High Throughput Detection of Drug-Metabolizing Enzyme Polymorphisms by Allele-Specific Fluorogenic 5' Nuclease Chain Reaction Assay

抄録

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.