This work was supported by a grant from the National Cancer Institute, CA131967.

1. BrdU Incorporation (Terminal Labeling)
<ol>
 <li>Plate cells to about 70% confluence in a 150 mm tissue culture dish 24 hr prior to the addition of BrdU.</li>
 <li>Replace media with fresh complete media containing 20 &mu;g/ml BrdU (Sigma) at appropriate time points prior to harvest. The length of time cells are cultured in media with BrdU will vary with cell type and species, typically the G2 phase lasts between 2 and 5 hr (Figure 1).</li>
</ol>
2. Chromosome Harvest of Monolayer Cells Cultures
<ol>
 <li>Remove culture media from the plate and collect 10 ml in a 15 ml conical centrifuge tube. Discard remaining media.</li>
 <li>Rinse cells once with 10 ml Versine [or HBSS (Sigma)].</li>
 <li>Add 5 ml 0.25% trypsin in Versine (or HBSS) to the plate. Incubate at room temperature until cells are detached from the plate.</li>
 <li>Add trypsinized cell suspension to the tube containing the 10 ml of media from step 2.1.</li>
 <li>Centrifuge at 400 x g for 10 min to pellet the cells. Aspirate supernatant, leaving 0.5 ml of media plus the cell pellet.</li>
 <li>Resuspend cells thoroughly by pipetting with a Pasteur pipette.</li>
 <li>Add 3 drops of hypotonic solution (0.075 M KCl (Sigma) warmed to 37 &deg;C); mix the cell suspension with a Pasteur pipette. Add 0.5 ml hypotonic solution and mix again with a Pasteur pipette. Bring volume of hypotonic solution to 5 ml and mix once again. Incubate at 37 &deg;C for 20-45 min, depending on the cell type.</li>
 <li>Centrifuge at 400 x g for 10 min. Aspirate supernatant, leaving 0.5 ml of media plus the cell pellet.</li>
 <li>Resuspend the cell pellet by gently flicking the tube. The osmotically swollen cells will be fragile at this point, so care should be taken not to disrupt the cytoplasmic membranes.</li>
 <li>Add 3 drops of Carnoy's fixative (1:3 Glacial Acetic Acid: Methanol); mix by gently pipetting with a Pasteur pipette. Add 0.5 ml fixative and mix by gently pipetting with a Pasteur pipette. Bring volume of fixative to 5 ml and mix again by gently pipetting with a Pasteur pipette. Fixed cells can be stored in the dark at -20 &deg;C for several months.</li>
 <li>Centrifuge and aspirate as in step 2.8.</li>
 <li>Resuspend the cell pellet in fresh fixative; estimate the volume of the pellet and add ~10x the volume of Carnoy's fixative.</li>
 <li>Add the cell suspension drop-wise on wet, ice-cold microscope slides; hold the slide at an ~45&deg; angle and allow the cell suspension to flow over the surface of the slide. Lay the slide flat on paper towels and flood the slide with fixative and allow to air dry. Check slides for the presence of mitotic spreads using an inverted microscope.</li>
</ol>
3. RNase Treatment and Ethanol Dehydration
<ol>
 <li>Place 200 &mu;l of 10 &mu;g/ml RNase A (Sigma) in 2x SSC to each slide; incubate at 37 &deg;C for 1 hr.</li>
 <li>Wash slides 3 times with 2x SSC, pH 7.0 at room temperature for 3 min each.</li>
 <li>Dehydrate slides through an EtOH (Sigma) series (70%, 90% and 100%) at room temperature for 3 min each.</li>
 <li>Air dry the slides at room temperature.</li>
</ol>
4. Preparation of Probe Cocktails for BAC Plus Chromosome-specific Centromere Enumeration Probe (CEP) or for Whole Chromosome Paints In situ Hybridization
<ol>
