New strategy for identification of novel Cry-type genes from Bacillus thuringiensis strains.
We designed five degenerate primers for detection of novel cry genes from Bacillus thuringiensis strains. An efficient strategy was developed based on a two-step PCR approach with these primers in five pair combinations. In the first step, only one of the primer pairs is used in the PCR, which allows amplification of DNA fragments encoding protein regions that include consensus domains of representative proteins belonging to different Cry groups. A second PCR is performed by 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 phylogenetic distance and the detection and characterization of cry-related sequences from novel B. thuringiensis isolates.
Berón
Corina M
CM
Centro de Investigaciones Biológicas, Fundación para Investigaciones Biológicas Aplicadas, C.C. 1348, 7600 Mar del Plata, Argentina.
Curatti
Leonardo
L
Salerno
Graciela L
GL
D001413:Bacillus thuringiensis / Q000235:genetics / Q000472:pathogenicity; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000235:genetics*; D001427:Bacterial Toxins / Q000235:genetics*; D003001:Cloning, Molecular; D017931:DNA Primers; D004731:Endotoxins / Q000235:genetics*; D006460:Hemolysin Proteins; D008969:Molecular Sequence Data; D010802:Phylogeny; D016133:Polymerase Chain Reaction / Q000379:methods*; D017422:Sequence Analysis, DNA
D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D017931:DNA Primers; D004731:Endotoxins; D006460:Hemolysin Proteins; C030956:insecticidal crystal protein, Bacillus Thuringiensis