We would like to thank Prof Michael Bittner for the kind gift of chromosome arm painting probes. LG was supported by EU funded EURO-Laminopathies project and the Brunel Progeria Research Fund.

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The authors have nothing to disclose.

The DNA halo method is an excellent method of choice when analyzing interactions between the nucleoskeleton and genome, however, there are some critical steps that must be adhered too. One of the most important parameters is the optimization of the cell seeding density. If cells become over confluent, then the DNA halos will overlap with neighboring cells making it impossible to perform the analysis. The CSK and extraction buffers must always be made fresh on the day of use with spermine, spermidine and digitonin being added to the extraction buffer at the end of the preparation process to maintain their biological activity. If performing Halo-FISH it is extremely important to use the correct denaturation temperature of the DNA halos to enable the probe or paint to subsequently hybridize.
Electron microscopy has been used to visualize the nuclear matrix, with filamentous structures being identified20. However, electron microscopy is limited as matrix associations with chromatin cannot easily be deduced. Indeed, the DNA Halo method is more versatile compared with electron microscopy as specific genes, chromosomes and cell states can all be examined. Furthermore, proteomic analysis of nuclear matrix proteins is being studied21,22. This method is good for comparing nuclear matrix components, particularly when comparing diseased cells, however, it doesn&#39;t provide the spatial distribution and attachments highlighted by the standard DNA Halo technique.
DNA Halo assays do have limitations. Firstly, as the matrix is extracted, this can only be performed on fixed cells so live imaging is not possible. Although the DNA Halo method is relatively quick and easy to perform, the overall process may be time consuming when cell culture, probe generation, Halo-FISH and analysis is all taken into account.
Image capture of DNA Halos and HALO-FISH using super-resolution microscopy would greatly improve the resolution of DNA specific probes and antibodies. In addition, as fluorochromes can be more easily spectrally resolved, it may be possible to use a number of DNA probes in a single experiment, providing even more information. Improvements in molecular biology techniques such as chromosome conformation capture (3C) have been used to determine interactions of gene loci and analyze the spatial organization on chromatin in the cell. DNA Halo assays and 3C can be combined, a term known as M3C23, again demonstrating the adaptability of the DNA Halo technique.
The original data presented here are to demonstrate the possibilities for genome behavior interrogation and how to present those data. With these data we have demonstrated that it is possible to determine significant differences in genome attachment using (1) chromosome painting probes, in this study revealing chromosome 18 being the least attached chromosome out of those analysed (Figure 3); (2) Gene loci with significant differences between two gene loci and (Figure 4) (3) Telomeres, which are less strongly attached in quiescent cells compared to proliferating and senescent cells (Figure 5). We are able to differentiate between proliferating and non-proliferating cells via the presence of the proliferation marker Ki67 antigen which is an insoluble protein so remains with the residual nuclei or using the incorporation of nucleotides to highlight cells that have been through S-phase within a specific time period (Figure 2). This technique has also enabled us to analyze genome behavior in cells that are compromised in their nucleoskeletons i.e. laminopathy cells and here and in Bikkul et al., 2018 we reveal that the genome can be less tightly attached when compared to control cells and can be restored when treating with specific drugs that ameliorate the effect of the lamin A mutation in classical HGPS cells15. However, we show new data here for the atypical HGPS AGO8466 cells, lacking a lamin A mutation but containing an unusual form of the LINC complex protein SUN119 that chromosome 13 is less tightly attached in (Figure 6).
HALO-FISH is a unique method by enabling the study of genomic interactions with the nucleoskeleton in combination with indirect immunofluorescence to resolve proteins not removed from the extraction procedure. It has been demonstrated that the nucleoskeleton is modified in various diseases such as certain cancer types19 and the importance of some nucleoskeleton-associated proteins as diagnostic biomarkers24,25. Thus, this technique has an important role in examining the effect of the nucleoskeleton on chromatin organization/disorganization in disease15,24,25,27 and is not restricted to human cells, with chromosomal painting probes from other animals, the same DNA-halo protocol could be employed28.

An erratum was issued for:&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/t/62017">Fluorescence In Situ Hybridization on DNA Halo Preparations to Reveal Whole Chromosomes, Telomeres and Gene Loci</a>. The Authors section was updated from:
Lauren S. Godwin1 
Joanna M. Bridger1 
Helen A. Foster2 
1Laboratory of Nuclear and Genomic Health, Centre for Genome Engineering and Maintenance, Division of Biosciences, Department of Life Sciences, College of Health, Medicine and Life Sciences, Brunel University London 
2Biosciences, Department of Clinical, Pharmaceutical and Biological Science, School of Life and Medical Sciences, University of Hertfordshire
to:
Lauren S. Godwin1 
Emily Roberts2 
Joanna M. Bridger1 
Helen A. Foster2 
1Laboratory of Nuclear and Genomic Health, Centre for Genome Engineering and Maintenance, Division of Biosciences, Department of Life Sciences, College of Health, Medicine and Life Sciences, Brunel University London 
2Biosciences, Department of Clinical, Pharmaceutical and Biological Science, School of Life and Medical Sciences, University of Hertfordshire

An erratum was issued for:&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/t/62017">Fluorescence In Situ Hybridization on DNA Halo Preparations to Reveal Whole Chromosomes, Telomeres and Gene Loci</a>. The Authors section was updated.

Erratum: Fluorescence In Situ Hybridization on DNA Halo Preparations to Reveal Whole Chromosomes, Telomeres and Gene Loci

