Department of Medical Genetics, Pathology and Oncology, National Taiwan University Hospital and Graduate Institutes of Clinical Medicine and Cancer Research Center, National Taiwan University, Taipei, Taiwan.
Abstract
Single nucleotide polymorphisms (SNP) in the human IL-6, IL-10, TNFalpha and TNFbeta genes have been associated with gene function and susceptibility to disease. In this study, primers containing mismatches at 1-3 nucleotide positions were designed to incorporate a new restriction site recognized by endonucleases AlwNI, BcgI, BglI, BsaBI, BslI, BstXI, EcoNI or XcmI for genotyping SNPs in the IL-6 gene (position - 174), IL-10 gene (positions -592 and -1082), TNFalpha gene (positions -238, - 308 and -863) and TNFbeta gene (position + 249) by mismatched polymerase chain reaction and restriction fragment length polymorphism (PCR/RFLP). Our results show that appropriately designed BslI-based mismatched PCR/RFLP assays can be successfully used to determine the genotypes for approximately 40% of SNPs. The mismatched PCR strategy can be coupled with multiplex-amplification to enable simple and rapid determination of several SNP genotypes in a single reaction.