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

1. DNAビオチン化<ol><li> ターミナルトランスフェラーゼを使用して平滑末端DNAのビオチン化酵素（TdT） 注：平滑末端DNAで使用T4 DNA（166 kbpの）を、。 <ol><li> 2.5mmののCoCl 2、10×反応緩衝液5μl、10mMのビオチン-11-dUTPを0.5μlの、ターミナルトランスフェラーゼの0.5μlの（10単位）、およびT4 DNA0.5μlの（0.5μgの/μl）を5μLを追加反応混合物。水38.5μLを添加することによって50μlの最終容積にしてください。 </li><li> 1時間37℃で反応混合物をインキュベートします。 注：延長反応時間、 すなわち 、二重係留DNAを得ることができ、ビオチンが両端にタグ付けされていることが可能です。 </li><li> 0.5 M EDTA、pHが8の5μLを加えて反応を停止します。 </li><li> 4℃でチューブを保管してください。 </li></ol></li><li> DNAリガーゼを用いて粘着性のエンドDNAのビオチン化 注意：使用λDNA（48.5 kbpの）、粘着末端のDNAです。 &lt;オール&gt; <li> T4 DNAリガーゼを1μl、10×ライゲーションバッファー5μlの、25 ngの/μlのλファージDNAを1μlを追加し、50μlの最終容量を作るために水の43μlを添加します。 </li><li> 4℃でチューブを保管してください。 </li></ol></li></ol> 2.官能の表面誘導体化注： 図1に示すように、ガラス表面上のDNA分子の末端をつなぐためには、第1級アミン基をビオチンPEGコーティングに続いてカバースリップ上にシラン化され、このPEG化プロセスがあるため、単一分子DNAの画像化のために重要です。著しく表面上の望ましくない分子の結合によって作成されたランダムノイズを低減することができます。 <ol><li> ピラニアクリーニング 注：ピラニア・ソリューションは、したがって、それは適切な安全ガイドラインに従って、取り扱いに注意する必要があり、有機物質と激しく反応します。 <ol><li>場所は、ポリテトラフルオロエチレン（PTFE）ラックにカバースリップ、およびbはそれらを保持しますyのPTFE長手方向シールテープ、ラックの表面のエッジと半分に半分。ラッピングした後、洗浄手順の間にラックを処理するためのテープの長い一片（〜5センチ）のままにします。 </li><li> SO H 2 O 2の4および150mlをヒュームフード内でピラニア溶液を作製するためにH 2 350 mlを1 Lのガラスビーカーを埋めます。 2時間ピラニア溶液中でラックを配置します。 </li><li>ビーカー内の水のpHになるまで脱イオン水で完全に空のビーカーからのピラニア溶液およびリンスカバースリップを中性に達します。 pH試験紙ストリップを使用してください。 </li><li> 30分間、水を含有するビーカーにカバースリップのラックを超音波処理。ビーカーからわずかアミノシラン化の前に空の水。ガラス基板をきれいにし、誘導体化するために超音波処理電力の75 Wを使用してください。 </li></ol></li><li> ガラス表面にAminosilanization <ol><li> N 2 mlを加え- [3-（トリメトキシシリル）プロピル]エチレンジアミン及び200mlまで氷酢酸10mlaminosilanization溶液を調製するための清浄なポリプロピレン容器中でメチルアルコール。 </li><li>ポリプロピレン容器中で洗浄カバースリップを置きます。 30分間100rpmで、室温でそれらを振ります。 </li><li> W.は、少なくとも30分間、100rpmで、室温でそれらを振る75で15分間、誘導体化されたカバースリップを入れたビーカーを超音波処理します。 </li><li>エチルアルコールで二回ビーカーからの溶液を空にし、メチルアルコールで一回、慎重にカバースリップをすすぎ、と。ストアエチルアルコール中のカバースリップとは、2週間以内にそれらを使用しています。 </li></ol></li><li> カバーガラスのPEG化 <ol><li> 0.1 M炭酸水素ナトリウム（NaHCO 3）の10ミリリットルを加えます。 0.22μmのシリンジフィルターで溶液をフィルタリングします。 </li><li>ビオチンPEGスクシンイミジルカーボネート（ビオチン-PEG-SC）およびNaHCO 3の350μlの中のPEGスクシンイミジル吉草酸80mgの（MPEG-SVG）の2ミリグラムを溶かします。