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 Monola...

<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>

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

Research

JoVE Journal

Biology

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.