Wednesday, 9 November 2005 - 9:00 AM
274-3

Soil Microbial Community Analysis Using Terminal Restriction Fragment Length Polymorphisms: Separating Signals from the Noise.

Janice Thies, Cornell University, 719 Bradfield Hall, Dept. of Crop & Soil Science, Ithaca, NY 14853

Terminal restriction fragment length polymorphism (T-RFLP) analysis is a PCR fingerprinting method that enables community DNA profiles, obtained from different samples, to be compared. The technique has gained in popularity in recent years because it is highly reproducible and yields more information than most other PCR fingerprinting methods. Sample DNA is extracted and amplified by PCR using primers labeled at one end with a fluorescent dye. Resulting PCR products are of a similar size. To separate them and obtain a fingerprint, the PCR products are digested with a selected restriction enzyme. Amplified DNA from different organisms containing different restriction sites will yield terminally-labeled fragments of different sizes. These terminal restriction fragments (TRFs) can be sized on an automated DNA sequencer using either gel-based or capillary methods thus yielding a fingerprint that is characteristic of the community from which the DNA was extracted. For bacteria, the 16S rRNA gene has been the most commonly used target due to the phylogenetic information it may yield. However, sizing of the TRFs is not sufficiently robust to enable taxonomic placement to be derived from them. Instead, fingerprints from different samples are compared using multivariate statistics to determine if community shifts in response to chosen variables are occurring. Many statistical methods limit the information that can be obtained from these fingerprints because they yield a low signal relative to the noise common in these data. We have found that use of the Additive Main Effects Multiplicative Interaction (AMMI) model is most useful for revealing trends in T-RFLP data, thus allowing for more in-depth pattern analysis. This presentation will focus on how T-RFLP data are derived, the most robust means of analysis and how the method can be used in a variety of experiments to provide useful information about the behavior of microbial populations in soil.

Back to Symposium--Molecular Based Approaches to Soil Microbiology
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Back to The ASA-CSSA-SSSA International Annual Meetings (November 6-10, 2005)