Name: synthesis of wavelength-shifting dna hybridization probes by using photostable cyanine dyes
Uploaded: 2016-07-06T13:00:00
Description: Watch this Scientific Journal Video about Synthesis of Wavelength-shifting DNA Hybridization Probes by Using Photostable Cyanine Dyes at JoVE.com

Financial support by the Deutsche Forschungsgemeinschaft (DFG, Wa 1386/17-1), the Research Training Group GRK 2039 (funded by DFG) and KIT is gratefully acknowledged.

Caution: Please consult all relevant material safety data sheets (MSDS) before use. Several of the chemicals used in these syntheses are toxic and carcinogenic. Please use all appropriate safety practices that are typically required in organic chemistry laboratories, such as wearing a laboratory coat, safety glasses and gloves.
1. Synthesis of the Dyes
Note: Both dyes can be synthesized by the same types of reaction. Figure 2 shows an overview of thes...

<ol>
	<li>Kobayashi, H., Ogawa, M., Alford, R., Choyke, P. L., Urano, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+strategies+for+fluorescent+probe+design+in+medical+diagnostic+imaging.">New strategies for fluorescent probe design in medical diagnostic imaging.</a> Chem Rev. 110 (5), 2620-2640 (2010).</li><li>Berezin, M. Y., Achilefu, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+Lifetime+Measurements+and+Biological+Imaging.">Fluorescence Lifetime Measurements and Biological Imaging.</a> Chem. Rev. 110 (5), 2641-2684 (2010).</li><li>Lee, J. S., Vendrell, M., Chang, Y. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diversity-oriented+optical+imaging+probe+development.">Diversity-oriented optical imaging probe development.</a> Curr. Opin. Chem. Biol. 15 (6), 760-767 (2011).</li><li>Tyagi, S., Bratu, D. P., Kramer, F. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multicolor+molecular+beacons+for+allele+discrimination.">Multicolor molecular beacons for allele discrimination.</a> Nat. Biotechnol. 16 (1), 49-53 (1998).</li><li>Holzhauser, C., Wagenknecht, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=#34;DNA+Traffic+Lights&amp;#34;:+Concept+of+Wavelength-Shifting+DNA+Probes+and+Application+in+an+Aptasensor.">#34;DNA Traffic Lights&amp;#34;: Concept of Wavelength-Shifting DNA Probes and Application in an Aptasensor.</a> ChemBioChem. 13 (8), 1136-1138 (2012).</li><li>Holzhauser, C., Wagenknecht, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+and+RNA+&amp;#34;Traffic+Lights&amp;#34;:+Synthetic+Wavelength-Shifting+Fluorescent+Probes+Based+on+Nucleic+Acid+Base+Substitutes+for+Molecular+Imaging.">DNA and RNA &amp;#34;Traffic Lights&amp;#34;: Synthetic Wavelength-Shifting Fluorescent Probes Based on Nucleic Acid Base Substitutes for Molecular Imaging.</a> J. Org. Chem. 78 (15), 7373-7379 (2013).</li><li>Berndl, S., Wagenknecht, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+Color+Readout+of+DNA+Hybridization+with+Thiazole+Orange+as+an+Artificial+DNA+Base.">Fluorescent Color Readout of DNA Hybridization with Thiazole Orange as an Artificial DNA Base.</a> Angew. Chem. Int. Ed. 48 (13), 2418-2421 (2009).</li><li>Bohl&#228;nder, P. R., Wagenknecht, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+of+a+Photostable+Energy-Transfer+Pair+for+&amp;#34;DNA+Traffic+Lights&amp;#34.">Synthesis of a Photostable Energy-Transfer Pair for &amp;#34;DNA Traffic Lights&amp;#34.</a> Eur. J. Org. Chem. 34, 7547-7551 (2014).</li><li>Walter, H. K., Bohl&#228;nder, P. R., Wagenknecht, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+Wavelength-Shifting+Fluorescent+Module+for+the+Adenosine+Aptamer+Using+Photostable+Cyanine+Dyes.">Development of a Wavelength-Shifting Fluorescent Module for the Adenosine Aptamer Using Photostable Cyanine Dyes.</a> ChemistryOpen. 4 (2), 92-96 (2015).</li><li>Gierlich, J., Burley, G. A., Gramlicj, P. M. E., Hammond, D. M., Carell, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Click+chemistry+as+a+reliable+method+for+the+high-density+postsynthetic+functionalization+of+alkyne-modified+DNA.">Click chemistry as a reliable method for the high-density postsynthetic functionalization of alkyne-modified DNA.</a> Org. Lett. 8 (17), 3639-3642 (2006).</li><li>Matteucci, M. D., Caruthers, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+of+deoxyoligonucleotides+on+a+polymer+support.">Synthesis of deoxyoligonucleotides on a polymer support.</a> J. Am. Chem. Soc. 103 (11), 3185-3191 (1981).</li><li>Fasman, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Handbook of Biochemistry and Molecular Biology, Volume 1: Nucleic Acids. , 589 (1975).</li><li>Puglisi, J. D., Tinoco, J. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Absorbance+melting+curves+of+RNA.">Absorbance melting curves of RNA.</a> Meth. Enzymol. 180, 304-325 (1989).</li><li>Johansson, M. K., Fidder, H., Dick, D., Cook, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intramolecular+Dimers:+A+New+Strategy+to+Fluorescence+Quenching+in+Dual-Labeled+Oligonucleotide+Probes.">Intramolecular Dimers: A New Strategy to Fluorescence Quenching in Dual-Labeled Oligonucleotide Probes.</a> J. Am. Chem. Soc. 124, 6950-6956 (2002).</li><li>Barrois, S., W&#246;rner, S., Wagenknecht, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Role+of+Duplex+Stability+for+Wavelength-Shifting+Fluorescent+DNA+Probes:+Energy+Transfer+vs+Excition+Interactions+in+DNA+&amp;#34;Traffic+Lights&amp;#34;,+Photochem.">The Role of Duplex Stability for Wavelength-Shifting Fluorescent DNA Probes: Energy Transfer vs Excition Interactions in DNA &amp;#34;Traffic Lights&amp;#34;, Photochem.</a> Photobiol. Sci. 13, 1126-1129 (2014).</li></ol>

