Cargando…

Quantitative analysis of genomic element interactions by molecular colony technique

Distant genomic elements were found to interact within the folded eukaryotic genome. However, the used experimental approach (chromosome conformation capture, 3C) enables neither determination of the percentage of cells in which the interactions occur nor demonstration of simultaneous interaction of...

Descripción completa

Detalles Bibliográficos
Autores principales: Gavrilov, Alexey A., Chetverina, Helena V., Chermnykh, Elina S., Razin, Sergey V., Chetverin, Alexander B.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Oxford University Press 2014
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3950710/
https://www.ncbi.nlm.nih.gov/pubmed/24369423
http://dx.doi.org/10.1093/nar/gkt1322
_version_ 1782307037514825728
author Gavrilov, Alexey A.
Chetverina, Helena V.
Chermnykh, Elina S.
Razin, Sergey V.
Chetverin, Alexander B.
author_facet Gavrilov, Alexey A.
Chetverina, Helena V.
Chermnykh, Elina S.
Razin, Sergey V.
Chetverin, Alexander B.
author_sort Gavrilov, Alexey A.
collection PubMed
description Distant genomic elements were found to interact within the folded eukaryotic genome. However, the used experimental approach (chromosome conformation capture, 3C) enables neither determination of the percentage of cells in which the interactions occur nor demonstration of simultaneous interaction of >2 genomic elements. Each of the above can be done using in-gel replication of interacting DNA segments, the technique reported here. Chromatin fragments released from formaldehyde–cross-linked cells by sodium dodecyl sulfate extraction and sonication are distributed in a polyacrylamide gel layer followed by amplification of selected test regions directly in the gel by multiplex polymerase chain reaction. The fragments that have been cross-linked and separate fragments give rise to multi- and monocomponent molecular colonies, respectively, which can be distinguished and counted. Using in-gel replication of interacting DNA segments, we demonstrate that in the material from mouse erythroid cells, the majority of fragments containing the promoters of active β-globin genes and their remote enhancers do not form complexes stable enough to survive sodium dodecyl sulfate extraction and sonication. This indicates that either these elements do not interact directly in the majority of cells at a given time moment, or the formed DNA–protein complex cannot be stabilized by formaldehyde cross-linking.
format Online
Article
Text
id pubmed-3950710
institution National Center for Biotechnology Information
language English
publishDate 2014
publisher Oxford University Press
record_format MEDLINE/PubMed
spelling pubmed-39507102014-03-12 Quantitative analysis of genomic element interactions by molecular colony technique Gavrilov, Alexey A. Chetverina, Helena V. Chermnykh, Elina S. Razin, Sergey V. Chetverin, Alexander B. Nucleic Acids Res Distant genomic elements were found to interact within the folded eukaryotic genome. However, the used experimental approach (chromosome conformation capture, 3C) enables neither determination of the percentage of cells in which the interactions occur nor demonstration of simultaneous interaction of >2 genomic elements. Each of the above can be done using in-gel replication of interacting DNA segments, the technique reported here. Chromatin fragments released from formaldehyde–cross-linked cells by sodium dodecyl sulfate extraction and sonication are distributed in a polyacrylamide gel layer followed by amplification of selected test regions directly in the gel by multiplex polymerase chain reaction. The fragments that have been cross-linked and separate fragments give rise to multi- and monocomponent molecular colonies, respectively, which can be distinguished and counted. Using in-gel replication of interacting DNA segments, we demonstrate that in the material from mouse erythroid cells, the majority of fragments containing the promoters of active β-globin genes and their remote enhancers do not form complexes stable enough to survive sodium dodecyl sulfate extraction and sonication. This indicates that either these elements do not interact directly in the majority of cells at a given time moment, or the formed DNA–protein complex cannot be stabilized by formaldehyde cross-linking. Oxford University Press 2014-03 2013-12-24 /pmc/articles/PMC3950710/ /pubmed/24369423 http://dx.doi.org/10.1093/nar/gkt1322 Text en © The Author(s) 2013. Published by Oxford University Press. http://creativecommons.org/licenses/by/3.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Gavrilov, Alexey A.
Chetverina, Helena V.
Chermnykh, Elina S.
Razin, Sergey V.
Chetverin, Alexander B.
Quantitative analysis of genomic element interactions by molecular colony technique
title Quantitative analysis of genomic element interactions by molecular colony technique
title_full Quantitative analysis of genomic element interactions by molecular colony technique
title_fullStr Quantitative analysis of genomic element interactions by molecular colony technique
title_full_unstemmed Quantitative analysis of genomic element interactions by molecular colony technique
title_short Quantitative analysis of genomic element interactions by molecular colony technique
title_sort quantitative analysis of genomic element interactions by molecular colony technique
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3950710/
https://www.ncbi.nlm.nih.gov/pubmed/24369423
http://dx.doi.org/10.1093/nar/gkt1322
work_keys_str_mv AT gavrilovalexeya quantitativeanalysisofgenomicelementinteractionsbymolecularcolonytechnique
AT chetverinahelenav quantitativeanalysisofgenomicelementinteractionsbymolecularcolonytechnique
AT chermnykhelinas quantitativeanalysisofgenomicelementinteractionsbymolecularcolonytechnique
AT razinsergeyv quantitativeanalysisofgenomicelementinteractionsbymolecularcolonytechnique
AT chetverinalexanderb quantitativeanalysisofgenomicelementinteractionsbymolecularcolonytechnique