High throughput detection of drug-metabolizing enzyme polymorphisms by allele-specific fluorogenic 5' nuclease chain reaction assay.
We have developed an allele-specific fluorogenic 5' nuclease chain reaction assay for detecting polymorphisms in the following human drug-metabolizing enzyme genes: CYP2C9 (CYP2C9*2 and *3), CYP2C19 (CYP2C19*2 and *3), CYP2D6 (CYP2D6*4, *10, *14, *18, and *21(C8)), N-acetyltransferase 2 (NAT2*5B, *6A, and *7B), thiopurine methyltransferase (TPMT*3C), and aldehyde dehydrogenase2 (ALDH2*2). This method is a marriage of two emerging technologies, the use of allele-specific amplification primers for target DNA and hybridization of the TaqMan probe. The TaqMan probe is labeled with both a fluorescent reporter dye and a quencher dye. Genotypes are separated according to the different threshold cycles of the wild-type and mutant primers. All assays are performed using a single thermocycling protocol. This genotyping method is rapid and highly sensitive and yields a high throughput. It could be applied toward automated large-scale genotyping.
Hiratsuka
M
M
Department of Pharmaceutical Sciences, Tohoku University Hospital, Sendai, Japan.
Agatsuma
Y
Y
Omori
F
F
Narahara
K
K
Inoue
T
T
Kishikawa
Y
Y
Mizugaki
M
M
D000444:Aldehyde Dehydrogenase / Q000235:genetics; D000483:Alleles; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics; D004247:DNA / Q000235:genetics; D004798:Enzymes / Q000235:genetics*; D043211:Exodeoxyribonuclease V; D005090:Exodeoxyribonucleases / Q000235:genetics*; D005838:Genotype; D007564:Japan; D008780:Methyltransferases / Q000235:genetics; D009841:Oligonucleotides; D004364:Pharmaceutical Preparations / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic / Q000235:genetics
D004798:Enzymes; D009841:Oligonucleotides; D004364:Pharmaceutical Preparations; D004247:DNA; D003577:Cytochrome P-450 Enzyme System; D000444:Aldehyde Dehydrogenase; D008780:Methyltransferases; C022745:thiopurine methyltransferase; D005090:Exodeoxyribonucleases; D043211:Exodeoxyribonuclease V