The &#34;nuclear&#160;matrix&#34; was first described by Berezney and Coffey in 19741. After performing extractions with high salt molarities and nuclease treatment on rat liver nuclei, they identified a proteinaceous structural framework. The DNA halo procedure was subsequently adapted from this method and involves the removal of soluble proteins so that only the nuclear matrix (NM) and NM-associated proteins and chromosomes persist. DNA attachment regions are located at the base of DNA loops and are called matrix attached regions (MARs) or scaffold attachment regions (SARs), which are resistant to extraction with high salt concentrations and ionic detergent lithium-3,5-diiodosalicylate (LIS) respectively. In DNA halos, DNA associated with MARs/SARs are bound within the residual nucleus whereas the DNA loops extend away from these sites and form the DNA halo. We know now that the genome is anchored via lamina associated domains (LADs) to the nuclear lamina and through nucleolar associated regions (NADs) and possibly through other nuclear structures such as specific nuclear bodies.
The DNA halo method can be used for physical mapping of DNA, genes, and chromosomal regions as the extended DNA and chromatin provides a greater resolution because the chromatin is stripped of histones and the DNA is stretched out2,3,4,5,6. However, there are some limitations when using DNA halos for this application. For instance, DNA tightly associated with residual nuclei of DNA halos can be inaccessible to probes thus precluding it from analysis and physical mapping6. Other techniques such as fiber-FISH2,4,5,7 and molecular combing8 also enable physical mapping and have the advantage of being relatively quick and easy to perform. Both are preferentially used for DNA mapping of genes over DNA halos. These methods extract chromatin fibers via the use of solvent or salt extractions from the nucleus, however, molecular combing tends to have better reproducibility8,9.
There is an increasing evidence that the nucleoskeleton has a role in supporting key nuclear processes, such as attachment sites for DNA, chromatin remodeling, DNA transcription, DNA repair and DNA replication11,12. As such, the DNA halo technique was developed to investigate the interactions between the nucleoskeleton and genome during these cellular activities and has been routinely used and reported in research. This technique has also been used to investigate interactions between the genome and nucleoskeleton in relation to disease progression with malignancy-associated changes in nuclear structure being identified11.
The DNA halo technique has also been used to investigate the relationship between the genome and nucleoskeleton during development and differentiation12. A number of studies have used a variation of the DNA halo technique known as halosperm13 or SpermHalo-FISH if coupled with FISH14. Spermatozoa chromatin is tightly bound to proteins known as protamines and this technique was developed to improve access to the sperm DNA. Halosperm has been used to investigate the integrity of spermatozoa DNA and determine if DNA damage is present. Spermatozoa with less DNA damage correlate to a larger DNA halo size, whereas spermatozoa with increased levels of fragmented and damaged DNA had either small halos or none at all. Thus, halosperm can be used as a potential prognostic marker of embryo quality and successful pregnancy with IVF13. This example emphasizes the potential clinical applications of this technique. In our work we have used HALO-FISH to assess changes in genome behavior and the effect of specific drug treatments in the premature ageing disease Hutchinson-Gilford Progeria Syndrome (HGPS)15.
Together these, and other studies, highlight the breadth of processes/applications that the DNA halo technique can be used to study and utility of the technique.

