Name: visualization of surface-tethered large dna molecules with a fluorescent protein dna binding peptide
Uploaded: 2016-06-23T15:00:00
Description: Watch this Scientific Journal Video about Visualization of Surface-tethered Large DNA Molecules with a Fluorescent Protein DNA Binding Peptide at JoVE.com

This work was supported by the Sogang University Research Grant of 201410036.

1. DNA Biotinylation <ol><li> Biotinylation av stump ended DNA ved hjelp av Terminal transferase (TdT) Merk: Bruk T4 DNA (166 kbp), som er en stump endet DNA. <ol><li> Tilsett 5 ul av 2,5 mM CoCl 2, 5 ul av 10 x reaksjonsbuffer, 0,5 ul 10 mM biotin-11-dUTP, 0,5 mL av terminal transferase (10 enheter), og 0,5 pl T4 DNA (0,5 ug / mL) til reaksjonsblandingen. Gjør til et endelig volum på 50 ul ved tilsetning av 38,5 ul vann. </li><li> Inkuber reaksjonsblandingen ved 37 ° C i 1 time. ...

<ol>
	<li>Smith, S. B., Finzi, L., Bustamante, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+Mechanical+Measurements+of+the+Elasticity+of+Single+DNA-Molecules+by+Using+Magnetic+Beads.">Direct Mechanical Measurements of the Elasticity of Single DNA-Molecules by Using Magnetic Beads.</a> Science. 258 (5085), 1122-1126 (1992).</li><li>Finkelstein, I. J., Visnapuu, M. -. L., Greene, E. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-molecule+imaging+reveals+mechanisms+of+protein+disruption+by+a+DNA+translocase.">Single-molecule imaging reveals mechanisms of protein disruption by a DNA translocase.</a> Nature. 468 (7326), 983-987 (2010).</li><li>Doyle, P. S., Ladoux, B., Viovy, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+a+tethered+polymer+in+shear+flow.">Dynamics of a tethered polymer in shear flow.</a> Phys. Rev. Lett. 84 (20), 4769-4772 (2000).</li><li>Perkins, T. T., Quake, S. R., Smith, D. E., Chu, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relaxation+of+a+Single+DNA+Molecule+Observed+by+Optical+Microscopy.">Relaxation of a Single DNA Molecule Observed by Optical Microscopy.</a> Science. 264 (5160), 822-826 (1994).</li><li>Cai, W., 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=Ordered+restriction+endonuclease+maps+of+yeast+artificial+chromosomes+created+by+optical+mapping+on+surfaces.">Ordered restriction endonuclease maps of yeast artificial chromosomes created by optical mapping on surfaces.</a> Proc. Natl. Acad. Sci. U.S.A. 92 (11), 5164-5168 (1995).</li><li>Kim, Y., Jo, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutravidin+coated+surfaces+for+single+DNA+molecule+analysis.">Neutravidin coated surfaces for single DNA molecule analysis.</a> Chem. Comm. 47 (22), 6248-6250 (2011).</li><li>Kim, Y., 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=Nanochannel+confinement:+DNA+stretch+approaching+full+contour+length.">Nanochannel confinement: DNA stretch approaching full contour length.</a> Lab on a Chip. 11 (10), 1721-1729 (2011).</li><li>Glazer, A. N., Rye, H. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable+dye-DNA+intercalation+complexes+as+reagents+for+high-sensitivity+fluorescence+detection.">Stable dye-DNA intercalation complexes as reagents for high-sensitivity fluorescence detection.</a> Nature. 359 (6398), 859-861 (1992).</li><li>Graneli, A., Yeykal, C. C., Prasad, T. K., Greene, E. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organized+arrays+of+individual+DNA+molecules+tethered+to+supported+lipid+bilayers.">Organized arrays of individual DNA molecules tethered to supported lipid bilayers.</a> Langmuir. 22 (1), 292-299 (2006).</li><li>Lee, 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=DNA+binding+fluorescent+proteins+for+the+direct+visualization+of+large+DNA+molecules.">DNA binding fluorescent proteins for the direct visualization of large DNA molecules.</a> Nucleic Acids Res. , (2015).</li><li>Roy, R., Hohng, S., Ha, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+practical+guide+to+single-molecule+FRET.">A practical guide to single-molecule FRET.</a> Nat Methods. 5 (6), 507-516 (2008).</li><li>Chandradoss, S. D., 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=Surface+passivation+for+single-molecule+protein+studies.">Surface passivation for single-molecule protein studies.</a> J. Vis. Exp. (86), e50549 (2014).</li></ol>

Her presenterer vi en plattform for å visualisere lange DNA-molekyler biotinylert for forankring på overflater. Vi har rapportert en tilnærming for DNA-molekyler tjoret på en avidin protein belagt overflate med biotinylert bovin serum albumin. 6 I tidligere tilnærmingen, har vi funnet en avgjørende spørsmålet om DNA foto spalting forårsaket av bis-interkaleringsforbindelser fargestoffer som flekker DNA-molekyler bundet på flate. Ettersom disse stadig eksiterte fluoroforer har en høy sannsynlighet f...

