Simple and rapid detection of uncoupling protein-2 −866G/A polymorphism by mutagenically separated polymerase chain reaction

Abstract

Background: Uncoupling protein-2 (UCP2), a recently identified member of the mitochondrial transporter superfamily, is a candidate gene for obesity. A common G/A polymorphism in the UCP2 promoter region is associated with enhanced adipose tissue mRNA expression in vivo. Methods: We developed a rapid and simple method, mutagenically separated polymerase chain reaction (MS-PCR) for genotyping UCP2 −866G/A polymorphism. Two reverse mutagenic allele-specific primers of different lengths for the UCP2 −866G/A polymorphic site were paired with the same forward primer in the same PCR reaction. Results: Agarose gel electrophoresis (3.5%) showed at least one of the two allelic products and provided a within-assay quality control to exclude false-negative results. The 203-bp fragment of the PCR products was A allele-specific and the 183-bp fragment was G allele-specific. The frequencies of the UCP2 −866G/A genotypes in 72 Japanese subjects were AA: 21 (29.2%), AG: 32 (44.4%), and GG: 19 (26.4%). The results were confirmed by the PCR-RFLP genotyping method, in which a 360-bp fragment of PCR products was cut into 290- and 70-bp fragments by the restriction enzyme MluI when the G allele was present. This Japanese group showed a higher frequency of the AA genotype, which is associated with a low prevalence of obesity, than Caucasian populations. Conclusions: The MS-PCR technique is a simple, rapid, and reliable method for genotyping UCP2 −866G/A polymorphism.