Multiplex PCR-pyrosequencing assay for genotyping CYP3A5 polymorphisms

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

Research output: Contribution to journalArticlepeer-review

9 Scopus citations


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.

Original languageEnglish (US)
Pages (from-to)195-198
Number of pages4
JournalClinica Chimica Acta
Issue number1-2
StatePublished - Oct 2006
Externally publishedYes


  • CYP3A5
  • Pharmacogenetic
  • Pyrosequencing

ASJC Scopus subject areas

  • Biochemistry
  • Clinical Biochemistry
  • Biochemistry, medical


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