Genetic Diversity of cry Gene Sequences of Bacillus thuringiensis Strains Analyzed by Denaturing Gradient Gel Electrophoresis Marı ´a E. Vidal-Domı ´nguez • Macarena Perez-Cenci • Graciela L. Salerno • Corina M. Bero ´n Received: 3 May 2010 / Accepted: 8 September 2010 / Published online: 4 November 2010 Ó Springer Science+Business Media, LLC 2010 Abstract 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 dem- onstrated 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. Introduction Cry proteins from the Gram-positive bacterium Bacillus thuringiensis have been used as a successful biological insecticide and are extensively used worldwide in pro- grams for controlling insect pests [18]. The significant amount of available Cry proteins is the result of a sys- tematic international effort to isolate and characterize new strains of B. thuringiensis searching for toxins with novel properties, particularly appropriate for the control of ag- ronomical or medically important pests [15]. To date, a great amount of proteins has been described, however, to find new Cry proteins with novel receptor specificities in pests will be a way to expand the host ranges of the available strains. On the other hand, it will be a useful alternative after the appearance of insect resistance, mainly due to the use of transgenic crops. DGGE is an electrophoretic method capable of detecting differences between DNA fragments of the same size but with different sequences. This is because the fragments can be separated in a denaturing gradient gel based on their differential denaturation (melting) profile. Separation is based on the decreased electrophoretic mobility of a par- tially melted double stranded DNA molecule in poly- acrylamide gels containing a linear gradient of DNA denaturants. The melting of DNA fragments proceeds in discrete, ‘‘so called’’, melting domains: stretches of base pairs with an identical melting temperature. While DGGE was originally developed to detect point mutations in DNA sequences, Muyzer et al. [12] expanded its use to study microbial genetic diversity in a marine ecosystem. For this purpose, total bacterial DNA was extracted from natural samples, segments of the 16S or 18S rRNA genes were amplified in the polymerase chain reaction and the indi- vidual products were subsequently separated by DGGE. The result was a pattern of bands, for which their number corresponded to the number of predominant members in the microbial communities allowing to compare them in different environments and to monitor changes in the composition of abundant members of a specific community through out the time. In the last years, DGGE of PCR amplified 16S rRNA and 18 rRNA genes fragments has been applied to profile the distribution of microbial popu- lations in a great variety of environments like soil, M. E. Vidal-Domı ´nguez Á M. Perez-Cenci Á G. L. Salerno Á C. M. Bero ´n (&) Centro de Estudios de Biodiversidad y Biotecnologı ´a - Centro de Investigaciones Biolo ´gicas - Fundacio ´n para Investigaciones Biolo ´gicas Aplicadas (CEBB-CIB FIBA), C.C. 1348, 7600 Mar del Plata, Argentina e-mail: cberon@fiba.org.ar 123 Curr Microbiol (2011) 62:866–870 DOI 10.1007/s00284-010-9776-1