Visualisering av store DNA-molekyler tjoret på glass eller perle overflater har blitt brukt for å undersøke DNA-protein interaksjoner, protein dynamikk på DNA-substrat, 1,2 og polymer fysikk. 3,4 A-plattformen for single-forankrede store DNA-molekyler har noen distinkt fordelene i forhold til andre DNA-immobilisering metoder. 5 Først, et stort DNA-molekyl bundet på overflaten har en naturlig tilfeldig kveil-konformasjon uten en skjærstrømning, noe som er kritisk viktig for en DNA-bindende protein for å gjenkjenne dets bindingssete. For det andre er det meget enkelt å endre den kjemiske miljøet rundt DNA-molekyler for en serie av enzymatiske reaksjoner i et strømningskammer. For det tredje, induserer en mikrofluidskjærstrømnings DNA-molekyl som strekker seg opp til 100% av full lengde kontur, noe som er svært vanskelig å oppnå ved bruk av alternative DNA forlengelse tilnærminger, for eksempel overflate immobilisering 6 og nanochannel innesperring. 7 Et fullt stretched DNA-molekylet gir også posisjons informasjon som kan være nyttig for å overvåke enzymatiske bevegelser på genomisk kartet. Ikke desto mindre, har DNA-tilknytning tilnærmingen en kritisk mangel ved at interkalerende fargestoff slik som YOYO-en generelt fører til binding av DNA-molekyler for å være lett brytes av fluorescenseksitasjon lys. Vanligvis store DNA-molekyler som måtte være farget med et fluorescerende fargestoff for visualisering under et fluorescerende mikroskop. For dette formålet, YOYO-en eller andre TOTO serie fargestoffer er først og fremst brukt fordi disse fargestoffer fluor bare når de intercalate dobbelttrådet DNA. 8 Men det er velkjent at bis-innføyings fargestoff fører lett-indusert DNA foto-spalting fordi av intercalation av fluoroforer. 9 videre fluorescens farget DNA-molekyler tjoret på overflaten er mer skjøre siden skjærstrømninger kan utøve bryte krefter på fritt bevegelige DNA-molekyler. Derfor utviklet vi FP-DBP som en roman DNA-farging protein fargestoff for avbilding av store DNA molekyler tjoret på overflaten. Fordelen med å bruke FP-DBP er at det ikke forårsaker foto-spalting av DNA-molekylene som det binder seg til. 10 I tillegg, FP-DBP øker ikke konturen lengden av DNA, mens bis-interkalerende fargestoff øke konturen lengde med omtrent 33%. Denne videoen metoden introduserer eksperimentell tilnærming for tethering store DNA-molekyler til en PEG-biotin overflaten. Figur 1 viser ulike tilnærminger til tethering DNA med butte ender og klebrige ender. Således kan denne fargemetoden anvendes på hvilken som helst type DNA-molekylet. Figur 2 viser en skjematisk representasjon av strømningskammermontasjen som kan styres ved hjelp av en sprøytepumpe for å generere skjærstrømninger for å strekke DNA-molekyler, så vel som å laste kjemisk og enzym løsninger. Figur 3 viser mikrografer av helt strukket DNA-molekyler tjoret på PEGylated overflaten 11 og farget med FP-DBP.

