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
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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.
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