A Mg2+-dependent high-yield method for extracting high-molecular-weight genomic DNA from a single planarian specimen
Abstract Background The isolation of intact, high-molecular-weight genomic DNA (HMW gDNA) is essential for achieving complete genome assemblies. However, extracting HMW gDNA from a single individual of Dugesia japonica remains a technical challenge using the standard protocol, probably due to the pr...
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BMC
2025-05-01
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| Series: | BMC Genomics |
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| Online Access: | https://doi.org/10.1186/s12864-025-11731-6 |
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| author | Ao Li Bingrui Sun Ying Zhang Ping Yu Jicheng Qu Hongkuan Deng Qiuxiang Pang Fengtang Yang |
| author_facet | Ao Li Bingrui Sun Ying Zhang Ping Yu Jicheng Qu Hongkuan Deng Qiuxiang Pang Fengtang Yang |
| author_sort | Ao Li |
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| description | Abstract Background The isolation of intact, high-molecular-weight genomic DNA (HMW gDNA) is essential for achieving complete genome assemblies. However, extracting HMW gDNA from a single individual of Dugesia japonica remains a technical challenge using the standard protocol, probably due to the presence of abundant polysaccharides and nucleases. Results In this study, we have developed a more robust protocol for preparing HMM gDNA, with high yields and quality, from a single D. japonica. The key step in our protocol involves the use of a Mg2+-dependent lysis buffer, rather than using metal cation chelation to block the activities of DNase I as in the standard protocol. Using this approach were able to achieve a yield of about 10–15 µg of HWM gDNA per worm. Our method showed species- and region-specific effectiveness, with optimal results observed at 20 mM Mg2+ for our local D. japonica specimens. The extracted HMW gDNA is fully compatible with advanced long-read sequencing platforms such as PacBio HiFi and Oxford Nanopore. However, when applied to Schmidtea mediterranea and D. japonica specimens from Beijing, the method was ineffective and led to progressive gDNA degradation. Conclusions This protocol offers a simple and high-yield solution for isolating HMW gDNA from D. japonica. It also provides an alternative for organisms whose gDNA consistently exhibits unexplained degradation using established protocols. |
| format | Article |
| id | doaj-art-22f25b3f6dd84a0f8b6604dd23d9f1a6 |
| institution | Kabale University |
| issn | 1471-2164 |
| language | English |
| publishDate | 2025-05-01 |
| publisher | BMC |
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| spelling | doaj-art-22f25b3f6dd84a0f8b6604dd23d9f1a62025-08-20T03:47:24ZengBMCBMC Genomics1471-21642025-05-0126111010.1186/s12864-025-11731-6A Mg2+-dependent high-yield method for extracting high-molecular-weight genomic DNA from a single planarian specimenAo Li0Bingrui Sun1Ying Zhang2Ping Yu3Jicheng Qu4Hongkuan Deng5Qiuxiang Pang6Fengtang Yang7School of Life Sciences and Medicine, Shandong University of TechnologySchool of Life Sciences and Medicine, Shandong University of TechnologySchool of Life Sciences and Medicine, Shandong University of TechnologySchool of Life Sciences and Medicine, Shandong University of TechnologySchool of Life Sciences and Medicine, Shandong University of TechnologySchool of Life Sciences and Medicine, Shandong University of TechnologySchool of Life Sciences and Medicine, Shandong University of TechnologySchool of Life Sciences and Medicine, Shandong University of TechnologyAbstract Background The isolation of intact, high-molecular-weight genomic DNA (HMW gDNA) is essential for achieving complete genome assemblies. However, extracting HMW gDNA from a single individual of Dugesia japonica remains a technical challenge using the standard protocol, probably due to the presence of abundant polysaccharides and nucleases. Results In this study, we have developed a more robust protocol for preparing HMM gDNA, with high yields and quality, from a single D. japonica. The key step in our protocol involves the use of a Mg2+-dependent lysis buffer, rather than using metal cation chelation to block the activities of DNase I as in the standard protocol. Using this approach were able to achieve a yield of about 10–15 µg of HWM gDNA per worm. Our method showed species- and region-specific effectiveness, with optimal results observed at 20 mM Mg2+ for our local D. japonica specimens. The extracted HMW gDNA is fully compatible with advanced long-read sequencing platforms such as PacBio HiFi and Oxford Nanopore. However, when applied to Schmidtea mediterranea and D. japonica specimens from Beijing, the method was ineffective and led to progressive gDNA degradation. Conclusions This protocol offers a simple and high-yield solution for isolating HMW gDNA from D. japonica. It also provides an alternative for organisms whose gDNA consistently exhibits unexplained degradation using established protocols.https://doi.org/10.1186/s12864-025-11731-6High-molecular-weight genomic DNA extractionHigh yieldPlanarianDeoxyribonuclease IIMg2+ |
| spellingShingle | Ao Li Bingrui Sun Ying Zhang Ping Yu Jicheng Qu Hongkuan Deng Qiuxiang Pang Fengtang Yang A Mg2+-dependent high-yield method for extracting high-molecular-weight genomic DNA from a single planarian specimen BMC Genomics High-molecular-weight genomic DNA extraction High yield Planarian Deoxyribonuclease II Mg2+ |
| title | A Mg2+-dependent high-yield method for extracting high-molecular-weight genomic DNA from a single planarian specimen |
| title_full | A Mg2+-dependent high-yield method for extracting high-molecular-weight genomic DNA from a single planarian specimen |
| title_fullStr | A Mg2+-dependent high-yield method for extracting high-molecular-weight genomic DNA from a single planarian specimen |
| title_full_unstemmed | A Mg2+-dependent high-yield method for extracting high-molecular-weight genomic DNA from a single planarian specimen |
| title_short | A Mg2+-dependent high-yield method for extracting high-molecular-weight genomic DNA from a single planarian specimen |
| title_sort | mg2 dependent high yield method for extracting high molecular weight genomic dna from a single planarian specimen |
| topic | High-molecular-weight genomic DNA extraction High yield Planarian Deoxyribonuclease II Mg2+ |
| url | https://doi.org/10.1186/s12864-025-11731-6 |
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