用于CYP3A5基因多态性基因分型的多重PCR-焦磷酸测序分析

Multiplex PCR-pyrosequencing assay for genotyping CYP3A5 polymorphisms.

作者信息

Aquilante Christina L, Langaee Taimour Y, Anderson Peter L, Zineh Issam, Fletcher Courtney V

机构信息

University of Colorado at Denver and Health Sciences Center, School of Pharmacy, Department of Clinical Pharmacy, Denver, CO 80262, USA.

出版信息

Clin Chim Acta. 2006 Oct;372(1-2):195-8. doi: 10.1016/j.cca.2006.02.046. Epub 2006 May 15.

DOI:10.1016/j.cca.2006.02.046

PMID:16701601

Abstract

BACKGROUND

The cytochrome P450 (CYP) 3A5 enzyme contributes to the metabolism of many drugs. Single nucleotide polymorphisms in the CYP3A5 gene (CYP3A5()3C and CYP3A5()6) are associated with decreased CYP3A5 expression in the liver. We designed a multiplex genotyping assay to detect the CYP3A5()3C and CYP3A5()6 polymorphisms in a single polymerase chain reaction (PCR) and a single pyrosequencing reaction.

METHODS

A multiplex PCR assay was designed to simultaneously amplify 2 fragments, one containing the CYP3A5()3C polymorphism and the other containing the CYP3A5()6 polymorphism. Following PCR, multiplex genotyping was performed with pyrosequencing analysis.

RESULTS

Patient samples (n=69) were analyzed for the CYP3A5()3C and CYP3A5()6 polymorphisms using the multiplex PCR-pyrosequencing assay. Genotypes obtained by the multiplex reaction were in 100% concordance with genotypes obtained using simplex PCR-pyrosequencing (n=69) and direct DNA sequencing (n=29).

CONCLUSIONS

The advantage of this method is that the CYP3A5()3C and CYP3A5()6 polymorphism can be amplified in a single PCR reaction and genotyped in a single pyrosequencing reaction. This combined approach improves the time-efficiency and decreases the cost of CYP3A5 genotyping.

摘要

背景

细胞色素P450（CYP）3A5酶参与多种药物的代谢。CYP3A5基因中的单核苷酸多态性（CYP3A5（）3C和CYP3A5（）6）与肝脏中CYP3A5表达降低有关。我们设计了一种多重基因分型检测方法，可在单个聚合酶链反应（PCR）和单个焦磷酸测序反应中检测CYP3A5（）3C和CYP3A5（）6多态性。