 <li>For the BAC/CEP simultaneous hybridization, prepare two separate probe cocktails for pre-hybridization. We have found that pre-hybridizing the BAC and CEP probes separately under different stringencies results in less background and greater signal intensities. We have also found that individual Fosmids can also be used in place of BAC clones. The cocktail formulation described below represents the volumes for a single slide. If processing multiple slides using the same probes simply increase the volumes appropriately.
 *BAC DNA probe cocktail: 
 11.5 &mu;l hybridization buffer (50% Formamide (Sigma) /2x SSC /Dextran Sulfate (Sigma)) 2 &mu;l dH2O
 2.5 &mu;l BAC/Cot1 DNA in dH2O (see below for labeling protocol) 15 &mu;l total volume 
 *CEP probe cocktail: 
 7 &mu;l CEP hybridization buffer (65% Formamide/2x SSC/Dextran Sulfate) 2 &mu;l dH2O 
 1 &mu;l CEP/Cot1 DNA (purchased prelabeled with Spectrum Orange/Cy3/Texas Red) 10 &mu;l total volume 
 *After denaturing and pre-hybridization (step 5.1) mix BAC probe with CEP probe at a 3:2 ratio 25 &mu;l/slide and mix probe cocktails. 
 OR</li>
 <li>Aliquot 20 &mu;l/slide of Whole Chromosome Paint probe (Cy3/Texas Red) into a tube. Proceed to denaturation and pre-hybridization step.</li>
</ol>
5. Slide and Probe Denaturation and In situ Hybridization
<ol>
 <li>Denature probe cocktail(s) at 75 &deg;C for 10 min. Pre-hybridize at 37 &deg;C for 30 min to allow the Cot1 DNA to hybridize to repetitive sequences and to prevent subsequent hybridization to metaphase chromosomes.</li>
 <li>Denature slides in Coplin jars in 70% Formamide/2x SSC, pH 7.0 at 72 &deg;C for 3 min.</li>
 <li>Immediately dehydrate slides through an EtOH series (70%, 90% and 100%) in Coplin jars at 4 &deg;C for 3 min each.</li>
 <li>Allow the slides to air dry at room temperature.</li>
 <li>Pre-warm slides to 45 &deg;C on slide warmer for the last 10 min of the probe pre-hybridzation (step 5.1). Mix pre-hybridized probe cocktails if needed and add 25 ul to each slide; gently place a coverslip on each slide and seal along all edges with rubber cement. Incubate overnight at 37 &deg;C in a humidified chamber to prevent evaporation.</li>
</ol>
6. Post Hybridization Washes
<ol>
 <li>Wash slides in Coplin jars 3 times in 50% formamide/2x SSC, pH 7.0 at 38-40 &deg;C for 3 min each. The temperature of the washes may vary between probes. If there is high background hybridization you can increase the temperature of the washes. Conversely, if the signal is faint and there is no background then you can decrease the temperature of these washes.</li>
 <li>Wash slides 1 time in PN buffer at room temperature for 3 min, and proceed to the BrdU detection step.</li>
</ol>
7. BrdU Detection
<ol>
 <li>Block slides with PNM buffer for 10 min (200 &mu;l/slide) at room temperature in the dark.</li>
 <li>Drain slides by turning on edge on a paper towel. Add 100 &mu;l of anti-BrdU-FITC (50 &mu;g/ml Roche or Millipore) in PNM buffer for 30 min at 37 &deg;C.</li>
 <li>Wash slides 3 times in PN buffer at room temperature for 3 min each.</li>
 <li>Drain excess PN buffer on slides one at a time by turning on edge on a paper towel. Add 20 &mu;l DAPI/antifade mounting solution (Invitrogen). Cover the slide with a paper towel and press down on the coverslip to force out air bubbles and excess mounting solution. Proceed to image analysis.</li>
</ol>
8. Capturing Images and Quantifying BrdU Incorporation
<ol>
