1c104869-89e5-4bfb-a853-7f07396bc8a8
title
Simple and rapid detection of  uncoupling protein-2   - 866G/A  polymorphism by mutagenically separated polymerase chain reaction.
b891cab4-58e8-442f-a9cf-ce5ee506defe
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.