Simultaneous Genotyping of Single Nucleotide Polymorphisms in the Il-6, Il-10, Tnf Alpha and Tnf Beta Genes

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