光保護管を使用してください。 </li><li> 1激しくチューブをボルテックス0秒、および気泡を除去するために1分間10,000×gで、それを遠心します。 </li><li>エチルアルコールですすぎ、続いてアセトンでスライドガラスを、洗浄します。完全に空気乾燥にスライドを許可します。 </li><li>きれいなガラススライド上のPEG溶液の滴（50μl）を配置します。任意の気泡を発生させることなく、アミノシラン化カバースリップで軽く滴をカバーしています。 </li><li> 3時間のための場所のスライドは一晩暗所で、室温で湿潤チャンバーを十分に平らにします。底に水で、チャンバとして空のピペットチップホルダーを使用します。チューブ内の残留PEG溶液を密封し、4℃で保管してください。 </li><li>脱イオン水で十分にペグ化されたカバースリップを洗い流します。使用するまで暗く乾燥した場所に保管してください。 </li></ol></li></ol> 3.フローチャンバーの組み立て<ol><li> 図2のように、パンチアクリルホルダーを製作。チューブインサートの直径は0.762ミリメートルであることを確認し、一つの孔を指定すると、入口であり、他方はであり、アウトレット（ 図2）。 </li><li>任意のチャンネル穴を摂動ない、入口と出口の穴に垂直に整列させ、アクリルホルダー（ 図2）に両面粘着テープ片（幅5〜6ミリメートル）を配置します。チャンバーを作るために、マルチチャネル壁としてこれらのテープストリップを使用してください。ピペットチップを使用してテープをスクラブ。 </li><li>フェイスダウンPEG化側で、フローチャンバーを作るために上にPEG化されたカバースリップを置きます。 </li><li>漏れる任意の溶液を防ぐために、両面テープが配置されている領域の上にカバーガラスの上部を押します。上部にある、ボトム（ 図2の黄色）でチャンバのエッジを閉じるには速乾エポキシ接着剤を追加します。 </li><li>ガスタイトシリンジにチューブ（外径、OD、0.042 &quot;）の短い断片（2.5センチ）を接続し、エポキシ接着剤で接合部をシールします。 </li><li>注射器にリンクされているチューブに、エポキシ接着剤で接合部をシールする：長いフレキシブルチューブ（0.03 &quot;OD）を接続します。 </li><li> TUBI塗りつぶしngのDI水で注射器にリンクされています。空気の泡がないことを確認してください。 </li><li>エポキシ接着剤で封止しフローチャンバーの穴にチューブを挿入します。 </li><li>貯水池などの他の穴に黄色の先端部（200μlの先端）を配置します。 </li></ol>フローチャンバー内に4.サンプルのロード注：ニュートラアビジンは、ストレプトアビジンのような他のアビジンのタンパク質で置き換えることができます。それは言及されない限り、全ての反応は、室温で行うことができます。黄色の先端に試料液を取り、アクリルホルダーの穴（ 図2b）上に溶液で先端をインストールします。 <ol><li> 50μl/分でシリンジポンプの流量を設定します。負荷アビジンタンパク質の20μlの（T50溶液中の25μgの/ mlで、10mMトリス、50mMのNaClを、pHを8）は、10分間それを維持します。 </li><li>負荷ビオチン化オリゴデオキシヌクレオチド（1×TE中100μM）を20μl、10分間それを維持します。ターミナルトランスフェラーゼを使用する場合は、ロードをスキップオリゴデオキシヌクレオチドの。 </li><li> 10μl/分の流速でフローチャンバーへのステップ1からの負荷のDNA溶液20μlを、30分間それを維持します。 </li><li> 1×TEとフローチャンバーを洗浄し、FP-DBP 10（〜80 nM）を40μlのをロードします。 </li><li> 60X対物レンズに1×TEで染色する分子の連続的な流れと蛍光顕微鏡下でDNAを観察します。 FP（EGFP）-dbpの励起に488nmの固体レーザーを使用してください。 DNA分子の完全なストレッチのために、使用されるDNAの長さに応じて異なる流量を適用します。例えば、T4 DNAの166 kbpのためのλDNAの48.5 kbpの、そして100μl/分を50μl/分を適用します。 <ol><li>アクリルホルダーのリサイクルのため、家庭用洗剤溶液12で組み立てフローチャンバーを浸します。かみそりの刃でテープとエポキシを削除し、それらを完全にオフに取るために手でこすります。注射針で穴を邪魔を除きます。さらに使用するまで脱イオン水にホルダーを保管してください。 </li></ol></li></ol>

