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In Situ Dark Adaptation Enhances the Efficiency of DNA Extraction from Mature Pin Oak (Quercus palustris) Leaves, Facilitating the Identification of Partial Sequences of the 18S rRNA and Isoprene Synthase (IspS) Genes

Mature oak (Quercus spp.) leaves, although abundantly available during the plants’ developmental cycle, are rarely exploited as viable sources of genomic DNA. These leaves are rich in metabolites difficult to remove during standard DNA purification, interfering with downstream molecular genetics app...

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Autores principales: Barta, Csengele E., Bolander, Bethany, Bilby, Steven R., Brown, Jeremy H., Brown, Reid N., Duryee, Alexander M., Edelman, Danielle R., Gray, Christina E., Gossett, Chandler, Haddock, Amie G., Helsel, Mackenzie M., Jones, Alyssa D., Klingseis, Marissa E., Leslie, Kalif, Miles, Edward W., Prawitz, Rachael A.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2017
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5750628/
https://www.ncbi.nlm.nih.gov/pubmed/29073736
http://dx.doi.org/10.3390/plants6040052
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author Barta, Csengele E.
Bolander, Bethany
Bilby, Steven R.
Brown, Jeremy H.
Brown, Reid N.
Duryee, Alexander M.
Edelman, Danielle R.
Gray, Christina E.
Gossett, Chandler
Haddock, Amie G.
Helsel, Mackenzie M.
Jones, Alyssa D.
Klingseis, Marissa E.
Leslie, Kalif
Miles, Edward W.
Prawitz, Rachael A.
author_facet Barta, Csengele E.
Bolander, Bethany
Bilby, Steven R.
Brown, Jeremy H.
Brown, Reid N.
Duryee, Alexander M.
Edelman, Danielle R.
Gray, Christina E.
Gossett, Chandler
Haddock, Amie G.
Helsel, Mackenzie M.
Jones, Alyssa D.
Klingseis, Marissa E.
Leslie, Kalif
Miles, Edward W.
Prawitz, Rachael A.
author_sort Barta, Csengele E.
collection PubMed
description Mature oak (Quercus spp.) leaves, although abundantly available during the plants’ developmental cycle, are rarely exploited as viable sources of genomic DNA. These leaves are rich in metabolites difficult to remove during standard DNA purification, interfering with downstream molecular genetics applications. The current work assessed whether in situ dark adaptation, to deplete sugar reserves and inhibit secondary metabolite synthesis could compensate for the difficulties encountered when isolating DNA from mature leaves rich in secondary metabolites. We optimized a rapid, commercial kit based method to extract genomic DNA from dark- and light-adapted leaves. We demonstrated that in situ dark adaptation increases the yield and quality of genomic DNA obtained from mature oak leaves, yielding templates of sufficiently high quality for direct downstream applications, such as PCR amplification and gene identification. The quality of templates isolated from dark-adapted pin oak leaves particularly improved the amplification of larger fragments in our experiments. From DNA extracts prepared with our optimized method, we identified for the first time partial segments of the genes encoding 18S rRNA and isoprene synthase (IspS) from pin oak (Quercus palustris), whose full genome has not yet been sequenced.
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spelling pubmed-57506282018-01-08 In Situ Dark Adaptation Enhances the Efficiency of DNA Extraction from Mature Pin Oak (Quercus palustris) Leaves, Facilitating the Identification of Partial Sequences of the 18S rRNA and Isoprene Synthase (IspS) Genes Barta, Csengele E. Bolander, Bethany Bilby, Steven R. Brown, Jeremy H. Brown, Reid N. Duryee, Alexander M. Edelman, Danielle R. Gray, Christina E. Gossett, Chandler Haddock, Amie G. Helsel, Mackenzie M. Jones, Alyssa D. Klingseis, Marissa E. Leslie, Kalif Miles, Edward W. Prawitz, Rachael A. Plants (Basel) Article Mature oak (Quercus spp.) leaves, although abundantly available during the plants’ developmental cycle, are rarely exploited as viable sources of genomic DNA. These leaves are rich in metabolites difficult to remove during standard DNA purification, interfering with downstream molecular genetics applications. The current work assessed whether in situ dark adaptation, to deplete sugar reserves and inhibit secondary metabolite synthesis could compensate for the difficulties encountered when isolating DNA from mature leaves rich in secondary metabolites. We optimized a rapid, commercial kit based method to extract genomic DNA from dark- and light-adapted leaves. We demonstrated that in situ dark adaptation increases the yield and quality of genomic DNA obtained from mature oak leaves, yielding templates of sufficiently high quality for direct downstream applications, such as PCR amplification and gene identification. The quality of templates isolated from dark-adapted pin oak leaves particularly improved the amplification of larger fragments in our experiments. From DNA extracts prepared with our optimized method, we identified for the first time partial segments of the genes encoding 18S rRNA and isoprene synthase (IspS) from pin oak (Quercus palustris), whose full genome has not yet been sequenced. MDPI 2017-10-24 /pmc/articles/PMC5750628/ /pubmed/29073736 http://dx.doi.org/10.3390/plants6040052 Text en © 2017 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Barta, Csengele E.
Bolander, Bethany
Bilby, Steven R.
Brown, Jeremy H.
Brown, Reid N.
Duryee, Alexander M.
Edelman, Danielle R.
Gray, Christina E.
Gossett, Chandler
Haddock, Amie G.
Helsel, Mackenzie M.
Jones, Alyssa D.
Klingseis, Marissa E.
Leslie, Kalif
Miles, Edward W.
Prawitz, Rachael A.
In Situ Dark Adaptation Enhances the Efficiency of DNA Extraction from Mature Pin Oak (Quercus palustris) Leaves, Facilitating the Identification of Partial Sequences of the 18S rRNA and Isoprene Synthase (IspS) Genes
title In Situ Dark Adaptation Enhances the Efficiency of DNA Extraction from Mature Pin Oak (Quercus palustris) Leaves, Facilitating the Identification of Partial Sequences of the 18S rRNA and Isoprene Synthase (IspS) Genes
title_full In Situ Dark Adaptation Enhances the Efficiency of DNA Extraction from Mature Pin Oak (Quercus palustris) Leaves, Facilitating the Identification of Partial Sequences of the 18S rRNA and Isoprene Synthase (IspS) Genes
title_fullStr In Situ Dark Adaptation Enhances the Efficiency of DNA Extraction from Mature Pin Oak (Quercus palustris) Leaves, Facilitating the Identification of Partial Sequences of the 18S rRNA and Isoprene Synthase (IspS) Genes
title_full_unstemmed In Situ Dark Adaptation Enhances the Efficiency of DNA Extraction from Mature Pin Oak (Quercus palustris) Leaves, Facilitating the Identification of Partial Sequences of the 18S rRNA and Isoprene Synthase (IspS) Genes
title_short In Situ Dark Adaptation Enhances the Efficiency of DNA Extraction from Mature Pin Oak (Quercus palustris) Leaves, Facilitating the Identification of Partial Sequences of the 18S rRNA and Isoprene Synthase (IspS) Genes
title_sort in situ dark adaptation enhances the efficiency of dna extraction from mature pin oak (quercus palustris) leaves, facilitating the identification of partial sequences of the 18s rrna and isoprene synthase (isps) genes
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5750628/
https://www.ncbi.nlm.nih.gov/pubmed/29073736
http://dx.doi.org/10.3390/plants6040052
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