Primer for simultaneous detection of BRCA 1/2 exon sequence and chemotherapy drug site, and application

An exon and locus technology, applied in DNA/RNA fragmentation, recombinant DNA technology, microbial determination/inspection, etc., can solve the problem of increased risk of breast cancer, high cost of direct sequencing, and large size of BRCA1/2 genes, etc. problems, to achieve the effect of short detection time, low cost, and increased detection range

Pending Publication Date: 2020-01-21
阔然生物医药科技(上海)有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The conventional method for early screening of BRCA1 / 2 gene mutations is direct Sanger sequencing, but the size of BRCA1 / 2 genes is large, and the cost of direct sequencing is high. Methods such as denaturing high-performance liquid chromatography analysis technology and high-resolution melting analysis are also often used.
Nowadays, next-generation sequencing (NGS) technology is becoming more and more high-throughput, low-cost, and diversified, and has become a widely used technology in the field of molecular diagnosis. At present, many laboratories use multiplex PCR technology to detect BRCA1 / BRCA2 mutations, such as , Rong Yan (Multiple PCR targeted sequencing-based mutation detection of breast cancer susceptibility gene BRCA1 / 2 whole exon region, master's thesis of Southeast University, 2017) studied and designed 58 pairs of primers, covering BRCA1 / 2 exon BRCA1 primers and splice site regions, including 25 pairs of BRCA1 primers and 33 pairs of BRCA2 primers, covering gene lengths of 17,763bp and 24,383bp respectively, amplicon length between 400-1500bp, a total of 24 mutations were detected, including 1 case of BRCA1 The pathogenic mutation rs80357303, the susceptibility mutation has been reported to increase the risk of breast cancer

Method used

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  • Primer for simultaneous detection of BRCA 1/2 exon sequence and chemotherapy drug site, and application
  • Primer for simultaneous detection of BRCA 1/2 exon sequence and chemotherapy drug site, and application
  • Primer for simultaneous detection of BRCA 1/2 exon sequence and chemotherapy drug site, and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Use primer3 to design primers with a target amplicon size of 220-300bp to ensure that all primers can cover all exon regions of BRCA1 / 2. Divide the primer pairs of BRCA1 and BRCA2 into two groups to ensure that each set of primers for the same gene There is no overlap of the amplified region inside. Among them, the first and second sets are BRCA1 exon amplification primers: the first set of primers are SEQ ID NO.3-SEQ ID NO.40, and the second set of primers are SEQ ID NO.41-SEQ ID NO. 72. The third and fourth groups are BRCA2 exon amplification primers: the third group of primers are SEQ ID NO.73-SEQ ID NO.130, and the fourth group of primers are SEQ ID NO.131-SEQ ID NO.180.

[0041] Number the 4 primer pairs respectively, number 1, 2, 3...n according to the genome coordinate sequence, and sort them into odd numbered primer pairs with SEQ ID.NO1 added to the 5'end of the upstream primer, and SEQ ID.NO2 added to the 5'end of the downstream primer. , The sequence is that e...

Embodiment 2

[0054] The primers of the present invention perform multiplex PCR amplification methods as follows:

[0055] 1) One round of PCR: use any set of primers from combinations I to IV for PCR reaction, the preferred PCR program is 95°C for 5min; 95°C for 30s, 60°C for 30s, 58°C for 30s, 56°C for 30s, 70°C for 1min (3cycles); 95℃30s, 68℃1min (15cycles); 72℃5min; 4℃∞. As shown in table 2:

[0056] Table 2. The first round of PCR amplification reaction system and amplification reaction conditions

[0057]

[0058]

[0059] 2) One round of purification, the PCR product is purified by Ampure beads for the first time, and then Beadsbuffer and water are used for the second purification; specifically: mix two tubes of PCR products into one tube; add 30μL (Ampurebeads) magnetic beads and mix Let it stand for 5 minutes and then place it on a magnetic stand. After clarification, aspirate the supernatant; wash both sides with 80% ethanol and dry; re-dissolve with 15 μL of distilled water for 3 minu...

Embodiment 3

[0068] A 5mL EDTA blood collection tube was used to collect the blood of a healthy volunteer, and the QIAGEN kit was used to extract blood cell gDNA as a DNA template for multiplex PCR amplification. The multiplex PCR technique in Example 2 was used for detection.

[0069] The concentration of the measured sample is 15ng / μL. Q-Sep results are as figure 1 Shown.

[0070] Table 4, evaluation of offline data quality

[0071] Sequencing raw reads (pieces) Number of reads remaining after quality control (pieces) Proportion of remaining data after quality control 2254879209780293.03％

[0072] The samples were sequenced using the Illumina sequencer. The total number of reads measured was not 2254879. After quality control (removal of unqualified reads), there were 2097802 reads remaining, and the qualified reads accounted for 93.03% of the total reads.

[0073] Table 5, Reference sequence alignment and depth data statistics

[0074]

[0075]

[0076] After the completion of the library ...

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Abstract

The invention discloses a primer for simultaneous detection of a BRCA1/2 exon sequence and a chemotherapy drug site. The primer comprises: an exon amplification primer pair designed for BRCA1, an exonamplification primer pair designed for BRCA2, and a specific primer pair designed for an SNP site, wherein each exon amplification primer pair is divided into two groups respectively, and no amplification region overlapping exists inside each group of primers of the same gene; in each group of the primer pairs, the 5' end of an upstream primer of a first part primer is added with SEQ ID.NO1, andthe 5' end of a downstream primer is added with SEQ ID.NO2; the 5' end of an upstream primer of a second part primer is added with SEQ ID.NO2, and the 5' end of a downstream primer is added with SEQ ID.NO1; a group of the primer pair of the BRCA1 gene and a group of the primer pair of the BRCA2 gene are randomly combined to form two primer group pairs; and the 5' end of an upstream primer of the specific primer is added with SEQ ID.NO1, and the 5' end of a downstream primer of the specific primer is added with SEQ ID.NO2. The cost is low, throughput is high, a detection range is wide, and a detection time is short; and BRCA1/2 and the cancer chemotherapy drug detection site can be simultaneously detected.

Description

Technical field [0001] The present invention relates to the field of biotechnology, in particular to a primer design method / primer set / kit for detecting BRCA1 / BRCA2 gene exon regions and chemotherapeutic drug sites and applications thereof. Background technique [0002] In 1990, researchers discovered a gene directly related to hereditary breast cancer, named Breast Cancer No. 1, or BRCA1 in English. In 1994, they discovered another gene related to breast cancer, called BRCA2. In many cases, people discuss the two genes collectively as BRCA1 / 2. BRCA1 is located on chromosome 17q221.31 with a gene size of 81.19 kb and a total of 24 exons. BRCA2 is located on chromosome 13q13.1 with a gene size of 84.19 kb and a total of 27 exons. [0003] BRCA1 / 2 are two genes that inhibit the occurrence of malignant tumors, and play an important role in regulating the replication of human cells, repairing genetic material DNA damage, and normal cell growth. Families with this gene mutation tend t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6886C12N15/11
CPCC12Q1/6886C12Q2600/156C12Q2600/106C12Q2600/16
Inventor 王雨倩江凌
Owner 阔然生物医药科技(上海)有限公司
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