Microbial Community Profiling: SSCP and T-RFLP Techniques

Abstract

Single-strand conformation polymorphism (SSCP) and terminal restriction fragment length polymorphism (T-RFLP) are two independent methods for fingerprinting/profiling the diversity of microbial communities by distinction of similar-sized DNA fragments, amplified from directly extracted environmental DNA with PCR primers, hybridizing to phylogenetically conserved regions of microbial genomes. Since their introduction to microbial ecology, both methods were mainly used to characterize microbial communities based on their rRNA genes and/or ITS (internal transcribed spacer) genomic regions. However, they can also be applied for profiling functional, i.e., protein-encoding genes. For efficient use of SSCP, one strand of the double-stranded DNA-PCR amplicon is enzymatically removed to avoid reannealing during electrophoresis. While SSCP requires tightly temperature-controlled electrophoresis conditions, T-RFLP can best be performed with automated capillary electrophoresis where terminal restriction fragments, previously labeled with a fluorescent dye, are detected as electropherograms. SSCP is typically conducted on rectangular thin gels, which limits the amounts of directly comparable samples but allows the isolation of DNA for further sequencing and identification. As an advantage, T-RFLP allows an unlimited number of samples to be compared. Both methods only capture the most dominant community members as selected by their PCR primers, and their future use in microbial ecology is now challenged by massively parallel high-throughput DNA amplicon sequencing. However, SSCP and T-RFLP remain highly useful tools for coping with the common challenge in ecological studies of distinguishing the background of natural variability from treatment or other effects and, thus, instructing on which samples to be studied by high-throughput DNA sequencing.