The recovery of nucleic acid from biomining and acid mine drainage microorganisms
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 t...
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| Format: | Journal Article |
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Elsevier Science BV
2011
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| Online Access: | http://hdl.handle.net/20.500.11937/15531 |
| _version_ | 1848748917919842304 |
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| author | Zammit, Carla Mutch, Lesley Watling, Helen Watkin, Elizabeth |
| author_facet | Zammit, Carla Mutch, Lesley Watling, Helen Watkin, Elizabeth |
| author_sort | Zammit, Carla |
| building | Curtin Institutional Repository |
| collection | Online Access |
| description | 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. |
| first_indexed | 2025-11-14T07:12:40Z |
| format | Journal Article |
| id | curtin-20.500.11937-15531 |
| institution | Curtin University Malaysia |
| institution_category | Local University |
| last_indexed | 2025-11-14T07:12:40Z |
| publishDate | 2011 |
| publisher | Elsevier Science BV |
| recordtype | eprints |
| repository_type | Digital Repository |
| spelling | curtin-20.500.11937-155312019-02-19T04:25:52Z The recovery of nucleic acid from biomining and acid mine drainage microorganisms Zammit, Carla Mutch, Lesley Watling, Helen Watkin, Elizabeth RNA extraction Acid mine drainage Bioleaching DNA extraction Microorganisms Biomining 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. 2011 Journal Article http://hdl.handle.net/20.500.11937/15531 10.1016/j.hydromet.2011.03.002 Elsevier Science BV fulltext |
| spellingShingle | RNA extraction Acid mine drainage Bioleaching DNA extraction Microorganisms Biomining Zammit, Carla Mutch, Lesley Watling, Helen Watkin, Elizabeth The recovery of nucleic acid from biomining and acid mine drainage microorganisms |
| title | The recovery of nucleic acid from biomining and acid mine drainage microorganisms |
| title_full | The recovery of nucleic acid from biomining and acid mine drainage microorganisms |
| title_fullStr | The recovery of nucleic acid from biomining and acid mine drainage microorganisms |
| title_full_unstemmed | The recovery of nucleic acid from biomining and acid mine drainage microorganisms |
| title_short | The recovery of nucleic acid from biomining and acid mine drainage microorganisms |
| title_sort | recovery of nucleic acid from biomining and acid mine drainage microorganisms |
| topic | RNA extraction Acid mine drainage Bioleaching DNA extraction Microorganisms Biomining |
| url | http://hdl.handle.net/20.500.11937/15531 |