title: Visualization and quantitative analysis of extrachromosomal telomere-repeat DNA in individual human cells by Halo-FISH.
abstract: Current methods for characterizing extrachromosomal nuclear DNA in mammalian cells do not permit single-cell analysis, are often semi-quantitative and frequently biased toward the detection of circular species. To overcome these limitations, we developed Halo-FISH to visualize and quantitatively analyze extrachromosomal DNA in single cells. We demonstrate Halo-FISH by using it to analyze extrachromosomal telomere-repeat (ECTR) in human cells that use the Alternative Lengthening of Telomeres (ALT) pathway(s) to maintain telomere lengths. We find that GM847 and VA13 ALT cells average ∼80 detectable G/C-strand ECTR DNA molecules/nucleus, while U2OS ALT cells average ∼18 molecules/nucleus. In comparison, human primary and telomerase-positive cells contain <5 ECTR DNA molecules/nucleus. ECTR DNA in ALT cells exhibit striking cell-to-cell variations in number (<20 to >300), range widely in length (<1 to >200 kb) and are composed of primarily G- or C-strand telomere-repeat DNA. Halo-FISH enables, for the first time, the simultaneous analysis of ECTR DNA and chromosomal telomeres in a single cell. We find that ECTR DNA comprises ∼15% of telomere-repeat DNA in GM847 and VA13 cells, but <4% in U2OS cells. In addition to its use in ALT cell analysis, Halo-FISH can facilitate the study of a wide variety of extrachromosomal DNA in mammalian cells.
authors: Genetics and Genome Biology Program, The Hospital for Sick Children, Toronto, Ontario, M5G 0A4, Canada.
authors: Martin
authors: M
authors: Komosa
authors: Genetics and Genome Biology Program, The Hospital for Sick Children, Toronto, Ontario, M5G 0A4, Canada.
authors: Heather
authors: H
authors: Root
authors: Genetics and Genome Biology Program, The Hospital for Sick Children, Toronto, Ontario, M5G 0A4, Canada Division of Clinical and Metabolic Genetics, The Hospital for Sick Children, Toronto, Ontario, M5G 1X8, Canada Department of Paediatrics, University of Toronto, Toronto, Ontario, M5S 1A8, Canada Department of Molecular Genetics, University of Toronto, Toronto, Ontario, M5S 1A8, Canada stephen.meyn@sickkids.ca.
authors: M Stephen
authors: MS
authors: Meyn
mesh_terms: D002453:Cell Cycle; D002460:Cell Line; D002467:Cell Nucleus / Q000235:genetics; D004247:DNA / Q000032:analysis* / Q000737:chemistry; D006801:Humans; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D012091:Repetitive Sequences, Nucleic Acid; D016615:Telomere / Q000737:chemistry*; D059505:Telomere Homeostasis
chemical_list: D004247:DNA