title: High-throughput detection of multiple genetic polymorphisms influencing drug metabolism with mismatch primers in allele-specific polymerase chain reaction.
abstract: Because of genetic polymorphisms of drug-metabolizing enzyme genes, the activities of the enzymes in humans vary widely and alter the metabolism of commonly used clinical agents. Severe adverse effects or resistance to therapy may result. We have developed a rapid and high-throughput genotyping method for detecting polymorphisms of the drug-metabolizing enzyme genes CYP2C9*3, CYP2C19*2, *3, CYP2D6*2, *4, *10, *14, *21, NAT2*5, *6, *7, and TPMT*3 using allele-specific polymerase chain reaction (PCR) with mismatch primers (ASPCR-MP) and CYP2D6*5, *36, and CYP2D6xN using stepdown PCR with detection by SYBR Green I. We analyzed genomic DNA from 139 Japanese volunteers. Identical genotyping results were obtained by using ASPCR-MP, stepdown PCR, and conventional PCR. We found that the methods clearly differentiate three specific profiles with no overlap in the signals. Moreover, both ASPCR-MP and stepdown PCR for genotyping took less than 3-4h. To our knowledge, this is the first report of successful simultaneous detection of multiple genetic polymorphisms with point mutations using ASPCR-MP or multiple genetic polymorphisms with large structural alterations using stepdown PCR. In conclusion, ASPCR-MP and stepdown PCR appear to be suitable for large clinical and epidemiological studies as methods that enable highly sensitive genotyping and yield a high-throughput.
authors.affiliation: Department of Pharmacy, University of Tokyo Hospital, Faculty of Medicine, Tokyo, 113-8655, Japan.
authors.forename: Akihiro
authors.initials: A
authors.lastname: Ishiguro
authors.forename: Takahiro
authors.initials: T
authors.lastname: Kubota
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Soya
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Sasaki
authors.forename: Osamu
authors.initials: O
authors.lastname: Yagyu
authors.forename: Yutaka
authors.initials: Y
authors.lastname: Takarada
authors.forename: Tatsuji
authors.initials: T
authors.lastname: Iga
mesh_terms: D000483:Alleles; D020137:Base Pair Mismatch / Q000235:genetics*; D019389:Cytochrome P-450 CYP2D6 / Q000235:genetics; D017931:DNA Primers / Q000235:genetics* / Q000378:metabolism; D005453:Fluorescence; D006801:Humans; D007564:Japan; D004364:Pharmaceutical Preparations / Q000378:metabolism*; D010597:Pharmacogenetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D012680:Sensitivity and Specificity
chemical_list: D017931:DNA Primers; D004364:Pharmaceutical Preparations; D019389:Cytochrome P-450 CYP2D6