T-RFLP

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Terminal restriction enzyme fragment length polymorphism (T-RFLP) is a method commonly employed for microbial ecological studies (T-RFLP Marsh 1999).

Contents

Principle

  1. Near complete 16S rRNA genes in a sample are amplified using a fluorescently-labelled primer to yield a mixture of labelled 16S rRNA genes.
  2. These amplification products are digested with restriction enzymes to produce labelled terminal restriction enzyme fragments (TRFs).
  3. These T-RFs are then denatured, and the single stranded DNA thus obtained is separated by electrophoresis under denaturing conditions (e.g. at high temperature).

Comparison of the migration time of the T-RFs to internal standards, labelled with a different fluorochrome, allows accurate sizing of the fragments to within ± 1 nucleotide. Ideally, each T-RF represents a single microorganism, though in practice microbes of different species often share one T-RF. Therefore, digestion with up to three different restriction enzymes is usually necessary to accurately identify a microorganism on the basis of T-RF size. The relative abundance of microorganisms represented by a T-RF can be determined by measuring the fluorescence of each T-RF relative to the sum of the fluorescence.

Advantages

  • T-RFLP analysis is a rapid method of assessing microbial communities;
  • Can be automated to process multiple samples in a short time-span;
  • Computer analysis of gene sequences can be carried out to determine the theoretical TRFs of known microorganisms for comparison with T-RFs obtained from environmental samples for identification of microorganisms in a sample;
  • Alternatively, a database of experimentally-derived T-RFs from known microorganisms can be created to allow for environmental microorganism identification;
  • The same primers as those typically used to make gene clone libraries are used for TRFLP analysis, making comparative information on microorganism abundance between the two techniques comparable and will also rapidly lead to the identification of microbes represented by unknown T-RFLPs.

Disadvantages

  • T-RFLP analysis suffers from the same drawbacks as all PCR-based community analysis techniques;
  • The variation in 16S rRNA gene copy number in different microbes makes this technique only “semi-quantitative”;
  • Reliable lower limit of detection of PCR products in a mixture is about 1%.

Current use in bioleaching studies

T-RFLP analysis has been rarely applied to bioleaching systems. It has been used to assess microbial communities in acid mine drainage streams (Hallberg et al., 2006) and also in mine tailings (Bryan et al., 2006).

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