High throughput detection of drug-metabolizing enzyme polymorphisms by allele-specific fluorogenic 5 ' nuclease chain reaction assay

We ha re developed an allele-specific fluorogenic 5' nuclease chain reactio n assay for detecting polymorphisms in the following human drug-metabolizin g enzyme genes: CYP2C9 (CYP2d9*2 and *3), CYP2C19 (CYP2C19*2 and *3), CYP2D 6 (CYP2D6*4, *10, *14, *18, and *21(C8)), N-acetyltransferase 2 (NAT2*5B, * 6A, and *7B), thiopurine methyltransferase (TPRIT*3C), and aldehyde dehydro genase2 (ALDH2*2). This method is a marriage of two emerging technologies, the use of allele-specific amplification primers for target DNA and hybridi zation of the TaqMan probe. The TaqMan probe is labeled with both a fluores cent reporter dye and a quencher die. Genotypes are separated according to the different threshold cycles of the wild-type and mutant primers. All ass ays are performed using a single thermocycling protocol. This genotyping me thod is rapid and highly sensitive and yields a high throughput. It could b e applied ton ard automated large-scale genotyping.