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*5...

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Bibliographic Details
Published in:Biological & pharmaceutical bulletin 2000/10/01, Vol.23(10), pp.1131-1135
Main Authors: HIRATSUKA, Masahiro, AGATSUMA, Yasuyuki, OMORI, Fumiko, NARAHARA, Kaori, INOUE, Tomoko, KISHIKAWA, Yukinaga, MIZUGAKI, Michinao
Format: Article
Language:English
Subjects:
Aldehyde Dehydrogenase - genetics
allel-specific amplification
Alleles
Biological and medical sciences
Biotechnology
Cytochrome P-450 Enzyme System - genetics
DNA - genetics
Enzymes - genetics
Exodeoxyribonuclease V
Exodeoxyribonucleases - genetics
Fundamental and applied biological sciences. Psychology
General pharmacology
Genetic engineering
Genetic technics
Genotype
genotyping
high-throughput
Japan
Medical sciences
Methods. Procedures. Technologies
Methyltransferases - genetics
Miscellaneous
Oligonucleotides
Pharmaceutical Preparations - metabolism
Pharmacokinetics. Pharmacogenetics. Drug-receptor interactions
Pharmacology. Drug treatments
Polymerase Chain Reaction
polymorphism
Polymorphism, Genetic - genetics
Synthetic digonucleotides and genes. Sequencing
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Summary: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.
ISSN:0918-6158
1347-5215
DOI:10.1248/bpb.23.1131