<table><tbody><tr><td>10X PBS</td><td>Thermo Fisher Scientific</td><td>10388739</td><td>Used to create DNA halos</td></tr><tr><td>5-bromo-2&amp;prime;-deoxy-uridine&amp;nbsp;&amp;nbsp;</td><td>Sigma-Aldrich</td><td>B5002-100MG</td><td>Labelled nucleotide</td></tr><tr><td>5-Fluoro-2&amp;prime;-deoxyuridine</td><td>Sigma-Aldrich</td><td>F0503-100MG</td><td>Labelled nucleotide</td></tr><tr><td>Agar Technical</td><td>Thermo Fisher Scientific</td><td>15562141</td><td>DNA isolation of BAC clones</td></tr><tr><td>Agarose</td><td>Sigma-Aldrich</td><td>A939-50G</td><td>Check product size of DOP-PCR and nick translation</td></tr><tr><td>Atypical type 2 HGPS fibroblasts (AG08466)</td><td>Coriell Institute</td><td>AG08466</td><td>Cell line</td></tr><tr><td>Bacto tryptone</td><td>Thermo Fisher Scientific</td><td>16269751</td><td>DNA isolation of BAC clones</td></tr><tr><td>Biotin-16-dUTP</td><td>Roche Diagnostics&amp;nbsp;</td><td>11093711103</td><td>Labelled nucleotides</td></tr><tr><td>Chloramphenicol</td><td>Sigma-Aldrich</td><td>C0378-25G</td><td>DNA isolation of BAC clones</td></tr><tr><td>Classical Hutchinson-Gilford progeria syndrome (HGPS) fibroblasts (AG06297)&amp;nbsp;</td><td>Coriell Institute</td><td>AG0297</td><td>Cell line</td></tr><tr><td>Coplin jar</td><td>Thermo Fisher Scientific</td><td>12608596</td><td>Holds 5 slides or 8 slides back to back</td></tr><tr><td>Cot-1 DNA</td><td>Thermo Fisher Scientific</td><td>15279011</td><td>Block nonspecific hybridization in HALO FISH</td></tr><tr><td>DEPC-treated water</td><td>Sigma-Aldrich</td><td>693520-1L</td><td>DNA isolation of BAC clones</td></tr><tr><td>Dextran sulphate</td><td>Sigma-Aldrich</td><td>S4030</td><td>Hybridisation mixture</td></tr><tr><td>Digitonin</td><td>Sigma-Aldrich</td><td>D141</td><td>Component of extraction buffer</td></tr><tr><td>Digoxigenin-11-dUTP&amp;nbsp;</td><td>Sigma-Aldrich</td><td>11093088910</td><td>Labelled nucleotides</td></tr><tr><td>Donkey anti-mouse Cy3</td><td>Jackson Laboratory</td><td>715-165-150</td><td>Secondary antibody</td></tr><tr><td>EDTA</td><td>Sigma-Aldrich</td><td>E6758</td><td>Component of extraction buffer</td></tr><tr><td>Ethanol</td><td></td><td></td><td>Component of extraction buffer</td></tr><tr><td>Ethanol</td><td>Sigma-Aldrich</td><td>443611</td><td>Probe precipitation and HALO FISH</td></tr><tr><td>Fetal bovine system</td><td>Thermo Fisher Scientific</td><td>26140079</td><td>Cell culture serum</td></tr><tr><td>Formamide</td><td>Thermo Fisher Scientific</td><td>10523525</td><td>2D FISH of DNA halos</td></tr><tr><td>Glass wool</td><td>Sigma-Aldrich</td><td>18421</td><td>Spin column</td></tr><tr><td>Herring sperm</td><td>Sigma-Aldrich</td><td>D7290</td><td>Probe precipitation</td></tr><tr><td>HXP&amp;trade; Lamp (metal halide microscope lamp)</td><td>OSRAM</td><td>HXP-R120W45C VIS</td><td>Image capture of DNA halos</td></tr><tr><td>Hydrochloric acid</td><td>Thermo Fisher Scientific</td><td>10313680</td><td>Cleaning microscope slides</td></tr><tr><td>Isopropanol</td><td>Sigma-Aldrich</td><td>I9516-25ML</td><td>DNA isolation of BAC clones</td></tr><tr><td>KAPA HiFi PCR Kit</td><td>KAPA Biosystems</td><td>KK2103</td><td>PCR Kit</td></tr><tr><td>Leica DM4000 fluorescent microscope with DFC365 FX camera and LAS AF (Version: 4.5.0) image acquisition software.</td><td>Leica Microsystems</td><td></td><td>Image capture of DNA halos</td></tr><tr><td>Luria-Bertani agar&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>13274843</td><td>DNA isolation of BAC clones</td></tr><tr><td>Magnesium chloride</td><td>Sigma-Aldrich</td><td>M8266</td><td>Component of CSK buffer</td></tr><tr><td>Methanol</td><td>Thermo Fisher Scientific</td><td>10284580</td><td>Cleaning and sterilizing microscope slides</td></tr><tr><td>Mouse anti-BrdU antibody</td><td>BD Pharmingen</td><td>B2531-100UL</td><td>BrdU visualisation</td></tr><tr><td>Newborn calf serum</td><td>Thermo Fisher Scientific</td><td>16010159</td><td>Cell culture serum and blocking reagent</td></tr><tr><td>Nick translation kit</td><td>Invitrogen</td><td></td><td></td></tr><tr><td>PCR grade water</td><td>Sigma-Aldrich</td><td>693520-1L</td><td>PCR and DNA isolation of BAC clones</td></tr><tr><td>PCR Primers</td><td>Sigma-Aldrich</td><td></td><td></td></tr><tr><td>PIPES</td><td>Sigma-Aldrich</td><td>P1851</td><td>Component of CSK and extraction buffers</td></tr><tr><td>Potassium acetate</td><td>Sigma-Aldrich</td><td>P1190-100G</td><td>DNA isolation of BAC clones</td></tr><tr><td>QuadriPERM&lt;sup&gt;&amp;reg;&lt;/sup&gt; 4 X 12</td><td>SARSTEDT</td><td>94.6077.307</td><td>Square cell culture dish, polysterene with four compartments.&amp;nbsp; This has hydrophobic surface, is sterile, non-pyrogenic/endotoxin-fee and non-cytotoxic.</td></tr><tr><td>Rabbit Anti-Ki67 antibody</td><td>Sigma-Aldrich</td><td>ZRB1007-25UL</td><td>Proliferation marker</td></tr><tr><td>Rnase A</td><td>Sigma-Aldrich</td><td>R6513</td><td>DNA isolation of BAC clones</td></tr><tr><td>Rubber cement</td><td>Halford&#039;s</td><td>101836</td><td>2D FISH of DNA halos</td></tr><tr><td>Sephadex G-50</td><td>Sigma-Aldrich</td><td>S6022-25G</td><td>Spin column</td></tr><tr><td>Sodium acetate</td><td>Sigma-Aldrich</td><td>S2889</td><td>Probe precipitation</td></tr><tr><td>Sodium chloride</td><td>Sigma-Aldrich</td><td>S5886</td><td>Component of CSK, extraction and SSC buffers</td></tr><tr><td>Sodium citrate</td><td>Sigma-Aldrich</td><td>C8532</td><td>Component of SSC buffer</td></tr><tr><td>Sodium dodecyl sulphate&amp;nbsp;</td><td></td><td>L3771-100G</td><td>DNA isolation of BAC clones</td></tr><tr><td>Sodium hydroxide</td><td>Sigma-Aldrich</td><td>S8045-500G</td><td>DNA isolation of BAC clones</td></tr><tr><td>Spermidine</td><td>Sigma-Aldrich</td><td>S2626</td><td>Component of extraction buffer</td></tr><tr><td>Spermine</td><td>Sigma-Aldrich</td><td>S4264</td><td>Component of extraction buffer</td></tr><tr><td>Streptavidin-Cy3&amp;nbsp;</td><td>Amersham Life Sciences Ltd, Scientific Laboratory Supplies</td><td>pa43001</td><td>Probe antibody</td></tr><tr><td>Sucrose</td><td>Sigma-Aldrich</td><td>S0389</td><td>Component of CSK buffer</td></tr><tr><td>Sucrose</td><td>Sigma-Aldrich</td><td>S0389</td><td>CSK buffer+A66:D68</td></tr><tr><td>SuperFrost&amp;trade; microscope slides</td><td>Thermo Fisher Scientific</td><td>12372098</td><td>Microscope slides: 1 mm thickness, 76 mm length, 26 mm width. Uncoated.</td></tr><tr><td>Swine anti-rabbit TRITC&amp;nbsp;</td><td>Dako</td><td></td><td></td></tr><tr><td>TELO-PNA FISH KIT</td><td>Agilent Dako</td><td>K532511-8</td><td>Delineation of telomeres</td></tr><tr><td>Tris-HCl</td><td>Sigma-Aldrich</td><td>T3253-100G</td><td>Column buffer</td></tr><tr><td>Triton&amp;trade; X-100</td><td>Sigma-Aldrich</td><td>T9284</td><td>Component of CSK buffer</td></tr><tr><td>Tryptone</td><td>Thermo Fisher Scientific</td><td>10158962</td><td>DNA isolation of BAC clones</td></tr><tr><td>Tween-20</td><td>Sigma-Aldrich</td><td>P9416- 100ML</td><td>Detergent</td></tr><tr><td>Vectashield mountant containing DAPI</td><td>Vector Laboratories</td><td>H-1200</td><td>2D FISH of DNA halos</td></tr><tr><td>Whole human chromosome probes</td><td>Calbiochem</td><td></td><td>2D FISH of DNA halos</td></tr><tr><td>Yeast extract</td><td>Thermo Fisher Scientific</td><td>10108202</td><td>DNA isolation of BAC clones</td></tr></tbody></table>

fluorescence in situ hybridization on dna halo preparations to reveal whole chromosomes, telomeres and gene loci

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&#39;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&#160;cells and in disease progression such as with cancer.

