Method for detecting mutant gene
An allele and gene polymorphism technology, applied in the allele field, can solve the problems of mutual pollution, complicated operation, time-consuming, etc., and achieve the effect of clinical application.
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Embodiment 1
[0074] High-sensitivity detection of the T790M mutant allele in exon 20 of the EGFR gene (1)
[0075] By using the DNA extracted and purified from the NCI-H1975 cell line as the DNA of the T790M mutation of exon 20 of the human EGFR gene, and using the DNA extracted and purified from the K562 cell line as the DNA without the mutation, it was evaluated to ensure that the DNA designed according to the present invention Concentration range of the first primer set for measuring system performance.
[0076] Primer
[0077] The first primer set (SEQ ID NO:3 and 4) for the first PCR reaction was set in the region flanking the T790M mutation, and its reverse primer was designed to match the second primer set (SEQ ID NO:4 and 5) A common primer for the reverse primer. The forward primer of the second primer set is an allele-specific primer corresponding to the T790M (2369C->T) mutation, and has the base (T) from the 3' end corresponding to the mutated nucleotide of the mutation. The...
Embodiment 2
[0105] High-sensitivity detection of the T790M mutant allele in exon 20 of the EGFR gene (2)
[0106] By using DNA extracted and purified from the NCI-H1975 cell line as the DNA of the T790M mutation of exon 20 of the human EGFR gene, the assay sensitivity of the assay system designed according to the present invention was compared with that of the assay system that did not include the forward primer used for the first PCR. The conventional ASP-PCR method was compared.
[0107] ·Preparation of template DNA
[0108] The template of this example was prepared and used so that the concentration of DNA (mutant) extracted from the NCI-H1975 cell line was 0.01 ng / μL and 0.003 ng / μL.
[0109] ·Reagents
[0110] Twenty-five (25) μL of reaction solutions containing the following reagents were prepared and amplified using a thermal cycler device CFX96 (Bio-Rad).
[0111] (1) ASP-PCR method (conventional technology)
[0112] [Table 2]
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[0114]
[0115] (2) In the case ...
Embodiment 3
[0128] High-sensitivity detection of the T790M mutant allele in exon 20 of the EGFR gene (3)
[0129] By using the DNA extracted and purified from the NCI-H1975 cell line as the DNA with the T790M mutation of exon 20 of the human EGFR gene, and the DNA extracted and purified from the K562 cell line as the DNA without the mutation, the DNA designed according to the present invention was evaluated. The measurement sensitivity of the measurement system.
[0130] ·Preparation of template DNA
[0131]The template DNA of this example was prepared by mixing together DNA extracted from the NCI-H1975 cell line (mutant, mu) and DNA extracted from the K562 cell line (wild) in respective ratios so that the total concentration was 30 ng / μL . For the respective preparations, wild-derived DNA and mutant-derived DNA were mixed at mixing ratios (mu / wild %) of 0.1%, 0.03%, 0.01% and 0% (control). In addition, DNA (mutation; mu) extracted from the NCI-H1975 cell line was prepared at 0.003 ng / ...
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