Bacterial diversity of soils assessed by DGGE, T-RFLP and SSCP fingerprints of PCR-amplified 16S rRNA gene fragments: do the different methods provide similar results?

Bacterial communities of four arable soils--pelosol, gley, para brown soil, and podsol brown soil--were analysed by fingerprinting of 16S rRNA gene fragments amplified from total DNA of four replicate samples for each soil type. Fingerprints were generated in parallel by denaturing gradient gel electrophoresis (DGGE), terminal restriction fragment length polymorphism (T-RFLP), and single strand conformation polymorphism (SSCP) to test whether these commonly applied techniques are interchangeable. PCR amplicons could be separated with all three methods resulting in complex ribotype patterns.

A new semi-nested PCR protocol to amplify large 18S rRNA gene fragments for PCR-DGGE analysis of soil fungal communities.

The analysis of soil fungal communities by molecular fingerprinting and subsequent identification of the underlying populations require the amplification of a phylogenetically informative gene fragment. In this study we tested the reliability and suitability of the previously published fungal primer combination (NS1/FR1-GC) that amplifies almost the entire 18S rRNA gene for the DGGE analysis of fungal communities in soil samples from 36 sites. This direct PCR system failed to amplify the fragment of interest from the total DNA extracted from most of the soils tested.