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

ABSTRACT: 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 t

SUBMITTER: Torres Montaguth OE 

PROVIDER: S-EPMC8599736 | biostudies-literature | 2021 Nov

REPOSITORIES: biostudies-literature