Cargando…
Using acetone for rapid PCR‐amplifiable DNA extraction from recalcitrant woody plant taxa
PREMISE: Quick and effective DNA extraction from plants for subsequent PCR amplification is sometimes challenging when working across diverse plant taxa that may contain a variety of inhibitory compounds. Time‐consuming methods may be needed to overcome these inhibitory effects as well as the effect...
Autores principales: | , , |
---|---|
Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
John Wiley and Sons Inc.
2020
|
Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7742202/ https://www.ncbi.nlm.nih.gov/pubmed/33344094 http://dx.doi.org/10.1002/aps3.11403 |
_version_ | 1783623932626075648 |
---|---|
author | Gouker, Fred E. Guo, Yonghong Pooler, Margaret R. |
author_facet | Gouker, Fred E. Guo, Yonghong Pooler, Margaret R. |
author_sort | Gouker, Fred E. |
collection | PubMed |
description | PREMISE: Quick and effective DNA extraction from plants for subsequent PCR amplification is sometimes challenging when working across diverse plant taxa that may contain a variety of inhibitory compounds. Time‐consuming methods may be needed to overcome these inhibitory effects as well as the effects of various preservation and collection methods to extract DNA from leaf samples. Our objective was to develop a rapid DNA extraction protocol that could be used with diverse plant taxa to produce high‐quality DNA suitable for downstream PCR applications. METHODS AND RESULTS: We tested the efficacy of acetone in extracting DNA from fresh, frozen, oven‐dried, acetone‐fixed, and herbarium leaf material of 22 species from 16 woody and herbaceous plant families. An improved simplified DNA extraction protocol was developed using acetone‐fixed leaf material. The addition of 1% sodium dodecyl sulfate solution resulted in the optimal extraction from all tissue samples. The DNA resulting from the extraction protocol was readily amplified using real‐time PCR assays. CONCLUSIONS: The protocol described here resulted in the extraction of DNA from recalcitrant plant species that was of sufficient quality and quantity for PCR amplification, as indicated by the low threshold cycle values from real‐time assays. This method is simple, fast, and cost‐effective, and is a reliable tool for extracting high‐quality DNA from plant material containing PCR inhibitors. |
format | Online Article Text |
id | pubmed-7742202 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2020 |
publisher | John Wiley and Sons Inc. |
record_format | MEDLINE/PubMed |
spelling | pubmed-77422022020-12-18 Using acetone for rapid PCR‐amplifiable DNA extraction from recalcitrant woody plant taxa Gouker, Fred E. Guo, Yonghong Pooler, Margaret R. Appl Plant Sci Protocol Note PREMISE: Quick and effective DNA extraction from plants for subsequent PCR amplification is sometimes challenging when working across diverse plant taxa that may contain a variety of inhibitory compounds. Time‐consuming methods may be needed to overcome these inhibitory effects as well as the effects of various preservation and collection methods to extract DNA from leaf samples. Our objective was to develop a rapid DNA extraction protocol that could be used with diverse plant taxa to produce high‐quality DNA suitable for downstream PCR applications. METHODS AND RESULTS: We tested the efficacy of acetone in extracting DNA from fresh, frozen, oven‐dried, acetone‐fixed, and herbarium leaf material of 22 species from 16 woody and herbaceous plant families. An improved simplified DNA extraction protocol was developed using acetone‐fixed leaf material. The addition of 1% sodium dodecyl sulfate solution resulted in the optimal extraction from all tissue samples. The DNA resulting from the extraction protocol was readily amplified using real‐time PCR assays. CONCLUSIONS: The protocol described here resulted in the extraction of DNA from recalcitrant plant species that was of sufficient quality and quantity for PCR amplification, as indicated by the low threshold cycle values from real‐time assays. This method is simple, fast, and cost‐effective, and is a reliable tool for extracting high‐quality DNA from plant material containing PCR inhibitors. John Wiley and Sons Inc. 2020-12-03 /pmc/articles/PMC7742202/ /pubmed/33344094 http://dx.doi.org/10.1002/aps3.11403 Text en © 2020 Gouker et al. Applications in Plant Sciences is published by Wiley Periodicals, LLC on behalf of the Botanical Society of America This is an open access article under the terms of the http://creativecommons.org/licenses/by/4.0/ License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. |
spellingShingle | Protocol Note Gouker, Fred E. Guo, Yonghong Pooler, Margaret R. Using acetone for rapid PCR‐amplifiable DNA extraction from recalcitrant woody plant taxa |
title | Using acetone for rapid PCR‐amplifiable DNA extraction from recalcitrant woody plant taxa |
title_full | Using acetone for rapid PCR‐amplifiable DNA extraction from recalcitrant woody plant taxa |
title_fullStr | Using acetone for rapid PCR‐amplifiable DNA extraction from recalcitrant woody plant taxa |
title_full_unstemmed | Using acetone for rapid PCR‐amplifiable DNA extraction from recalcitrant woody plant taxa |
title_short | Using acetone for rapid PCR‐amplifiable DNA extraction from recalcitrant woody plant taxa |
title_sort | using acetone for rapid pcr‐amplifiable dna extraction from recalcitrant woody plant taxa |
topic | Protocol Note |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7742202/ https://www.ncbi.nlm.nih.gov/pubmed/33344094 http://dx.doi.org/10.1002/aps3.11403 |
work_keys_str_mv | AT goukerfrede usingacetoneforrapidpcramplifiablednaextractionfromrecalcitrantwoodyplanttaxa AT guoyonghong usingacetoneforrapidpcramplifiablednaextractionfromrecalcitrantwoodyplanttaxa AT poolermargaretr usingacetoneforrapidpcramplifiablednaextractionfromrecalcitrantwoodyplanttaxa |