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(rsl 800849) 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.

作 者： WU Mei DU Zhen-wu LIU Jia-nan SONG Yang WANG Ya-li ZHANG Gui-zhen   作者單位： Department of Central Research,China-Japan Union Hospital of Jilin University,Changchun 130033,P.R.China  刊 名： 高等學(xué)校化學(xué)研究（英文版）  ISTIC SCI 英文刊名： CHEMICAL RESEARCH IN CHINESE UNIVERSITIES  年，卷(期)： 2010 26(2)  分類號： O6  關(guān)鍵詞： AS-PCR   Polymorphism   UCP3   SNP genotyping  

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