Formamide denaturation of double-stranded DNA for fluorescence in situ hybridization (FISH) distorts nanoscale chromatin structure.
PLoS One
; 19(5): e0301000, 2024.
Article
de En
| MEDLINE
| ID: mdl-38805476
ABSTRACT
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 fluorescence in situ hybridization (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.
Texte intégral:
1
Collection:
01-internacional
Base de données:
MEDLINE
Sujet principal:
ADN
/
Chromatine
/
Hybridation fluorescente in situ
/
Formamides
Limites:
Animals
Langue:
En
Journal:
PLoS One
Sujet du journal:
CIENCIA
/
MEDICINA
Année:
2024
Type de document:
Article
Pays d'affiliation:
États-Unis d'Amérique