The use of degenerate oligonucleotide primed polymerase chain reaction (DOP-PCR) technique to detect and isolate cytochrome P450 2E1 and 2A genes

Abstract

Cytochrome P450 (CYP, P450) enzymes are widely studied for their involvement in the metabolism of drugs as well as endogenous substrates. Assessing some forms of P450 such as P450 2E1 and 2A phenotype may be of great importance to predict organism’s susceptibility to xenobiotic and environmental pollutants which are metabolically activated by these isoenzymes. In the present study, we designed universal degenerate oligonucleotide primers sets based on the highly conserved nucleotide sequences at the 5’ and 3’ cDNA ends of P450 2E1 and 2A gene family from Camelus dromedaries (Arabian camel), Bos Taurus (cow), Capra hircus (domestic goat), Ovis aries (domestic sheep), Oryctolagus cuniculus (European rabbit), Xenopus tropicalis (Western clawed frog), Rattus norvegicus (Norway rat), Mus musculus (house mouse) and Mesocricetus auratus (golden hamster). We successfully demonstrated that these primers sets were able to detect the expression of P450 2E1 and 2A genes from these eukaryotic organisms. Moreover, we amplified 1070 and 1200 nucleotides of both P450 2E1 and 2A from these organisms and this represent two third of the actual size of these P450 isoforms. The nucleotide sequences of these genes showed percent identities ranging from 80 to 85%. The degenerate primers designed in this study allowed the detection and amplification of known and unknown P450s belonging to the gene families 2E1 and 2A from different organisms.

Key words: Cytochrome P450, xenobiotics, degenerate primers, mixed function oxidases.