PCR-enriched cDNA pool method for cloning of gene homologues

We have developed a PCR-enriched cDNA pooling method for enrichment of the complete 5'ends of the target homology cDNA fragments with just I conserved region needed. By using reverse transcription and a few rounds of PCR amplification, a full-length cDNA population flanked by T7 and M13 primers was generated. Multiple complete 5' ends of cDNA members of a gene family can subsequently be enriched via PCR with M 13 and degenerate primer mix priming at the 5' end and the conserved region, and they migrate as a single dense band when separated on an agarose gel. The enriched homologous cDNA fragments could be separated for subsequent cloning and sequencing. The main advantages of our method are its speediness, simplicity, and cost-effectiveness. The method has been successfully applied to the cloning of members of the cation-efflux family in Brassica Juncea L. and the natural resistance-associated macrophage protein family in Thlaspi caerulescens, which demonstrates that this novel approach permits rapid isolation of novel interspecific gene orthologues. It could also be easily adapted to highly specific cloning of gene homologues identified in target genomes.