'dirty' business: testing the limitations of terminal restriction fragment length polymorphism (TRFLP) analysis of soil fungi

Terminal restriction fragment length polymorphism (TRFLP) is an increasingly popular method in molecular ecology. However, several key limitations of this method have not been fully examined especially when used to study fungi. We investigated the impact of spore contamination, intracollection ribos...

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Bibliographic Details
Published in:Molecular ecology Vol. 15; no. 3; pp. 873 - 882
Main Authors: Avis, Peter G, Dickie, Ian A, Mueller, Gregory M
Format: Journal Article
Language:English
Published: Oxford, UK Oxford, UK : Blackwell Publishing Ltd 01.03.2006
Blackwell Publishing Ltd
Subjects:
analysis
Base Sequence
community ecology
Deoxyribonucleic acid
DNA
DNA Restriction Enzymes
DNA, Fungal
DNA, Fungal - analysis
DNA, Fungal - genetics
DNA, Ribosomal Spacer
DNA, Ribosomal Spacer - genetics
ectomycorrhizae
Enzymes
Fluorescence
Fungi
Fungi - genetics
Genetic Variation
genetics
internal transcribed spacers
ITS variation
loci
Molecular Sequence Data
mycorrhizal fungi
Polymerase Chain Reaction
Polymorphism
Polymorphism, Restriction Fragment Length
restriction enzyme inefficiency
restriction enzyme recognition loci
restriction fragment length polymorphism
ribosomal DNA
Soil analysis
soil ecology
soil fungi
Soil Microbiology
Soils
Spores
terminal restriction fragment length polymorphism
TRFLP
ISSN:0962-1083, 1365-294X
Online Access:Get full text
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Summary:Terminal restriction fragment length polymorphism (TRFLP) is an increasingly popular method in molecular ecology. However, several key limitations of this method have not been fully examined especially when used to study fungi. We investigated the impact of spore contamination, intracollection ribosomal DNA internal transcribed spacer (ITS) region variation, and conserved restriction enzyme recognition loci on the results produced by TRFLP to characterize soil fungal communities. We find that (i) the potential for nontarget structures such as spores to contribute DNA to target sample extractions is high; (ii) multiple fragments (i.e. 'extra peaks') per PCR primer-restriction enzyme combination can be detected that are caused by restriction enzyme inefficiency and intracollection ribosomal DNA ITS variation; and (iii) restriction enzyme digestion in conserved vs. variable gene regions leads to different characterizations of community diversity. Based on these results, we suggest that studies employing TRFLP need to include information from known, identified fungi from sites within which studies take place and not to rely only on TRFLP profiles as a short cut to fungal community description.
Bibliography:http://dx.doi.org/10.1111/j.1365-294X.2005.02842.x
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ArticleID:MEC2842
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ISSN:0962-1083
1365-294X
DOI:10.1111/j.1365-294X.2005.02842.x