Isolation of distantly related members in a multigene family using the polymerase chain reaction technique

We have designed a strategy to isolate and identify genes (cDNAs) coding for distantly-related members within a large multigene family. We have used limited protein sequence information data to delineate conserved regions where members of a supergene family are related. Comparison of the nucleotide sequences of such conserved areas defined consensus sequences that were used for the synthesis of deoxynucleotide primers. Two forward and two reverse primers were synthesized, and four separate pairs of primer combinations were used under low stringency in polymerase chain reactions (PCR) to generate amplified DNA products. The PCR products were directionally cloned into the phage vector M13mp18. Each of four libraries was screened with radiolabeled PCR product generated using a pair of primers different from those used to generate the library. Using this approach on the supergene family of ligand-gated ion channels, we were able to isolate and identify two novel subunits of neurotransmitter-operated ion channels.