Improved Allele-specific Polymerase Chain Reaction for Single Nucleotide Polymorphism Genotyping

An improved allele-specific PCR(AS-PCR) approach was applied to investigating -55C/T polymorphism in promoter region of the uncoupling protein 3(UCP3)gene. AS-PCR is a competitive PCR method which is based on positioning the 3' base of a PCR primer to match one single nucleotide polymorphism(SNP) allele and accurately extend only the correctly matched primer. But it is limited in use because of its poor specificity. In this study, we improved the specificity of AS-PCR by introducing additional mismatch at the penultimate base of 3' end of AS-PCR primer in combination with decreasing the level of dNTP in the reaction mixture. Sensitivity, specificity and reliability of this method were assessed for both simple plasmid model and complex human genomic SNP targets. The -55C/T(rs1800849) polymorphism of the UCP3 gene was analyzed via this AS-PCR and restriction fragment length polymorphism(RFLP), the latter was used as a gold standard. The results suggest that the increase in AS-PCR discrimination with this method should facilitate the use of this simple, rapid, and inexpensive technique for SNP genotyping application.