This method of DNA halo preparation has helped us in our endeavors to determine differences in genome behavior within young and old cells, but also in cells derived from premature ageing diseases with aberrant nucleoskeletal proteins15. Figure 1 displays examples of DNA halos where it is possible to see the edge of a residual nucleus, the DNA remaining within the residual nucleus and the unattached DNA that has spooled out into the surrounding area creating a DNA halo. It also depicts the analysis showing how the residual nucleus is obtained and the NE and CTE measurements. It is possible to differentiate between proliferating and non-proliferating cells by either incorporating a labeled nucleotide such as BrdU when cells are in S-phase or employing the diagnostic proliferation marker anti-pKi67, which reveals nucleoli, and regions of heterochromatin in G1 cells17,18. Primary cells grown in high serum without achieving confluency, that are negative for the proliferation markers, are assumed to be senescent. Primary cells grown in low serum or have become confluent i.e., contact inhibited that are negative for the proliferation markers are deemed quiescent and would be able to reenter the proliferative cell cycle given the correct nutrients and situation. Being able to differentiate between Ki67 positive and negative cells has enabled us to determine differences between proliferating, quiescent and senescent human dermal fibroblasts. Figure 2 displays DNA halos of proliferating human dermal fibroblasts created from cells where BrdU was incorporated into them during DNA replication, a mechanism that does not occur in non-proliferating cells, and subsequently stained with anti-BrdU antibody. Staining with the proliferative marker anti-pKi67 antibody is also visible in Figure 2. This is a robust antigen and survives the FISH protocol and so can be stained for post-FISH and pre-mounting. Thus, proliferating cells are positive (red) for BrdU and anti-pKi67 (red) in the left-hand column and non-proliferating cells, indeed senescent cells in Figure 2 are displayed in the right-hand column. The green signals are individual telomeres revealed with a telomere PNA FISH/FITC kit. Combining immunofluorescence with DNA halos enables analysis during different cell states, as shown in Figure 2 when investigating proliferating, quiescent and senescent cells. Depending on the antibody chosen other conditions can be examined, such as differentiation, DNA damage via irradiation etc.
Chromosome territories can also be visualized within DNA halos using FISH. Due to the preparation permitting spooling of DNA out the nuclei, the chromosome territory shape can be disturbed, with smaller or larger amounts of the chromosome found in the DNA halo, depending on the anchorage of the genome inside the residual nucleus and its structures. Figure 3 reveals a panel of DNA halos whereby individual chromosomes have been revealed with specific whole arm chromosome painting probes (red) for chromosomes 1, 13, 17 and 18. Anti-pKi67 (green) has been used to mark proliferating cells and its absence within the same culture, upon the same slide, denoting senescent cells. It is very obvious from the images and the data presented as CTE/NE that the small gene-poor chromosome 18 is a chromosome that has few attachments and spools further out into the DNA halo away from the residual nuclei and is significantly further from the center of the residual nuclei than the other chromosomes. However, this is also true for chromosome 1 as well. Using the proliferative marker anti-pKi67 it has also been possible to compare proliferating with senescent cells, within the same culture, and on the same slide, and this analysis has revealed that chromosomes within these two very different cell statuses are not significantly different from one another, with respect to attachment with the residual nuclear structures.
Interestingly, genes also are showing statistically significant differences between proliferating and senescent cells with respect to remaining within a residual nucleus or being located in the DNA Halo. Figure 4 demonstrates this with gene loci delineated by labeled BAC probes in red and anti-Ki67 in green. There are no significant differences between gene locations in the proliferating versus the senescent cells, after a DNA Halo preparation. However, there are significantly more catenin alpha 1 CTNNA1 loci within the DNA halo than cyclin D1 CNDD1 loci, where there are very few. Figure 5 displays DNA halo preparations with telomeres in green. The background is left deliberately high to enable telomere signals to be visualized within the DNA halo. In this set of data quiescent cells i.e., cells that have been serum starved for 7 days have been included and interestingly there are significantly more telomeres unattached and located within the DNA halos in quiescent cells than for proliferating and senescent cells. In Figure 5a the proportion of telomeres in the DNA halo can be observed, particularly for the image &#39;Experiment 2&#39;. This corresponds with Figure 5b where the mean percentage of telomeres in DNA halo is approximately 17% in quiescent cells. There is some evidence that not all telomeres in senescent cells can be seen as some of them maybe very short.
This method of DNA halo has been successful for us to investigate genome interaction alterations within nuclei in diseased cells15. Figure 6 demonstrates differences in chromosome attachment in primary control fibroblasts and in diseased cells with typical (lamin A mutation) and atypical Hutchinson-Gilford Progeria Syndrome, expressing a different SUN1 isoform and no lamin A mutation19. Chromosomes 1 and 13 show statistically significant differences in their attachment within the residual nuclei when compared to control DNA halos. Figure 6b correlates the position of the whole chromosome territory to the residual nucleus and DNA Halo. Values of 1 or less indicates the chromosome is located within the residual nucleus and values over 1 demonstrate chromosomes or portions of chromosomes within the DNA Halo.
Overall, this highlights the utility of HALO-FISH in investigating genomic interactions of whole chromosomes, specific genes and telomeres under a variety of conditions that affect the cell cycle (proliferation, quiescence and senescence) or within disease cells e.g., progeria and cancer cell lines. Indeed, the differences in interactions between these states implies the nucleoskeleton has an important role in regulating key processes within the nucleus.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62017/62017fig01.jpg" /> 
Figure 1:&#160;HDF extracted nucleus displaying the residual nucleus and DNA halo and overview of analysis method. (a) An HDF nucleus prepared via DNA halo assay and counterstained with DAPI. The brightly stained residual nucleus shows DNA anchored to the nucleoskeleton and this is surrounded by the non-attached DNA which forms a halo of DNA. Magnification = x 100; scale bar 10 &#181;m. (b) The blue channel captures the DAPI-stained nucleus and surrounding DNA. The residual nucleus is selected and removed using ImageJ. The arrow depicts the distance from the nuclear center to the residual nuclear edge (NE). (c) The red channel shows the probe signal. (d) The image denoted &#39;Result&#39; is the outcome of superimposing the red channel on the blue channel image; this allows the distance from the nuclear center to the furthest chromosome territory edge (CTE). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62017/62017fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62017/62017fig02.jpg" /> 
