Internal Transcribed Sequence - Restriction Fragment Length Polymorphism
RFLP is a molecular genotypic technique which can be used for bacterial speciation. A gene, genomic sequence or nucleotide sequence is digested with specific restriction endonuclease enzymes and run through gel electrophoresis. Different fragment patterns will be generated depending on the location of restriction endonuclease sites in the nucleotide sequence. When DNA sequence with polymorphic sites is known, PCR is used to selectively amplify the region for the analysis. Organisms are distinguished due to differing locations of restriction endonuclease sites.
ITS-RFLP analysis is used in bacterial identification and speciation. In ITS-RFLP analysis, the 16S-23S rDNA ITS region is PCR amplified, digested with specific restriction endonucleases, and run through gel electrophoresis after which restriction fragment lengths are estimated. RFLP banding patterns are then compared among each other to differentiate organisms into different species.
ITS-RFLP analysis for University of Delaware Bradyrhizobium Culture Collection
ITS-RFLP analysis was carried out for 371 UDBCC accessions. ITS amplicons from each of 371 accessions were digested with MspI (restriction enzyme) and run through gel electrophoresis. A total 19 different banding patterns were observed for the 371 accessions (figure 1). Banding patterns were clustered to form three main clusters A, B, and C corresponding to B. japonicum, B. diazoefficiens, and B. elkanii respectively. A dendrogram was built to show the relationship between the 19 banding patterns (figure 2).
