A Novel Multiplex PCR-RFLP Method for Simultaneous Genotyping of CYP3A4*4 A>G, CYP3A4*18B G>A and CYP3A4*22 C>T

Background: Cytochrome P450 3A enzymes exhibit a variety of physiological roles and have been reported to be the most predominant enzymes involved in drugs metabolism. Single nucleotide polymorphisms (SNPs) in the genes that code for these enzymes may result in functional changes that affect enzyme activity. CYP3A4 is an important enzyme in the metabolism of many important drugs used in the treatment of breast cancer.
Methods: A total of 94 post-menopausal breast cancer patients were recruited for the study and their DNA was isolated for polymerase chain reaction (PCR). The primers were designed using Primer3 software with primer specificities checked via the Basic Local Alignment Tool (BLAST) database. The primer specificity, functionality and annealing temperature were first investigated using uniplex PCR protocols, followed by a single multiplex polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. The digested amplification fragments were analysed by gel electrophoresis and subsequently validated by sequencing.
Results: A multiplex PCR-RFLP method was successfully developed for simultaneous detection of CYP3A4*4, CYP3A4*18B and CYP3A4*22 in a population of post-menopausal breast cancer patients.
Conclusion: The technique is simple, cost-effective, time-saving and can be routinely applied in the identification of SNPs and determination of allelic and genotypic frequencies of CYP3A4*4, CYP3A4*18B and CYP3A4*22.

Keywords
multiplex PCR-RFLP; single nucleotide polymorphisms; CYP3A4*4; CYP3A4*18B; CYP3A4*22