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.
These relatively high throughput methods were developed in order to reduce the cost and effort in analyzing microbial communities using a clone library.
The method was first described by Avaniss-Aghajani et al in 1994 [1] and later by Liu in 1997[2] which employed the amplification of the 16S rDNA target gene from the DNA of several isolated bacteria as well as environmental samples.
The result of a T-RFLP profiling is a graph called electropherogram which is an intensity plot representation of an electrophoresis experiment (gel or capillary).
Another modification which is sometimes used is to fluorescently label the reverse primer as well using a different dye, again resulting in two parallel profiles per sample each resolving a different number of variants.
In pattern comparison the general shapes of electropherograms of different samples are compared for changes such as presence-absence of peaks between treatments, their relative size, etc.
In order to perform multivariate statistical analysis on T-RFLP data, the data must first be converted to table known as a “sample by species table“ which depicts the different samples (T-RFLP profiles) versus the species (T-RFS) with the height or area of the peaks as values.
The major advantage of T-RFLP is the use of an automated sequencer which gives highly reproducible results for repeated samples.
The use of an automated sequencer which outputs the results in a digital numerical format also enables an easy way to store the data and compare different samples and experiments.
Because T-RFLP relies on DNA extraction methods and PCR, the biases inherent to both will affect the results of the analysis.
Although this phenomenon makes the T-RFLP results easier to handle, it naturally introduces biases and oversimplification of the real diversity.
Attempts to minimize (but not overcome) this problem are often done by applying several restriction enzymes and/ or labeling both primers with a different fluorescent dye.