Genetic Diversity of cry Gene Sequences of Bacillus thuringiensis Strains Analyzed by Denaturing Gradient Gel Electrophoresis
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PCR has been widely used to identify cry-type genes, to determine their distribution, to detect new such genes and to predict insecticidal activities. We describe here a molecular approach to analyze the genetic diversity of B. thuringiensis cry-like genes based on denaturing gradient gel electrophoresis (DGGE). This analysis demonstrated that different B. thuringiensis isolates can be distinguished according to its PCR-DGGE profile of cry-like genes. Identification of the resolvable DNA fragments was easy to accomplish by DNA sequencing, which was confirmed in this work. Importantly, the strategy allowed the identification of unknown B. thuringiensis cry-like sequences present in a single strain that remained cryptic after PCR analysis using degenerate primers. The method developed in this work contributes to the availability of molecular techniques for both B. thuringiensis strains and cry-like genes identification and discovery.
KeywordsDegenerate Primer Thuringiensis Strain Deoxyinosine cry24Ca Gene Mastercycler Thermal Cycler
This investigation was supported by Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT PICT-2007-02069) and by the Universidad Nacional de Mar del Plata (grant 15E/329 EXA 382/07). G.L.S. and C.M.B. are career investigators, and M.E.V.D. and M.P-C. are fellows at the Consejo Nacional de Investigaciones Científicas y Tecnológicas (CONICET), Argentina. We especially thank Dr. Leonardo Curatti and Prof. Ana Tassi for critical revision of the manuscript and Ing. Agr. Clara Fernández for technical assistance.
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