Figure 2:&#160;DNA halo preparation with telomere PNA FISH on proliferating and senescent HDFs. Telomere PNA FISH on HDFs subjected to DNA halo assay. Telomere signals are visualized in green (FITC), residual and halo DNA was counterstained using DAPI (blue) and proliferating nuclei were detected using either anti-BrdU or anti-pKi67 antibodies via indirect immunofluorescence in red (TRITC). Magnification = x 100; scale bar 10 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62017/62017fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62017/62017fig03.jpg" /> 
Figure 3:&#160;Nucleoskeleton-chromosome interactions and analysis using DNA halo assay. (a) 2D-FISH with probes specific for chromosomes 1, 13, 15, 17 and 18 was performed on HDFs subjected to DNA halo preparation. Whole chromosomes were painted in red (Cy3) and nuclei were probed with pKi67 to determine if they were proliferating or senescent. Proliferating cells (pKi67+) were delineated in green (FITC), whereas senescent cells remained unstained (pKi67-) i.e. no green signal detected. Magnification = x 100; scale bar 10 &#181;m. (b) Chromosome anchorage by the nucleoskeleton in proliferating and senescent HDFs that had undergone HALO-FISH. Measurements show the ratio of the furthest chromosome territory edge (CTE) to respective nuclear edge (NE) for chromosomes 1, 13, 15, 17 and 18 in proliferating (pKi67+) and senescent (pKi67-) cells. Error bars represent &#177; SEM. (c) Modified box plot representation of chromosome territory edge (CTE) to respective nuclear edge (NE) of specific chromosomes in pKi67+ and pKi67- nuclei. Q1 = lower quartile; Min = lowest value recorded; Med = median; Max = maximum value recorded; Q3 = upper quartile. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62017/62017fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/62017/62017fig04.jpg" /> 
Figure 4:&#160;Gene-specific interactions in HDFs using HALO-FISH. (a) DNA halo extracted nuclei were probed with gene specific probes (CCND1 and CTNNA1) to investigate their anchorage to the NM on proliferating and senescent cells. The gene signals are shown in red (Cy3) and anti-pKi67 depicts proliferating cells and signal is visualized in green (FITC). For the proliferating CCND1 image, the residual nucleus is enclosed within the white circle, and the space between the white and green circle depicts the DNA Halo. Magnification = x 100; scale bar 10 &#181;m. (b) Gene-specific signals for CCND1 and CTNNA1 are compared between the residual nucleus and DNA halo, and also, between proliferating and senescent cells. Error bars represent &#177; SEM. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62017/62017fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/62017/62017fig05.jpg" /> 
Figure 5:&#160;DNA halo assay on quiescent HDFs probed with telomere PNA-FISH. (a) Quiescence of HDFs was induced by culture in low serum medium for 7 days. The DNA halo assay was performed, and PNA-FISH enabled visualization of telomeres by FITC signal (green) and the residual nucleus and surrounding DNA halo was counterstained with DAPI (blue). Cells were also stained with anti-pKi67 antibody to ensure nuclei were non-proliferating. This was repeated on two separate occasions. Magnification = x 100; scale bar 10 &#181;m. (b) Comparison of the mean percentage of telomeres localized within the DNA halo in proliferating, senescent and quiescent HDF cells. Error bars represent &#177; SEM. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62017/62017fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/62017/62017fig06.jpg" /> 
Figure 6: Examining whole chromosome anchorage to the nucleoskeleton in HGPS cells using HALO-FISH26. (a) Control HDF (2DD), classical HGPS (AG06297) and atypical type 2 HGPS (AG08466) nuclei underwent DNA halo preparation and then 2D-FISH using whole chromosome paints for chromosome 1, 13, 15 and 17. Whole chromosomes are depicted in green (FITC) and DNA was counterstained with DAPI (blue). Magnification = x 100; scale bar 10 &#181;m. (b) Positioning of chromosomes within extracted nuclei was determined by measuring the ratio of the mean chromosome territory edge (CTE) to the nuclear edge (NE). A ratio above 1 demonstrates that the furthest CTE lies outside the corresponding NE within the DNA halo, while a ratio below 1 signifies that the furthest CTE lies within the NE within the residual nucleus. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62017/62017fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Constituents</td>
			<td>Volume(&#956;L)</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>5XDOP-PCRbuffer</td>
			<td>10</td>
		</tr>
		<tr>
			<td>dNTPmix(withoutdTTP)(2mM)</td>
			<td>5</td>
		</tr>
		<tr>
			<td>dTTP(2mM)</td>
			<td>2</td>
		</tr>
		<tr>
			<td>Biotin-16-dUTPorDigoxigenin-11-dUTP</td>
			<td>10</td>
		</tr>
		<tr>
			<td>DOPprimer(20&#956;M)</td>
			<td>5</td>
		</tr>
		<tr>
			<td>TaqDNAPolymerase(1U/&#956;L)</td>
			<td>1</td>
		</tr>
		<tr>
			<td>PCRgradewater</td>
			<td>12</td>
		</tr>
		<tr>
			<td>Template</td>
			<td>5</td>
		</tr>
	</tbody>
</table>
Table 1: Table showing the DOP-PCR components and volumes for a 1x reaction
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Step&#160;</td>
			<td>Cycles&#160;</td>
			<td>Temp (degree Centigrade)</td>
			<td>Time&#160;</td>
		</tr>
		<tr>
			<td>Initial Denaturation&#160;</td>
			<td>1</td>
			<td>95</td>
			<td>3 min</td>
		</tr>
		<tr>
			<td>Denaturation&#160;</td>
			<td rowspan="3">34</td>
			<td>98</td>
			<td>20 s</td>
		</tr>
		<tr>
			<td>Primer Annealing&#160;</td>
			<td>62</td>
			<td>1 min</td>
		</tr>
		<tr>
			<td>Extension&#160;</td>
			<td>72</td>
			<td>30 s</td>
		</tr>
		<tr>
			<td>Final Extension&#160;</td>
			<td>1</td>
			<td>72</td>
			<td>5 min&#160;</td>
		</tr>
		<tr>
			<td>Cooling&#160;</td>
			<td></td>
			<td>4</td>
			<td>Hold</td>
		</tr>
	</tbody>
</table>
Table 2: Table showing the DOP-PCR cycle, temperature, and time profile.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Constituent</td>
			<td>Volume(&#956;L)</td>
		</tr>
		<tr>
			<td>10x NT buffer (0.5M Tris-HCl pH 8,50 mM MgCl2, 0.5 mg/ml BSA)</td>
			<td>5</td>
		</tr>
		<tr>
			<td>0.1 M beta-mercaptoethanol</td>
			<td>5</td>
		</tr>
		<tr>
			<td>10X Nucleotide stock (0.5 mM dATP, 0.5 mM dCTP, 0.5 mM dGTP, 0.5 mM dTTP, 0.5 mg/ml biotin-16-dUTP)</td>
			<td>5</td>
		</tr>
		<tr>
			<td>Dnase I (1 ng/ml)</td>
			<td>2</td>
		</tr>
		<tr>
			<td>DNApolymerase I</td>
			<td>5U per &#956;g of DNA</td>
		</tr>
		<tr>
			<td>DNAtemplate (1 &#956;g)</td>
			<td>1</td>
		</tr>
		<tr>
			<td>DEPC-treated water</td>
			<td>To 50 &#956;L</td>
		</tr>
	</tbody>
</table>
Table 3: Table showing the nick translation components and volumes for a one probe.

