Kit and method for detecting KRAS genetic mutation
A kit and gene technology, applied in biochemical equipment and methods, microbial measurement/inspection, fluorescence/phosphorescence, etc., can solve the problems of complex operation, difficult clinical diagnosis products, low sensitivity, etc., and achieve detection sensitivity and specificity High, avoid cross-contamination, low reagent cost effect
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[0027] Example 1. Detection of the G12D (GGT>GAT) mutation at codon 12 of the KRAS gene in clinically collected colon cancer tumor tissue samples
[0028] In this embodiment, the method of the present invention is used to detect whether the gene of colon cancer tumor tissue sample is mutated at codon 12. The experimental plan is as follows:
[0029]1. Sample processing and genomic DNA extraction: accept colon cancer tumor tissue samples from clinical surgical resection, cut the tumor tissue into small pieces, and perform genomic DNA extraction according to the instructions of the QIAamp? DNA Mini Kit (Qiajie Biotechnology (Shanghai) Co., Ltd.) extract. Adjust the DNA concentration to 4 ng / μL and store at -20°C for later use.
[0030] 2. Specific amplification of sample DNA KRAS gene (LightCycler 480 real-time fluorescent quantitative PCR instrument of Roche Diagnostic Products (Shanghai) Co., Ltd.):
[0031] Preparation of reaction system:
[0032] ingredient...
Example Embodiment
[0043] Example 2. Detection of KRAS gene mutation at codon 13 G13D (GGC>GAC) in paraffin tissue samples of rectal cancer collected clinically
[0044] In this embodiment, the method of the present invention is used to detect whether the gene of the rectal cancer tumor tissue sample is mutated at codon 13. The experimental plan is as follows:
[0045] 1. Sample processing and genomic DNA extraction: accept paraffin tissue samples of rectal cancer from the clinic, cut the samples into 5 μm, and perform genomic DNA extraction according to the instructions of the QIAamp? DNA FFPE Tissue Kit (Qiagen Biotechnology (Shanghai) Co., Ltd.) . Adjust the DNA concentration to 2 ng / μl and store at -20°C for later use.
[0046] 2. Specific amplification of sample DNA KRAS gene (LightCycler 480 real-time fluorescent quantitative PCR instrument of Roche Diagnostic Products (Shanghai) Co., Ltd.):
[0047] Preparation of reaction system:
[0048] ingredients volume 2...
Example Embodiment
[0059] Example 3. Detection of KRAS gene mutation at codon 12 G12C (GGT>TGT) in clinically collected non-small cell lung cancer tumor tissue samples
[0060] In this embodiment, the method of the present invention is used to detect whether a mutation occurs at codon 13 in a tumor tissue sample of non-small cell lung cancer. The experimental plan is as follows:
[0061] 1. Sample processing and genomic DNA extraction: accept paraffin tissue samples from clinical non-small cell lung cancer, cut the samples into 5 μm, and perform genome analysis according to the instructions of QIAamp? DNA FFPE Tissue Kit (Qiaagen Biotechnology (Shanghai) Co., Ltd.) DNA extraction. Adjust the DNA concentration to 5 ng / μL and store at -20°C for later use.
[0062] 2. Specific amplification of sample DNA KRAS gene (LightCycler 480 real-time fluorescent quantitative PCR instrument of Roche Diagnostic Products (Shanghai) Co., Ltd.):
[0063] Preparation of reaction system:
[0064] ...
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