Detection Of Single Nucleotide Polymorphisms By Conformation-Difference Gel Electrophoresis And Its Clinical Application

With the accomplishment of Human Genome Project, more and more researches are being aware of the importance for the application of gene variation on medicine. Baccal mucosa epithelial cells are most common sources for DNA extraction in molecular epidemiology, forensic medicine, and genetic studies. Although there are different methods for DNA extraction from buccal mucosa epithelial cells, none of which can meet the standards of safety, rapidity, convenience, economy and reliability. Salting out method is a commonly used method for genomic DNA extraction from blood, which is characteristic for its lots of advantages. Whether salting out method can be used for genomic DNA extraction from buccal mucosa epithelial cells remains uncertain.Single nucleotide polymorphisms (SNPs), as one of the most common genetic variation in human genome, gains more and more attention on its relationship with the occurrence, development and prognosis of diseases, drugs efficacy and toxic side effects during disease therapy. Therefore, accurate detection of SNPs is helpful for early diagnosis and appropriate treatment of disease. There are many SNPs genotyping methods in which either complicated process, specific equipment, expensive reagents or experienced skills are needed. Therefore, if normal polyacrylamide gel electrophoresis (PAGE) can be applied for directly SNPs genotyping, it will significantly reduce cost and increase detection efficiency.Objects:To establish a rapid, simple, economic and reliable method for genomic DNA extraction from buccal mucosa swabs.To set up an accurate, simple, fast, low-cost and high-throughput SNPs genotyping method which can be applied in normal laboratories.Clinical samples would be analysed by the new method.Methods:10buccal mucosa swabs from volunteers in our lab were collected and DNA extracted with reagents made ourselves. Buccal mucosa epithelial cells were digested with cell lysate solution and proteinase K solution. Then the proteins were removed by salting out and centrifugation and DNA was precipitated with isopropyl alcohol, The precipitations of DNA were washed with70%ethanol, resuspended in TE. DNA was detected by UV spectrophotometer. The rs1042522loci of TP53gene and rs12910984loci of CHRNA3gene were genotyped by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique. The samples with different genotypes were confirmed by direct sequencing analysis.Genomic DNA was extracted from130volunteers’ baccal swabs with previously well constructed salting out method.13SNPs (MTHFR gene A1298C, CYP19A1gene rs10046and rs4646, CYP3A5gene A6986G, TP53gene rs1042522, NQO1gene C609T, GCKR gene rs780094, RGS4gene rs951436, CHRNA3gene rs12910984, MYOC-1081A/G and-998C/G, MDR1gene3489+59T>G and3489+80C>T) were analyzed by PAGE.5of these13SNPs (TP53gene rs1042522, GCKR gene rs780094, NQO1gene rs1800566, MTHFR gene A1298C, and CHRNA3gene rs12910984), where had endonuclease restriction sites, were digested by restriction endonucleases. The own factors of DNA fragments and the conditions of PCR amplification and electrophoresis which might affect SNPs genotyping were optimized, to set up a method to analyze SNPs using normal PAGE. Based on the optimization of detecting conditions,13SNPs were detected. For high-throughput detection, samples were multiplex amplificated and loaded into10layers on1single gel. SNPs were confirmed by direct sequencing analysis. Then the results of PAGE, enzyme digestion and direct sequencing analysis were compared with each other.3SNPs(MTHFR gene A1298C、MDR1gene3489+59T>G and3489+80CT) were genotyped in104advanced gastric cancer samples in the treatment of fluorouracil based chemotherapy with the new method. The relationship between genotypes and response rate of chemotherapy were analysed.Results:Time and cost for the established salting out method for DNA extraction from baccal swabs were about1h and1.5Yuan per single buccal swab, respectively. The DNA yield of single buccal swab ranged from0.68μg to2.56μg; the OD26o/OD28o value ranged from1.77to1.94. After PCR amplification and enzyme digestion, two SNPs of10samples were clearly genotyped. The results of PCR-RFLP agreed well with the results of direct sequencing.5SNPs of enzyme digesting samples were clearly genotyped. The optimization results of analyzing SNPs with PAGE showed that, GC content and base composition had little effect on the electrophoresis, while the positions of SNPs in fragments and the length of amplified fragments might be the key influencing factors of SNPs genotyping with PAGE. For PCR amplification, template amount, DNA polymerase, annealing temperature and number of cycles might all affect the results of SNPs genotyping. Becaused this SNPs genotyping method is based on conformational differences, we call it conformation-difference gel electrophoresis (CDGE).13SNPs with CDGE showed that8of them were genotyped directly, while2SNPs could not directly be genotyped, and3SNPs were not clear due to the small sample size. In addition, one new SNPs was found in CHRNA3gene. Based on the results of electrophoresis with sample-loading10layers of NQO1C609T PCR pruducts of10samples, it showed that at least sample-loading with7layers could clearly separate three kinds of genotypes. The results of multiplex amplification of4SNPs were also clear. Due to the good results of direct sequencing, it was confirmed that the amplified fragments were the target ones. Based on the results of CDGE agreeing well with the results of enzyme digestion and direct sequencing, it confirmed the reliability of CDGE for SNPs genotyping. In comparision with enzyme digestion, CDGE did not need restriction endonucleases and was low-cost. And in contrast with single-strand conformation polymorphism (SSCP), CDGE was low-cost and convenient owing to no need for denature of PCR products and direct sample loading after PCR amplification.Different genotypes of3SNPs were distinguished clearly on CDGE.The genotypes distribution of3SNPs were accorded with Hardy-Weinberg equilibrium. Of all the104cases, the overall response rate was41.3%. The relationship between different genotypes and the response rate:①MTHFR gene A1298C, the response rates to chemotherapy of AA, AC, CC genotypes were34.8%,50.0%and66.7%, respectively.②MDR1gene3489+59T>G, the response rates to chemotherapy of GG, GT, TT genotypes were34.8%,50.0%and66.7%, respectively.③MDR1gene3489+80C>T, the response rates to chemotherapy of CC, CT, TT genotypes were27.3%,36.2%and48.9%, respectively. No significant difference in response rate to chemotherapy was observed according to the3SNPs genotypes.Conclusions:High quality DNA can be rapidly obtained with salting out method from baccal swabs and applied in molecular epidemiology, forensic medicine, and genetic studies.CDGE is an accurate, simple, fast, low-cost and high-throughput SNPs genotyping method which can be used in normal laboratories.No significant difference in response rate to advanced gastric cancer samples in the treatment of fluorouracil based chemotherapy was observed according to the MTHFR gene A1298C, MDR1gene3489+59T>G and3489+80C>T genotypes.