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https://hdl.handle.net/11147/15541
Title: | Improved Isolation of Ultra-High Genomic Dna Suitable for Third-Generation Sequencing | Authors: | Ova, Ayse Oyku Joffre, Enrique Shafagh, Reza Zandi Assuncao, Mariana F. G. Sidorov, Roman Y. Santos, Lilia M. A. Romling, Ute |
Keywords: | Bacteria Dna Isolation Incubation Chamber Third-Generation Sequencing |
Publisher: | Mdpi | Abstract: | Although a variety of protocols to isolate high-molecular-weight genomic DNA exist, the isolation and preservation of ultra-high-molecular-weight genomic DNA of sufficient quality and length for error-free third-generation sequencing remains challenging. Inspired by the isolation of high-molecular-weight DNA in agarose plugs suitable to be separated by pulsed-field gel electrophoresis, we report the construction of an incubation chamber for DNA isolation. The chamber is flanked by semi-permeable polycarbonate membranes of variable pore size for the selective diffusion of compounds and components, which allows efficient cell lysis and the subsequent isolation of ultra-high-molecular-weight genomic DNA without shearing. The designed experimental approach is simple, but effective in isolating high-quality ultra-long genomic DNA that is suitable for third-generation sequencing by Oxford Nanopore Technology from challenging bacterial samples. We envisage that genomic DNA isolation using the incubation chamber, or variations thereof, will facilitate the regular sequencing of megabasepair-long DNA fragments, with a multitude of applications in microbiology, immunology, oncology, paleontology and forensic science. | URI: | https://doi.org/10.3390/microorganisms13030534 https://hdl.handle.net/11147/15541 |
ISSN: | 2076-2607 |
Appears in Collections: | PubMed İndeksli Yayınlar Koleksiyonu / PubMed Indexed Publications Collection Scopus İndeksli Yayınlar Koleksiyonu / Scopus Indexed Publications Collection WoS İndeksli Yayınlar Koleksiyonu / WoS Indexed Publications Collection |
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