Application of PCR-based Restriction Fragment Length Polymorphisms (RFLP) of Genes Encoding 16S rRNA in the Study of Bacterial Diversity in Antibiotic Bioreactors

The PCR-based restriction fragment length polymorphisms (PCR-based RFLP) is a method applied in molecular biology which leverage the differences in cells’ homologous DNA molecules from different locations on sites of action of restriction enzyme. RFLP acts as a molecular marker and is specific to a single clone or combination of restriction enzyme. This method involves the PCR amplification of 16S rRNA genes, a highly conserved region with variable regions for bacterial species differentiation. The PCR method uses two primers to amplify the 16S rRNA genes isolated from total bacterial community DNA. The amplicons from the PCR are subjected to restriction enzymes digest. The resulting restriction fragments are resolved according to their size using gel electrophoresis. The patterns are visualized and used to classify the bacterial community into diverse groups. Representative amplicons of each group are sequenced to determine their identity. In this study, PCR-based RFLP analysis was employed to study the effect of different antibiotics at varying concentrations on bacterial community in various bioreactors. Following the standard procedures, the method was able to classify and give the identity of 254 selected bacterial isolates from sludge samples of the various reactors into 7 groups, which includes Pseudomonas gessardii group, Staphylococcus saprophyticus group, Acinetobacter sp. group, Bacillus sp. group, Myroides marinus group, and Enterobacter sp. From their different patterns and subsequent sequencing. Generally, our result shows that PCR-RFLP is a veritable tool for studying and identifying the diversity of bacterial communities and can be employed in studying the recombination rate providing genetic distance between loci.