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Multiplex-terminal restriction fragment length polymorphism

Nature Protocols volume 1, pages 2428–2433 (2006)Cite this article

Abstract

A novel method called “multiplex-terminal restriction fragment length polymorphism (M-TRFLP)” has been recently developed which can be used for simultaneous analysis of the community composition of two or more microbial taxa (up to four). This method can also be used for microbial diagnostic purposes. For M-TRFLP analysis, primers specific to different target genes are used for multiplex-PCR, with one primer for each target being labeled with a unique fluorescent dye at its 5′ end. Restriction digestion of the amplified products followed by fragment size analysis on a DNA sequencer produces profiles for targeted genes, which can be distinguished from each other by the color of the terminal fragments imparted by the unique fluorescent dye used for primer labeling. In contrast to current protocols, M-TRFLP allows multiple communities or multiple targets (genes) data to be obtained in just one reaction and therefore saves time, cost and labor. This protocol can be completed in 5–8 h.

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Figure 1: Schematic outline of the protocol of M-TRFLP for community analysis and microbial diagnostics.
Figure 2: Outputs obtained for M-TRFLP after analysis by the sequencer software.

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Acknowledgements

We thank Loic Nazaries and Stacey Munro for initial technical support on this method. We also thank Mrs Laura Price (Unilever, Safety & Environmental Assurance Centre, Bedfordshire), Dr. Catriona Macdonald (Environmental Science Research, Porirua, New Zealand), Dr. Oliver Price (Cambridge Environmental Assessment, Boxworth, Cambridge) and Dr. Ian Anderson and Dr. Colin Campbell (Macaulay Institute), three anonymous referees and Dr. K. Barnes for their valuable suggestions to improve the quality of the manuscript. A patent application for commercial use of this method is under consideration by the British Patent Office.

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Authors and Affiliations

  1. Macaulay Institute, Craigiebuckler, Aberdeen, UK

    Brajesh K Singh & Nadine Thomas

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  1. Brajesh K Singh
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  2. Nadine Thomas
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Correspondence to Brajesh K Singh.

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Author declares competing financial interests: a patent application is under consideration which has been mentioned in the Acknowledgements section

Supplementary information

Supplementary Table 1

List of primers used in the present study. (PDF 50 kb)

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Singh, B., Thomas, N. Multiplex-terminal restriction fragment length polymorphism. Nat Protoc 1, 2428–2433 (2006). https://doi.org/10.1038/nprot.2006.392

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