A simple multiplex PCR method for the concurrent detection of three CYP2C8 variants.
BACKGROUND
Cytochrome P450 (CYP) 2C8 is a principle enzyme responsible for the metabolism of many clinically important drugs as well as endogenous compounds such as arachidonic acid. The enzyme is genetically polymorphic but a simple method is not available to study its genetic polymorphism. We developed and optimized a variant-specific PCR techniques to detect CYP2C8*2, CYP2C8*3 and CYP2C8*4.


METHODS
Genomic DNA was extracted from blood using standard extraction methods. A two-step PCR method was developed to detect simultaneously three CYP2C8 variants. In the first PCR (PCR1), specific regions from exons 3, 5 and 8 of the CYP2C8 gene were amplified. The products were used as templates in parallel alleles-specific PCR (PCR2). This method was tested against DNA samples obtained from 57 healthy Malaysian volunteers.


RESULTS
The bands of interest were successfully amplified. This method showed specific and reproducible results when tested on healthy volunteers. DNA sequencing further confirmed genotype results obtained from current method.


CONCLUSIONS
We have successfully developed and optimized a multiplex PCR method suitable for use in population studies of CYP2C8 polymorphism.
Muthiah
Y D
YD
Pharmacogenetics Study Group, Institute for Research in Molecular Medicine and Department of Pharmacology, School of Medical Sciences, Universiti Sains Malaysia, 16150 Kota Bharu, Kelantan, Malaysia.
Lee
W L
WL
Teh
L K
LK
Ong
C E
CE
Salleh
M Z
MZ
Ismail
R
R
D000483:Alleles; D001189:Aryl Hydrocarbon Hydroxylases / Q000032:analysis / Q000235:genetics*; D065727:Cytochrome P-450 CYP2C8; D017931:DNA Primers; D005091:Exons; D005787:Gene Frequency; D014644:Genetic Variation; D005838:Genotype; D006801:Humans; D008296:Malaysia / Q000453:epidemiology; D011110:Polymorphism, Genetic; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*
D017931:DNA Primers; D001189:Aryl Hydrocarbon Hydroxylases; C450259:CYP2C8 protein, human; D065727:Cytochrome P-450 CYP2C8