Gene mutant and application thereof
A gene and nucleic acid technology, applied in the field of gene mutant constructs and recombinant cells, can solve problems such as protein loss, repair of DNA damage, chromosome instability, etc.
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Embodiment 1
[0058] Example 1 Detection of BRCA2 gene mutation c.2389delA in hereditary breast cancer
[0059] 1. Sample information: a family with hereditary breast cancer, including a proband and 5 family members, see Table 2 for specific information
[0060] Main instrument information: pipette, PCR instrument, centrifuge, vortex shaker, electrophoresis instrument, 2.0, DNA interrupter, magnetic frame, dry constant temperature metal bath, Complete Genomics sequencer
[0061] Main reagents: QIAGEN Blood DNA Extraction Kit, Complete Genomics platform library building reagents, liquid phase probe of about 600Kb target region in the human genome (hg19) (Roche, Nimblegen EZ chip)
[0062] 2. Sample collection
[0063] Hereditary breast cancer family contains 5 members, including 2 breast cancer patients ( figure 2 . A breast cancer patient in the family was selected as a region capture sequencing sample (Table 1), and 5 ml of peripheral blood samples were collected from each sample, an...
Embodiment 2
[0086] Example 2 Sanger method sequencing to verify the pathogenic mutation of breast cancer
[0087] Due to a certain degree of false positives in the sequencing of the target region, the inventors used the Sanger sequencing method to verify the newly discovered mutation sites that may have pathogenic significance.
[0088] Design primers for the sequence of the new mutation site detected in Example 1, and then obtain the relevant sequence of the above mutation by PCR amplification, product purification and sequencing. Mutation or homozygous mutation, and the correlation between sequence and phenotype and breast cancer.
[0089] The specific method steps are as follows:
[0090] 1. DNA extraction
[0091] According to the method for extracting DNA described in Example 1, the genomic DNA in the peripheral venous blood of subjects (including breast cancer patients and normal people) was extracted and prepared respectively, and set aside.
[0092] 2. Primer design and PCR rea...
Embodiment 4
[0113] Example 4 Analysis of relevant family members and family history carried by the mutation
[0114] Using PCR combined with Sanger sequencing method, the family members of the proband with a breast cancer susceptibility gene mutation were verified for the corresponding mutation site to determine whether the mutation site was co-segregated with the disease. The family diagram is as follows figure 2 shown.
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