Terminal restriction fragment length polymorphism (T-RFLP) analysis: Characterizing the unseen

Abstract

The golden era of microbiology during early 1900s was based on the isolation and characterization of purified single cultures. However, since the understanding of limitations of culturing methods to decipher the majority of the microbial diversity, microbiologists have been keen to assess the abundance and distribution of microbial diversity by alternative methods¹. The quest has been further fueled by the idea of relating such information about the community structure to the ecosystem function. Therefore, the goal of new cohort of modern day microbiologists is to understand microbial community structure and dynamics within their natural habitats. Consequently, a wide variety of culture independent approaches and methods for microbial community structure determination have been developed and applied to varied ecosystems. By far the majority of such methods use direct isolation of genetic content from the environmental samples and PCR amplification of genes of interest for bypassing the culturing biases. Existing methods include Amplified Ribosomal DNA Restriction Analysis (ARDRA), Single Stranded Conformation Polymorphism (SSCP), Thermal and Denaturing Gradient Gel Electrophoresis (TGGE and DGGE), Amplified Length Heterogeneity (ALH) analysis and Terminal Restriction Fragment Length Polymorphism (T-RFLP) analysis².