<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>

The authors have nothing to disclose.

ここでは、表面上に固定するためにビオチン化長いDNA分子を可視化するためのプラットフォームを提示します。私たちは、ビオチン化ウシ血清アルブミンとアビジンタンパク質コーティングされた表面上の係留DNA分子のためのアプローチを報告している。6以前のアプローチでは、我々は上の係留DNA分子を染色ビス-インターカレーション染料によって引き起こされるDNAの光開裂の重要...

ガラスまたはビーズ表面に係留大きなDNA分子の可視化DNA基質、1,2および高分子物理学上のDNAタンパク質相互作用、タンパク質の動力学を調査するために利用されてきた。3,4-単一係留大型DNA分子のためのプラットフォームは、いくつかの異なるを有します他のDNA固定化方法に比べて利点が5まず、表面につなが大きいDNA分子は、その結合部位を認識するDNA結合タンパク質のための非常に重要であるせん断流れずに自然なランダムコイルコンフォメーションを有します。第二に、フローチャンバー内の一連の酵素反応のためのDNA分子の周りの化学的環境を変更することは非常に簡単です。第三に、マイクロ流体せん断流がDNA分子、このような表面固定化6及びナノチャネルの狭窄などの代替的なDNA伸長手法を用いて達成することは非常に困難である完全な輪郭の長さの100％にまで延伸を誘発する。7 A完全stretcheDのDNA分子は、ゲノムマップ上の酵素の動きを監視するために有用であることができる位置情報を提供します。 それにもかかわらず、DNAテザリングアプローチは、YOYO-1のような挿入色素は、一般的に容易に蛍光励起光によって破壊される係留DNA分子を引き起こすという重大な欠点を有しています。一般的に、大規模なDNA分子は蛍光顕微鏡下で可視化のための蛍光色素で染色されなければなりません。これらの色素は、それらが二本鎖DNAにインターカレートしたときにのみ蛍光を発するので、この目的のために、YOYO-1または他のTOTO系色素が主に使用されている。8しかし、ビス-インターカレート色素があるため、光誘導されるDNA光切断を引き起こすことがよく知られています蛍光体のインターカレーション。9更に、表面につなが蛍光染色されたDNA分子は、剪断流れが自由にDNA分子の移動の力を壊す発揮することができるため、より脆弱です。したがって、我々は新たなDとFP-DBPを開発しました表面に係留大型DNA分子を画像化するためのNA-染色タンパク質色素。 FP-DBPを使用する利点は、それが結合するDNA分子の光開裂を起こさないことである。加えて、10のビス-インターカレート色素は輪郭長さを増加しながら、FP-DBPは、DNAの輪郭の長さを増加させません約33％です。 このビデオ方式は、PEG-ビオチン表面に大きなDNA分子を係留するための実験的アプローチを紹介する。 図1は、平滑末端および付着末端を有するDNAを係留の異なるアプローチを示しています。したがって、この染色方法は、DNA分子の任意のタイプに適用することができる。 図2は、DNA分子を伸長するだけでなく、化学的および酵素をロードするためにせん断流を生成するために、シリンジポンプによって制御することができるフローチャンバアセンブリの概略図を示していますソリューション。 図3は、PEGylatにつなが完全に伸張したDNA分子の顕微鏡写真を示していますエド面11とFP-DBPで染色しました。

<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).

