دورية أكاديمية
أعرض في EDS

ENDO-Pore: high-throughput linked-end mapping of single DNA cleavage events using nanopore sequencing.

التفاصيل البيبلوغرافية
العنوان: ENDO-Pore: high-throughput linked-end mapping of single DNA cleavage events using nanopore sequencing.
المؤلفون: Torres Montaguth OE; DNA-Protein Interactions Unit, School of Biochemistry, Faculty of Life Sciences, University of Bristol, Bristol BS8 1TD, UK., Cross SJ; Wolfson Bioimaging Facility, Faculty of Life Sciences, University of Bristol, Bristol BS8 1TD, UK., Ingram KWA; DNA-Protein Interactions Unit, School of Biochemistry, Faculty of Life Sciences, University of Bristol, Bristol BS8 1TD, UK., Lee L; DNA-Protein Interactions Unit, School of Biochemistry, Faculty of Life Sciences, University of Bristol, Bristol BS8 1TD, UK., Diffin FM; DNA-Protein Interactions Unit, School of Biochemistry, Faculty of Life Sciences, University of Bristol, Bristol BS8 1TD, UK., Szczelkun MD; DNA-Protein Interactions Unit, School of Biochemistry, Faculty of Life Sciences, University of Bristol, Bristol BS8 1TD, UK.
المصدر: Nucleic acids research [Nucleic Acids Res] 2021 Nov 18; Vol. 49 (20), pp. e118.
نوع المنشور: Journal Article; Research Support, Non-U.S. Gov't
اللغة: English
بيانات الدورية: Publisher: Oxford University Press Country of Publication: England NLM ID: 0411011 Publication Model: Print Cited Medium: Internet ISSN: 1362-4962 (Electronic) Linking ISSN: 03051048 NLM ISO Abbreviation: Nucleic Acids Res Subsets: MEDLINE
أسماء مطبوعة: Publication: 1992- : Oxford : Oxford University Press
Original Publication: London, Information Retrieval ltd.
مواضيع طبية MeSH: DNA Cleavage*, High-Throughput Nucleotide Sequencing/*methods , Nanopore Sequencing/*methods, DNA/chemistry ; DNA/genetics ; DNA Restriction Enzymes/metabolism ; Nucleotide Motifs
مستخلص: Mapping the precise position of DNA cleavage events plays a key role in determining the mechanism and function of endonucleases. ENDO-Pore is a high-throughput nanopore-based method that allows the time resolved mapping single molecule DNA cleavage events in vitro. Following linearisation of a circular DNA substrate by the endonuclease, a resistance cassette is ligated recording the position of the cleavage event. A library of single cleavage events is constructed and subjected to rolling circle amplification to generate concatemers. These are sequenced and used to produce accurate consensus sequences. To identify the cleavage site(s), we developed CSI (Cleavage Site Investigator). CSI recognizes the ends of the cassette ligated into the cleaved substrate and triangulates the position of the dsDNA break. We firstly benchmarked ENDO-Pore using Type II restriction endonucleases. Secondly, we analysed the effect of crRNA length on the cleavage pattern of CRISPR Cas12a. Finally, we mapped the time-resolved DNA cleavage by the Type ISP restriction endonuclease LlaGI that introduces random double-strand breaks into its DNA substrates.
(© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)
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معلومات مُعتمدة: BB/S001239/1 United Kingdom BB_ Biotechnology and Biological Sciences Research Council
المشرفين على المادة: 9007-49-2 (DNA)
EC 3.1.21.- (DNA Restriction Enzymes)
تواريخ الأحداث: Date Created: 20210821 Date Completed: 20211222 Latest Revision: 20211222
رمز التحديث: 20240628
مُعرف محوري في PubMed: PMC8599736
DOI: 10.1093/nar/gkab727
PMID: 34417616
قاعدة البيانات: MEDLINE
الوصف
تدمد:1362-4962
DOI:10.1093/nar/gkab727