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Method and primer for detecting mutation sites of all exon sequences of human BRCA1 and BRCA2 genes

A mutation site and whole exon technology, which is applied in the field of detection of mutation sites in the whole exon sequence of human BRCA1 and BRCA2 genes, can solve the problems of inability to sequence the coding sequences of BRCA1 and BRCA2 genes, so as to reduce the risk of disease and improve the sensitivity The effect of high, high specificity and accuracy

Active Publication Date: 2015-04-22
上海润达榕嘉生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] Domestic manufacturers have carried out sequencing of breast cancer-related BRCA1 and BRCA2 gene mutations, but most of the tests only sequence the partial sequences in the BRCA1 and BRCA2 genes, and it is not yet possible to sequence the entire coding sequences of the BRCA1 and BRCA2 genes

Method used

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  • Method and primer for detecting mutation sites of all exon sequences of human BRCA1 and BRCA2 genes
  • Method and primer for detecting mutation sites of all exon sequences of human BRCA1 and BRCA2 genes
  • Method and primer for detecting mutation sites of all exon sequences of human BRCA1 and BRCA2 genes

Examples

Experimental program
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Effect test

Embodiment 1

[0041] Primer sequences for detection of exon mutations in BRCA1 and BRCA2 genes

[0042] (1) Primers for detection of BRCA1 gene exon mutation sites

[0043] Including the sequence shown in SEQ ID NO: 001-062, it is the forward and reverse primers for amplifying and covering the exon mutation site of BRCA1 gene.

[0044] In the detection, first use the above-mentioned forward and reverse primers to amplify the DNA fragment covering the exon mutation site of BRCA1 gene to obtain the amplified products, and then use the above-mentioned amplification primers to sequence the amplified products respectively to obtain the amplified The gene sequence of the augmented product.

[0045] (2) Primers for detection of BRCA2 gene exon mutation sites

[0046] Including the sequence shown in SEQ ID NO: 063-142, it is the forward and reverse primers for amplifying and covering the exon mutation site of BRCA2 gene.

[0047] In the detection, first use the above-mentioned forward and revers...

Embodiment 2

[0049] Kit for detecting mutation sites in exons of BRCA1 and BRCA2 genes

[0050] Including: tissue DNA extraction kit (for example, using Capgemini’s extraction kit); absolute ethanol; detection system PCR reaction solution, sequencing system reaction solution, positive control substance, negative control substance and blank control substance. The detection system PCR reaction solution includes: 2x PCR Buffer; 2mM dNTPs; Roche DNA Polymerase (1U / μl); forward and reverse primers covering the whole exons of BRCA1 and BRCA2 genes shown in SEQ ID NO: 001-142.

[0051] The sequencing system includes: sequencing purification solution, EDTA (1.25mmol), absolute ethanol, 75% ethanol, HIDI (highly deionized formamide), covering all exons of BRCA1 and BRCA2 genes shown in SEQ ID NO: 001-142 Forward and reverse amplification primers of 3.2 μm each, and Bigdye Terminator V3.1 (purchased from Applied Biosystems, USA), wherein the sequencing purification solution includes shrimp alkaline ...

Embodiment 3

[0053] Method for detecting BRCA1 gene and BRCA2 gene mutation sites

[0054] (1) Genomic DNA extraction from blood:

[0055] 1) Draw 500 μL of blood and add 1000 μl of red blood cell lysate, invert and mix well, and place at room temperature for 5 min, during which time, invert and mix several times. Centrifuge at 3000rpm for 5min, suck off the supernatant, leave the white blood cell pellet, add 200μl buffer GA, shake until thoroughly mixed.

[0056] 2) Add 20 μl proteinase K solution and mix well.

[0057] 3) Add 200 μl of buffer solution, mix thoroughly by inversion, place at 70°C for 10 minutes, the solution should become clear, and briefly centrifuge to remove water droplets on the inner wall of the tube cap.

[0058] 4) Add 200 μl of absolute ethanol, vortex fully for 15 seconds, at this time flocculent precipitation may appear, briefly centrifuge to remove water droplets on the inner wall of the tube cap.

[0059] 5) Add the solution and flocculent precipitate obtain...

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Abstract

The invention relates to the technical field of gene detection, and provides a method and a primer for detecting mutation sites of all exon sequences of human BRCA1 and BRCA2 genes. The specific primers include forward and backward primers for amplifying all 24 exons covering the BRCA1 gene, and base sequences of the primers are as shown in SEQ ID NO: 001-062, as well as forward and backward primers for amplifying all 27 exons covering the BRCA2 gene, and base sequences of the primers are as shown in SEQ ID NO: 063-142. By virtue of a Sanger sequencing method, the method and the primer disclosed by the invention can be used for detecting the mutation of all exons of the BRCA1 gene and the BRCA2 gene and can be used for extending all exons of the entire BRCA1 and BRCA2 genes, covering all to-be-detected mutation sites. The method and the primer disclosed by the invention are quite high in specificity and accuracy, simple to operate and low in cost, and can be used for greatly enhancing the risk assessment of hereditary breast cancer and effectively reducing the onset risk of the breast cancer.

Description

technical field [0001] The invention relates to the technical field of gene detection, in particular to a method and primers for detecting mutation sites in whole exon sequences of human BRCA1 and BRCA2 genes. Background technique [0002] Breast cancer is one of the most common malignant tumors in women. Every year, there are 1.3 million new breast cancer patients worldwide, and more than 460,000 patients die of breast cancer. In recent years, the incidence of breast cancer in my country ranks first among female malignant tumors, and it is increasing year by year at a rate of more than 3%. It is conservatively estimated that more than 40,000 women die from this disease every year in the country. It is currently believed that most hereditary breast cancers are caused by gene mutations, among which the tumor suppressor genes BRCA1 and BRCA2 are closely related to the onset of breast cancer. Studies have confirmed that the probability of developing breast cancer before the age...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6869C12Q2535/101
Inventor 钱学庆
Owner 上海润达榕嘉生物科技有限公司
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