 <li>Images are captured with an Olympus BX61 Fluorescent Microscope and Olympus CCD Camera, 100x objective, automatic filter-wheel and Cytovision software (Applied Imaging). BrdU is captured using an FITC filter; chromosome paints (Texas Red, Metasystems or Cytocell), BAC's (Cy3) and CEP (Vysis Spectrum Orange) probes are captured using a Texas Red or Cy3 filter; DAPI is captured with a DAPI Filter (see Figures 2 and 3).</li>
 <li>Individual chromosomes of interest are identified with BAC/CEP or chromosome-specific paint probes (Figures 2B and 3B). Utilizing the Cytovision software, each chromosome of interest is "cut-out" from the metaphase spread as a whole, a line is drawn through the along the length of the chromosome from short arm to long arm (Figures 2C and 3C). The Cytovision software is used to quantify the pixel intensity profile along the length of each chromosome. The Cytovision software calculates the area occupied by the pixels and quantifies the intensity of the pixels represented by the BrdU or DAPI signals on each isolated chromosome. The average pixel intensity represented by each chromosome is then multiplied by the area occupied by those pixels to obtain the total number of pixels (Table 1 and Figure 4). In addition, this protocol allows for the visualization of the latest replicating regions of chromosomes. Accordingly, the banded pattern of BrdU incorporation allows for the detection of actively replicating regions of chromosomes, and differences in replication timing between chromosome pairs are seen as differences in this banding pattern (Figures 2 and 3). Finally, these differences in binding patterns can be compared to known replication timing maps for each chromosome6,7, which allows for an estimate of the replication timing difference between homologous chromosomes or chromosome arms4. For example, the two chromosome 6's in Figure 3A display a difference in banding pattern of &gt;2 hr when compared to the normal replication timing of chromosome 64. 
 In addition, a similar procedure to the one described here has been successfully used for the study of the asynchronous replication timing between the inactive and active X chromosomes8,9. The active and inactive X chromosomes were identified using X chromosome paints. Images were acquired using Quips mFISH software (Vysis) and the number of pixels occupied by the X chromosomes and the number of pixels occupied by fluorescently labeled BrdU were then calculated using NIH Image (<a href="http://rsb.info.nih.gov/nihimage" target="_blank">http://rsb.info.nih.gov/nih-image/index.html</a>).</li>
</ol>
9. Nick Translation of BAC DNA for Fluorescent Labeling (Vysis)
<ol>
 <li>Add the following probe mix to chilled a microfuge tube: 
 70 &mu;l (4 &mu;g DNA) in dH2O 
 10 &mu;l .2 mM dUTP-Orange or Green 
 20 &mu;l .1 mM dTTP 
 20 &mu;l 10x Nick Translation Buffer 
 40 &mu;l NT dNTP's (minus dTTP) 
 40 &mu;l nick Translation enzyme 
 200 &mu;l total volume 
 Incubate @ 16 &deg;C O/N. Stop reaction by heating at 70 &deg;C for 10 min. Chill on ice for 5 min.</li>
 <li>Precipitate the DNA with ethanol by adding the following to the probe mix: 
 200 &mu;l probe solution 
 48 &mu;l 3 M NaOAc 
 160 &mu;l Cot1DNA (.25 &mu;g/ul) 
 1,200 &mu;l 100% EtOH</li>
 <li>Store at -80 &deg;C for 10 min to O/N.</li>
 <li>Spin in cold at 12,000 x g for 20 min. Decant off EtOH. Wash 1 time with 70% EtOH. Spin as before. Decant off EtOH. Air dry just until EtOH evaporates. Resuspend in 40 &mu;l dH2O for final concentration of 100 ng/&mu;l of BAC DNA.</li>
</ol>

