TRFLP

Terminal Restriction Fragment Length Polymorphism (T-RFLP) is a technique that is used to determine the diversity of microbial communities in an ecosystem. It is based on the position of a restriction site closest to a labeled end (with a fluorochrome) of an amplified gene. The method is based on digesting a mixture of PCR amplified variants of a single gene using one or more restriction enzymes and detecting the size of each of the individual resulting terminal fragments using a DNA sequencer.
 * T-RFLP **

In the result depicted in the figure above (under number 5), we can see a graph image – called an electropherogram – where the X axis represents the sizes of the labeled fragments and the Y axis represents their fluorescence intensity. In this way, T-RFLP is one of several molecular methods used to generate a fingerprint of an unknown microbial community.

In addition, T-RFLP is based on PCR amplification of a target gene. The amplification is performed with one or both the primers having their 5’ end labeled with a fluorescent molecule. The most widely used fluorescent dye is 6-FAM. The mixture of amplicons is then subjected to a restriction reaction. Following the restriction reaction, the mixture of fragments is separated using electrophoresis in a DNA sequencer and the sizes of the different terminal fragments are determined by the fluorescence detector. Because the excised mixture of amplicons is analyzed in a sequencer, only the terminal fragments (i.e the labeled end or ends of the amplicon) are read while all other fragments are ignored. Reference **
 * Liu W, Marsh T, Cheng H & Forney L ** (1997) Characterization of microbial diversity by determining terminal restriction fragment length polymorphisms of genes encoding 16S rRNA. Appl. Environ. Microbiol. **63**: 4516-4522.