A polymerase chain reaction-based method for cloning novel members of a gene family using a combination of degenerate and inhibitory primers

Abstract

We have developed a novel method for cloning gene family members by using a polymerase chain reaction technique. The method is based on the amplification of a broad range of homologous genes in combination with the specific inhibition of already cloned genes. To accomplish this, we designed degenerate primers to highly conserved regions among the gene family members, and inhibitory primers to the divergent region at the 3′-margin of each degenerate primer. The 5′-end of the inhibitory primer, the 3′-end of which was aminated, had 3-4 bases overlapping the 3′-end of the degenerate primer. The potential of this method was demonstrated by the successful cloning of a novel member of the yeast MKC7/YAP3 gene family homologue from a filamentous fungus, Aspergillus oryzae, by inhibiting amplification of an already cloned homologue, opsB. © 2002 Elsevier Science B.V. All rights reserved.