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Multiplex PCR-pyrosequencing assay for genotyping CYP3A5 polymorphisms.

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

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AllelesBase SequenceCytochrome P-450 CYP3ACytochrome P-450 Enzyme SystemDNA PrimersGenotypeLiverPolymerase Chain ReactionPolymorphism, GeneticJournal ArticleResearch Support, N.I.H., Extramural

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BibTeXRIS

Christina L Aquilante, Taimour Y Langaee, Peter L Anderson, Issam Zineh, Courtney V Fletcher. 2006-05-15. Multiplex PCR-pyrosequencing assay for genotyping CYP3A5 polymorphisms.. https://doi.org/10.1016/j.cca.2006.02.046

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