This protocol shows the complete procedure to label DNA post-synthetically via CuAAC by azide-modified fluorescent dyes. This includes the synthesis of the dyes and the alkyne-modified DNA as well as the labeling procedure.
The synthesis of the dyes follows four steps. All products can be obtained by a rather simple precipitation due to their positive charge and no time consuming column chromatography is needed. The introduction of the azide functionalities before the central coupling steps sh...

Molecular imaging represents a fundamental technique for understanding biological processes within living cells.1-3 The development of fluorescent nucleic acid based probes for such chemical-biological applications has become an expanding research field. These fluorescent probes need to meet a few requirements to become suitable tools for cell imaging. Firstly, the applied dyes should exhibit fluorescence with high quantum yields, large Stokes&#39; shifts and, most importantly, high photostabilities to allow long-term in vivo imaging. And secondly, they should show a reliable fluorescence readout. Conventional chromophore-quencher-systems are based on the readout of a single fluorescence color by simple changes in fluorescence intensities.4 This approach bears the risk of false positive or false negative results due to autofluorescence of intracellular components or low signal-to-noise ratios due to undesired quenching by other components.4
We recently reported on the concept of &#34;DNA traffic lights&#34; that show dual fluorescence color readouts by using two different chromophores.5-6 The concept is based on the energy transfer (ET) from the donor dye to the acceptor dye which changes the fluorescence color (see Figure 1). This allows a more reliable readout and thereby provides a powerful tool for fluorescent imaging probes. Labelling of oligonucleotides with fluorescent dyes can be achieved by two different approaches. Dyes can be incorporated during the chemical DNA synthesis on a solid phase by using correspondingly modified phosphoramidite building blocks.7 This method is limited to dyes that are stable under standard phosphoramidite and deprotection conditions. As an alternative, post-synthetic modification methodologies were established in oligonucleotide chemistry. Here, we demonstrate the synthesis of one of our new photostable energy transfer pairs8,9 and the post-synthetic labelling of DNA by using copper-catalyzed 1,3-cycloaddition between azides and alkynes (CuAAC).10