図1は、DNA分子の末端構造に応じて2つの異なるDNAテザリング方法を示している。 図1aは、スティッキーエンドのDNA分子は、アビジン被覆PEG表面上に固定化された補完的なビオチン化オリゴヌクレオチドとハイブリダイズされている様子を示す。 図1bは、の追加を示していますビオチン化のddNTPまたはターミナルトランスフェラー?...

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.

ペプチドを結合蛍光タンパク質のDNAを用いた表面係留大型DNA分子の可視化

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

visualization-surface-tethered-large-dna-molecules-with-fluorescent

Research

JoVE Journal

Biology

10.7K ビュー.  Sogang University. 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. 

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

Visualizing Single-molecule DNA Replication with Fluorescence Microscopy

We describe a simple fluorescence microscopy-based real-time method for observing DNA replication at the single-molecule level. A circular, forked DNA template is attached to a functionalized glass coverslip and replicated extensively after introduction of replication proteins and nucleotides (Figure 1). The growing product double-strand DNA (dsDNA) is extended with laminar flow and visualized by using an intercalating dye. Measuring the position of the growing DNA end in real time allows precise determination of replication rate (Figure 2). Furthermore, the length of completed DNA products reports on the processivity of replication. This experiment can be performed very easily and rapidly and requires only a fluorescence microscope with a reasonably sensitive camera.


This protocol demonstrates a simple single-molecule fluorescence microscopy technique for visualizing DNA replication by individual replisomes in real time.

蛍光顕微鏡による可視化単分子のDNAの複製

We describe a simple fluorescence microscopy-based real-time method for observing DNA replication at the single-molecule level. A circular, forked DNA ...

生物学

Direct Observation of Enzymes Replicating DNA Using a Single-molecule DNA Stretching Assay

We describe a method for observing real time replication of individual DNA molecules mediated by proteins of the bacteriophage replication system. Linearized &lambda; DNA is modified to have a biotin on the end of one strand, and a digoxigenin moiety on the other end of the same strand. The biotinylated end is attached to a functionalized glass coverslip and the digoxigeninated end to a small bead. The assembly of these DNA-bead tethers on the surface of a flow cell allows a laminar flow to be applied to exert a drag force on the bead. As a result, the DNA is stretched close to and parallel to the surface of the coverslip at a force that is determined by the flow rate (Figure 1). The length of the DNA is measured by monitoring the position of the bead. Length differences between single- and double-stranded DNA are utilized to obtain real-time information on the activity of the replication proteins at the fork. Measuring the position of the bead allows precise determination of the rates and processivities of DNA unwinding and polymerization (Figure 2).

We describe a method for observing real time replication of individual DNA molecules mediated by proteins of the bacteriophage replication system.

アッセイをストレッチ単一分子のDNAを用いて酵素の複製DNAの直接観察

We describe a method for observing real time replication of individual DNA molecules mediated by proteins of the bacteriophage replication system. ...

DNA Stable-Isotope Probing (DNA-SIP)

DNA stable-isotope probing (DNA-SIP) is a powerful technique for identifying active microorganisms that assimilate particular carbon substrates and nutrients into cellular biomass. As such, this cultivation-independent technique has been an important methodology for assigning metabolic function to the diverse communities inhabiting a wide range of terrestrial and aquatic environments. Following the incubation of an environmental sample with stable-isotope labelled compounds, extracted nucleic acid is subjected to density gradient ultracentrifugation and subsequent gradient fractionation to separate nucleic acids of differing densities. Purification of DNA from cesium chloride retrieves labelled and unlabelled DNA for subsequent molecular characterization (e.g. fingerprinting, microarrays, clone libraries, metagenomics). This JoVE video protocol provides visual step-by-step explanations of the protocol for density gradient ultracentrifugation, gradient fractionation and recovery of labelled DNA. The protocol also includes sample SIP data and highlights important tips and cautions that must be considered to ensure a successful DNA-SIP analysis.

