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Laboratory Protocols in Fungal Biology pp 289–298Cite as

Semi-Nested PCR Approach to Amplify Large 18S rRNA Gene Fragments for PCR-DGGE Analysis of Soil Fungal Communities

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Part of the book series: Fungal Biology ((FUNGBIO))

Abstract

Denaturing gradient gel electrophoresis (DGGE) of 18S rRNA gene fragments PCR-amplified from total community DNA is a powerful tool for the parallel comparative analysis of environmental fungal communities. The 18S rRNA gene has the advantages of universality, high phylogenetic information content, and a large number of sequences in the data banks. The comparative analysis of soil fungal communities from large numbers of samples by PCR-DGGE requires consistent amplification and separation efficiency, as achieved by the following semi-nested PCR-DGGE protocol based on two-step PCR of 1,650 bp rRNA gene fragments from bulk soil DNA and their separation in DGGE.

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Author information

Authors and Affiliations

  1. Julius Kühn Institut, Institute for Epidemiology and Pathogen Diagnostics, Zimmerstrasse 26, Braunschweig, Lower Saxony, 38106, Germany

    Miruna Oros-Sichler

  2. Julius Kühn Institut, Federal Research Centre for Cultivated Plants, Institute for Epidemiology and Pathogen Diagnostics, Messeweg 11-12, Braunschweig, Lower Saxony, 38104, Germany

    Kornelia Smalla

Authors
  1. Miruna Oros-Sichler
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  2. Kornelia Smalla
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Corresponding author

Correspondence to Kornelia Smalla .

Editor information

Editors and Affiliations

  1. , Department of Biochemistry, National University of Ireland Galway, University Road, Galway, N/A, Ireland

    Vijai Kumar Gupta

  2. , Department of Biochemistry, National University of Ireland Galway, University Road, Galway, N/A, Ireland

    Maria G. Tuohy

  3. , Department of Biochemistry, National University of Ireland Galway, University Road, Galway, N/A, Ireland

    Manimaran Ayyachamy

  4. , Manufacturing Sciences and Technology, Pfizer Ireland Pharmaceuticals, The Pfizer Biotech Campus at Grange Castle, Clondalkin, N/A, Dublin, Ireland

    Kevin M. Turner

  5. , School of Natural Sciences, National University of Ireland Galway, University Road, Galway, N/A, Ireland

    Anthonia O’Donovan

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Oros-Sichler, M., Smalla, K. (2013). Semi-Nested PCR Approach to Amplify Large 18S rRNA Gene Fragments for PCR-DGGE Analysis of Soil Fungal Communities. In: Gupta, V., Tuohy, M., Ayyachamy, M., Turner, K., O’Donovan, A. (eds) Laboratory Protocols in Fungal Biology. Fungal Biology. Springer, New York, NY. https://doi.org/10.1007/978-1-4614-2356-0_23

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