for 1.5-2.5 min, then at 90-100 ℃ for 8-12 seconds, at 60-70 ℃ for 25-35 seconds and at 68-75 ℃ for 25-35 seconds for 12-18 cycles, and then the treatment is carried out at 68-75 ℃ for 3-5 minutes and then the storage is carried out.

10. The method for constructing a sequencing library of the coding sequences of human breast cancer susceptibility genes BRCA1 and BRCA2 as claimed in claim 8 or 9, further comprising the step of isolating and purifying the sequencing library by using magnetic bead purification and/or purification column purification methods.