<ol>
	<li>Payer, B., Lee, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=X+chromosome+dosage+compensation:+how+mammals+keep+the+balance.">X chromosome dosage compensation: how mammals keep the balance.</a> Annu. Rev. Genet. 42, 733-772 (2008).</li><li>Breger, K. S., Smith, L., Turker, M. S., Thayer, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ionizing+radiation+induces+frequent+translocations+with+delayed+replication+and+condensation.">Ionizing radiation induces frequent translocations with delayed replication and condensation.</a> Cancer Research. 64, 8231-8238 (2004).</li><li>Smith, L., Plug, A., Thayer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delayed+Replication+Timing+Leads+to+Delayed+Mitotic+Chromosome+Condensation+and+Chromosomal+Instability+of+Chromosome+Translocations.">Delayed Replication Timing Leads to Delayed Mitotic Chromosome Condensation and Chromosomal Instability of Chromosome Translocations.</a> Proc. Natl. Acad. Sci. U.S.A. 98, 13300-13305 (2001).</li><li>Stoffregen, E. P., Donley, N., Stauffer, D., Smith, L., Thayer, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+autosomal+locus+that+controls+chromosome-wide+replication+timing+and+mono-allelicexpression.">An autosomal locus that controls chromosome-wide replication timing and mono-allelicexpression.</a> Hum. Mol. Genet. 20, 2366-2378 (2011).</li><li>Breger, K. S., Smith, L., Thayer, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+translocations+with+delayed+replication:+evidence+for+cis+control+of+chromosome+replication+timing.">Engineering translocations with delayed replication: evidence for cis control of chromosome replication timing.</a> Hum. Mol. Genet. 14, 2813-2827 (2005).</li><li>Camargo, M., Cervenka, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterns+of+DNA+replication+of+human+chromosomes.+II.+Replication+map+and+replication+model.">Patterns of DNA replication of human chromosomes. II. Replication map and replication model.</a> Am. J. Hum. Genet. 34, 757-780 (1982).</li><li>Cohen, S. M., Cobb, E. R., Cordeiro-Stone, M., Kaufman, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+chromosomal+bands+replicating+early+in+the+S+phase+of+normal+human+fibroblasts.">Identification of chromosomal bands replicating early in the S phase of normal human fibroblasts.</a> Exp. Cell Res. 245 (98), 321-329 (1998).</li><li>Diaz-Perez, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+element(s)+at+the+nontranscribed+Xist+locus+of+the+active+X+chromosome+controls+chromosomal+replication+timing+in+the+mouse.">The element(s) at the nontranscribed Xist locus of the active X chromosome controls chromosomal replication timing in the mouse.</a> Genetics. 171, 663-672 (2005).</li><li>Diaz-Perez, S. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+deletion+at+the+mouse+Xist+gene+exposes+trans-effects+that+alter+the+heterochromatin+of+the+inactive+X+chromosome+and+the+replication+time+and+DNA+stability+of+both+X+chromosomes.">A deletion at the mouse Xist gene exposes trans-effects that alter the heterochromatin of the inactive X chromosome and the replication time and DNA stability of both X chromosomes.</a> Genetics. 174, 1115-1133 (2006).</li><li>Salic, A., Mitchison, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+chemical+method+for+fast+and+sensitive+detection+of+DNA+synthesis+in+vivo.">A chemical method for fast and sensitive detection of DNA synthesis in vivo.</a> Proc. Natl. Acad. Sci. U.S.A. 105, 2415-2420 (2008).</li><li>Schlesinger, S., Selig, S., Bergman, Y., Cedar, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Allelic+inactivation+of+rDNA+loci.">Allelic inactivation of rDNA loci.</a> Genes Dev. 23, 2437-2447 (2009).</li><li>Branzei, D., Foiani, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+checkpoint+response+to+replication+stress.">The checkpoint response to replication stress.</a> DNA Repair (Amst). 8, 1038-1046 (2009).</li></ol>

