Multiplex PCR-pyrosequencing assay for genotyping CYP3A5 polymorphisms.
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.
Aquilante
Christina L
CL
University of Colorado at Denver and Health Sciences Center, School of Pharmacy, Department of Clinical Pharmacy, Denver, CO 80262, USA. Christina.aquilante@UCHSC.edu
Langaee
Taimour Y
TY
Anderson
Peter L
PL
Zineh
Issam
I
Fletcher
Courtney V
CV
D000483:Alleles; D001483:Base Sequence; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics*; D017931:DNA Primers; D005838:Genotype; D008099:Liver / Q000201:enzymology; D016133:Polymerase Chain Reaction / Q000379:methods*; D011110:Polymorphism, Genetic
D017931:DNA Primers; D003577:Cytochrome P-450 Enzyme System; C510164:CYP3A5 protein, human; D051544:Cytochrome P-450 CYP3A