SS2.06 Microbial Diversity in Time and Space
Date: Tuesday, June 11, 2002
Time: 10:30:00 AM
Location: Lecture Theatre
 
StephensAQ, University of California, Santa Cruz, USA, stephens@es.ucsc.edu
Steward, G, F, University of California, Santa Cruz, USA, gsteward@cats.ucsc.edu
Zehr, J, P, University of California, Santa Cruz, USA, zehrj@cats.ucsc.edu
 
COMPUTER SIMULATION OF TRFLP USING FUNCTIONAL GENE SEQUENCES AND IMPLICATIONS FOR MICROBIAL COMMUNITY ANALYSIS
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Terminal Restriction Fragment Length Polymorphism (TRFLP) is a PCR and restriction enzyme based technique used in characterizing microbial communities. Effective use of this technique requires the selection of appropriate restriction enzymes. We developed a software package for TRFLP simulations, and used it to investigate several databases of functional gene sequences. A set of 207 restriction enzymes was evaluated for the ability to uniquely distinguish individual nitrogenase (nifH) and nitrite reductase (nirS) DNA sequences. Sequence discrimination was found to range from 0 - 48 % for different enzymes. As the size of the database (or environmental complexity) increased, sequence discrimination decreased. Enzyme performance varied among target genes and environmental contexts. Multi-enzyme digests run in parallel produced better discrimination than single-enzyme digests, with an accompanying increase in complexity of interpretation. These results imply that TRFLP is best applied in well-characterized environments, and that computer analysis can improve the effectiveness of the technique by optimizing enzyme selection and simplifying the interpretation of results.