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 journalArticle

9 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)195-198
Number of pages4
JournalClinica Chimica Acta
Volume372
Issue number1-2
DOIs
StatePublished - Oct 1 2006

Fingerprint

Cytochrome P-450 CYP3A
Multiplex Polymerase Chain Reaction
Polymerase chain reaction
Polymorphism
Assays
Polymerase Chain Reaction
Genotype
DNA Sequence Analysis
Metabolism
Liver
Cytochrome P-450 Enzyme System

Keywords

  • CYP3A5
  • Pharmacogenetic
  • Pyrosequencing

ASJC Scopus subject areas

  • Biochemistry
  • Clinical Biochemistry
  • Biochemistry, medical

Cite this

Multiplex PCR-pyrosequencing assay for genotyping CYP3A5 polymorphisms. / Aquilante, Christina L.; Langaee, Taimour Y.; Anderson, Peter L.; Zineh, Issam; Fletcher, Courtney V.

In: Clinica Chimica Acta, Vol. 372, No. 1-2, 01.10.2006, p. 195-198.

Research output: Contribution to journalArticle

Aquilante, Christina L. ; Langaee, Taimour Y. ; Anderson, Peter L. ; Zineh, Issam ; Fletcher, Courtney V. / Multiplex PCR-pyrosequencing assay for genotyping CYP3A5 polymorphisms. In: Clinica Chimica Acta. 2006 ; Vol. 372, No. 1-2. pp. 195-198.
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