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  • Formamide denaturation of double-stranded DNA for fluorescence in situ hybridization (FISH) distorts nanoscale chromatin structure
Title: Formamide denaturation of double-stranded DNA for fluorescence in situ hybridization (FISH) distorts nanoscale chromatin structure
As imaging techniques rapidly evolve to probe nanoscale genome organization at higher resolution, it is critical to consider how the reagents and procedures involved in sample preparation affect chromatin at the relevant length scales. Here, we investigate the effects of fluorescent labeling of DNA sequences within chromatin using the gold standard technique of three-dimensional fluorescencein situhybridization (3D FISH). The chemical reagents involved in the 3D FISH protocol, specifically formamide, cause significant alterations to the sub-200 nm (sub-Mbp) chromatin structure. Alternatively, two labeling methods that do not rely on formamide denaturation, resolution after single-strand exonuclease resection (RASER)-FISH and clustered regularly interspaced short palindromic repeats (CRISPR)-Sirius, had minimal impact on the three-dimensional organization of chromatin. We present a polymer physics-based analysis of these protocols with guidelines for their interpretation when assessing chromatin structure using currently available techniques.  more » « less
Award ID(s):
1830961
PAR ID:
10555922
Author(s) / Creator(s):
; ; ; ; ; ; ; ; ; ; ; ; ;
Editor(s):
Ren, Xiaojun
Publisher / Repository:
PLoS
Date Published:
Journal Name:
PLOS ONE
Volume:
19
Issue:
5
ISSN:
1932-6203
Page Range / eLocation ID:
e0301000
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
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