Name: simple method for fluorescence dna in situ hybridization to squashed chromosomes
Uploaded: 2015-01-06T10:15:00
Description: Watch this Scientific Journal Video about Simple Method for Fluorescence DNA In Situ Hybridization to Squashed Chromosomes at JoVE.com

We thank Zhaohua Irene Tang in the W. M. Keck Science Department for the use of her epifluorescence microscope and the Werren lab for donating Nasonia for dissections. This work was supported in part by an NIH-NRSA fellowship (5F32GM105317-02) to AML.

1. Tissue Dissection and Fixation (60 min)
<ol>
	<li>For Drosophila brains, place 3rd instar larvae in a drop of 1x PBS (phosphate buffered saline). Choose large 3rd instar larvae that are actively crawling from vials or bottles that are not overcrowded.
	<ol>
		<li>Use one ultrafine tweezer pair to grab hold of the mouth hooks and another tweezer pair to grab 2/3 down the length of the body (Figure 1A, B). Pull gently on the mouth hooks to expose the brain, ventral ganglia, saliv...

<ol>
	<li>Blattes, R., Kas, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+in+situ+hybridization+(FISH)+on+diploid+nuclei+and+mitotic+chromosomes+from+Drosophila+melanogaster+larval+tissues.">Fluorescent in situ hybridization (FISH) on diploid nuclei and mitotic chromosomes from Drosophila melanogaster larval tissues.</a> Cold Spring Harbor Protocols. 2009 (9), (2009).</li><li>Dimitri, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+in+situ+hybridization+with+transposable+element+probes+to+mitotic+chromosomal+heterochromatin+of+Drosophila.">Fluorescent in situ hybridization with transposable element probes to mitotic chromosomal heterochromatin of Drosophila.</a> Methods in Molecular Biology. 260, 29-39 (2004).</li><li>Pardue, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+hybridization+to+polytene+chromosomes+in+Drosophila+using+digoxigenin-labeled+probes.">In situ hybridization to polytene chromosomes in Drosophila using digoxigenin-labeled probes.</a> Cold Spring Harbor Protocols. 2011 (8), 1003-1006 (2011).</li><li>Hoskins, R. A., 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=Heterochromatic+sequences+in+a+Drosophila+whole-genome+shotgun+assembly.">Heterochromatic sequences in a Drosophila whole-genome shotgun assembly.</a> Genome Biology. 3 (12), (2002).</li><li>Hoskins, R. A., 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=Sequence+finishing+and+mapping+of+Drosophila+melanogaster+heterochromatin.">Sequence finishing and mapping of Drosophila melanogaster heterochromatin.</a> Science. 316 (58331), 1625-1628 (2007).</li><li>Treangen, T. J., Salzberg, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Repetitive+DNA+and+next-generation+sequencing:+computational+challenges+and+solutions.">Repetitive DNA and next-generation sequencing: computational challenges and solutions.</a> Nature Reviews Genetics. 13 (1), 36-46 (2012).</li><li>He, B., 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=Mapping+the+pericentric+heterochromatin+by+comparative+genomic+hybridization+analysis+and+chromosome+deletions+in+Drosophila+melanogaster.">Mapping the pericentric heterochromatin by comparative genomic hybridization analysis and chromosome deletions in Drosophila melanogaster.</a> Genome Research. 22 (12), 2507-2519 (2012).</li><li>Pimpinelli, S., Bonaccorsi, S., Fanti, L., Gatti, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+in+situ+hybridization+(FISH)+of+mitotic+chromosomes+from+Drosophila+larval+brain.">Fluorescent in situ hybridization (FISH) of mitotic chromosomes from Drosophila larval brain.</a> Cold Spring Harbor Protocols. 2010 (3), (2010).</li><li>Larracuente, A. M., Noor, M. A., Clark, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translocation+of+Y-linked+genes+to+the+dot+chromosome+in+Drosophila+pseudoobscura.">Translocation of Y-linked genes to the dot chromosome in Drosophila pseudoobscura.</a> Molecular Biology and Evolution. 27 (7), 1612-1620 (2010).</li><li>Ferree, P. M., Barbash, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Species-specific+heterochromatin+prevents+mitotic+chromosome+segregation+to+cause+hybrid+lethality+in+Drosophila.">Species-specific heterochromatin prevents mitotic chromosome segregation to cause hybrid lethality in Drosophila.</a> PLoS Biology. 7 (10), e1000234 (2009).</li><li>Williams, B. C., Karr, T. L., Montgomery, J. M., Goldberg, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Drosophila+l(1)zw10+gene+product,+required+for+accurate+mitotic+chromosome+segregation,+is+redistributed+at+anaphase+onset.">The Drosophila l(1)zw10 gene product, required for accurate mitotic chromosome segregation, is redistributed at anaphase onset.</a> The Journal of Cell Biology. 118 (4), 759-773 (1992).</li><li>Gatti, M., Bonaccorsi, S., Pimpinelli, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Looking+at+Drosophila+Mitotic+Chromosomes.">Looking at Drosophila Mitotic Chromosomes.</a> Methods in Cell Biology. 44, 371-391 (1994).</li><li>Werren, J. H., Stouthamer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PSR+(paternal+sex+ratio)+chromosomes:+the+ultimate+selfish+genetic+elements.">PSR (paternal sex ratio) chromosomes: the ultimate selfish genetic elements.</a> Genetica. 117 (1), 85-101 (2003).</li></ol>