DNA Stable-Isotope Probing DNA-SIP

DNA stable-isotope probing is a cultivation-independent method to identify and characterize active communities of microorganisms that are capable of utilizing specific substrates. Assimilation of substrate enriched in heavy isotope leads to incorporation of labelled atoms into microbial biomass. Density gradient ultracentrifugation retrieves labelled DNA for downstream molecular analyses.

プロービングDNA安定同位体（DNA - SIP）

DNA stable-isotope probing (DNA-SIP) is a powerful technique for identifying active microorganisms that assimilate particular carbon substrates and ...

Visualization of DNA Replication in the Vertebrate Model System DT40 using the DNA Fiber Technique

Maintenance of replication fork stability is of utmost importance for dividing cells to preserve viability and prevent disease. The processes involved not only ensure faithful genome duplication in the face of endogenous and exogenous DNA damage but also prevent genomic instability, a recognized causative factor in tumor development.
Here, we describe a simple and cost-effective fluorescence microscopy-based method to visualize DNA replication in the avian B-cell line DT40. This cell line provides a powerful tool to investigate protein function in vivo by reverse genetics in vertebrate cells1. DNA fiber fluorography in DT40 cells lacking a specific gene allows one to elucidate the function of this gene product in DNA replication and genome stability. Traditional methods to analyze replication fork dynamics in vertebrate cells rely on measuring the overall rate of DNA synthesis in a population of pulse-labeled cells. This is a quantitative approach and does not allow for qualitative analysis of parameters that influence DNA synthesis. In contrast, the rate of movement of active forks can be followed directly when using the DNA fiber technique2-4. In this approach, nascent DNA is labeled in vivo by incorporation of halogenated nucleotides (Fig 1A). Subsequently, individual fibers are stretched onto a microscope slide, and the labeled DNA replication tracts are stained with specific antibodies and visualized by fluorescence microscopy (Fig 1B). Initiation of replication as well as fork directionality is determined by the consecutive use of two differently modified analogues. Furthermore, the dual-labeling approach allows for quantitative analysis of parameters that influence DNA synthesis during the S-phase, i.e. replication structures such as ongoing and stalled forks, replication origin density as well as fork terminations. Finally, the experimental procedure can be accomplished within a day, and requires only general laboratory equipment and a fluorescence microscope.

DT40, a model vertebrate genetic system, provides a powerful tool to analyze protein function. Here we describe a simple method that allows qualitative analysis of parameters that influence DNA synthesis during the S-phase in DT40 cells at the single molecule level.

DNAの繊維技術を用いた脊椎動物のモデルシステムのDT40のDNA複製の可視化

Maintenance of replication fork stability is of utmost importance for dividing cells to preserve viability and prevent disease. The processes involved not ...

