Determining a minimum detection threshold in terminal restriction fragment length polymorphism analysis |
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Authors: | Courtney Kevin C Bainard Luke D Sikes Benjamin A Koch Alexander M Maherali Hafiz Klironomos John N Hart Miranda M |
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Institution: | a Department of Integrative Biology, University of Guelph, 50 Stone Road East, Guelph, ON, Canada N1G 2 W1b Section of Integrative Biology, The University of Texas, 1 University Station, Austin, TX 78712, USAc Biology, The University of British Columbia Okanagan, 3333 University Way, Kelowna, BC, Canada V1V 1 V7 |
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Abstract: | Terminal restriction fragment length polymorphism (T-RFLP) analysis is a common technique used to characterize soil microbial diversity. The fidelity of this technique in accurately reporting diversity has not been thoroughly evaluated. Here we determine if rare fungal species can be reliably detected by T-RFLP analysis. Spores from three arbuscular mycorrhizal fungal species were each mixed at a range of concentrations (1%, 10%, 50%, and 100%) with Glomus irregulare to establish a minimum detection threshold. T-RFLP analysis was capable of detecting diagnostic peaks of rare taxa at concentrations as low as 1%. The relative proportion of the target taxa in the sample and DNA concentration influenced peak detection reliability. However, low concentrations produced small, inconsistent electropherogram peaks contributing to difficulty in differentiating true peaks from signal noise. The results of this experiment suggest T-RFLP is a reproducible and high fidelity procedure, which requires careful data interpretation in order to accurately characterize sample diversity. |
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Keywords: | AM Arbuscular mycorrhizal ANOVA Analysis of Variance INVAM International Culture Collection of (Vesicular) Arbuscular Mycorrhizal Fungi LSU Large ribosomal subunit PCR Polymerase Chain Reaction P/E Primer/enzyme T-RFLP Terminal restriction fragment length polymorphism |
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