title: Direct assessment of cytochrome P450 2D6 genotypes by high-resolution melting analysis and DNA sequencing.
abstract: We developed a CYP2D6 genotyping method that required only one polymerase chain reaction (PCR) followed by a high-resolution melting curve analysis (HRM) and DNA sequencing. DNA was extracted from peripheral blood samples obtained from 100 normal individuals. From the HRM analysis using three fragments of amplicons (exons 1, 6, and 9), we successfully identified four common CYP2D6 gene polymorphisms (100C>T, 2850C>T, 2988G>A, and 4180G>C). Exons 3 and 7 were also screened by HRM analysis. The heteroduplexes, wild-type homoduplexes, and homoduplexes of compound mutations showed distinct melting plots. The other four exons (exons 2, 4, 5, and 8) were directly analyzed by DNA sequencing. In conclusion, we developed an HRM and DNA sequencing based method to assess the CYP2D6 gene directly without the need for nested PCR. This method is quick and cost-effective; it reduces the chance of PCR contamination and is suitable for clinical application.
authors.affiliation: Epigenome Research Center, China Medical University Hospital, Taichung, Taiwan; Department of Laboratory Medicine, China Medical University Hospital, Taichung, Taiwan.
authors.forename: Ya-Sian
authors.initials: YS
authors.lastname: Chang
authors.affiliation: Epigenome Research Center, China Medical University Hospital, Taichung, Taiwan.
authors.forename: Chien-Chin
authors.initials: CC
authors.lastname: Lee
authors.affiliation: Epigenome Research Center, China Medical University Hospital, Taichung, Taiwan; Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan.
authors.forename: Ting-Yuan
authors.initials: TY
authors.lastname: Liu
authors.affiliation: Epigenome Research Center, China Medical University Hospital, Taichung, Taiwan; Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan.
authors.forename: Yu-Chia
authors.initials: YC
authors.lastname: Chen
authors.affiliation: Department of Laboratory Medicine, China Medical University Hospital, Taichung, Taiwan.
authors.forename: Hsiu-Chin
authors.initials: HC
authors.lastname: Lu
authors.affiliation: Epigenome Research Center, China Medical University Hospital, Taichung, Taiwan; Department of Laboratory Medicine, China Medical University Hospital, Taichung, Taiwan; School of Medicine, China Medical University, Taichung, Taiwan. Electronic address: d6781@mail.cmuh.org.tw.
authors.forename: Jan-Gowth
authors.initials: JG
authors.lastname: Chang
mesh_terms: D044466:Asian People; D019389:Cytochrome P-450 CYP2D6 / Q000235:genetics*; D005091:Exons; D005838:Genotype; D006801:Humans; D009154:Mutation; D020641:Polymorphism, Single Nucleotide; D017422:Sequence Analysis, DNA / Q000191:economics / Q000379:methods*; D013624:Taiwan
chemical_list: D019389:Cytochrome P-450 CYP2D6
keywords: DNA sequencing; Direct CYP2D6 genotyping; High-resolution melting curve analysis