<table border="0" cellpadding="0" cellspacing="0" width="885">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:409px;">synthesis</td>
			<td style="width:180px;"></td>
			<td style="width:132px;"></td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">4-Picoline</td>
			<td>Sigma Aldrich</td>
			<td>239615</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1,3-Diiodopropane</td>
			<td>Sigma Aldrich</td>
			<td>238414</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Acetonitrile</td>
			<td>Fisher Scientific</td>
			<td>10660131</td>
			<td>HPLC grade</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ethyl acetate</td>
			<td>Fisher Scientific</td>
			<td>10456870</td>
			<td>technical grade</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium azide</td>
			<td>Sigma Aldrich</td>
			<td>71290</td>
			<td>p.a. grade</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dichloromethane</td>
			<td>Fisher Scientific</td>
			<td>10626642</td>
			<td>technical grade</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Indole-3-carboxaldehyde; 98%</td>
			<td>ABCR</td>
			<td>AB112969</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Potassium carbonate, 99+%</td>
			<td>Acros</td>
			<td>424081000</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">dimethylcarbonate</td>
			<td>Sigma Aldrich</td>
			<td>517127</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">N,N-Dimethylformamide, 99.8%, Extra Dry over Molecular Sieve</td>
			<td>Acros</td>
			<td>348435000</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium sulfate</td>
			<td>Bernd Kraft</td>
			<td>12623.46</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ethanol, 99.5%</td>
			<td>Acros</td>
			<td>397690010</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Piperidine, 99%</td>
			<td>Acros</td>
			<td>147181000</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Diethylether</td>
			<td>Fisher Scientific</td>
			<td>10407830</td>
			<td>technical grade</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">2-Phenylindole-3-carboxaldehyde; 97%</td>
			<td>ABCR</td>
			<td>AB125050</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">4-Methylquinoline</td>
			<td>ABCR</td>
			<td>AB117222</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DNA synthesis</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Expedite 8909 Nucleic Acid Synthesizer</td>
			<td>Applied Biosystems</td>
			<td>&#160;-</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DMT-dA(bz) Phosphoramidite</td>
			<td>Sigma Aldrich</td>
			<td>A111081</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DMT-dT Phosphoramidite</td>
			<td>Sigma Aldrich</td>
			<td>T111081</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DMT-dG(dmf) Phosphoramidite</td>
			<td>Sigma Aldrich</td>
			<td>G11508</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DMT-dC(bz) Phosphoramidite</td>
			<td>Sigma Aldrich</td>
			<td>C11108</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Amidite Diluent for DNA synthesis</td>
			<td>Sigma Aldrich</td>
			<td>L010010</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ultrapure Acetonitrile for DNA synthesis</td>
			<td>Sigma Aldrich</td>
			<td>L010400</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cap A</td>
			<td>Sigma Aldrich</td>
			<td>L840000</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cap B</td>
			<td>Sigma Aldrich</td>
			<td>L850000</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CPG dT Column 1.0 &#181;mole</td>
			<td>Proligo Reagents</td>
			<td>T461010</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CPG dA(bz) Column 1.0 &#181;mole</td>
			<td>Proligo Reagents</td>
			<td>A461010</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CPG dG(ib) Column 1.0 &#181;mole</td>
			<td>Proligo Reagents</td>
			<td>G461010</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CPG dC(bz) Column 1.0 &#181;mole</td>
			<td>Proligo Reagents</td>
			<td>C461010</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ammonia (aqueous solution)&#160;</td>
			<td>Fluka Analytical</td>
			<td>318612</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">centrifugal devices nanosep 0.45 &#181;m</td>
			<td>Pall</td>
			<td>ODGHPC34</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">5-(Benzylthio)-1H-tetrazole (Activator)</td>
			<td>Sigma Aldrich</td>
			<td>75666</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">2&#39;-O-propargyl deoxyuridinephosphoramidite</td>
			<td>Chem Genes</td>
			<td>ANP-7754</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">workup</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">vacuum concentrator</td>
			<td>Christ</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">clicking procedure</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tetrakis(acetonitrile)copper(I) hexafluorophosphate</td>
			<td>Sigma Aldrich</td>
			<td>346276</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium acetate</td>
			<td>Sigma Aldrich</td>
			<td>S2889</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">(+)-Sodium L-ascorbate</td>
			<td>Sigma Aldrich</td>
			<td>A7631</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">EDTA disodium salt</td>
			<td>Sigma Aldrich</td>
			<td>E5134</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TBTA-ligand</td>
			<td>&#160;-</td>
			<td>&#160;-</td>
			<td>synthesized according to a literature procedure [1]</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HPLC</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HPLC-system</td>
			<td>Shimadzu</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MALDI-Biflex-IV spectrometer</td>
			<td>Bruker Daltonics</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">LC-318 C18 column</td>
			<td>Supelcosil via Sigma Aldrich</td>
			<td>58368</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">determination of concentration</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ND 1000 Spectrophotometer</td>
			<td>nanodrop</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">sample preparation and spectroscopy</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cary 100 Bio</td>
			<td>Varian</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fluoromax-3 fluorimeter</td>
			<td>Jobin-Yvon</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:409px;">[1] R. Chan Timothy, R. Hilgraf, K. B. Sharpless, V. Fokin Valery, Org Lett 2004, 6, 2853-2855.</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

