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Improved methods for creating migratable Holliday junction substrates

Previously, we published a method for creating a novel DNA substrate, the double Holliday junction substrate. This substrate contains two Holliday junctions that are mobile, topologically constrained and separated by a distance comparable with conversion tract lengths. Although useful for studying l...

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Autores principales: Chen, Stefanie Hartman, Plank, Jody L., Willcox, Smaranda, Griffith, Jack D., Hsieh, Tao-shih
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Oxford University Press 2013
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3597647/
https://www.ncbi.nlm.nih.gov/pubmed/23275569
http://dx.doi.org/10.1093/nar/gks1343
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author Chen, Stefanie Hartman
Plank, Jody L.
Willcox, Smaranda
Griffith, Jack D.
Hsieh, Tao-shih
author_facet Chen, Stefanie Hartman
Plank, Jody L.
Willcox, Smaranda
Griffith, Jack D.
Hsieh, Tao-shih
author_sort Chen, Stefanie Hartman
collection PubMed
description Previously, we published a method for creating a novel DNA substrate, the double Holliday junction substrate. This substrate contains two Holliday junctions that are mobile, topologically constrained and separated by a distance comparable with conversion tract lengths. Although useful for studying late stage homologous recombination in vitro, construction of the substrate requires significant effort. In particular, there are three bottlenecks: (i) production of large quantities of single-stranded DNA; (ii) the loss of a significant portion of the DNA following the recombination step; and (iii) the loss of DNA owing to inefficient gel extraction. To address these limitations, we have made the following changes to the protocol: (i) use of a helper plasmid, rather than exogenous helper phage, to produce single-stranded DNA; (ii) use of the unidirectional ϕC31 integrase system in place of the bidirectional Cre recombinase reaction; and (iii) gel extraction by DNA diffusion. Here, we describe the changes made to the materials and methods and characterize the substrates that can be produced, including migratable single Holliday junctions, hemicatenanes and a quadruple Holliday junction substrate.
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spelling pubmed-35976472013-03-15 Improved methods for creating migratable Holliday junction substrates Chen, Stefanie Hartman Plank, Jody L. Willcox, Smaranda Griffith, Jack D. Hsieh, Tao-shih Nucleic Acids Res Methods Online Previously, we published a method for creating a novel DNA substrate, the double Holliday junction substrate. This substrate contains two Holliday junctions that are mobile, topologically constrained and separated by a distance comparable with conversion tract lengths. Although useful for studying late stage homologous recombination in vitro, construction of the substrate requires significant effort. In particular, there are three bottlenecks: (i) production of large quantities of single-stranded DNA; (ii) the loss of a significant portion of the DNA following the recombination step; and (iii) the loss of DNA owing to inefficient gel extraction. To address these limitations, we have made the following changes to the protocol: (i) use of a helper plasmid, rather than exogenous helper phage, to produce single-stranded DNA; (ii) use of the unidirectional ϕC31 integrase system in place of the bidirectional Cre recombinase reaction; and (iii) gel extraction by DNA diffusion. Here, we describe the changes made to the materials and methods and characterize the substrates that can be produced, including migratable single Holliday junctions, hemicatenanes and a quadruple Holliday junction substrate. Oxford University Press 2013-03 2012-12-26 /pmc/articles/PMC3597647/ /pubmed/23275569 http://dx.doi.org/10.1093/nar/gks1343 Text en © The Author(s) 2012. Published by Oxford University Press. http://creativecommons.org/licenses/by-nc/3.0 This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Methods Online
Chen, Stefanie Hartman
Plank, Jody L.
Willcox, Smaranda
Griffith, Jack D.
Hsieh, Tao-shih
Improved methods for creating migratable Holliday junction substrates
title Improved methods for creating migratable Holliday junction substrates
title_full Improved methods for creating migratable Holliday junction substrates
title_fullStr Improved methods for creating migratable Holliday junction substrates
title_full_unstemmed Improved methods for creating migratable Holliday junction substrates
title_short Improved methods for creating migratable Holliday junction substrates
title_sort improved methods for creating migratable holliday junction substrates
topic Methods Online
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3597647/
https://www.ncbi.nlm.nih.gov/pubmed/23275569
http://dx.doi.org/10.1093/nar/gks1343
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