RESUMO
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.
Assuntos
Bacillus thuringiensis/genética , Proteínas de Bactérias/genética , Reação em Cadeia da Polimerase/métodos , Toxinas de Bacillus thuringiensis , Proteínas de Bactérias/classificação , Clonagem Molecular , Primers do DNA/genética , DNA Bacteriano/genéticaRESUMO
The strain 273 B, the type strain of a H serotype of Bacillus thuringiensis not yet characterized: B. thuringiensis subsp. cameroun, serotype H32, was isolated from soil samples collected in Cameroon. This strain produces cuboidal parasporal bodies composed of two major proteins of 53 kDa and 35 kDa. N-terminal sequences of the major proteins share no homology with published sequences. Only the 35 kDa protein is susceptible to digestion by trypsin. A complex array of 9 plasmids was revealed.
