Genotyping and mapping assay of single-nucleotide polymorphisms in CYP3A5 using DNA-binding zinc(II) complexes

Objective: It is clinically important to detect the single-nucleotide polymorphism (SNP) of CYP3A5*3 (6986A>G) associated with enzymatic activity for drug metabolism. The aim of this study was to establish an accurate strategy for SNP screening. Design and methods: Polyacrylamide gel electrophoresis (PAGE) using zinc(II) complexes were applied for SNP detection. Genomic analyses of 19 healthy subjects were conducted by using both Zn2+-Phos-tag-PAGE and Zn2+-cyclen-PAGE methodologies. Results: Zn2+-Phos-tag PAGE permitted identification of the following allele genotypes: the A/A homozygote (CMA5*1/*1) in 3 individuals, the G/G homozygote (CMA5*3/*3) in 14 individuals, and the A/G heterozygote (CMA5*1/*3) in 2 individuals. Zn2+-cyclen PAGE demonstrated not only reproducibility of the genotyping but also existence of a novel heterozygous SNP (6929G>A) in the subject with CMA5*1/*1. Conclusion: We demonstrated reliable SNP genotyping and mapping in CYP3A5 using the combination method of Zn2+-Phos-tag PAGE and Zn2+-cyclen PAGE. (C) 2009 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.