Simple and rapid detection of uncoupling protein-2 - 866G/A polymorphism by mutagenically separated polymerase chain reaction.
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.
Zhang
Bo
B
Department of Cardiology, Fukuoka University School of Medicine, 7-45-1 Nanakuma Jonan, Fukuoka 814-0180, Japan. bozhang@fukuoka-u.ac.jp
Tanaka
Hiroaki
H
Saku
Keijiro
K
D000293:Adolescent; D000328:Adult; D004252:DNA Mutational Analysis / Q000379:methods*; D017931:DNA Primers; D005787:Gene Frequency; D005838:Genotype; D006801:Humans; D007473:Ion Channels; D007564:Japan / Q000453:epidemiology; D026901:Membrane Transport Proteins / Q000235:genetics*; D024101:Mitochondrial Proteins / Q000235:genetics*; D017720:Molecular Epidemiology; D009765:Obesity / Q000453:epidemiology / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods / Q000592:standards; D020641:Polymorphism, Single Nucleotide; D011786:Quality Control; D000071246:Uncoupling Protein 2
D017931:DNA Primers; D007473:Ion Channels; D026901:Membrane Transport Proteins; D024101:Mitochondrial Proteins; C000604389:UCP2 protein, human; D000071246:Uncoupling Protein 2