In Vivo Genomic Supercoiling Mapping Using Psora-seq.

Autor:	Visser BJ; Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA., Bates D; Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA. bates@bcm.edu.
Jazyk:	angličtina
Zdroj:	Methods in molecular biology (Clifton, N.J.) [Methods Mol Biol] 2024; Vol. 2819, pp. 147-156.
DOI:	10.1007/978-1-0716-3930-6_8
Abstrakt:	Supercoiling is a fundamental property of DNA that governs all strand opening reactions, including DNA replication, transcription, and homologous recombination. However, traditional genomic supercoiling assays are difficult and lack sensitivity. Building on prior assays using the DNA intercalator psoralen, we developed a supercoil mapping assay that is robust and sensitive to a wide range of supercoiling while requiring only commercially available reagents and common laboratory equipment. This method, psoralen affinity purification with genomic sequencing (Psora-seq), utilizes biotinylated psoralen and streptavidin-conjugated magnetic beads to facilitate efficient pull-down of psoralen-bound DNA, followed by deep sequencing to identify and quantify supercoiling at 1 kb resolution. Psora-seq overcomes two major bottlenecks associated with existing psoralen pull-down assays, inefficient photo-binding of psoralen-bound molecules, and poor recovery of cross-linked DNA. (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)
Databáze:	MEDLINE
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