Measuring Peptide Translocation into Large Unilamellar Vesicles

There is an active interest in peptides that readily cross cell membranes without the assistance of cell membrane receptors1. Many of these are referred to as cell-penetrating peptides, which are frequently noted for their potential as drug delivery vectors1-3. Moreover, there is increasing interest in antimicrobial peptides that operate via non-membrane lytic mechanisms4,5, particularly those that cross bacterial membranes without causing cell lysis and kill cells by interfering with intracellular processes6,7. In fact, authors have increasingly pointed out the relationship between cell-penetrating and antimicrobial peptides1,8. A firm understanding of the process of membrane translocation and the relationship between peptide structure and its ability to translocate requires effective, reproducible assays for translocation. Several groups have proposed methods to measure translocation into large unilamellar lipid vesicles (LUVs)9-13. LUVs serve as useful models for bacterial and eukaryotic cell membranes and are frequently used in peptide fluorescent studies14,15. Here, we describe our application of the method first developed by Matsuzaki and co-workers to consider antimicrobial peptides, such as magainin and buforin II16,17. In addition to providing our protocol for this method, we also present a straightforward approach to data analysis that quantifies translocation ability using this assay. The advantages of this translocation assay compared to others are that it has the potential to provide information about the rate of membrane translocation and does not require the addition of a fluorescent label, which can alter peptide properties18, to tryptophan-containing peptides. Briefly, translocation ability into lipid vesicles is measured as a function of the Foster Resonance Energy Transfer (FRET) between native tryptophan residues and dansyl phosphatidylethanolamine when proteins are associated with the external LUV membrane (Figure 1). Cell-penetrating peptides are cleaved as they encounter uninhibited trypsin encapsulated with the LUVs, leading to disassociation from the LUV membrane and a drop in FRET signal. The drop in FRET signal observed for a translocating peptide is significantly greater than that observed for the same peptide when the LUVs contain both trypsin and trypsin inhibitor, or when a peptide that does not spontaneously cross lipid membranes is exposed to trypsin-containing LUVs. This change in fluorescence provides a direct quantification of peptide translocation over time.

This protocol details a method for the quantitative measure of peptide translocation into large unilamellar lipid vesicles. This method also provides information about the rate of membrane translocation and can be used to identify peptides that efficiently and spontaneously cross lipid bilayers.

大型単層小胞へのペプチドの転座の測定

There is an active interest in peptides that readily cross cell membranes without the assistance of cell membrane receptors1.  Many of these are referred ...

Preparation of DNA-crosslinked Polyacrylamide Hydrogels

Mechanobiology is an emerging scientific area that addresses the critical role of physical cues in directing cell morphology and function. For example, the effect of tissue elasticity on cell function is a major area of mechanobiology research because tissue stiffness modulates with disease, development, and injury. Static tissue-mimicking materials, or materials that cannot alter stiffness once cells are plated, are predominately used to investigate the effects of tissue stiffness on cell functions. While information gathered from static studies is valuable, these studies are not indicative of the dynamic nature of the cellular microenvironment in vivo. To better address the effects of dynamic stiffness on cell function, we developed a DNA-crosslinked polyacrylamide hydrogel system (DNA gels). Unlike other dynamic substrates, DNA gels have the ability to decrease or increase in stiffness after fabrication without stimuli. DNA gels consist of DNA crosslinks that are polymerized into a polyacrylamide backbone. Adding and removing crosslinks via delivery of single-stranded DNA allows temporal, spatial, and reversible control of gel elasticity. We have shown in previous reports that dynamic modulation of DNA gel elasticity influences fibroblast and neuron behavior. In this report and video, we provide a schematic that describes the DNA gel crosslinking mechanisms and step-by-step instructions on the preparation DNA gels.

Our laboratory has developed DNA-crosslinked polyacrylamide hydrogels, a dynamic hydrogel system, to better understand the effects of modulating tissue stiffness on cell function. Here, we provide schematics, descriptions, and protocols to prepare these hydrogels.

DNA架橋ポリアクリルアミドハイドロゲルの調製

Mechanobiology is an emerging scientific area that addresses the critical role of physical cues in directing cell morphology and function. For example, ...

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 development of diverse techniques to visualize individual RNA transcripts in single living cells. One promising technique that has recently been described utilizes an oligonucleotide-based optical probe, ratiometric bimolecular beacon (RBMB), to detect RNA transcripts that were engineered to contain at least four tandem repeats of the RBMB target sequence in the 3&#8217;-untranslated region. RBMBs are specifically designed to emit a bright fluorescent signal upon hybridization to complementary RNA, but otherwise remain quenched. The use of a synthetic probe in this approach allows photostable, red-shifted, and highly emissive organic dyes to be used for imaging. Binding of multiple RBMBs to the engineered RNA transcripts results in discrete fluorescence spots when viewed under a wide-field fluorescent microscope. Consequently, the movement of individual RNA transcripts can be readily visualized in real-time by taking a time series of fluorescent images. Here we describe the preparation and purification of RBMBs, delivery into cells by microporation and live-cell imaging of single RNA transcripts.

