Culture-independent microbial community analysis with terminal restriction fragment length polymorphism

Terminal restriction fragment length polymorphism is a polymerase chain reaction (PCR)-based technique that has been used to effectively interrogate microbial communities to determine the diversity of both phylogenetic and functional markers. It requires the isolation of community DNA and knowledge of the target sequence. PCR amplification, performed with fluorescently labeled primers, is followed with restriction digestion and size selection on automated sequencing systems. The fluorescent tag identifies the terminal fragment, and the length polymorphism of the terminal fragments reveals a fraction of the phylogenetic diversity within the target sequence. Because the technique has high-throughput capabilities, it performs well in surveys where a large number of samples must be interrogated to ascertain spatial or temporal changes in community structure.