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...
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| Format: | Article |
| Language: | English |
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Public Library of Science (PLoS)
2024-01-01
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| Series: | PLoS ONE |
| Online Access: | https://doi.org/10.1371/journal.pone.0301000 |
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| author | Anne R Shim Jane Frederick Emily M Pujadas Tiffany Kuo I Chae Ye Joshua A Pritchard Cody L Dunton Paola Carrillo Gonzalez Nicolas Acosta Surbhi Jain Nicholas M Anthony Luay M Almassalha Igal Szleifer Vadim Backman |
| author_facet | Anne R Shim Jane Frederick Emily M Pujadas Tiffany Kuo I Chae Ye Joshua A Pritchard Cody L Dunton Paola Carrillo Gonzalez Nicolas Acosta Surbhi Jain Nicholas M Anthony Luay M Almassalha Igal Szleifer Vadim Backman |
| author_sort | Anne R Shim |
| collection | DOAJ |
| description | 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. |
| format | Article |
| id | doaj-art-89f2c803491e4e51a3af9fa3dee2c896 |
| institution | OA Journals |
| issn | 1932-6203 |
| language | English |
| publishDate | 2024-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj-art-89f2c803491e4e51a3af9fa3dee2c8962025-08-20T02:22:25ZengPublic Library of Science (PLoS)PLoS ONE1932-62032024-01-01195e030100010.1371/journal.pone.0301000Formamide denaturation of double-stranded DNA for fluorescence in situ hybridization (FISH) distorts nanoscale chromatin structure.Anne R ShimJane FrederickEmily M PujadasTiffany KuoI Chae YeJoshua A PritchardCody L DuntonPaola Carrillo GonzalezNicolas AcostaSurbhi JainNicholas M AnthonyLuay M AlmassalhaIgal SzleiferVadim BackmanAs 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.https://doi.org/10.1371/journal.pone.0301000 |
| spellingShingle | Anne R Shim Jane Frederick Emily M Pujadas Tiffany Kuo I Chae Ye Joshua A Pritchard Cody L Dunton Paola Carrillo Gonzalez Nicolas Acosta Surbhi Jain Nicholas M Anthony Luay M Almassalha Igal Szleifer Vadim Backman Formamide denaturation of double-stranded DNA for fluorescence in situ hybridization (FISH) distorts nanoscale chromatin structure. PLoS ONE |
| title | Formamide denaturation of double-stranded DNA for fluorescence in situ hybridization (FISH) distorts nanoscale chromatin structure. |
| title_full | Formamide denaturation of double-stranded DNA for fluorescence in situ hybridization (FISH) distorts nanoscale chromatin structure. |
| title_fullStr | Formamide denaturation of double-stranded DNA for fluorescence in situ hybridization (FISH) distorts nanoscale chromatin structure. |
| title_full_unstemmed | Formamide denaturation of double-stranded DNA for fluorescence in situ hybridization (FISH) distorts nanoscale chromatin structure. |
| title_short | Formamide denaturation of double-stranded DNA for fluorescence in situ hybridization (FISH) distorts nanoscale chromatin structure. |
| title_sort | formamide denaturation of double stranded dna for fluorescence in situ hybridization fish distorts nanoscale chromatin structure |
| url | https://doi.org/10.1371/journal.pone.0301000 |
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