Ratiometric bimolecular beacons (RBMBs) can be used to image single engineered RNA transcripts in living cells. Here, we describe the preparation and purification of RBMBs, delivery of RBMBs into cells by microporation and fluorescent imaging of single RNA transcripts in real-time.

レシオメトリック二分子ビーコンを使用して、生細胞における単エンジニアのRNA転写物のリアルタイムイメージング

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

Reduced-gravity Environment Hardware Demonstrations of a Prototype Miniaturized Flow Cytometer and Companion Microfluidic Mixing Technology

Until recently, astronaut blood samples were collected in-flight, transported to earth on the Space Shuttle, and analyzed in terrestrial laboratories. If humans are to travel beyond low Earth orbit, a transition towards space-ready, point-of-care (POC) testing is required. Such testing needs to be comprehensive, easy to perform in a reduced-gravity environment, and unaffected by the stresses of launch and spaceflight. Countless POC devices have been developed to mimic laboratory scale counterparts, but most have narrow applications and few have demonstrable use in an in-flight, reduced-gravity environment. In fact, demonstrations of biomedical diagnostics in reduced gravity are limited altogether, making component choice and certain logistical challenges difficult to approach when seeking to test new technology. To help fill the void, we are presenting a modular method for the construction and operation of a prototype blood diagnostic device and its associated parabolic flight test rig that meet the standards for flight-testing onboard a parabolic flight, reduced-gravity aircraft. The method first focuses on rig assembly for in-flight, reduced-gravity testing of a flow cytometer and a companion microfluidic mixing chip. Components are adaptable to other designs and some custom components, such as a microvolume sample loader and the micromixer may be of particular interest. The method then shifts focus to flight preparation, by offering guidelines and suggestions to prepare for a successful flight test with regard to user training, development of a standard operating procedure (SOP), and other issues. Finally, in-flight experimental procedures specific to our demonstrations are described.

Spaceflight blood diagnostics need innovation. Few demonstrations have been published illustrating in-flight, reduced-gravity health diagnostic technology. Here we present a method for construction and operation of a parabolic flight test rig for a prototype point-of-care flow-cytometry design, with components and preparation strategies adaptable to other setups.

プロトタイプ小型化フローサイトメーターおよびコンパニオンマイクロ流体混合技術の縮小重力環境ハードウェアデモンストレーション

Until recently, astronaut blood samples were collected in-flight, transported to earth on the Space Shuttle, and analyzed in terrestrial laboratories. If ...

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

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

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

A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.

膜タンパク質の緑色蛍光タンパク質ベースの発現スクリーニングで<em&gt;エシェリヒア·コリ</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)

The limits to optical resolution and the challenge of identifying specific protein populations in transmission electron microscopy have been obstacles in cell biology. Many phenomena cannot be explained by in vitro analysis in simplified systems and need additional structural information in situ, particularly in the range between 1 nm and 0.1 &#181;m, in order to be fully understood. Here, electron spectroscopic imaging, a transmission electron microscopy technique that allows simultaneous mapping of the distribution of proteins and nucleic acids, and an expression tag, miniSOG, are combined to study the structure and organization of DNA double-strand break repair foci.

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

Electron spectroscopic imaging can image and distinguish nucleic acid from protein at nanometer resolution. It can be combined with the miniSOG system, which is able to specifically label tagged proteins in transmission electron microscopy samples. We illustrate the use of these technologies using double-strand break repair foci as an example.

電子分光イメージングと組み合わせたminiSOGタグ付きのDNA修復タンパク質の可視化（ESI）

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