阅读说明：本技术 同时检测brca1/2外显子序列和化疗用药位点的引物及应用 (Primer for simultaneously detecting BRCA1/2 exon sequence and chemotherapy drug site and application ) 是由 王雨倩 江凌 于 2019-08-13 设计创作，主要内容包括：一种同时检测BRCA1/2外显子序列和化疗用药位点位点的引物,包括：为BRCA1设计的外显子扩增引物对、为BRCA2设计的外显子扩增引物对、针对SNP位点设计的特异性引物对；每一种外显子扩增引物对分别分为两组,且同一基因的每一组引物内部无扩增区域重叠；每一组引物对中,第一部分引物上游引物5’端添加SEQ ID.NO1,下游5’端添加SEQ ID.NO2；第二部分引物上游引物5’端添加SEQ ID.NO2,下游5’端添加SEQ ID.NO1；BRCA1基因的一组引物对与BRCA2基因的一组引物对随机组合,组成两个引物组对；所述特异性引物上游引物的5’端添加SEQ ID.NO1,在所述特异性引物下游引物5’端添加SEQ ID.NO2。本申请成本低,通量高,检测范围广,检测时间短；可同时检测BRCA1/2和癌症化疗用药检测位点。(A primer for simultaneously detecting BRCA1/2 exon sequences and chemotherapy application site sites comprises: an exon amplification primer pair designed for BRCA1, an exon amplification primer pair designed for BRCA2 and a specific primer pair designed aiming at SNP loci; each exon amplification primer pair is divided into two groups, and each group of primers of the same gene has no amplification region overlapping; in each group of primer pairs, the 5 'end of the upstream primer of the first part of primers is added with SEQ ID.NO1, and the 5' end of the downstream primer of the first part of primers is added with SEQ ID.NO2; the 5 'end of the upstream primer of the second part of primers is added with SEQ ID.NO2, and the 5' end of the downstream primer is added with SEQ ID.NO1; combining a group of primer pairs of the BRCA1 gene and a group of primer pairs of the BRCA2 gene randomly to form two primer pairs; and the 5 'end of the upstream primer of the specific primer is added with SEQ ID.NO1, and the 5' end of the downstream primer of the specific primer is added with SEQ ID.NO2. The method has the advantages of low cost, high flux, wide detection range and short detection time; can simultaneously detect BRCA1/2 and the cancer chemotherapy drug detection site.)

1. A primer for simultaneously detecting BRCA1/2 exon sequences and chemotherapy application sites, comprising:

an exon amplification primer pair designed for BRCA1, an exon amplification primer pair designed for BRCA 2; wherein, the exon amplification primer pair designed for BRCA1 can cover the whole exon region of BRCA1, and the exon amplification primer pair designed for BRCA2 can cover the whole exon region of BRCA 2; in addition, the exon amplification primer pair designed for BRCA1 and the exon amplification primer pair designed for BRCA2 are divided into two groups respectively, and no amplification region is overlapped in each group of primers of the same gene; in each group of primer pairs, the 5 'end of the upstream primer of the first part of primers is added with SEQ ID.NO1, and the 5' end of the downstream primer of the first part of primers is added with SEQ ID.NO2; the 5 'end of the upstream primer of the second part of primers is added with SEQ ID.NO2, and the 5' end of the downstream primer is added with SEQ ID.NO1; combining a group of primer pairs of the BRCA1 gene and a group of primer pairs of the BRCA2 gene randomly to form two primer pairs;

specific primers designed for the SNP sites as follows: rs1801133, rs2072671, rs60369023, rs1934951, rs1113129, rs183484, rs9937, rs1042858, rs2306283, rs11045879, rs116855232, rs4646, rs8060157, rs2232228, rs1042522, rs 183964, rs151264360, rs25487, rs1799782, rs13181, rs1052555, rs1056836, rs1872328, rs 10497297203, rs7582141, rs6432512, rs 010010010681, rs 686892, rs 3892092097, rs 7309, rs2228001, rs 1801011011019, rs924607, rs1801394 1394, rs442767, rs1142345, rs1800460, rs 223462237993, rs 409380, rs 1214568, rs 104777943799, rs 1047942, rs 56500884; and adding SEQ ID.NO1 at the 5 'end of the upstream primer of the specific primer and adding SEQ ID.NO2 at the 5' end of the downstream primer of the specific primer.

2. The primer for simultaneously detecting the BRCA1/2 exon sequence and the chemotherapeutic drug site as claimed in claim 1, further comprising a universal library primer.

3. The primer for simultaneously detecting the BRCA1/2 exon sequence and the chemotherapy administration site according to claim 2, wherein the universal library-building primer is selected from any one or more of SEQ ID No.267 and SEQ ID No. 268.