Watch this Scientific Journal Video about Fluorescence In Situ Hybridization on DNA Halo Preparations to Reveal Whole Chromosomes, Telomeres and Gene Loci at JoVE.com

Fluorescence In Situ Hybridization on DNA Halo Preparations to Reveal Whole Chromosomes, Telomeres and Gene Loci

Combining DNA halo preparations with fluorescence in situ hybridization enables high-resolution analysis of genomic interactions with the nucleoskeleton. Attached genome leads to hybridized fluorescent signals located within the residual extracted nuclei, whereas non-attached genome is in the halo of DNA surrounding the residual nuclei.

The genome is associated with several structures inside cell nuclei, in order to regulate its activity and anchor it in specific locations. These ...

fluorescence-situ-hybridization-on-dna-halo-preparations-to-reveal

Nanyang Technological University

Research

JoVE Journal

Biology

2.8K Views.  Brunel University London. Dit protocol kan worden gebruikt voor het beoordelen van aanhechtingen aan het nucleoskeleton in plaats van DamID-sequencing te gebruiken, en het is een test met één cel. Het belangrijkste voordeel van het gebruik van deze methode is dat het kosteneffectief en gemakkelijk toegankelijk is. Het kan functionele verschillen tussen genen en chromosomen benadrukken die normaal gesproken niet voorkomen met andere visualisatietechnieken.Ruimtelijke en functionele organisaties binnen het geno...

Video: Fluorescence In Situ Hybridization on DNA Halo Preparations to Reveal Whole Chromosomes, Telomeres and Gene Loci

3D Multicolor DNA FISH Tool to Study Nuclear Architecture in Human Primary Cells

A major question in cell biology is genomic organization within the nuclear space and how chromatin architecture can influence processes such as gene expression, cell identity and differentiation. Many approaches developed to study the 3D architecture of the genome can be divided into two complementary categories: chromosome conformation capture based technologies (C-technologies) and imaging. While the former is based on capturing the chromosome conformation and proximal DNA interactions in a population of fixed cells, the latter, based on DNA fluorescence in situ hybridization (FISH) on 3D-preserved nuclei, allows contemporary visualization of multiple loci at a single cell level (multicolor), examining their interactions and distribution within the nucleus (3D multicolor DNA FISH). The technique of 3D multicolor DNA FISH has a limitation of visualizing only a few predetermined loci, not permitting a comprehensive analysis of the nuclear architecture. However, given the robustness of its results, 3D multicolor DNA FISH in combination with 3D-microscopy and image reconstruction is a possible method to validate C-technology based results and to unambiguously study the position and organization of specific loci at a single cell level. Here, we propose a step by step method of 3D multicolor DNA FISH suitable for a wide range of human primary cells and discuss all the practical actions, crucial steps, notions of 3D imaging and data analysis needed to obtained a successful and informative 3D multicolor DNA FISH within different biological contexts.

3D multicolor DNA FISH represents a tool to visualize multiple genomic loci within 3D preserved nuclei, unambiguously defining their reciprocal interactions and localization within the nuclear space at a single cell level. Here, a step by step protocol is described for a wide spectrum of human primary cells.

A major question in cell biology is genomic organization within the nuclear space and how chromatin architecture can influence processes such as gene ...

Genetics

Using Fluorescence In Situ Hybridization (FISH) to Monitor the State of Arm Cohesion in Prometaphase and Metaphase I Drosophila Oocytes

In humans, chromosome segregation errors in oocytes are responsible for the majority of miscarriages and birth defects. Moreover, as women age, their risk of conceiving an aneuploid fetus increases dramatically and this phenomenon is known as the maternal age effect. One requirement for accurate chromosome segregation during the meiotic divisions is maintenance of sister chromatid cohesion during the extended prophase period that oocytes experience. Cytological evidence in both humans and model organisms suggests that meiotic cohesion deteriorates during the aging process. In addition, segregation errors in human oocytes are most prevalent during meiosis I, consistent with premature loss of arm cohesion. The use of model organisms is critical for unraveling the mechanisms that underlie age-dependent loss of cohesion. Drosophila melanogaster offers several advantages for studying the regulation of meiotic cohesion in oocytes. However, until recently, only genetic tests were available to assay for loss of arm cohesion in oocytes of different genotypes or under different experimental conditions. Here, a detailed protocol is provided for using fluorescence in situ hybridization (FISH) to directly visualize defects in arm cohesion in prometaphase I and metaphase I arrested Drosophila oocytes. By generating a FISH probe that hybridizes to the distal arm of the X chromosome and collecting confocal Z stacks, a researcher can visualize the number of individual FISH signals in three dimensions and determine whether sister chromatid arms are separated. The procedure outlined makes it possible to quantify arm cohesion defects in hundreds of Drosophila oocytes. As such, this method provides an important tool for investigating the mechanisms that contribute to cohesion maintenance as well as the factors that lead to its demise during the aging process.

Using Fluorescence In Situ Hybridization FISH to Monitor the State of Arm Cohesion in Prometaphase and Metaphase I Drosophila Oocytes

This manuscript presents a detailed method for generating X-chromosome arm probes and performing fluorescence in situ hybridization (FISH) to examine the state of sister chromatid cohesion in prometaphase and metaphase I arrested Drosophila oocytes. This protocol is suitable for determining whether meiotic arm cohesion is intact or disrupted in different genotypes.

In humans, chromosome segregation errors in oocytes are responsible for the majority of miscarriages and birth defects. Moreover, as women age, their risk ...