No conflicts of interest declared.

The preparation of chromosome spreads is a critical step for the successful replication-timing assay described here. The inclusion of a colcemid pretreatment step prior to the hypotonic treatment may aid in the frequency and spreading of mitotic cells. We typically expose cells to colcemdi for 1-3 hr prior to harvest, and use colcemid at a final concentration of 10 ug/ml. However, the inclusion of a colcemid pretreatment step may alter the length of G2 and consequently may alter the apparent replication timing and state ...

<table border="1">
 <tbody>
 <tr>
 <td>Name of Reagent</td>
 <td>Company</td>
 <td>Catalogue Number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td>Anti-BrdU-FITC</td>
 <td>Roche Millipore</td>
 <td>11202593001 MAB326F</td>
 <td>50 &mu;g/&mu;l</td>
 </tr>
 <tr>
 <td>Nick Translation Kit</td>
 <td>Abbott Molecular (Vysis)</td>
 <td>07J00-0001</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Spectrum Orange dUTP</td>
 <td>Abbott Molecular (Vysis)</td>
 <td>02N33-050</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>CEP</td>
 <td>Abbott Molecular (Vysis)</td>
 <td>Varies</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>LSI/WCP hybridization buffer</td>
 <td>Abbott Molecular (Vysis)</td>
 <td>06J67-011</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>CEP hybridization buffer</td>
 <td>Abbott Molecular (Vysis)</td>
 <td>07J36-001</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Chromosome paints</td>
 <td>MetaSystems Group</td>
 <td>D-14NN-050-TR</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Olympus BX61 Fluorescent Microscope</td>
 <td>Olympus</td>
 <td>BX61TRF-1-5</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Microscope imaging software system</td>
 <td>Applied Imaging</td>
 <td>Cytovision 3.93.1</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Digital Camera</td>
 <td>Olympus</td>
 <td>UCMAD3</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>
 IN SITU HYBRIDIZATION RECIPES 
 Formamide Solutions 
 70% Formamide/2x SSC
 35 ml Formamide* (Sigma) 
 10 ml 10x SSC 
 5 ml d2H20 
 pH to 7.0 with HCl (Sigma)
 * It is important to use formamide that has been stored at -20 &deg;C. Prolonged room temperature storage will generate formic acid and the pH will be too low.
 50% Formamide/2x SSC
 25 ml formamide (Sigma) 
 10 ml 10x SSC 
 15 ml dH2O 
 pH to 7.0 with HCl (Sigma)
 20x SSC, 4 L
 702 g NaCl (Sigma) 
 358 g Na Citrate (Sigma) 
 dH2O to volume
 PN Buffer [0.1 M NaP04 0.1% NP_40 (Sigma)]
 Make a 0.1 M solution each of sodium phosphate (Filter sterilize and store in 500 ml aliquots).
 0.1 M NaH2P04 , 1 L
 13.8 g NaH2P04 (Sigma): 
 dH2O to volume
 0.1 M NaH2P04 1 L
 14.2 g NaH2P04 (Sigma) 
 dH2O to volume.
 PN: Adjust pH of 0.1 M Na2HP04 to pH 8.0 with .1 M NaH2P04. Filter sterilize and add 1 ml of NP-40.
 PNM 50 ml
 1.25 g Non-fat dry milk (Sigma) 
 25 ml PN buffer (Recipe above)
 Mix for 15-20 min with constant stirring. Spin 2 times at 400 x g for 10 min. Use supernatant, and make sure not to disturb precipitated milk proteins.</td>
 </tr>
 </tbody>
</table>

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.

An example of the replication timing analysis for human chromosome 6 is shown in Figure 2. Cells containing a deletion of the ASAR6 gene4, located at 6q16.1, were exposed to BrdU for 5 hr, harvested for mitotic cells and processed for FISH with a chromosome 6 paint probe (Vysis) and for BrdU incorporation. Note that there is a significant difference in the BrdU banding pattern between the two 6's, which is consistent with a delay in replication timing of &gt;2 hr for one of the chromosome 6's ...

Watch this Scientific Journal Video about Chromosome Replicating Timing Combined with Fluorescent In situ Hybridization at JoVE.com

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.

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 ...

chromosome-replicating-timing-combined-with-fluorescent-situ

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13.9K Views.  Oregon Health & Science University. 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.