<table border="0" cellpadding="0" cellspacing="0" width="925">
	<tbody>
		<tr height="21">
			<td colspan="4" height="21" style="height:21px;width:925px;">1. DNA Biotinylation</td>
		</tr>
		<tr height="21">
			<td colspan="4" height="21" style="height:21px;width:925px;">1.1) Biotinylation of blunt-end DNA using Terminal Transferase (TdT)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Terminal Transferase</td>
			<td style="width:167px;">New England Biolabs</td>
			<td style="width:133px;">M0315S</td>
			<td>Provided with 10x reaction buffer, 2.5 mM Cobalt chloride</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Biotin-11-dUTP</td>
			<td style="width:167px;">Invitrogen</td>
			<td style="width:133px;">R0081</td>
			<td>Biotin-ddNTP is also available</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">T4GT7 Phage DNA</td>
			<td style="width:167px;">Nippon Gene</td>
			<td style="width:133px;">318-03971</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Ethylenediaminetetraacetic acid</td>
			<td style="width:167px;">Sigma-Aldrich</td>
			<td style="width:133px;">E6758</td>
			<td>EDTA</td>
		</tr>
		<tr height="21">
			<td colspan="4" height="21" style="height:21px;width:925px;">1.2) Biotinylation of sticky end DNA Using DNA Ligase</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">T4 DNA Ligase</td>
			<td style="width:167px;">New England Biolabs</td>
			<td style="width:133px;">M0202S</td>
			<td>Provided with 10x reaction buffer</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:287px;">Lambda Phage DNA</td>
			<td style="width:167px;">Bioneer</td>
			<td style="width:133px;">D-2510</td>
			<td>Also available at New England Biolabs</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:925px;">2. Functionalized Surface Derivatization</td>
		</tr>
		<tr height="21">
			<td colspan="4" height="21" style="height:21px;width:925px;">2.1) Piranha Cleaning</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Coverslip</td>
			<td style="width:167px;">Marienfeld-Superior</td>
			<td style="width:133px;">0101050</td>
			<td>22x22 mm, No. 1 Thickness</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Teflon rack</td>
			<td style="width:167px;"></td>
			<td style="width:133px;"></td>
			<td>Custom Fabrication</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:287px;">PTFE Thread Seal Tape</td>
			<td style="width:167px;">Han Yang Chemical Co. Ltd.</td>
			<td style="width:133px;">3032292</td>
			<td>Teflon&#8482; tape</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Sulferic acid</td>
			<td style="width:167px;">Jin Chemical Co. Ltd.</td>
			<td style="width:133px;">S280823</td>
			<td>H2SO4, 95 % Purity</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Hydrogen peroxide</td>
			<td style="width:167px;">Jin Chemical Co. Ltd.</td>
			<td style="width:133px;">H290423</td>
			<td>H2O2, 35 % in water</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:287px;">Sonicator</td>
			<td style="width:167px;">Daihan Scientific Co. Ltd.</td>
			<td style="width:133px;">WUC-A02H</td>
			<td>Table-top Ultrasonic Cleaner</td>
		</tr>
		<tr height="21">
			<td colspan="4" height="21" style="height:21px;width:925px;">2.2) Aminosilanization on Glass Surface</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:287px;">N-[3-(Trimethoxysilyl)propyl] 
			ethylenediamine</td>
			<td style="width:167px;">Sigma-Aldrich</td>
			<td style="width:133px;">104884</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Glacial Acetic Acid</td>
			<td style="width:167px;">Duksan Chemicals</td>
			<td style="width:133px;">414</td>
			<td>99 % Purity</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Methyl Alcohol</td>
			<td style="width:167px;">Jin Chemical Co. Ltd.</td>
			<td style="width:133px;">M300318</td>
			<td>99.9 % Purity</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Polypropylene Container</td>
			<td style="width:167px;">Qorpak</td>
			<td style="width:133px;">PLC-04907</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Ethyl Alcohol</td>
			<td style="width:167px;">Jin Chemical Co. Ltd.</td>
			<td style="width:133px;">A300202</td>
			<td>99.9 % Purity</td>
		</tr>
		<tr height="21">
			<td colspan="4" height="21" style="height:21px;width:925px;">2.3) PEGylation of the coverslip</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Sodium Bicarbonate</td>
			<td style="width:167px;">Sigma-Aldrich</td>
			<td style="width:133px;">S5761</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Syringe Filter</td>
			<td style="width:167px;">Sartorius</td>
			<td style="width:133px;">16534----------K</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:287px;">Biotin-PEG-SC</td>
			<td style="width:167px;">Laysan Bio</td>
			<td style="width:133px;">Biotin-PEG-SC-5000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">mPEG-SVG</td>
			<td style="width:167px;">Laysan Bio</td>
			<td style="width:133px;">MPEG-SVA-5000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Acetone</td>
			<td style="width:167px;">Jin Chemical Co. Ltd.</td>
			<td style="width:133px;">A300129</td>
			<td>99 % Purity</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Microscope Slides</td>
			<td style="width:167px;">Marienfeld-Superior</td>
			<td style="width:133px;">1000612</td>
			<td>~76x26x1 mm</td>
		</tr>
		<tr height="21">
			<td colspan="4" height="21" style="height:21px;width:925px;">3. Assembling a Flow Chamber</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Acrylic Support</td>
			<td style="width:167px;"></td>
			<td style="width:133px;"></td>
			<td>Custom Fabrication</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Double-sided Tape</td>
			<td style="width:167px;">3M</td>
			<td style="width:133px;"></td>
			<td>Transparent type</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Quick-dry Epoxy</td>
			<td style="width:167px;">3M</td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:287px;">Polyethylene Tubing</td>
			<td style="width:167px;">Cole-Parmer</td>
			<td style="width:133px;">06417-11, 06417-21</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Gas Tight 250 &#181;l Syringe</td>
			<td style="width:167px;">Hamilton</td>
			<td style="width:133px;">81165</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:287px;">Syringe Pump</td>
			<td style="width:167px;">New Era Pump Systems Inc.</td>
			<td style="width:133px;">NE-1000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td colspan="4" height="21" style="height:21px;width:925px;">4. Sample Loading into Flow Chamber</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Neutravidin</td>
			<td style="width:167px;">Thermo Scientific</td>
			<td style="width:133px;">31000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Tris base</td>
			<td style="width:167px;">Sigma-Aldrich</td>
			<td style="width:133px;">T1503-5KG</td>
			<td>Trizma base</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Microscope</td>
			<td style="width:167px;">Olympus</td>
			<td style="width:133px;">IX70</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">EMCCD Camera</td>
			<td style="width:167px;">Q Imaging</td>
			<td style="width:133px;">Rolera EM-C2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Solid-state Laser (488 nm)</td>
			<td style="width:167px;">Oxxius</td>
			<td style="width:133px;">LBX488</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Alconox</td>
			<td style="width:167px;">Alconox Inc.</td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

