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  • Title: Multiplex PCR-pyrosequencing assay for genotyping CYP3A5 polymorphisms.
    Author: Aquilante CL, Langaee TY, Anderson PL, Zineh I, Fletcher CV.
    Journal: Clin Chim Acta; 2006 Oct; 372(1-2):195-8. PubMed ID: 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.
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