Method for detecting mononucleotide polymorphism by conformational difference gel electrophoresis
A technology of single nucleotide polymorphism and gel electrophoresis, which is applied in the direction of biochemical equipment and methods, and the determination/inspection of microorganisms. The effect of easy operation
Inactive Publication Date: 2012-11-07
NANCHANG UNIV
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Compared with ordinary polyacrylamide gel electrophoresis, conformation-sensitive gel electrophoresis uses an expensive cross-linking agent 1,4-bisacryloylpiperazine, a denaturant is added to the gel, and 0.5 ×TTE is used as the electrophoresis buffer, which increases the cost of the experiment, and the electrophoresis time is longer (usually 16h)
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[0026] like figure 1 , figure 2 , image 3 , Figure 4 As shown, oral swabs were provided by volunteers. After each person rinsed their mouths with clean water, they wiped the buccal mucosa 10 times with a sterile cotton swab, and stored them in a 5ml sterile centrifuge tube at room temperature. Genomic DNA was extracted from buccal swabs using the salting-out method according to the literature, and 50 μL TE was added to dissolve them. According to the results of agarose gel electrophoresis, the DNA was diluted to 10 ng / μl as a template for PCR amplification.
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A method for detecting mononucleotide polymorphism by conformational difference gel electrophoresis. The method comprises the following specific steps: (1) collecting samples; (2) extracting DNA of a sample genome; (3) selecting single nucleotide polymorphisms (SNPs) associated with disease occurrence, drug efficacy and toxicity for a polymerase chain reaction (PCR ) amplification; (4) carrying out conformational difference gel electrophoresis analysis on PCR products, and determining different genotypes according to position and number of bands; and (5) carrying out DNA sequencing on PCR products of samples with different band types, and determining specific genotypes of different samples. According to the invention, the analysis of SNPs is analysis of double-stranded PCR products, without requiring endonuclease; samples can be loaded directly for gel electrophoresis without any prior processing; and no expensive equipment or reagent is required; therefore the method has advantages of simple operation and low cost. This invention provides a fast, accurate, simple, and low-cost detection means for SNPs related to disease susceptibility and individualized drug therapy, and lays foundation for clinical application of the method.
Description
technical field [0001] The invention relates to a method for detecting single nucleotide polymorphisms, in particular to a method for detecting single nucleotide polymorphisms by conformational difference gel electrophoresis. Background technique [0002] With the completion of the Human Genome Project, more and more studies have begun to focus on the application of genetic polymorphisms in medicine. As the third generation of genetic markers, single nucleotide polymorphisms may be closely related to the occurrence, development and prognosis of diseases, and may also be related to the efficacy and side effects of drugs during disease treatment. Therefore, accurate typing of SNPs Contribute to the early diagnosis, prevention and treatment of diseases. [0003] At present, there are many SNPs typing methods, but some methods need to go through a complicated process, some methods require specific equipment, some methods require expensive reagents, and some methods require skil...
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IPC IPC(8): C12Q1/68
Inventor 朱伟锋邓燕揭克敏万福生易敬林张泉罗达亚余乐涵
Owner NANCHANG UNIV