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Kit and method for detecting mutation sites of BRCA 1 gene, BRCA 2 gene and PALB 2 gene

A technology of mutation sites and kits, applied in biochemical equipment and methods, DNA/RNA fragments, recombinant DNA technology, etc., can solve the problems of limited detection ability and time-consuming, and achieve short time-consuming, pollution-reducing, and high-precision high effect

Pending Publication Date: 2017-11-21
上海伯豪生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The invention is a fluorescent PCR detection method based on the ARMS-qPCR system, which can overcome the shortcomings of existing sequencing technologies such as low throughput, time-consuming, and limited detection capabilities, and has the advantages of rapidity, high throughput, and high sensitivity.

Method used

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  • Kit and method for detecting mutation sites of BRCA 1 gene, BRCA 2 gene and PALB 2 gene
  • Kit and method for detecting mutation sites of BRCA 1 gene, BRCA 2 gene and PALB 2 gene
  • Kit and method for detecting mutation sites of BRCA 1 gene, BRCA 2 gene and PALB 2 gene

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Experimental program
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Effect test

Embodiment 1

[0064] Use the plasmid templates (corresponding to each site to be tested) containing the mutation sites of exon 10 and exon 23 of the BRCA1 gene, exon 11 of the BRCA2 gene, and exon 4 of the PALB2 gene, using Table 2 The primers and probes were used for qPCR to optimize and select the best ARMS primers.

[0065] Wherein, the wild-type plasmid and the mutant-type plasmid involved in the following can be prepared according to the conventional plasmid construction method and the PCR cloning method.

[0066] 1) Plasmid treatment and extraction:

[0067] Plasmid extraction was carried out using the plasmid extraction kit of TIANGEN (HighPure Plasmid Kit, DP116). For details, please refer to the product manual. The extracted DNA was dissolved in Tris-HCl (10mmol / L, pH8.0), and the quality of the sample was detected by an ultraviolet spectrophotometer and the concentration was determined. Then, dilute the sample to 2000copies / uL. Take 5 μL for PCR reaction.

[0068] 2) Perform P...

Embodiment 2

[0086] Using the present invention to detect clinical samples, after extracting DNA from 97 blood samples, use the specific ARMS primers and fluorescent probe PCR system of the present invention to detect them, and perform sequencing verification at the same time.

[0087] Proceed as follows:

[0088] 1) Sample processing and DNA extraction:

[0089] Use a commercial DNA extraction kit to extract sample DNA, and refer to the kit instructions for specific operations.

[0090] Samples were diluted to 10ng / μL.

[0091] 2) Perform PCR amplification according to the following amplification system (total volume 40 μL)

[0092]

[0093]Wherein, the primers in the above PCR amplification are ARMS primers shown in SEQ ID NO.1-12, and the probes are the probes shown in SEQ ID NO.20-23.

[0094] In addition, 2×Premix Ex Taq (Probe qPCR) in PCR amplification was from TAKARA Company, and water was from LIFETECH Company.

[0095] 3) The PCR reaction conditions are: 95°C pre-denaturat...

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Abstract

The invention discloses a kit for detecting mutation sites of BRCA 1 gene, BRCA 2 gene and PALB 2 gene, comprising ARMS primers shown as SEQ ID NO. 1 to 12 and probes as SEQ ID NO. 20 to 23. In addition, the invention further discloses a method for detecting the mutation sites of the BRCA 1 gene, the BRCA 2 gene and the PALB 2 gene, which comprises the steps of 1) extracting a DNA (deoxyribonucleic acid) sample to be used as a DNA template; 2) performing fluorescent PCR (polymerase chain reaction) amplification on the DNA template by using the ARMS primers shown as the SEQ ID NO. 1 to 12 and the probes shown as SEQ ID NO. 20 to 23; 3) detecting the fluorescence intensity of a reaction system in the fluorescent PCR amplification, and judging the mutation of the mutation sites, namely the 10th exon and the 23th exon of the BRCA 1 gene, the 11th exon of the BRCA 2 gene and the 4th exon of the PALB 2 gene. The kit and the method provided by the invention are capable of overcoming the defects of low flux, long time consumption, limited detecting capability and the like in the existing sequencing technique, and have the advantages of quickness, high flux, good specificity, high sensitivity and the like.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a detection kit, in particular to a detection kit for detecting mutation sites of BRCA1 gene, BRCA2 gene and PALB2 gene; in addition, the present invention also relates to detection of BRCA1 gene, BRCA2 gene and PALB2 gene method of mutation. Background technique [0002] Breast cancer (mammary carcinoma) is the most important malignant tumor in women. In North America, Western Europe and other developed countries, the incidence of breast cancer ranks first in the incidence of female malignant tumors. According to the American Cancer Society, in 2003, 124.2 out of every 100,000 women in the United States developed breast cancer. In recent years, the incidence of breast cancer in my country has also increased significantly. The incidence of breast cancer in China has increased at a rate of 3% per year, becoming the fastest growing tumor. Shanghai, Beijing, Tianjin and co...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6886C12Q1/686C12Q2600/156C12Q2535/137C12Q2563/107C12Q2545/114
Inventor 徐晓晶赵莹贺华姚琴琴陆凌佳
Owner 上海伯豪生物技术有限公司
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