Developmental Biology

Chromosomics: Detection of Numerical and Structural Alterations in All 24 Human Chromosomes Simultaneously Using a Novel OctoChrome FISH Assay

Fluorescence in situ hybridization (FISH) is a technique that allows specific DNA sequences to be detected on metaphase or interphase chromosomes in cell nuclei1. The technique uses DNA probes with unique sequences that hybridize to whole chromosomes or specific chromosomal regions, and serves as a powerful adjunct to classic cytogenetics. For instance, many earlier studies reported the frequent detection of increased chromosome aberrations in leukemia patients related with benzene exposure, benzene-poisoning patients, and healthy workers exposed to benzene, using classic cytogenetic analysis2. Using FISH, leukemia-specific chromosomal alterations have been observed to be elevated in apparently healthy workers exposed to benzene3-6, indicating the critical roles of cytogentic changes in benzene-induced leukemogenesis. 
Generally, a single FISH assay examines only one or a few whole chromosomes or specific loci per slide, so multiple hybridizations need to be conducted on multiple slides to cover all of the human chromosomes. Spectral karyotyping (SKY) allows visualization of the whole genome simultaneously, but the requirement for special software and equipment limits its application7. Here, we describe a novel FISH assay, OctoChrome-FISH, which can be applied for Chromosomics, which we define here as the simultaneous analysis of all 24 human chromosomes on one slide in human studies, such as chromosome-wide aneuploidy study (CWAS)8. The basis of the method, marketed by Cytocell as the Chromoprobe Multiprobe System, is an OctoChrome device that is divided into 8 squares, each of which carries three different whole chromosome painting probes (Figure 1). Each of the three probes is directly labeled with a different colored fluorophore, green (FITC), red (Texas Red), and blue (Coumarin). The arrangement of chromosome combinations on the OctoChrome device has been designed to facilitate the identification of the non-random structural chromosome alterations (translocations) found in the most common leukemias and lymphomas, for instance t(9;22), t(15;17), t(8;21), t(14;18)9. Moreover, numerical changes (aneuploidy) in chromosomes can be detected concurrently. The corresponding template slide is also divided into 8 squares onto which metaphase spreads are bound (Figure 2), and is positioned over the OctoChrome device. The probes and target DNA are denatured at high-temperature and hybridized in a humid chamber, and then all 24 human chromosomes can be visualized simultaneously. 
OctoChrome FISH is a promising technique for the clinical diagnosis of leukemia and lymphoma and for detection of aneuploidies in all chromosomes. We have applied this new Chromosomic approach in a CWAS study of benzene-exposed Chinese workers8,10.

A novel fluorescence in situ hybridization (FISH) method that simultaneously examines both numerical and structural chromosome alterations, particularly the specific chromosomal translocations associated with leukemia and lymphoma, of all 24 human chromosomes on a single device in one hybridization, is described.

Fluorescence in situ hybridization (FISH) is a technique that allows specific DNA sequences to be detected on metaphase or interphase chromosomes in cell ...

Chromosome Replicating Timing Combined with Fluorescent In situ Hybridization

Mammalian DNA replication initiates at multiple sites along chromosomes at different times during S phase, following a temporal replication program. The specification of replication timing is thought to be a dynamic process regulated by tissue-specific and developmental cues that are responsive to epigenetic modifications. However, the mechanisms regulating where and when DNA replication initiates along chromosomes remains poorly understood. Homologous chromosomes usually replicate synchronously, however there are notable exceptions to this rule. For example, in female mammalian cells one of the two X chromosomes becomes late replicating through a process known as X inactivation1. Along with this delay in replication timing, estimated to be 2-3 hr, the majority of genes become transcriptionally silenced on one X chromosome. In addition, a discrete cis-acting locus, known as the X inactivation center, regulates this X inactivation process, including the induction of delayed replication timing on the entire inactive X chromosome. In addition, certain chromosome rearrangements found in cancer cells and in cells exposed to ionizing radiation display a significant delay in replication timing of &gt;3 hours that affects the entire chromosome2,3. Recent work from our lab indicates that disruption of discrete cis-acting autosomal loci result in an extremely late replicating phenotype that affects the entire chromosome4. Additional 'chromosome engineering' studies indicate that certain chromosome rearrangements affecting many different chromosomes result in this abnormal replication-timing phenotype, suggesting that all mammalian chromosomes contain discrete cis-acting loci that control proper replication timing of individual chromosomes5.
Here, we present a method for the quantitative analysis of chromosome replication timing combined with fluorescent in situ hybridization. This method allows for a direct comparison of replication timing between homologous chromosomes within the same cell, and was adapted from6. In addition, this method allows for the unambiguous identification of chromosomal rearrangements that correlate with changes in replication timing that affect the entire chromosome. This method has advantages over recently developed high throughput micro-array or sequencing protocols that cannot distinguish between homologous alleles present on rearranged and un-rearranged chromosomes. In addition, because the method described here evaluates single cells, it can detect changes in chromosome replication timing on chromosomal rearrangements that are present in only a fraction of the cells in a population.

Chromosome Replicating Timing Combined with Fluorescent In situ Hybridization

A quantitative method for the analysis of chromosome replication timing is described. The method utilizes BrdU incorporation in combination with fluorescent in situ hybridization (FISH) to assess replication timing of mammalian chromosomes. This technique allows for the direct comparison of rearranged and un-rearranged chromosomes within the same cell.

Mammalian DNA replication initiates at multiple sites along chromosomes at different times during S phase, following a temporal replication program. The ...

Combined Immunofluorescence and DNA FISH on 3D-preserved Interphase Nuclei to Study Changes in 3D Nuclear Organization

Fluorescent in situ hybridization using DNA probes on 3-dimensionally preserved nuclei followed by 3D confocal microscopy (3D DNA FISH) represents the most direct way to visualize the location of gene loci, chromosomal sub-regions or entire territories in individual cells. This type of analysis provides insight into the global architecture of the nucleus as well as the behavior of specific genomic loci and regions within the nuclear space. Immunofluorescence, on the other hand, permits the detection of nuclear proteins (modified histones, histone variants and modifiers, transcription machinery and factors, nuclear sub-compartments, etc). The major challenge in combining immunofluorescence and 3D DNA FISH is, on the one hand to preserve the epitope detected by the antibody as well as the 3D architecture of the nucleus, and on the other hand, to allow the penetration of the DNA probe to detect gene loci or chromosome territories 1-5. Here we provide a protocol that combines visualization of chromatin modifications with genomic loci in 3D preserved nuclei.

Here we describe a protocol for simultaneous detection of histone modifications by immunofluorescence and DNA sequences by DNA FISH followed by 3D microscopy and analyses (3D immuno-DNA FISH).

Fluorescent in situ hybridization using DNA probes  on 3-dimensionally preserved nuclei followed by 3D confocal microscopy (3D DNA  FISH) represents the ...