visualization of surface-tethered large dna molecules with a fluorescent protein dna binding peptide

Large DNA molecules tethered on the functionalized glass surface have been utilized in polymer physics and biochemistry particularly for investigating interactions between DNA and its binding proteins. Here, we report a method that uses fluorescent microscopy for visualizing large DNA molecules tethered on the surface. First, glass coverslips are biotinylated and passivated by coating with biotinylated polyethylene glycol, which specifically binds biotinylated DNA via avidin protein linkers and significantly reduces undesirable binding from non-specific interactions of proteins or DNA molecules on the surface. Second, the DNA molecules are biotinylated by two different methods depending on their terminals. The blunt ended DNA is tagged with biotinylated dUTP at its 3' hydroxyl terminus, by terminal transferase, while the sticky ended DNA is hybridized with biotinylated complimentary oligonucleotides by DNA ligase. Finally, a microfluidic shear flow makes single DNA molecules stretch to their full contour lengths after being stained with fluorescent protein-DNA binding peptide (FP-DBP).

Figur 1 viser to forskjellige DNA fortøyningsmetoder avhengig av terminale strukturer i DNA-molekylet. Figur 1a viser hvordan de klebrige endede DNA-molekyler blir hybridisert med komplementære biotinylerte oligonukleotider, som er immobilisert på avidin-belagte PEG overflate. Figur 1b viser tilsetning av biotinylert ddNTP eller dNTP til 3&#39;-hydroksylgruppen i en stump endet DNA ved hjelp av terminal transferase. Vi har lagt fleksi...

Watch this Scientific Journal Video about Visualization of Surface-tethered Large DNA Molecules with a Fluorescent Protein DNA Binding Peptide at JoVE.com

Visualization of Surface-tethered Large DNA Molecules with a Fluorescent Protein DNA Binding Peptide

We present an approach for visualizing fluorescent protein DNA binding peptide (FP-DBP)-stained large DNA molecules tethered on the polyethylene glycol (PEG) and avidin-coated glass surface and stretched with microfluidic shear flows.

Visualisering av Surface-tjoret Large DNA-molekyler med et fluorescerende protein DNA Binding Peptide

Large DNA molecules tethered on the functionalized glass surface have been utilized in polymer physics and biochemistry particularly for investigating ...

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Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

Grønn Fluorescent Protein-baserte Expression Screening av membran proteiner i<em&gt; Escherichia coli</em

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...

Visualization of miniSOG Tagged DNA Repair Proteins in Combination with Electron Spectroscopic Imaging (ESI)

Visualization of miniSOG Tagged DNA Repair Proteins in Combination with Electron Spectroscopic Imaging ESI

Visualisering av miniSOG Tagged DNA Repair Proteiner i kombinasjon med Electron Spektroskopiske Imaging (ESI)

The limits to optical resolution and the challenge of identifying specific protein populations in transmission electron microscopy have been obstacles in ...

Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo

Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo

Chromatin Immunutfelling analyse for identifikasjon av Arabidopsis Protein-DNA interaksjoner<em&gt; I Vivo</em

Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and ...