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

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  • High Throughput Detection of Drug-Metabolizing Enzyme Polymorphisms by Allele-Specific Fluorogenic 5' Nuclease Chain Reaction Assay.
  • High throughput detection of drug-metabolizing enzyme polymorphisms by fluorogenic 5′ nuclease chain reaction assay

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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.

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