DNA ISH is frequently used to map specific sequences to chromosomes. We have described a simple method for DNA ISH optimized for high copy number, heterochromatic sequences. Rather than using washes in a formamide solution, which is a requirement in other existing DNA ISH protocols, we place tissue-mounted slides directly on a pre-heated block to denature DNA. This method circumvents the use of large amounts of formamide. One critical step for producing crisp hybridization signals is to use freshly made fixation solution...

DNA in situ hybridization (DNA ISH) is a commonly used method for mapping sequences to specific chromosome regions. Probes to single copy regions within euchromatin can be generated through a handful of approaches, including nick-translation or end-labeling of long DNA products1,2 and the incorporation of deoxygenin (DIG)-attached nucleotides and their recognition through a wide variety of group-conjugated antibodies1-3. Visualization of euchromatic sequences in few or single copy number requires the use of either single, large probes with high specific activity or a cocktail of multiple, smaller probes that collectively enhance signal.
In contrast, highly repetitive sequences found in heterochromatin, such as satellite DNAs, are easier targets for DNA ISH because they normally exist as tens to thousands of repeats clustered in single chromosome regions known as blocks. Transposable elements also can be found at high copy numbers at distinct chromosomal loci2. In these cases, single probes with low specific activity can effectively label heterochromatic sequences due to their hybridization at multiple sites. Probes to repetitive sequences can be synthesized commercially as short oligonucleotides (30-50 bp) and chemically conjugated with any of multiple different fluorescent groups. Mapping repetitive sequences within heterochromatin by using genome-sequencing technologies is difficult due to challenges encountered in building scaffolds within highly repetitive satellite blocks4-6,7. Currently, ISH stands as the most effective way of mapping these sequences at the sub-chromosome level. This strategy is important for mapping large numbers of repetitive sequences that are being uncovered by ongoing genome and transcriptome sequencing studies.
The efficiency and ease of mapping repetitive sequences on slide-mounted chromosomes would be greatly enhanced by a simplified protocol for DNA ISH. For example, existing protocols for DNA ISH involve multiple washes of hybridized tissues in formamide solution2,8, thus adding substantially to the required time for mapping sequences and also producing large amounts of chemical waste for this costly reagent. Here we describe a revised DNA ISH method that circumvents the need for formamide washes and utilizes basic laboratory equipment and reagents. This method was originally designed for the rapid mapping of highly repetitive DNA sequences in heterochromatic regions of Drosophila larval neuroblasts by using commercially synthesized oligos that are conjugated with fluorescence dyes. However, this method also works for mapping repetitive sequences by using larger probes synthesized through other means9,10 and across multiple different tissue and chromosome types. Additionally, this method can be used to map euchromatic sequences by using longer or multiple, short probes within the euchromatic sequence of interest.