4. The primer for simultaneously detecting the exon sequences of BRCA1/2 and the site of chemotherapy according to claim 1, wherein a specific primer designed for said SNP site is used as one set in combination with one of the two primer sets.

5. The primer for simultaneously detecting the exon sequences of BRCA1/2 and the chemotherapeutic application site as claimed in claim 1, wherein the first set of primer pairs consists of any two or more primers selected from SEQ ID no3 to SEQ ID no40, the second set of primer pairs consists of any two or more primers selected from SEQ ID no41 to SEQ ID no72, the third set of primer pairs consists of any two or more primers selected from SEQ ID no73 to SEQ ID no130, the fourth set of primer pairs consists of any two or more primers selected from SEQ ID no131 to SEQ ID no180, and the specific primers consists of any two or more primers selected from SEQ ID no181 to SEQ ID no 266.

6. A method for designing primers capable of simultaneously detecting BRCA1/2 and a cancer chemotherapy detection site, comprising:

designing BRCA1 exon amplification primer pairs which can cover all exon regions of BRCA1, and designing BRCA2 exon amplification primer pairs which can cover all exon regions of BRCA 2; dividing BRCA1 exon amplification primer pairs and BRCA2 exon amplification primer pairs into two groups respectively, wherein no amplification region is overlapped in each group of primers of the same gene; in each group of primer pairs, the 5 'end of the upstream primer of the first part of primers is added with SEQ ID.NO1, and the 5' end of the downstream primer of the first part of primers is added with SEQ ID.NO2; the 5 'end of the upstream primer of the second part of primers is added with SEQ ID.NO2, and the 5' end of the downstream primer is added with SEQ ID.NO1; a group of primer pairs of the BRCA1 gene and a group of primer pairs of the BRCA2 gene are randomly combined to form two primer pairs;

specific primers designed for the SNP sites as follows: rs1801133, rs2072671, rs60369023, rs1934951, rs1113129, rs183484, rs9937, rs1042858, rs2306283, rs11045879, rs116855232, rs4646, rs8060157, rs2232228, rs1042522, rs 183964, rs151264360, rs25487, rs1799782, rs13181, rs1052555, rs1056836, rs1872328, rs 10497297203, rs7582141, rs6432512, rs 010010010681, rs 686892, rs 3892092097, rs 7309, rs2228001, rs 1801011011019, rs924607, rs1801394 1394, rs442767, rs1142345, rs1800460, rs 223462237993, rs 409380, rs 1214568, rs 104777943799, rs 1047942, rs 56500884; and adding SEQ ID.NO1 at the 5 'end of the upstream primer of the specific primer and adding SEQ ID.NO2 at the 5' end of the downstream primer of the specific primer.

7. A nucleotide sequence for ligation to the BRCA1 exon amplification primer pair, BRCA1 exon amplification primer pair, said specific primers of claim 1, comprising SEQ id No.1, SEQ id No. 2.

8. A kit capable of simultaneously detecting BRCA1/2 and a cancer chemotherapy detection site, which is characterized by comprising the primer capable of simultaneously detecting BRCA1/2 and the cancer chemotherapy detection site.

9. The kit of claim 8, further comprising any one or more of: distilled water, one-round PCR amplification enzyme and two-round PCR amplification enzyme.

10. The kit of claim 8, wherein the kit further comprises a gDNA extraction reagent or a gDNA extraction kit.

Technical Field

The invention relates to the technical field of biology, in particular to a method/primer group/kit for designing exon regions of BRCA1/BRCA2 genes and a detection primer of chemotherapy drug sites and application thereof.

Background

In 1990, researchers discovered a gene directly related to hereditary breast cancer, named as breast cancer gene No.1, which is abbreviated as BRCA 1. In 1994, another gene associated with breast cancer was discovered, designated BRCA2, and in many cases both genes were discussed together under the general designation BRCA 1/2. BRCA1 maps to chromosome 17q221.31 with a gene size of 81.19kb for a total of 24 exons, and BRCA2 maps to chromosome 13q13.1 with a gene size of 84.19kb for a total of 27 exons.

BRCA1/2 is two genes with effects of inhibiting malignant tumor, and has important effects in regulating human cell replication, repairing genetic material DNA injury, and normal cell growth. Families with this gene mutation tend to have a high incidence of breast cancer, usually at a younger age, with both breasts being true and ovarian cancer at the same time. The life-long risks of cancer associated with mutations in BRCA1 and BRCA2 genes were summarized, indicating that the risk for breast and ovarian cancer was 50% -85% and 15% -45%, respectively, for those with mutations in BRCA1, and 50% -85% and 10% -20%, respectively, for those with mutations in BRCA 2. Therefore, the BRCA1/2 gene mutation site detection of high-risk population has important significance for the prevention and early inspection of cancer.

