Aquilante Christina L, Langaee Taimour Y, Anderson Peter L, Zineh Issam, Fletcher Courtney V
University of Colorado at Denver and Health Sciences Center, School of Pharmacy, Department of Clinical Pharmacy, Denver, CO 80262, USA.
Clin Chim Acta. 2006 Oct;372(1-2):195-8. doi: 10.1016/j.cca.2006.02.046. Epub 2006 May 15.
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.
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.
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).
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.
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。
设计了一种多重PCR检测方法，以同时扩增2个片段，一个包含CYP3A5（）3C多态性，另一个包含CYP3A5（）6多态性。PCR后，通过焦磷酸测序分析进行多重基因分型。
使用多重PCR-焦磷酸测序检测方法对69例患者样本进行CYP3A5（）3C和CYP3A5（）6多态性分析。多重反应获得的基因型与使用单重PCR-焦磷酸测序（n = 69）和直接DNA测序（n = 29）获得的基因型100%一致。
该方法的优点是CYP3A5（）3C和CYP3A5（）6多态性可在单个PCR反应中扩增，并在单个焦磷酸测序反应中进行基因分型。这种联合方法提高了时间效率，降低了CYP3A5基因分型的成本。
