DNA fiber combing protocol using in-house reagents and coverslips to analyze replication fork dynamics in mammalian cells

Gemma Moore; Judit Jimenez Sainz; Ryan B Jensen

doi:10.1016/j.xpro.2022.101371

DNA fiber combing protocol using in-house reagents and coverslips to analyze replication fork dynamics in mammalian cells

STAR Protoc. 2022 May 5;3(2):101371. doi: 10.1016/j.xpro.2022.101371. eCollection 2022 Jun 17.

Authors

Gemma Moore¹, Judit Jimenez Sainz¹, Ryan B Jensen¹

Affiliation

¹ Department of Therapeutic Radiology, Yale University School of Medicine, 15 York Street, New Haven, CT 06520, USA.

Abstract

DNA fiber combing is a versatile technique that provides insight into replication fork dynamics at single-molecule resolution. DNA fibers are bound to silanized coverslips and combed, which straightens and aligns the fibers along a single axis. Here, we present a DNA fiber combing protocol that does not use commercial kits; we detail the steps to prepare all materials, reagents, and silanized coverslips. We describe the use of DLD-1 cells, but the protocol is amenable to other cell types.

Keywords: Cell Biology; Molecular Biology; Single-molecule Assays.

Publication types

Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't

MeSH terms

Animals
DNA Replication*
DNA*
Indicators and Reagents
Mammals / genetics

Substances

Indicators and Reagents
DNA

Abstract

Publication types

MeSH terms

Substances

Grants and funding