( Richard Hastings Jon Sa...)Monitoring Bacterial Genetic Diversity in a Freshwater Lake Using TTGE and DNA Sequence Analysis

Richard Hastings, Jon Saunders and Alan McCarthy
School of Biological Sciences, University of Liverpool, Liverpool, UK

Introduction
Microbial ecology is now firmly focused on the genotypic analysis of naturally-occurring assemblages as facilitated by the development of molecular biological techniques that enable amplification and sequencing of DNA extracted directly from the environment. The ubiquitous prokaryotic 16S ribosomal RNA gene has been the most widely targeted molecule for these studies because regions of nucleotide sequence conservation allow PCR* amplification to generate DNA fragments incorporating the variable sequence regions from which phylogeny can be inferred. In this paper, we describe the use of Temporal Temperature Gradient Gel Electrophoresis (TTGE) as the method of choice for sequence variation studies. In the commonly used Denaturing Gradient Gel Electrophoresis,⁵ electrophoretic separation is performed at high temperature in the presence of a chemical gradient. In TTGE, the denaturing environment is formed by a constant concentration of denaturant in the gel in combination with a gradual increase in temperature over the course of the separation. Thus, by using TTGE, one avoids pouring chemical gradient gels.⁶

Temporal Temperature Gradient Gel Electrophoresis is an attractive technique for the molecular microbial ecologist, as it separates similar length PCR products according to sequence variation typical of 16S rRNA genes. PCR products separated by TTGE into discrete bands, in theory, represent individual DNA species from a microbial consortium, so banding profiles provide a rapid insight into genetic diversity in situ. Excision
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