Primers, kit and method for detection of human BRCA1 and BRCA2 gene mutation

A gene and primer pair technology, which is applied in the field of primers and kits for detecting human BRCA1 and BRCA2 gene mutations, can solve the problems that BRCA1 cannot be completely covered, gene detection cannot be used, and experimental operations are cumbersome. The effect of fast speed and high detection throughput

Active Publication Date: 2016-05-18
AMOY DIAGNOSTICS CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] At present, there are many methods for detecting BRCA1/2 gene mutations, mainly including: fluorescent quantitative PCR technology, which has high sensitivity and strong specificity, but it can only detect one type of mutation each time, and cannot completely cover the entirety of BRCA1 and BRCA2 genes. Coding region; restriction small fragment length polymorphism analysis (RFLP method), used to detect genes with altered restriction site

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  • Primers, kit and method for detection of human BRCA1 and BRCA2 gene mutation
  • Primers, kit and method for detection of human BRCA1 and BRCA2 gene mutation
  • Primers, kit and method for detection of human BRCA1 and BRCA2 gene mutation

Examples

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Embodiment 1

[0050] Example 1: Detection of BRCA1 and BRCA2 gene mutations

[0051] Using the system of the present invention to detect 100 healthy voluntary blood donor whole blood samples, the method for detecting BRCA1 / 2 gene mutation using the above two-step PCR amplification is as follows:

[0052] (1) DNA extraction and quality control

[0053] AmoyDxBloodDNAKit was used to extract genomic DNA from whole blood, and AmoyDxFFPEDNAKit was used to extract genomic DNA from paraffin wax sections. The specific steps were according to the kit instructions. The extracted DNA was dissolved in Tris-HCL, and the quality of the extraction was detected by a UV spectrophotometer. After the DNA concentration of the sample was detected by Qubit2.0, each sample was diluted to a concentration of 5 ng / μL.

[0054] (2) The first step of PCR amplification:

[0055] The first step PCR amplification reaction system is

[0056]

[0057] Description: The specific primer mixture with Tag sequence (8 tube...

Embodiment 2

[0089] Embodiment 2: Detection of breast cancer or ovarian cancer patient experiment

[0090] 5 peripheral blood samples from patients with breast cancer or ovarian cancer, 2 BRCA1 or BRCA2 gene positive cell lines, respectively BT474 and HCT15 (both can be purchased from ATCC).

[0091] (1) The method for detecting BRCA1 or BRCA2 gene mutation by two-step amplification method is the same as that in Example 1.

[0092] (2) The samples (whole blood, paraffin section) of 5 breast cancer / ovarian cancer patients are shown in Table 3. In Table 3, the mutation name is gene number + exon number + mutated base sequence + amino acid change, the gene name refers to the gene where the detected site is located, and the chromosome refers to the chromosome position where the detected site is located. The initial position refers to the initial position on the chromosome where the detected site is located, and the RS number is the unified number of the SNP database on the NCBI website.

[0...

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Abstract

The invention discloses primers, a kit and a method for detection of human BRCA1 and BRCA2 gene mutation. The upstream primers are in a sequence shown as SEQ ID NO: 1,3,5,7,9-193, and meanwhile the universal primer Tag1 is added to the 5' end of the sequence; the downstream primers are in a sequence shown as SEQ ID NO: 2,4,6,8,10-194, and meanwhile the universal primer Tag2 is added to the 5' end of the sequence. The upstream primers with a P5 sequence and a sequence shown as SEQ ID NO: 221-236 and the downstream primers with a P7 sequence and a sequence shown as SEQ ID NO: 197-220 are also included. Through the primers, the kit and the method, mutation of all exons of BRCA1 and BRCA2 genes, a connection region between the exons and introns, an untranslated region and a promoter region can be detected rapidly, accurately, easily and conveniently.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to primers, kits and methods for detecting human BRCA1 and BRCA2 gene mutations. Background technique [0002] Breast cancer and ovarian cancer are common malignant tumors in women. The lifetime risk of breast cancer and ovarian cancer in the general population is 13% and 1.5%, respectively. Increased at a rate of more than 3% year by year (AntoniouA, PharoahPD, NarodS, et al. At present, it is believed that most hereditary breast cancers are caused by gene mutations, and the mutations of BRCA1 and BRCA2 genes are closely related to the onset of breast cancer. Studies have shown that women carrying BRCA1 or BRCA2 gene pathogenic mutations have a 5-30-fold increased risk of disease after the age of 70, as high as 75% and 46% respectively (CaoW, WangX, LiJC. Hereditary breast cancer the Han Chinese population. J Epidemiol. 2013; 23:75–84). [0003] The BRCA1 gene is located on human ch...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6869C12Q1/6886C12Q2600/156C12Q2600/16C12Q2531/113C12Q2535/122
Inventor 涂泽华林清华翁颖康雅君葛会娟李旭超阮力郑立谋
Owner AMOY DIAGNOSTICS CO LTD
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