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Keywords:

  • directed T-RFLP;
  • design software;
  • diagnostic tool;
  • environmental monitoring

Summary

1. T-RFLP is an established tool for high-throughput studies of microbial communities, which can, with care and practical validation, be enhanced to aid identification of specific organisms in a community by associating T-RFs from experimental runs with predicted T-RFs from a set of existing sequences. A barrier to this approach is the laborious process of selecting diagnostic restriction enzyme(s) for further validation.

2. Here, we describe directed terminal restriction analysis tool (DRAT), a software tool that aids the design of directed terminal-restriction fragment length polymorphism (DT-RFLP) strategies, to separate DNA targets based on restriction enzyme polymorphisms. The software assesses multiple user-supplied DNA sequences, ranks optimal restriction endonucleases for separating targets and provides summary information including the length of diagnostic terminal restriction fragments. A worked example suggesting enzymes uniquely separating selected arbuscular mycorrhizal fungal groups is presented.

3. This tool greatly facilitates identification of diagnostic restriction enzymes for user-designated groups within complex populations and provides expected product sizes for all designated groups.