Abstract
Publisher Summary This chapter focuses on the sampling of P450 diversity by cloning the polymerase chain reaction (PCR) products obtained with degenerate primers. Molecular cloning of P450 genes and their heterologous expression and reconstitution has so far provided the best method for the biochemical characterization of insect P450s. Molecular probes may also be needed to investigate a myriad of biological problems, such as pesticide resistance, induction by foreign chemicals, and hormonal or developmental control. One strategy is to use the conserved region around the conserved cysteine liganding the heme as a forward primer. Vector primers (for library screening) or oligo(dT) primers (for cDNA) are used as reverse primers. Another strategy is to use the region surrounding the conserved cysteine to design a reverse primer and to use as forward primer a sequence derived from another conserved region in the P450 protein. Thus, versatile methods are now available to clone P450 fragments from virtually any organism.
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