TY - JOUR
T1 - Fluorescence In Situ Hybridization on DNA Halo Preparations to Reveal Whole Chromosomes, Telomeres and Gene Loci
AU - Godwin, Lauren S
AU - Bridger, Joanna M
AU - Foster, Helen
N1 - © 2021 JoVE. Open Access Attribution License. https://creativecommons.org/licenses/by/4.0/
PY - 2021/3/4
Y1 - 2021/3/4
N2 - The genome is associated with several structures inside cell nuclei, in order to regulate its activity and anchor it in specific locations. These structures are collectively known as the nucleoskeleton and include the nuclear lamina, the nucleoli, and nuclear bodies. Although many variants of fluorescence in situ hybridization (FISH) exist to study the genome and its organization, these are often limited by resolution and provide insufficient information on the genome's association with nuclear structures. The DNA halo method uses high salt concentrations and nonionic detergents to generate DNA loops that remain anchored to structures within nuclei through attachment regions within the genome. Here, soluble nuclear proteins, such as histones, lipids, and DNA not tightly bound to the nuclear matrix, are extracted. This leads to the formation of a halo of unattached DNA surrounding a residual nucleus which itself contains DNA closely associated with internal nuclear structures and extraction-resistant proteins. These extended DNA strands enable increased resolution and can facilitate physical mapping. In combination with FISH, this method has the added advantage of studying genomic interactions with all the structures that the genome is anchored by. This technique, termed HALO-FISH, is highly versatile whereby DNA halos can be coupled with nucleic acid probes to reveal gene loci, whole chromosomes, alpha satellite, telomeres and even RNA. This technique provides an insight into nuclear organization and function in normal cells and in disease progression such as with cancer.
AB - The genome is associated with several structures inside cell nuclei, in order to regulate its activity and anchor it in specific locations. These structures are collectively known as the nucleoskeleton and include the nuclear lamina, the nucleoli, and nuclear bodies. Although many variants of fluorescence in situ hybridization (FISH) exist to study the genome and its organization, these are often limited by resolution and provide insufficient information on the genome's association with nuclear structures. The DNA halo method uses high salt concentrations and nonionic detergents to generate DNA loops that remain anchored to structures within nuclei through attachment regions within the genome. Here, soluble nuclear proteins, such as histones, lipids, and DNA not tightly bound to the nuclear matrix, are extracted. This leads to the formation of a halo of unattached DNA surrounding a residual nucleus which itself contains DNA closely associated with internal nuclear structures and extraction-resistant proteins. These extended DNA strands enable increased resolution and can facilitate physical mapping. In combination with FISH, this method has the added advantage of studying genomic interactions with all the structures that the genome is anchored by. This technique, termed HALO-FISH, is highly versatile whereby DNA halos can be coupled with nucleic acid probes to reveal gene loci, whole chromosomes, alpha satellite, telomeres and even RNA. This technique provides an insight into nuclear organization and function in normal cells and in disease progression such as with cancer.
KW - DNA halo
KW - Fluorescence in situ hybridization
KW - Nuclear matrix
KW - Chromosome territories
KW - Telomeres
KW - Gene Loci
UR - http://www.scopus.com/inward/record.url?scp=85103473795&partnerID=8YFLogxK
U2 - 10.3791/62017
DO - 10.3791/62017
M3 - Article
SN - 1940-087X
JO - Journal of Visualised Experiments
JF - Journal of Visualised Experiments
IS - 169
M1 - e62017
ER -