Isolating Cytochrome P450 cDNA and Genomic Clones: Library Screening with Synthetic DNA Oligomers

This chapter discusses the isolating cytochrome P450 complementary DNA (cDNA) and genomic clones. Cytochrome P450 enzymes are encoded by a large and complex superfamily of genes. Current P450 gene pools are believed to be the products of extensive duplication events, ultimately descended from one or more ancestral genes. Notable regions of conservation and divergence are apparent within P450 structures. Amino acid sequence conservation is found near a carboxy-terminal cysteine residue; this region is thought to function as the ligand to heme at the enzyme active site. Traditional methods of library screening fall within two categories: (1) antigenic detection using antibodies, and (2) homology to radiolabeled nucleic acid probes. Identification of target cDNAs by the interaction of antigens with antibody probe requires that the cloned DNA be inserted into an expression vector; the insert DNA must be in the correct orientation and reading frame.