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Clone Libraries of Ribosomal RNA Gene Sequences for Characterization of Microbial Communities

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Hydrocarbon and Lipid Microbiology Protocols

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Abstract

Constructing clone libraries from phylogenetic marker genes is one approach for assessing microbial community composition and diversity. Although more expensive and time-consuming than community fingerprinting techniques, sequence analysis of clone libraries provides a high level of phylogenetic resolution due to long read lengths generated by Sanger sequencing. In this chapter, we provide the principles and methodologies of clone library construction and sequence analyses for the purpose of investigating bacterial, archaeal, and fungal community composition. Protocols for generating libraries from either ribosomal RNA (rRNA) genes or rRNA transcripts are provided. Each of the multiple steps involved in cloning is discussed, including DNA extraction, PCR amplification of phylogenetic marker genes, cleanup and preparation of the insert, ligation of the insert into a plasmid vector, transformation of competent Escherichia coli cells, and screening of clones to verify the presence of cloned inserts, followed by DNA sequence analyses. Primers useful for phylogenetic analyses of bacterial, archaeal, and fungal communities are provided.

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Acknowledgments

Stefan Green is thanked for constructive comments on this protocol. MBL was supported by the National Science Foundation under Award 0626544. DLT was supported by the National Science Foundation under Grant no. 0632332, and JDN acknowledges a Discovery Grant from the Natural Sciences and Engineering Research Council of Canada (NSERC).

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Authors and Affiliations

  1. Institute of Arctic Biology, University of Alaska Fairbanks, Fairbanks, AK, 99775-7000, USA

    Mary Beth Leigh

  2. Department of Biology, University of New Mexico, Albuquerque, NM, 87131-0001, USA

    Lee Taylor

  3. Department of Biology, University of Waterloo, Waterloo, ON, N2L 3G1, Canada

    Josh D. Neufeld

Authors
  1. Mary Beth Leigh
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  2. Lee Taylor
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  3. Josh D. Neufeld
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Corresponding authors

Correspondence to Mary Beth Leigh , Lee Taylor or Josh D. Neufeld .

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Editors and Affiliations

  1. University of Essex Dept. Biological Sciences, Colchester, Essex, United Kingdom

    Terry J. McGenity

  2. Institute of Microbiology, Technical University Braunschweig, Braunschweig, Germany

    Kenneth N. Timmis

  3. Dept. Biologia, Univ de les Illes Balears, Palma de Mallorca, Spain

    Balbina Nogales

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Leigh, M.B., Taylor, L., Neufeld, J.D. (2015). Clone Libraries of Ribosomal RNA Gene Sequences for Characterization of Microbial Communities. In: McGenity, T., Timmis, K., Nogales , B. (eds) Hydrocarbon and Lipid Microbiology Protocols. Springer Protocols Handbooks. Springer, Berlin, Heidelberg. https://doi.org/10.1007/8623_2015_120

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  • DOI: https://doi.org/10.1007/8623_2015_120

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