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    The recovery of nucleic acid from biomining and acid mine drainage microorganisms

    154613_154613.pdf (161.2Kb)
    Access Status
    Open access
    Authors
    Zammit, Carla
    Mutch, Lesley
    Watling, Helen
    Watkin, Elizabeth
    Date
    2011
    Type
    Journal Article
    
    Metadata
    Show full item record
    Citation
    Zammit, Carla M. and Mutch, Lesley A. and Watling, Helen R. and Watkin, Elizabeth L.J. 2011. The recovery of nucleic acid from biomining and acid mine drainage microorganisms. Hydrometallurgy. 108 (1-2): pp. 87-92.
    Source Title
    Hydrometallurgy
    DOI
    10.1016/j.hydromet.2011.03.002
    ISSN
    0304386X
    School
    School of Biomedical Sciences
    Remarks

    NOTICE: This is the author’s version of a work that was accepted for publication in Hydrometallurgy. Changes resulting from the publishing process, such as peer review, editing, corrections, structural formatting, and other quality control mechanisms may not be reflected in this document. Changes may have been made to this work since it was submitted for publication. A definitive version was subsequently published in Hydrometallurgy [108, 1-2, 2011] DOI 10.1016/j.hydromet.2011.03.002

    URI
    http://hdl.handle.net/20.500.11937/15531
    Collection
    • Curtin Research Publications
    Abstract

    The cornerstone of biological molecular techniques is the extraction of the intra- or extra-cellular component of interest. However, inefficiencies in the extraction method can lead to results that are not representative of the microbial population under investigation. It is particularly difficult to extract clean and pure samples of the cellular component of interest from the microbial inhabitants of low-pH, sulfidic environments, such as those found in biomining or acid mine drainage (AMD). A number of nucleic acid (NA) extraction methods were tested using microorganisms commonly found in biomining and AMD environments, including archaea and Gram-negative and Gram-positive bacteria. The different stages in the methods of NA extraction were investigated separately, including the: (i) removal of cells from pyrite, (ii) cell lysis and nucleic acid extraction, (iii) nucleic acid precipitation and (iv) RNA purification using, as appropriate, microscopy, spectrophotometry, agarose gel electrophoresis of NA, PCR, quantitative-PCR and quantitative reverse transcriptase-PCR to assess the quality and quantity of the DNA and RNA.The relative percentage of NA recovered from each microorganism using the optimised method discussed in this paper returned the following percentage of NA per cell: At. ferrooxidans 91% ± 1.4%; At. caldus 91% ± 2.7%; L. ferriphilum 98% ± 1.2%; F. acidiphilum 83% ± 2.9%; and S. thermosulfidooxidans 79% ± 0.7%. Differences in lysis methods and NA precipitation greatly impacted the quality and quantity of the extracted NA. A method for the reliable, representative and reproducible extraction of NA from five strains of biomining and AMD microorganisms from pyrite and from liquid culture is described.

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