Telomeres and NextGen CO-FISH: Directional Genomic Hybridization (Telo-dGH™)

Abstract

The cytogenomics-based methodology of Directional Genomic Hybridization (dGH™) emerged from the concept of strand-specific hybridization, first made possible by Chromosome Orientation FISH (CO-FISH), the utility of which was demonstrated in a variety of early applications, often involving telomeres. Similar to standard whole chromosome painting (FISH), dGH™ is capable of identifying inter-chromosomal rearrangements (translocations between chromosomes), but its distinctive strength stems from its ability to detect intra-chromosomal rearrangements (inversions within chromosomes), and to do so at higher resolution than previously possible. dGH™ brings together the strand specificity and directionality of CO-FISH with sophisticated bioinformatics-based oligonucleotide probe design to unique sequences. dGH™ serves not only as a powerful discovery tool-capable of interrogating the entire genome at the megabase level-it can also be used for high-resolution targeted detection of known inversions, a valuable attribute in both research and clinical settings. Detection of chromosomal inversions, particularly small ones, poses a formidable challenge for more traditional cytogenetic approaches, especially when they occur near the ends or telomeric regions. Here, we describe Telo-dGH™, a strand-specific scheme that utilizes dGH™ in combination with telomere CO-FISH to differentiate between terminal exchange events, specifically terminal inversions, and an altogether different form of genetic recombination that often occurs near the telomere, namely sister chromatid exchange (SCE).

Endogenous feline leukemia virus long terminal repeat integration site diversity is highly variable in related and unrelated domestic cats

“…After a series of dehydration steps, 30 µL of enFeLV FISH probes were applied to the slide, followed by Dapi, and then visualized with a Coolsnap ES 2 camera and running MetaVue Imaging System software (Molecular Devices, San Jose, CA). Telomere-FISH was used as a positive control for the FISH protocol following previously reported methods (telomere probes generously provided by Dr. Susan Bailey; [ 29 ]).…”

Monitoring Genomic Structural Rearrangements Resulting from Gene Editing

“…Directional genomic hybridization (dGH) identifies structural variants within single cells, providing information on genomic heterogeneity and the distribution of chromosomal rearrangements within edited and/or exposed cell populations [66,67]. In addition to the detection and measurement of structural variants arising from radiation exposure or induced by various methods of genome editing, dGH has been used for a variety of other applications, including oncogenic fusion gene tracking, genetic disease characterization, the detection of instability, and characterization of cancer cell lines [15,16,[68][69][70].…”

Elevated retrotransposon activity and genomic instability in primed pluripotent stem cells

“…Although telomerase activity was high in both states, the difference in telomere length dynamics suggests that mechanism other than telomerase activity could be involved in telomere maintenance of these cells. Alternative lengthening of telomeres (ALT) is a telomerase-independent mechanism to elongate telomeres rapidly [58], in association with frequent telomere sister chromatic exchange (T-SCE), which can indicate the recombinational rate of telomeres [59][60][61]. We assessed T-SCE in naïve and primed cells by telomere chromosome orientation FISH (CO-FISH) analysis [59].…”