Duplex polymerase chain reaction with confronting two-pair primers (PCR-CTPP) for genotyping alcohol dehydrogenase
Akiko Tamakoshi1, Nobuyuki Hamajima, Haruya Kawase
1Department of Preventive Medicine/Biostatistics and Medical Decision Making, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan.
Aims:
Alcohol dehydrogenase beta subunit (ADH2) Arg47His and aldehyde dehydrogenase 2 (ALDH2) Glu487Lys were genotyped by a duplex polymerase chain reaction (PCR) with confronting two-pair primers (PCR-CTPP), which allows DNA amplification with one-tube PCR including eight primers, and subsequent electrophoresis.
Methods:
Several PCR conditions were tested to establish the optimal conditions for distinguishing the allele-specific bands for the two polymorphisms. Under the optimal PCR conditions, 454 Japanese health check-up examinees were genotyped.
Results:
The allele-specific bands were successfully amplified under the optimal conditions of the duplex PCR-CTPP. The genotype distributions were within the Hardy-Weinberg equilibrium. The bands produced by the duplex PCR-CTPP genotyping were clearer than those produced by PCR-CTPP, conducted solely for ADH2.
Conclusions:
ADH2 Arg47His and ALDH2 Glu487Lys were successfully genotyped by this newly developed duplex PCR-CTPP, an inexpensive and time-saving genotyping tool, which will be useful in epidemiological studies on alcoholism, as well as risk estimation of alcohol-related diseases.
