Profiling the Diversity of Microbial Communities with Single-Strand Conformation Polymorphism (SSCP)

Abstract

Genetic fingerprinting techniques for microbial community analysis have evolved over the last decade into standard applications for efficient and fast differentiation of microbial communities based on their diversity. These techniques commonly analyze the diversity of PCR products amplified from extracted environmental DNA usually utilizing primers hybridizing to suspected conserved regions of the targeted genes. In comparison to the more commonly applied terminal restriction fragment length polymorphism (TRFLP) or denaturing gradient gel electrophoresis (DGGE) techniques, the here-described single-strand conformation polymorphism (SSCP) fingerprinting technique features some advantageous key characteristics. (1) Primers for the polymerase chain reaction (PCR) do only need minimal 5′-end alterations; (2) SSCP is adaptable to high throughput applications in automated sequencers; and (3) a second dimension in the SSCP gel electrophoresis can be implemented to obtain high resolution 2D gels. One central key requirement for SSCP gel electrophoresis is a tight temperature control. Gels that run at different temperatures will produce entirely different fingerprints. This can be exploited for an improved analysis of highly diverse communities by running the same template at different temperatures or by 2D-SSCP gel electrophoresis.