DTGGE (DENATURING AND TEMPERATURE GRADIENT GEL ELECTROPHRESIS) A NEW APPROACH FOR THE DETECTION OF 16S rDNA SEQUENCE VARIATION IN RIZOBIA POPULATION

M. VERONICA LOPEZ; M. AGUILAR

Viña del Mar, Chile

Congreso; Reunion anual de la SAIB N XXXVI; 2000

Since its initial application to environmental 16S ribosomal DNA (rDNA) by Muyzer et al, denaturing gradient gel electrophoresis (DGGE) and temperature gradient gel electrophoresis (TGGE) had been an attractive technique in molecular microbial ecology. In most studies were investigated uncultured bacteria by fingerprints generated either by TGGE or DGGE from DNA environmental samples. The aim of this work was to found a region target in 16S rDNA gene that show significant difference in sequence into rizobia species and studied the melting behavior in denaturing gels. Firstly, the alignment and comparison of 16S rDNA sequences of known species of rhizobia and bradyrhizobia were performed in order to identify regions with high variability. A sequence of 260 bp was found to be more diverse and therefore exploited to design primers. A GC-clamp was added in the 5’-end of forward primer. Secondly, we applied these primers to obtain PCR-product with DNA samples of reference strains. Thirdly, we studied the behavior of these PCR-product in denaturing gel and found that the combination of both technique temperature and denaturant environment (a new technique, DTGGE) result in the optimal resolution of the DNA-fragment. We found that was possible differentiate species R. etli, R. leguminosarum, S. teranga, R, tropici B, R. galegae, B. japonicum and B. elkanii by DTGGE-patterns. Results of analysis of most species were a good agreement with DNA CG content obtained from the sequence data.