Use of redundant exclusion PCR to identify a novel Bacillus thuringiensis Cry8 toxin gene from pooled genomic DNA

With the aim of optimizing the cloning of novel genes from a genomic pool containing many previously identified, homologous, genes we designed a redundant exclusion PCR technique. In RE-PCR a pair of generic amplification primers are combined with additional primers that are designed to specifically bind to redundant, unwanted genes that are a subset of those copied by the amplification primers. During RE-PCR the specific primer blocks amplification of the full length redundant gene. Using this method we managed to clone a number of cry8 or cry9 toxin genes from a pool of Bacillus thuringiensis genomic DNA while excluding amplicons for cry9Da, cry9Ea and cry9Eb. The method proved very efficient at increasing the number of rare genes in the resulting library. One such rare, and novel, cry8-like gene was expressed and the encoded toxin was shown to be toxic to Anomola corpulenta.