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 CYP3A53 (6986A>G) associated with enzymatic activity for drug metabolism. The aim of this study was to establish an accurate strategy for SNP screening.


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 Zn(2+)-Phos-tag-PAGE and Zn(2+)-cyclen-PAGE methodologies.


RESULTS
Zn(2+)-Phos-tag PAGE permitted identification of the following allele genotypes: the A/A homozygote (CYP3A51/1) in 3 individuals, the G/G homozygote (CYP3A53/3) in 14 individuals, and the A/G heterozygote (CYP3A51/3) in 2 individuals. Zn(2+)-cyclen PAGE demonstrated not only reproducibility of the genotyping but also existence of a novel heterozygous SNP (6929G>A) in the subject with CYP3A51/1.


CONCLUSIONS
We demonstrated reliable SNP genotyping and mapping in CYP3A5 using the combination method of Zn(2+)-Phos-tag PAGE and Zn(2+)-cyclen PAGE.
Kinoshita
Eiji
E
Department of Functional Molecular Science, Graduate School of Biomedical Sciences, Hiroshima University, Kasumi 1-2-3, Hiroshima 734-8553, Japan. kinoeiji@hiroshima-u.ac.jp
Kinoshita-Kikuta
Emiko
E
Nakashima
Hiromi
H
Koike
Tohru
T
D001483:Base Sequence; D002874:Chromosome Mapping; D051544:Cytochrome P-450 CYP3A / Q000235:genetics*; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D005838:Genotype; D006801:Humans; D007527:Isoenzymes / Q000235:genetics; D008969:Molecular Sequence Data; D020641:Polymorphism, Single Nucleotide; D011485:Protein Binding; D017422:Sequence Analysis, DNA; D015032:Zinc / Q000737:chemistry
D007527:Isoenzymes; D004247:DNA; C510164:CYP3A5 protein, human; D051544:Cytochrome P-450 CYP3A; D015032:Zinc