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Four PCR primers necessary for the detection of periplasmic nitrate reductase genes in all groups of Proteobacteria and in environmental DNA
Generic primers are available for detecting bacterial genes required for almost every reaction of the biological nitrogen cycle, the one notable exception being napA (gene for the molybdoprotein of the periplasmic nitrate reductase) encoding periplasmic nitrate reductases. Using an iterative approac...
Autores principales: | , , , |
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Formato: | Texto |
Lenguaje: | English |
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Portland Press Ltd.
2011
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3064403/ https://www.ncbi.nlm.nih.gov/pubmed/21265796 http://dx.doi.org/10.1042/BST0390321 |
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author | Klatte, Tobias Evans, Laura Whitehead, Rebekah N. Cole, Jeffrey A. |
author_facet | Klatte, Tobias Evans, Laura Whitehead, Rebekah N. Cole, Jeffrey A. |
author_sort | Klatte, Tobias |
collection | PubMed |
description | Generic primers are available for detecting bacterial genes required for almost every reaction of the biological nitrogen cycle, the one notable exception being napA (gene for the molybdoprotein of the periplasmic nitrate reductase) encoding periplasmic nitrate reductases. Using an iterative approach, we report the first successful design of three forward oligonucleotide primers and one reverse primer that, in three separate PCRs, can amplify napA DNA from all five groups of Proteobacteria. All 140 napA sequences currently listed in the NCBI (National Center for Biotechnology Information) database are predicted to be amplified by one or more of these primer pairs. We demonstrate that two pairs of these primers also amplify PCR products of the predicted sizes from DNA isolated from human faeces, confirming their ability to direct the amplification of napA fragments from mixed populations. Analysis of the resulting amplicons by high-throughput sequencing will enable a good estimate to be made of both the range and relative abundance of nitrate-reducing bacteria in any community, subject only to any unavoidable bias inherent in a PCR approach to molecular characterization of a highly diverse target. |
format | Text |
id | pubmed-3064403 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2011 |
publisher | Portland Press Ltd. |
record_format | MEDLINE/PubMed |
spelling | pubmed-30644032011-03-29 Four PCR primers necessary for the detection of periplasmic nitrate reductase genes in all groups of Proteobacteria and in environmental DNA Klatte, Tobias Evans, Laura Whitehead, Rebekah N. Cole, Jeffrey A. Biochem Soc Trans Biochemical Society Focused Meetings Generic primers are available for detecting bacterial genes required for almost every reaction of the biological nitrogen cycle, the one notable exception being napA (gene for the molybdoprotein of the periplasmic nitrate reductase) encoding periplasmic nitrate reductases. Using an iterative approach, we report the first successful design of three forward oligonucleotide primers and one reverse primer that, in three separate PCRs, can amplify napA DNA from all five groups of Proteobacteria. All 140 napA sequences currently listed in the NCBI (National Center for Biotechnology Information) database are predicted to be amplified by one or more of these primer pairs. We demonstrate that two pairs of these primers also amplify PCR products of the predicted sizes from DNA isolated from human faeces, confirming their ability to direct the amplification of napA fragments from mixed populations. Analysis of the resulting amplicons by high-throughput sequencing will enable a good estimate to be made of both the range and relative abundance of nitrate-reducing bacteria in any community, subject only to any unavoidable bias inherent in a PCR approach to molecular characterization of a highly diverse target. Portland Press Ltd. 2011-01-19 2011-02-01 /pmc/articles/PMC3064403/ /pubmed/21265796 http://dx.doi.org/10.1042/BST0390321 Text en © 2011 The Author(s) The author(s) has paid for this article to be freely available under the terms of the Creative Commons Attribution Non-Commercial Licence (http://creativecommons.org/licenses/by-nc/2.5/) which permits unrestricted non-commercial use, distribution and reproduction in any medium, provided the original work is properly cited. http://creativecommons.org/licenses/by-nc/2.5/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. |
spellingShingle | Biochemical Society Focused Meetings Klatte, Tobias Evans, Laura Whitehead, Rebekah N. Cole, Jeffrey A. Four PCR primers necessary for the detection of periplasmic nitrate reductase genes in all groups of Proteobacteria and in environmental DNA |
title | Four PCR primers necessary for the detection of periplasmic nitrate reductase genes in all groups of Proteobacteria and in environmental DNA |
title_full | Four PCR primers necessary for the detection of periplasmic nitrate reductase genes in all groups of Proteobacteria and in environmental DNA |
title_fullStr | Four PCR primers necessary for the detection of periplasmic nitrate reductase genes in all groups of Proteobacteria and in environmental DNA |
title_full_unstemmed | Four PCR primers necessary for the detection of periplasmic nitrate reductase genes in all groups of Proteobacteria and in environmental DNA |
title_short | Four PCR primers necessary for the detection of periplasmic nitrate reductase genes in all groups of Proteobacteria and in environmental DNA |
title_sort | four pcr primers necessary for the detection of periplasmic nitrate reductase genes in all groups of proteobacteria and in environmental dna |
topic | Biochemical Society Focused Meetings |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3064403/ https://www.ncbi.nlm.nih.gov/pubmed/21265796 http://dx.doi.org/10.1042/BST0390321 |
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