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 mitochondr...
| Main Authors: | , , , |
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| Format: | Article |
| Language: | English |
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Wiley
2018-09-01
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| Series: | Applications in Plant Sciences |
| Subjects: | |
| Online Access: | https://doi.org/10.1002/aps3.1183 |
| _version_ | 1818418187183587328 |
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| author | Najmeh Nasiri Ghorbanali Nematzadeh Vahid Shariati‐Joni Hamid Najafi‐Zarrini |
| author_facet | Najmeh Nasiri Ghorbanali Nematzadeh Vahid Shariati‐Joni Hamid Najafi‐Zarrini |
| author_sort | Najmeh Nasiri |
| collection | DOAJ |
| 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. |
| first_indexed | 2024-12-14T12:18:41Z |
| format | Article |
| id | doaj.art-6f277a590c324439879b483fe5fb7784 |
| institution | Directory Open Access Journal |
| issn | 2168-0450 |
| language | English |
| last_indexed | 2024-12-14T12:18:41Z |
| publishDate | 2018-09-01 |
| publisher | Wiley |
| record_format | Article |
| series | Applications in Plant Sciences |
| spelling | doaj.art-6f277a590c324439879b483fe5fb77842022-12-21T23:01:34ZengWileyApplications in Plant Sciences2168-04502018-09-0169n/an/a10.1002/aps3.1183NaOH low‐salt method for chloroplast isolation and highly pure cpDNA preparation from Aeluropus littoralisNajmeh Nasiri0Ghorbanali Nematzadeh1Vahid Shariati‐Joni2Hamid Najafi‐Zarrini3Department of Molecular Plant Breeding and Genetic Engineering University of Agricultural Sciences and Natural Resources Sari IranDepartment of Plant Genetics, Genetic and Agricultural Biotechnology Institute of Tabarestan (GABIT) University of Agricultural Sciences and Natural Resources Sari IranDepartment of Plant Molecular Biotechnology National Institute of Genetic Engineering and Biotechnology (NIGEB) Tehran IranDepartment of Plant Breeding and Biotechnology University of Agricultural Sciences and Natural Resources Sari IranPremise 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.https://doi.org/10.1002/aps3.1183chloroplast extractioncpDNA preparationhigh‐salt methodsPoaceaewhole genome sequencing |
| spellingShingle | Najmeh Nasiri Ghorbanali Nematzadeh Vahid Shariati‐Joni Hamid Najafi‐Zarrini NaOH low‐salt method for chloroplast isolation and highly pure cpDNA preparation from Aeluropus littoralis Applications in Plant Sciences chloroplast extraction cpDNA preparation high‐salt methods Poaceae whole genome sequencing |
| title | NaOH low‐salt method for chloroplast isolation and highly pure cpDNA preparation from Aeluropus littoralis |
| title_full | NaOH low‐salt method for chloroplast isolation and highly pure cpDNA preparation from Aeluropus littoralis |
| title_fullStr | NaOH low‐salt method for chloroplast isolation and highly pure cpDNA preparation from Aeluropus littoralis |
| title_full_unstemmed | NaOH low‐salt method for chloroplast isolation and highly pure cpDNA preparation from Aeluropus littoralis |
| title_short | NaOH low‐salt method for chloroplast isolation and highly pure cpDNA preparation from Aeluropus littoralis |
| title_sort | naoh low salt method for chloroplast isolation and highly pure cpdna preparation from aeluropus littoralis |
| topic | chloroplast extraction cpDNA preparation high‐salt methods Poaceae whole genome sequencing |
| url | https://doi.org/10.1002/aps3.1183 |
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