In the screening of early BRCA1/2 gene mutation, Sanger sequencing is directly carried out by a conventional method, but the BRCA1/2 gene has large size and high direct sequencing cost, and methods such as a denaturing high performance liquid chromatography analysis technology, a high-resolution melting analysis technology and the like are also frequently adopted. Nowadays, the second generation sequencing (NGS) technology is increasingly high-throughput, low-cost and diversified, and has become a widely applied technology in the field of molecular diagnosis, and many laboratories currently use multiplex PCR technology to detect mutations of BRCA1/BRCA2, for example, 58 pairs of primers are designed to cover positions of full exons of BRCA1/2 and splice sites of BRCA1/2 based on multiplex PCR targeted sequencing, the master thesis at southeast university, 2017, wherein the primer pair BRCA1 and the primer pair BRCA2, which respectively cover gene lengths of 17763bp and 24383bp, and the amplicon length is between 400 and 1500bp, and 24 mutations are detected in total, and 1 BRCA1 pathogenic mutation rs80357303 is one of the pathogenic mutations, which is reported to increase the risk of breast cancer.

Second generation sequencing technology has the characteristic of high throughput, and as sequencing time is gradually shortened, the feasibility of cheap and short-period universal screening by using second generation sequencing for BRCA1 and BRCA2 is enhanced.

Chemotherapy is a treatment method for inhibiting cell growth and reproduction, killing tumor cells or promoting tumor cell differentiation by using chemical drugs, and the method is widely applied to cancer treatment, but more and more clinical experiments show that the treatment effect of the chemotherapy drugs varies from one person to another. At present, the genotype of certain SNP loci has obvious correlation with the treatment effect of specific chemotherapeutic drugs, and if the SNP loci are detected before the treatment scheme is determined, the diagnosis and treatment scheme can be determined, the time of ineffective treatment is shortened, and the life cycle of patients is prolonged.

Therefore, the simultaneous detection of BRAC1/2 and chemotherapy-related SNP sites has great significance for the people with high risk of breast cancer.

Disclosure of Invention

The invention provides a primer design method, a primer combination, a detection method and a kit for simultaneously detecting BRCA1/2 exon sequences and chemotherapy-related SNP sites. The invention mainly uses multiplex PCR technology to enrich the target area and then carry out second-generation sequencing, and carries out identification through the exon sequence of BRCA1/2 in the bioinformatics section and carries out typing on chemotherapy SNP loci, thereby simultaneously realizing the prediction of susceptibility degree of breast cancer and susceptibility degree of chemotherapy drugs.

In a first aspect, the present application provides a primer for simultaneously detecting the exon sequence of BRCA1/2 and the chemotherapeutic drug site, comprising: an exon amplification primer pair designed for BRCA1, an exon amplification primer pair designed for BRCA 2; wherein, the exon amplification primer pair designed for BRCA1 can cover the whole exon region of BRCA1, and the exon amplification primer pair designed for BRCA2 can cover the whole exon region of BRCA 2; in addition, the exon amplification primer pair designed for BRCA1 and the exon amplification primer pair designed for BRCA2 are divided into two groups respectively, and no amplification region is overlapped in each group of primers of the same gene; in each group of primer pairs, the 5 'end of the upstream primer of the first part of primers is added with SEQ ID.NO1, and the 5' end of the downstream primer of the first part of primers is added with SEQ ID.NO2; the 5 'end of the upstream primer of the second part of primers is added with SEQ ID.NO2, and the 5' end of the downstream primer is added with SEQ ID.NO1; combining a group of primer pairs of the BRCA1 gene and a group of primer pairs of the BRCA2 gene randomly to form two primer pairs;

also comprises a specific primer pair designed aiming at the SNP sites as follows: rs1801133, rs2072671, rs60369023, rs1934951, rs1113129, rs183484, rs9937, rs1042858, rs2306283, rs11045879, rs116855232, rs4646, rs8060157, rs2232228, rs1042522, rs 183964, rs151264360, rs25487, rs1799782, rs13181, rs1052555, rs1056836, rs1872328, rs 10497297203, rs7582141, rs6432512, rs 010010010681, rs 686892, rs 3892092097, rs 7309, rs2228001, rs 1801011011019, rs924607, rs1801394 1394, rs442767, rs1142345, rs1800460, rs 223462237993, rs 409380, rs 1214568, rs 104777943799, rs 1047942, rs 56500884; and adding SEQ ID.NO1 at the 5 'end of the upstream primer of the specific primer and adding SEQ ID.NO2 at the 5' end of the downstream primer of the specific primer.

In a preferred embodiment, specific primers designed for the SNP sites as a set can be combined with one of the two primer sets.

