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NaOH low‐salt method for chloroplast isolation and highly pure cpDNA preparation from Aeluropus littoralis
PREMISE OF THE STUDY: High‐yield pure chloroplast DNA (cpDNA) is necessary for whole genome sequencing. Chloroplast extraction with traditional high‐salt methods causes damage to nuclei and destroys the integrity of organelles, which leads to high genomic contamination from the nucleus and mitochond...
Autores principales: | , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
John Wiley and Sons Inc.
2018
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6159644/ https://www.ncbi.nlm.nih.gov/pubmed/30276031 http://dx.doi.org/10.1002/aps3.1183 |
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author | Nasiri, Najmeh Nematzadeh, Ghorbanali Shariati‐Joni, Vahid Najafi‐Zarrini, Hamid |
author_facet | Nasiri, Najmeh Nematzadeh, Ghorbanali Shariati‐Joni, Vahid Najafi‐Zarrini, Hamid |
author_sort | Nasiri, Najmeh |
collection | PubMed |
description | PREMISE OF THE STUDY: High‐yield pure chloroplast DNA (cpDNA) is necessary for whole genome sequencing. Chloroplast extraction with traditional high‐salt methods causes damage to nuclei and destroys the integrity of organelles, which leads to high genomic contamination from the nucleus and mitochondria. To overcome this issue, we modified a traditional high‐salt method to obtain a new approach called the NaOH low‐salt method (NLS). METHODS AND RESULTS: The NLS method is based on the mild alkaline lysis of plant cells, followed by homogenization with ultrasonic waves and fractionation under reduced osmotic pressure. Results showed that this modified protocol worked efficiently to extract the intact chloroplast from Aeluropus littoralis and other grasses to obtain high‐quality pure cpDNA, which was confirmed by fluorescent microscopy, qPCR, and Illumina paired‐end sequencing analysis. CONCLUSIONS: Compared with high‐salt methods, the NLS method has proven robust for extraction of intact chloroplasts and preparation of high‐yield pure cpDNA from grasses. |
format | Online Article Text |
id | pubmed-6159644 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2018 |
publisher | John Wiley and Sons Inc. |
record_format | MEDLINE/PubMed |
spelling | pubmed-61596442018-10-01 NaOH low‐salt method for chloroplast isolation and highly pure cpDNA preparation from Aeluropus littoralis Nasiri, Najmeh Nematzadeh, Ghorbanali Shariati‐Joni, Vahid Najafi‐Zarrini, Hamid Appl Plant Sci Protocol Note PREMISE OF THE STUDY: High‐yield pure chloroplast DNA (cpDNA) is necessary for whole genome sequencing. Chloroplast extraction with traditional high‐salt methods causes damage to nuclei and destroys the integrity of organelles, which leads to high genomic contamination from the nucleus and mitochondria. To overcome this issue, we modified a traditional high‐salt method to obtain a new approach called the NaOH low‐salt method (NLS). METHODS AND RESULTS: The NLS method is based on the mild alkaline lysis of plant cells, followed by homogenization with ultrasonic waves and fractionation under reduced osmotic pressure. Results showed that this modified protocol worked efficiently to extract the intact chloroplast from Aeluropus littoralis and other grasses to obtain high‐quality pure cpDNA, which was confirmed by fluorescent microscopy, qPCR, and Illumina paired‐end sequencing analysis. CONCLUSIONS: Compared with high‐salt methods, the NLS method has proven robust for extraction of intact chloroplasts and preparation of high‐yield pure cpDNA from grasses. John Wiley and Sons Inc. 2018-09-27 /pmc/articles/PMC6159644/ /pubmed/30276031 http://dx.doi.org/10.1002/aps3.1183 Text en © 2018 Nasiri et al. Applications in Plant Sciences is published by Wiley Periodicals, Inc. on behalf of the Botanical Society of America This is an open access article under the terms of the http://creativecommons.org/licenses/by-nc/4.0/ License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes. |
spellingShingle | Protocol Note Nasiri, Najmeh Nematzadeh, Ghorbanali Shariati‐Joni, Vahid Najafi‐Zarrini, Hamid NaOH low‐salt method for chloroplast isolation and highly pure cpDNA preparation from Aeluropus littoralis |
title | NaOH low‐salt method for chloroplast isolation and highly pure cpDNA preparation from Aeluropus littoralis
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title_full | NaOH low‐salt method for chloroplast isolation and highly pure cpDNA preparation from Aeluropus littoralis
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title_fullStr | NaOH low‐salt method for chloroplast isolation and highly pure cpDNA preparation from Aeluropus littoralis
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title_full_unstemmed | NaOH low‐salt method for chloroplast isolation and highly pure cpDNA preparation from Aeluropus littoralis
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title_short | NaOH low‐salt method for chloroplast isolation and highly pure cpDNA preparation from Aeluropus littoralis
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title_sort | naoh low‐salt method for chloroplast isolation and highly pure cpdna preparation from aeluropus littoralis |
topic | Protocol Note |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6159644/ https://www.ncbi.nlm.nih.gov/pubmed/30276031 http://dx.doi.org/10.1002/aps3.1183 |
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