PCR-based approach for detection of novel Bacillus thuringiensis cry genes

A two-step strategy, named exclusive PCR or E-PCR, has been developed to overcome the main limitation of PCR, which is the detection of already-known sequences only. This strategy allows the ability to detect and further clone and sequence genes for which no specific primers are available and in which a variable region exists between two conserved regions. This approach has been applied to Bacillus thuringiensis cryI genes by the use of mixtures of degenerate and specific primers recognizing well-known sequences. The first step allows the accurate identification of already-characterized cryI genes by the use of three primers. During the second step, the same sets of primers are used to exclude known sequences and to positively detect cryI genes unrecognized by any specific primer. The method, as well as its application to detect, clone, and sequence a novel cryIB gene, is described in this article.