synthesis of wavelength-shifting dna hybridization probes by using photostable cyanine dyes

In this protocol, we demonstrate a method for the synthesis of 2&#39;-alkyne modified deoxyribonucleic acid (DNA) strands by automated solid phase synthesis using standard phosphoramidite chemistry. Oligonucleotides are post-synthetically labeled by two new photostable cyanine dyes using copper-catalyzed click-chemistry. The synthesis of both donor and acceptor dye is described and is performed in three consecutive steps. With the DNA as the surrounding architecture, these two dyes undergo an energy transfer when they are brought into close proximity by hybridization. Therefore, annealing of two single stranded DNA strands is visualized by a change of fluorescence color. This color change is characterized by fluorescence spectroscopy but can also be directly observed by using a handheld ultraviolet (UV) lamp. The concept of a dual fluorescence color readout makes these oligonucleotide probes excellent tools for molecular imaging especially when the described photostable dyes are used. Thereby, photobleaching of the imaging probes is prevented, and biological processes can be observed in real time for a longer time period.

Absorption and fluorescence spectra of the single and double stranded DNA are recorded as shown in Figure 4.
The recorded absorption spectra (Figure 4 right) show absorption maxima &#955;max at 465 nm for single-stranded DNA1 (dye 1) and 546 nm for single-stranded DNA2 (dye 2). The annealed DNA1_2 (dye 1 &#38; dye 2) shows maxima at both 469 nm and 567 nm. Both absorption maxima show ...

Watch this Scientific Journal Video about Synthesis of Wavelength-shifting DNA Hybridization Probes by Using Photostable Cyanine Dyes at JoVE.com

Synthesis of Wavelength-shifting DNA Hybridization Probes by Using Photostable Cyanine Dyes

Photostable cyanine dyes are attached to oligonucleotides to monitor hybridization by energy transfer.

In this protocol, we demonstrate a method for the synthesis of 2&#39;-alkyne modified deoxyribonucleic acid (DNA) strands by automated solid phase ...