In a preferred embodiment, the primers for simultaneously detecting the BRCA1/2 exon sequence and the site further comprise a universal library primer.

In a second aspect, the present invention provides a method for designing primers capable of simultaneously detecting BRCA1/2 and a cancer chemotherapy detection site (chemotherapy administration site), comprising:

designing BRCA1 exon amplification primer pairs which can cover all exon regions of BRCA1, and designing BRCA2 exon amplification primer pairs which can cover all exon regions of BRCA 2; dividing BRCA1 exon amplification primer pairs and BRCA2 exon amplification primer pairs into two groups respectively, wherein no amplification region is overlapped in each group of primers of the same gene; in each group of primer pairs, the 5 'end of the upstream primer of the first part of primers is added with SEQ ID.NO1, and the 5' end of the downstream primer of the first part of primers is added with SEQ ID.NO2; SEQ ID.NO2 is added to the 5 'end of the upstream primer of the second partial primer, and SEQ ID.NO1 is added to the 5' end of the downstream primer; a group of primer pairs of the BRCA1 gene and a group of primer pairs of the BRCA2 gene are randomly combined to form two primer pairs;

specific primers designed for the SNP sites as follows: rs1801133, rs2072671, rs60369023, rs1934951, rs1113129, rs183484, rs9937, rs1042858, rs2306283, rs11045879, rs116855232, rs4646, rs8060157, rs2232228, rs1042522, rs 183964, rs151264360, rs25487, rs1799782, rs13181, rs1052555, rs1056836, rs1872328, rs 10497297203, rs7582141, rs6432512, rs 010010010681, rs 686892, rs 3892092097, rs 7309, rs2228001, rs 1801011011019, rs924607, rs1801394 1394, rs442767, rs1142345, rs1800460, rs 223462237993, rs 409380, rs 1214568, rs 104777943799, rs 1047942, rs 56500884; and adding SEQ ID.NO1 at the 5 'end of the upstream primer of the specific primer and adding SEQ ID.NO2 at the 5' end of the downstream primer of the specific primer.

In a preferred embodiment, the method further comprises combining specific primers designed for the SNP site as one set with one of the two primer set pairs.

In a preferred embodiment, the first set of primer pairs consists of any two or more primers selected from SEQ id no3 to SEQ id no 40.

In a preferred embodiment, the second set of primer pairs consists of any two or more primers selected from SEQ id no41 to SEQ id no 72.

In a preferred embodiment, the third set of primer pairs consists of any two or more primers selected from SEQ id no73 to SEQ id no 130.

In a preferred embodiment, the fourth set of primer pairs consists of any two or more primers selected from SEQ id no131 to SEQ id no 180.

In a preferred embodiment, the specific primers consist of any two or more primers selected from SEQ id No.181 to SEQ id No. 266.

In a preferred embodiment, the universal library primer is selected from any one or more of SEQ id No.267, SEQ id No. 268.

The third aspect of the invention provides a kit capable of simultaneously detecting BRCA1/2 and a cancer chemotherapy detection site (chemotherapy drug application site), which comprises the primer capable of simultaneously detecting BRCA1/2 and the cancer chemotherapy detection site.

In a preferred embodiment, the kit may further comprise distilled water.

In a preferred embodiment, the kit further comprises a round of PCR amplification enzymes, preferably multiplex PCR amplification enzymes, more preferably Vazyme multiplex PCR amplification enzymes.

In a preferred embodiment, the kit further comprises a two-round PCR amplification enzyme, preferably PCR amplification enzyme Q5.

In a preferred embodiment, the kit may further comprise a gDNA extraction reagent or a gDNA extraction kit.

In a fourth aspect, the invention provides a nucleotide sequence for connecting to a BRCA1 exon amplification primer pair, a BRCA1 exon amplification primer pair and the specific primers, and the nucleotide sequence comprises SEQ ID No.1 and SEQ ID No. 2.

Compared with the prior invention, the method has the following advantages:

1) the cost is low, and the detection range is wide;

2) the flux is high;

3) the detection time is short;

4) the detection range is increased by adopting a mode of combining various PCR strategies;

5) the detection flexibility is high, and different primer groups can be set according to different requirements.

6) Can simultaneously detect BRCA1/2 and cancer chemotherapy detection sites.

Drawings

FIG. 1 shows the Q-Sep identification result of the product to be sequenced obtained by using the primers and the kit of the present application.

Detailed Description

For a clearer understanding of the objects, features and advantages of the present invention, reference is made to the following detailed description of the invention taken in conjunction with the accompanying drawings.

In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, but the present invention may be practiced in other ways than those specifically described herein, and thus the present invention is not limited to the specific embodiments disclosed below.