Robust 3D DNA FISH Using Directly Labeled Probes

3D DNA FISH has become a major tool for analyzing three-dimensional organization of the nucleus, and several variations of the technique have been published. In this article we describe a protocol which has been optimized for robustness, reproducibility, and ease of use. Brightly fluorescent directly labeled probes are generated by nick-translation with amino-allyldUTP followed by chemical coupling of the dye. 3D DNA FISH is performed using a freeze-thaw step for cell permeabilization and a heating step for simultaneous denaturation of probe and nuclear DNA. The protocol is applicable to a range of cell types and a variety of probes (BACs, plasmids, fosmids, or Whole Chromosome Paints) and allows for high-throughput automated imaging. With this method we routinely investigate nuclear localization of up to three chromosomal regions.

We describe a robust and versatile protocol for analyzing nuclear architecture by 3D DNA FISH using directly labeled fluorescent probes.

3D DNA FISH has become a major tool for  analyzing three-dimensional organization of the nucleus, and several variations  of the technique have been ...

Combined DNA-RNA Fluorescent In situ Hybridization (FISH) to Study X Chromosome Inactivation in Differentiated Female Mouse Embryonic Stem Cells

Fluorescent in situ hybridization (FISH) is a molecular technique which enables the detection of nucleic acids in cells. DNA FISH is often used in cytogenetics and cancer diagnostics, and can detect aberrations of the genome, which often has important clinical implications. RNA FISH can be used to detect RNA molecules in cells and has provided important insights in regulation of gene expression. Combining DNA and RNA FISH within the same cell is technically challenging, as conditions suitable for DNA FISH might be too harsh for fragile, single stranded RNA molecules. We here present an easily applicable protocol which enables the combined, simultaneous detection of Xist RNA and DNA encoded by the X chromosomes. This combined DNA-RNA FISH protocol can likely be applied to other systems where both RNA and DNA need to be detected.

Combined DNA-RNA Fluorescent In situ Hybridization FISH to Study X Chromosome Inactivation in Differentiated Female Mouse Embryonic Stem Cells

Fluorescent in situ hybridization (FISH) allows the detection of nucleic acids in their native environment within cells. We here describe a protocol for the combined, simultaneous detection of RNA and DNA by means of FISH, which can be used to study X chromosome inactivation in mouse embryonic stem cells.

Fluorescent in situ hybridization (FISH) is a molecular technique which enables the detection of nucleic acids in cells. DNA FISH is often used in ...

Simple Method for Fluorescence DNA In Situ Hybridization to Squashed Chromosomes

DNA in situ hybridization (DNA ISH) is a commonly used method for mapping sequences to specific chromosome regions. This approach is particularly effective at mapping highly repetitive sequences to heterochromatic regions, where computational approaches face prohibitive challenges. Here we describe a streamlined protocol for DNA ISH that circumvents formamide washes that are standard steps in other DNA ISH protocols. Our protocol is optimized for hybridization with short single strand DNA probes that carry fluorescent dyes, which effectively mark repetitive DNA sequences within heterochromatic chromosomal regions across a number of different insect tissue types. However, applications may be extended to use with larger probes and visualization of single copy (non-repetitive) DNA sequences. We demonstrate this method by mapping several different repetitive sequences to squashed chromosomes from Drosophila melanogaster neural cells and Nasonia vitripennis spermatocytes. We show hybridization patterns for both small, commercially synthesized probes and for a larger probe for comparison. This procedure uses simple laboratory supplies and reagents, and is ideal for investigators who have little experience with performing DNA ISH.

Simple Method for Fluorescence DNA In Situ Hybridization to Squashed Chromosomes

Here, we present a simple method for performing fluorescence DNA in situ hybridization (DNA ISH) to visualize repetitive heterochromatic sequences on slide-mounted chromosomes. The method requires minimal reagents and it is versatile for use with short or long probes, different tissues, and detection with fluorescence or non-fluorescence-based signals.

DNA in situ hybridization (DNA ISH) is a commonly used method for mapping sequences to specific chromosome regions. This approach is particularly ...

Chemical Dimerization-Induced Protein Condensates on Telomeres

Chromatin-associated condensates are implicated in many nuclear processes, but the underlying mechanisms remain elusive. This protocol describes a chemically-induced protein dimerization system to create condensates on telomeres. The chemical dimerizer consists of two linked ligands that can each bind to a protein: Halo ligand to Halo-enzyme and trimethoprim (TMP) to E. coli dihydrofolate reductase (eDHFR), respectively. Fusion of Halo enzyme to a telomere protein anchors dimerizers to telomeres through covalent Halo ligand-enzyme binding. Binding of TMP to eDHFR recruits eDHFR-fused phase separating proteins to telomeres and induces condensate formation. Because TMP-eDHFR interaction is non-covalent, condensation can be reversed by using excess free TMP to compete with the dimerizer for eDHFR binding. An example of inducing promyelocytic leukemia (PML) nuclear body formation on telomeres and determining condensate growth, dissolution, localization and composition is shown. This method can be easily adapted to induce condensates at other genomic locations by fusing Halo to a protein that directly binds to the local chromatin or to dCas9 that is targeted to the genomic locus with a guide RNA. By offering the temporal resolution required for single cell live imaging while maintaining phase separation in a population of cells for biochemical assays, this method is suitable for probing both the formation and function of chromatin-associated condensates.

This protocol illustrates a chemically induced protein dimerization system to create condensates on chromatin.&#160; The formation of promyelocytic leukemia (PML) nuclear body on telomeres with chemical dimerizers is demonstrated. Droplet growth, dissolution, localization and composition are monitored with live cell imaging, immunofluorescence (IF) and fluorescence in situ hybridization (FISH).

Chromatin-associated condensates are implicated in many nuclear processes, but the underlying mechanisms remain elusive. This protocol describes a ...

Cytogenetics

Cytogenetics is the field of study devoted to chromosomes, and involves the direct observation of a cell&#8217;s chromosomal number and structure, together known as its karyotype. Many chromosomal abnormalities are associated with disease. Each chromosome in a karyotype can be stained with a variety of dyes to give unique banding patterns. More recent techniques, including comparative genomic hybridization and fluorescence in situ hybridization (FISH), allow for detecting specific chromosomal features or abnormalities.This video will begin by examining the principles of these classical and modern cytogenetics techniques. This is followed by an examination of a general protocol for performing FISH. Finally, several examples of how karyotyping can be applied to various medical applications are presented.

Fluorescence In Situ Hybridization on DNA Halo Preparations to Reveal Whole Chromosomes, Telomeres and Gene Loci

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