The overall goal of this experiment is to visualize the energy transfer from one dye modified DNA strand to another. This method can help on the key questions in the field of molecular biology. Such as RNA interference by monitoring siRNA processing and integrity in living cells in real time.The main advantage of this technique, is the ease of monitoring the florescence reach out and the color change, giving a clear result. So false positive results are eliminated. The begin this procedure, add the appropriate reagents as indicated in the text protocol to a 20 milliliter, round bottom flask, equipped with a magnetic stir bar and reflux condenser.Then, add zero point zero six milliliters of piperidine. And heat the reaction mixture to 80 degrees celsius for four hours. After the reaction has cooled to room temperature, collect the resulting precipitate by filtration.And wash with diethyl ether three times. Next, add one hundred milliliters of diethyl ether to the supernatant. And collect the resulting precipitate by filtration.After washing three times with diethyl ether, combine the precipitate with isolated solid from the first filtration. Using the computer, connected to a DNA synthesizer, enter the desired DNA sequence and coupling method. Following the prompts from the manufacturer's protocol.Next, mount the column containing the controlled pour glass, that is modified with one micro mole of the first face into the synthesizer. Now, start the synthesis on the DNA synthesizer. Following workup of the DNA strands, add the appropriate regents as indicated in the text protocol to the diethylized alkyne, modified DNA sample.Incubate the sample at 60 degrees celsius for one point five hours. Then, transfer the sample to a 10 milliliter tube. After cooling to room temperature, add 150 microliters, of a zero point zero five molar EDTA disodium salt solution, and 450 microliters of a zero point three molar sodium acetate solution to the DNA sample.Following this, add 10 milliliters of ethanol to the sample. And keep it at minus 32 degrees celsius for 16 hours. Next, centrifuge the sample for 15 minutes at one thousand times G.When finished, remove the supernatant.Wash the resulting DNA pellet with two milliliters of cold 80%ethanol. Then dry the pellet under reduced pressure using a freeze drier. Purify the crude DNA pellet by HP LC.Monitor the run by checking 260 nanometers, and 459 nanometers for Dye one.Or 542 nanometers for Dye two. Collect the fractions that show absorption in both wave length channels. For single strand absorption spectroscopy measurements, record a blank measurement containing only 200 millimolar sodium chloride and 50 millimolar sodium phosphate buffer.Next, transfer the previously prepared DNA1 solution into a one centimeter, quartz glass cuvette. Record the absorption. Then determine the maximum value of dye absorption.For single strand fluorescent spectroscopy measurements, record a blank measurement containing only 200 millimolar sodium chloride and 50 millimolar sodium phosphate, using the excitation wave length of Dye one. Following this, place the cuvette with the DNA1 solution into the spectrometer. Record the florescence spectrum.To perform the double strand, fluorescent spectroscopy measurement, place the cuvette with the double strand of DNA1 and DNA2 solution into the spectrometer. Then record the florescent spectrum using the excitation wave length of Dye one. To gain a better understanding of what the recorded spectra are showing, irradiate the cuvettes with hand held UV lamps.Observe the change in florescence color from the single to the double stranded DNA. The recorded absorption spectra show the maxima at 465 nanometers for single stranded DNA1 and 546 nanometers for single stranded DNA2. The annealed double strand of DNA1 and DNA2 shows maxima at both 469 and 567 nanometers.Both absorption maxima show a bathaohromic shift. And the smallest spectroscopic changes are the result of excitonic interactions between the dyes within the double helical DNA architecture. The corresponding fluorescent spectra exhibit a maxima at 537 nanometers for single stranded DNA1.At 607 nanometers for single stranded DNA2. And at 615 nanometers for annealed, double strand of DNA1 and DNA2. Double stranded of DNA1 and DNA2, shows solely the emission maximum of Dye two, although Dye one is selectively excited, which evidences that the energy transfer occurs efficiently from Dye one to Dye two.After watching this video, you should have a good understanding of how to post synthetically modify DNA by a couple catalytic reaction of an alkyne labeled DNA strand with an A side.

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Alkynes

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Selective Labelling of Cell-surface Proteins using CyDye DIGE Fluor Minimal Dyes

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Detection of Viral RNA by Fluorescence in situ Hybridization (FISH)

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Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization (FISH)

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Robust 3D DNA FISH Using Directly Labeled Probes

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Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the ...