<table border="0" cellpadding="0" cellspacing="0" width="542">
	<tbody>
		<tr height="17">
			<td height="17" style="height:17px;width:285px;"></td>
			<td style="width:257px;">Company</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Materials&#160;</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;"></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Poly-L-lysine coated slides (regular slides also can be used)</td>
			<td>Sigma Aldrich</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Ultrafine tweezers (5 gauge)</td>
			<td>Dumont</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">22mmx22mm cover slips, Sigmacote-treated by immersion for 15 seconds, blotting dry, and wiping away all traces of Sigmacote so that cover slip is clear</td>
			<td>Fisher</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Sigmacote</td>
			<td>Sigma</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Filter paper (75-150mm)</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Paraffin wax paper</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Heat block with thermometer</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Dry incubator</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Razor blades</td>
			<td></td>
		</tr>
		<tr height="17">
			<td colspan="2" height="17" style="height:17px;">Humidity chamber (empty pipette tip box or Tupperware, lined with moistened paper towels or Kimwipes)</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Coplin jars (with slide grooves)</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Aluminum foil</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Pasteur pipettes</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">1.5 mL microfuge tubes</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Nail polish (clear or colored)</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">2-20 &#956;L micropipette and plastic tips</td>
			<td></td>
		</tr>
		<tr height="17">
			<td colspan="2" height="17" style="height:17px;">20-25 standard metal paperclips linked to form a chain</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;"></td>
			<td>Company/Notes</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Reagents</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;"></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">16% EM grade paraformaldehyde</td>
			<td>Electron Microscopy Reagents</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Acetic acid</td>
			<td>Sigma</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Liquid nitrogen</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;"></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">100% Ethanol, chemical grade</td>
			<td></td>
		</tr>
		<tr height="17">
			<td colspan="2" height="17" style="height:17px;">Commercially synthesized, fluorescently labeled oligos</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Long biotinylated probe (e.g. nick translated and biotinylated with BioNick from Invitrogen)</td>
			<td>Invitrogen; Alternative steps 2.7.1-2.7.3</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Rhodamine-Avidin (for detection of long biotinylated probe)</td>
			<td>Roche; Alternative steps 2.7.1-2.7.3</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Hybridization buffer</td>
			<td>Recipe below</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">4X SSCT (Saline-sodium citrate + Tween)</td>
			<td>Recipe below</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">0.1X SSC (Saline-sodium citrate)</td>
			<td>Recipe below</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Blocking solution</td>
			<td>Recipe below</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">SBT (SSC, Bovine serum albumin, Tween)</td>
			<td>Recipe below</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">1X PBT (Phosphate-buffered saline + Tween)</td>
			<td>Recipe below</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">1X PBS (Phosphate-buffered saline)</td>
			<td></td>
		</tr>
		<tr height="17">
			<td colspan="2" height="17" style="height:17px;">Hypotonic solution (0.5% sodium citrate in H2O)</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Formamide</td>
			<td>Sigma Aldrich</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Vectashield mounting medium with DAPI</td>
			<td>Vector laboratories</td>
		</tr>
	</tbody>
</table>

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.

To demonstrate this method, we hybridized a set of small commercially synthesized oligos that were chemically modified with fluorescent conjugates (Figure 2) and a longer biotinylated probe (made through nick translation of a PCR product; Figure 2B), to chromosomes from several different tissue types (see Table 1). The target sequences included satellite repeats located in pericentromeric (heterochromatic) regions of mitotic chromosomes from D. melanogaster larv...

Watch this Scientific Journal Video about Simple Method for Fluorescence DNA In Situ Hybridization to Squashed Chromosomes at JoVE.com

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.

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 ...

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