Terminal Restriction Fragment Length Polymorphism (t-RFLP) analysis

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t-RFLP is a method similar to DGGE in that it generates fingerprints of a populations, but unlike DGGE, the bands can (ideally) be assigned to specific organisms directly, without the need for sequencing.

Imagine the simplest case of a pure culture "unknown". You amplify the ssu-rRNA with some set of primers, e.g. 515F and 1492R, and one of the primers (515F in this example) is fluorescently labeled. You digest this ssu-rDNA with several different restriction enzymes and separate the products out on a sequencing gel:

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The sizes of the labeled fragments are compared to a database of potential fragments of ssu-rRNA sequences that would be generated from PCR products from those primers digested with those enzymes. If the restriction enzymes were carefully chosen, a computer program should be able to sift through the database and identify your organism based on the observed (from the gel) sizes of the labeled fragments. For example, there might be 100 organisms who's ssu-rDNA, if amplified with labeled 515F and unlabeled 1492R and digested with HaeIII, should give a 201bp fragment. There might also be another 100 organisms that would have a 571bp MspI fragment, but only one name on both lists - that's your organism. This identification might be verified by the presence of a predictable 823bp Sau3AI fragment.

This should be pretty easy, but now imagine doing the same thing with a population of organisms from a natural environment. Now you have several abundant organisms, some more common than others, creating a pattern of bands in each digest. However, the computer can, if the experiment is properly set up, sift through the peaks and determine what mixture of organisms would create that pattern of bands.

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Example data from Sakamoto et al, 2004 J. Med. Microbiol. 53:563-571.

Your ability to sift through the microbial population using t-RFLP is basically limited only by your choice of primers (what kind of organisms they'll amplify ssu-rDNA from), your ability to choose the best restriction enzymes to use, and the database you're fitting your data to. t-RFLP is an emerging technology, so there is plenty of room for improvement in all of these aspects, but this approach is already very useful, and incredibly promising. Probably the ultimate limitation will be PCR primers; this is a limitation shared by all of the molecular phylogenetic approaches we've talked about.