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Rapid detection of BRCA (Breast Cancer) genic mutation

A gene and exon technology, applied in the fields of biotechnology and medicine, can solve problems such as pollution, low accuracy, and cumbersome detection procedures of BRCA gene mutations

Active Publication Date: 2010-12-22
JIANGSU MICRODIAG BIOMEDICINE TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] The purpose of the present invention is to provide a rapid, accurate, easy-to-operate, and effectively pollution-free detection kit for BRCA gene mutations, which solves the cumbersome, expensive, time-consuming, and low-precision detection procedures for BRCA gene mutations in the prior art. and pollution issues

Method used

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  • Rapid detection of BRCA (Breast Cancer) genic mutation
  • Rapid detection of BRCA (Breast Cancer) genic mutation
  • Rapid detection of BRCA (Breast Cancer) genic mutation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0090] Design, synthesis and verification of embodiment 1 primer

[0091] In this embodiment, Primer 5 is used to design primers. First, the size of the primer fragments is optimized to obtain the electropherograms of PCR amplification of primers with different fragment sizes. The results are as follows figure 1 and as shown in the table below. figure 1 a is the electropherogram of the primer PCR amplification of 5-9bp, figure 1 B is the electropherogram of the primer PCR amplification of 13-16bp, figure 1 c is the electropherogram of the primer PCR amplification of 18-24bp, figure 1 d is the electropherogram of the primer PCR amplification of 25-29bp, figure 1 e is the electropherogram of the primer PCR amplification of 30-37bp, figure 1 f is the electropherogram of PCR amplification with 39-45bp primers. from the table below or figure 1 It can be seen that the optimal primer is a primer with a size of 18-24bp.

[0092] Primer size (bp)

5-9

13-16 ...

Embodiment 2

[0095] The making of embodiment 2 negative or positive control DNA

[0096] Extraction of Negative Control DNA:

[0097] sampling:

[0098] 1. Take 2.5ml of peripheral blood from normal people or people with known mutations and put it in a sodium citrate (1:9) anticoagulant tube.

[0099] 2. The blood in the anticoagulant tube was centrifuged at 2000rpm for 5 minutes to separate the blood cells and washed twice with PBS.

[0100] 3. DNA extraction. The blood was extracted with the Blood Mini kit (manufactured by QIAGEN, Germany). The extraction steps were carried out according to the instructions of the kit. Other related products were treated in the same way, and the extracted DNA was stored at -20°C.

[0101] Quantification: use Nano 1000 quantifier to measure DNA concentration,

[0102] Eligibility index: 1, in line with the requirements of OD value A260 / A230: 2.0-2.2; A260 / A280: 1.8-2.0, and then quantitatively dilute the DNA to 10ng / ul

[0103] Electrophoresis identi...

Embodiment 3

[0104] Example 3 Extraction of Tumor Tissue DNA

[0105] Sample collection:

[0106] Take 50-100 mg of fresh tissue block and wash it with PBS or normal saline. The tissue used must be cut to a thickness of less than 0.5 cm, put into a cryopreservation tube containing 1.5 ml of RNAlater, and mix well. (Complete within 30 minutes) Store at room temperature for 1-2 hours, freeze overnight at 4°C, and transfer to -20°C for long-term storage the next day.

[0107] DNA extraction;

[0108] DNA was extracted using the TIANamp Genomic DNA Kit (TIANGEN BIOTECH CO., LTD) Genomic DNA Extraction Kit and following the kit instructions.

[0109] DNA purification: (if the extracted DNA, OD value A260 / A230: 2.0-2.2; A260 / A280: 1.8-2.0, is not within the standard range, this step is required)

[0110] Add an equal volume of chloroform-isoamyl alcohol (25:1), turn down the centrifuge tube for 5 minutes to mix well, and centrifuge at 13000rpm for 10 minutes to carefully suck out the supernat...

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Abstract

The invention discloses a PCR (Polymerase Chain Reaction) kit for detecting BRCA (Breast Cancer) genic mutation, which is characterized by comprising a plurality of primer pairs used for specifically amplifying a BRCA gene targeting sequence, wherein each primer pair contains a continuous nucleotide sequence formed by at least 15 continuous nucleotides in the 2nd exon of a BRCA1 gene or the 20th exon of the BRCA1 gene or the 11th exon of a BRCA2 gene; the 2nd exon of the BRCA1 gene has a continuous nucleotide sequence of SEQ ID No:1; the 20th exon of the BRCA2 gene has a continuous nucleotide sequence of SEQ ID No:2; and the 11th exon of the BRCA2 gene has a continuous nucleotide sequence of SEQ ID No:3. The kit completes the judgment on the sample genotype by adopting a saturation probe and a high resolution dissolution curve analyzing technology, thereby providing the guidance for medication selection and genetic predisposition of familial breast cancer and ovarian cancer.

Description

technical field [0001] The invention relates to the fields of biotechnology and medicine, in particular to a rapid detection of BRCA gene mutations related to tumor drug selection and genetic susceptibility. Background technique [0002] Human breast cancer susceptibility genes (BRCA1 / 2) were first discovered in breast cancer families, and are susceptibility genes for breast and ovarian cancer with genetic predisposition. BRCA1 is located on human chromosome 17 q21. It is inherited in an autosomal dominant manner and has a high penetrance. BRCA1 is about 100kb long and contains 24 exons. Its gene product is a phosphorylated protein composed of 1863 amino acids. BRCA2 is located on chromosome 13 q12. The whole genome DNA is about 70kb in length. The coding region contains 10987bp. It is rich in ATf about 64%1, and its gene sequence has no obvious relationship with BRCA1. BRCA2 consists of 27 exons. The 11th exon is about 4932bpmRNA and about 10.2kb in length. The encoded BR...

Claims

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Application Information

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IPC IPC(8): C12Q1/68G01N21/64
Inventor 王弢陈菲秦勇王秀娟
Owner JIANGSU MICRODIAG BIOMEDICINE TECH CO LTD
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