Visualizing DNA replication by single-molecule analysis of replicated DNA
Summary: Single-molecule analysis of replicated DNA (SMARD) is a unique technique that enables visualization of DNA replication at specific genomic regions at single-molecule resolution. Here, we present a protocol for visualizing DNA replication by SMARD. We describe steps for pulse labeling DNA, f...
| Main Authors: | , |
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| Format: | Article |
| Language: | English |
| Published: |
Elsevier
2023-12-01
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| Series: | STAR Protocols |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S2666166723006883 |
| _version_ | 1797405364623245312 |
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| author | Advaitha Madireddy Jeannine Gerhardt |
| author_facet | Advaitha Madireddy Jeannine Gerhardt |
| author_sort | Advaitha Madireddy |
| collection | DOAJ |
| description | Summary: Single-molecule analysis of replicated DNA (SMARD) is a unique technique that enables visualization of DNA replication at specific genomic regions at single-molecule resolution. Here, we present a protocol for visualizing DNA replication by SMARD. We describe steps for pulse labeling DNA, followed by isolating and stretching of genomic DNA. We then detail the detection of the replication at chromosomal regions through immunostaining and fluorescence in situ hybridization. Using SMARD, we can visualize replication initiation, progression, termination, and fork stalling.For complete details on the use and execution of this protocol, please refer to Norio et al. (2001) and Gerhardt et al. (2014).1,2 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. |
| first_indexed | 2024-03-09T03:08:57Z |
| format | Article |
| id | doaj.art-559c3663b80e4f2fb23ece13c1a61704 |
| institution | Directory Open Access Journal |
| issn | 2666-1667 |
| language | English |
| last_indexed | 2024-03-09T03:08:57Z |
| publishDate | 2023-12-01 |
| publisher | Elsevier |
| record_format | Article |
| series | STAR Protocols |
| spelling | doaj.art-559c3663b80e4f2fb23ece13c1a617042023-12-04T05:24:07ZengElsevierSTAR Protocols2666-16672023-12-0144102721Visualizing DNA replication by single-molecule analysis of replicated DNAAdvaitha Madireddy0Jeannine Gerhardt1Rutgers Cancer Institute of New Jersey, New Brunswick, NJ, USA; Department of Pediatrics Hematology/Oncology, Robert Wood Johnson Medical School, Rutgers University, New Brunswick, NJ, USA; Corresponding authorThe Ronald O. Perelman and Claudia Cohen Center for Reproductive Medicine, Weill Cornell Medicine, New York, NY, USA; Department of Obstetrics and Gynecology, Weill Cornell Medicine, New York, NY, USA; Corresponding authorSummary: Single-molecule analysis of replicated DNA (SMARD) is a unique technique that enables visualization of DNA replication at specific genomic regions at single-molecule resolution. Here, we present a protocol for visualizing DNA replication by SMARD. We describe steps for pulse labeling DNA, followed by isolating and stretching of genomic DNA. We then detail the detection of the replication at chromosomal regions through immunostaining and fluorescence in situ hybridization. Using SMARD, we can visualize replication initiation, progression, termination, and fork stalling.For complete details on the use and execution of this protocol, please refer to Norio et al. (2001) and Gerhardt et al. (2014).1,2 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.http://www.sciencedirect.com/science/article/pii/S2666166723006883Cell BiologyIn Situ HybridizationMicroscopyMolecular BiologySingle Cell |
| spellingShingle | Advaitha Madireddy Jeannine Gerhardt Visualizing DNA replication by single-molecule analysis of replicated DNA STAR Protocols Cell Biology In Situ Hybridization Microscopy Molecular Biology Single Cell |
| title | Visualizing DNA replication by single-molecule analysis of replicated DNA |
| title_full | Visualizing DNA replication by single-molecule analysis of replicated DNA |
| title_fullStr | Visualizing DNA replication by single-molecule analysis of replicated DNA |
| title_full_unstemmed | Visualizing DNA replication by single-molecule analysis of replicated DNA |
| title_short | Visualizing DNA replication by single-molecule analysis of replicated DNA |
| title_sort | visualizing dna replication by single molecule analysis of replicated dna |
| topic | Cell Biology In Situ Hybridization Microscopy Molecular Biology Single Cell |
| url | http://www.sciencedirect.com/science/article/pii/S2666166723006883 |
| work_keys_str_mv | AT advaithamadireddy visualizingdnareplicationbysinglemoleculeanalysisofreplicateddna AT jeanninegerhardt visualizingdnareplicationbysinglemoleculeanalysisofreplicateddna |