New strategy for identification of novel cry-type genes from Bacillus thuringiensis strains.

BERÓN, C. M.; CURATTI, L.; SALERNO, G.L.

APPLIED AND ENVIRONMENTAL MICROBIOLOGY

American Society of Microbiology

Lugar: Washington D.C.; Año: 2005 vol. 71 p. 761 - 765

0099-2240

We have designed five degenerate primers for the detection of novel cry genes from Bacillus thuringiensis strains. An efficient strategy has been developed based on a two-step PCR approach using those primers in five pair combinations. In the first step, only one of the primer pairs is used in the PCR reaction, allowing the amplification of DNA fragments encoding protein regions that include consensus domains of representative proteins of different Cry groups. A second PCR-reaction is performed using the first-step amplification products as DNA templates and the set of five primer combinations. Cloning and sequencing of the last-step amplicons allow both the identification of known cry genes encoding Cry proteins covering a wide range of phylogenetic distance, and the detection and characterization of cry-related sequences from novel B. thuringiensis isolates.