Use of Redundant Exclusion PCR To Identify a Novel Bacillus thuringiensis Cry8 Toxin Gene from Pooled Genomic DNA

摘要

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 (RE-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 to be 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 Anomala corpulenta .IMPORTANCE Protein toxins from the bacterium Bacillus thuringiensis are being increasingly used as biopesticides against a wide range of insect pests, yet the search for new or improved toxins is becoming more difficult, as traditional methods for gene discovery routinely isolate previously identified clones. This paper describes an approach that we have developed to increase the success rate for novel toxin gene identification through reducing or eliminating the cloning of previously characterized genes.