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Single cell PCR amplification of diatoms using fresh and preserved samples
Single cell Chelex® DNA extraction and nested PCR amplification were used to examine partial gene sequences from natural diatom populations for taxonomic and phylogenetic studies at and above the level of species. DNA was extracted from cells that were either fresh collected or stored in RNAlater. E...
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Formato: | Online Artículo Texto |
Lenguaje: | English |
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Frontiers Media S.A.
2015
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Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4604310/ https://www.ncbi.nlm.nih.gov/pubmed/26528252 http://dx.doi.org/10.3389/fmicb.2015.01084 |
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author | Hamilton, Paul B. Lefebvre, Keely E. Bull, Roger D. |
author_facet | Hamilton, Paul B. Lefebvre, Keely E. Bull, Roger D. |
author_sort | Hamilton, Paul B. |
collection | PubMed |
description | Single cell Chelex® DNA extraction and nested PCR amplification were used to examine partial gene sequences from natural diatom populations for taxonomic and phylogenetic studies at and above the level of species. DNA was extracted from cells that were either fresh collected or stored in RNAlater. Extractions from Lugol's fixation were also attempted with limited success. Three partial gene sequences (rbcL, 18S, and psbA) were recovered using existing and new primers with a nested or double nested PCR approach with amplification and success rates between 70 and 96%. An rbcL consensus tree grouped morphologically similar specimens and was consistent across the two primary sample treatments: fresh and RNAlater. This tool will greatly enhance the number of microscopic diatom taxa (and potentially other microbes) available for barcoding and phylogenetic studies. The near-term increase in sequence data for diatoms generated via routine single cell extractions and PCR will act as a multiproxy validation of longer-term next generation genomics. |
format | Online Article Text |
id | pubmed-4604310 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2015 |
publisher | Frontiers Media S.A. |
record_format | MEDLINE/PubMed |
spelling | pubmed-46043102015-11-02 Single cell PCR amplification of diatoms using fresh and preserved samples Hamilton, Paul B. Lefebvre, Keely E. Bull, Roger D. Front Microbiol Microbiology Single cell Chelex® DNA extraction and nested PCR amplification were used to examine partial gene sequences from natural diatom populations for taxonomic and phylogenetic studies at and above the level of species. DNA was extracted from cells that were either fresh collected or stored in RNAlater. Extractions from Lugol's fixation were also attempted with limited success. Three partial gene sequences (rbcL, 18S, and psbA) were recovered using existing and new primers with a nested or double nested PCR approach with amplification and success rates between 70 and 96%. An rbcL consensus tree grouped morphologically similar specimens and was consistent across the two primary sample treatments: fresh and RNAlater. This tool will greatly enhance the number of microscopic diatom taxa (and potentially other microbes) available for barcoding and phylogenetic studies. The near-term increase in sequence data for diatoms generated via routine single cell extractions and PCR will act as a multiproxy validation of longer-term next generation genomics. Frontiers Media S.A. 2015-10-14 /pmc/articles/PMC4604310/ /pubmed/26528252 http://dx.doi.org/10.3389/fmicb.2015.01084 Text en Copyright © 2015 Hamilton, Lefebvre and Bull. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. |
spellingShingle | Microbiology Hamilton, Paul B. Lefebvre, Keely E. Bull, Roger D. Single cell PCR amplification of diatoms using fresh and preserved samples |
title | Single cell PCR amplification of diatoms using fresh and preserved samples |
title_full | Single cell PCR amplification of diatoms using fresh and preserved samples |
title_fullStr | Single cell PCR amplification of diatoms using fresh and preserved samples |
title_full_unstemmed | Single cell PCR amplification of diatoms using fresh and preserved samples |
title_short | Single cell PCR amplification of diatoms using fresh and preserved samples |
title_sort | single cell pcr amplification of diatoms using fresh and preserved samples |
topic | Microbiology |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4604310/ https://www.ncbi.nlm.nih.gov/pubmed/26528252 http://dx.doi.org/10.3389/fmicb.2015.01084 |
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