An evaluation of terminal-restriction fragment length polymorphism (T-RFLP) analysis for the study of microbial community structure and dynamics

A systematic evaluation of the value and potential of terminal-restriction fragment length polymorphism (T-RFLP) analysis for the study of microbial c ommunity structure has been undertaken. The reproducibility and robustness of the method has been assessed using environmental DNA samples isolated di rectly from PCB-polluted or pristine soil, and subsequent polymerase chain reaction (PCR) amplification of total community 16S rDNA, An initial invest igation to assess the variability both within and between different polyacr ylamide gel electrophoresis (PAGE) runs showed that almost identical commun ity profiles were consistently produced from the same sample. Similarly, ve ry little variability was observed as a result of variation between replica te restriction digestions, PCR amplifications or between replicate DNA isol ations. Decreasing concentrations of template DNA produced a decline in bot h the complexity and the intensity of fragments present in the community pr ofile, with no additional fragments detected in the higher dilutions that w ere not already present when more original template DNA was used. Reducing the number of cycles of PCR produced similar results. The greatest variatio n between profiles generated from the same DNA sample was produced using di fferent Tao DNA polymerases, while lower levels of variability were found b etween PCR products that had been produced using different annealing temper atures. Incomplete digestion by the restriction enzyme may, as a result of the generation of partially digested fragments, lead to an overestimation o f the overall diversity within a community, The results obtained indicate t hat, once standardized, T-RFLP analysis is a highly reproducible and robust technique that yields high-quality fingerprints consisting of fragments of precise sizes, which, in principle, could be phylogenetically assigned, on ce an appropriate database is constructed.