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

1. DNA 바이오 티 닐화 <ol><li> 터미널 트랜스퍼하여 블런트 엔드 DNA의 바이오 티 닐화 (TDT) 참고 : 무딘 엔드 DNA이다 사용 T4 DNA (166 KBP)를. <ol><li> 2.5 밀리미터의의 CoCl2, 10 × 반응 버퍼의 5 μL, 10 mM의 비오틴-11-dUTP를 0.5 μL, 터미널 전이 0.5 μL (10 대), 및 T4 DNA의 0.5 μL (0.5 μg의 / μL)을 5 μl를 추가 반응 혼합물에 관한 것이다. 물 38.5 μL를 첨가하여 50 ㎕의 최종 부피로 만든다. </li><li> 1 시간 동안 37 ℃에서 반응 혼합물을 인큐베이션. 주의 : 연장 반응 시간, 즉 이중 닿는 DNA를 얻을 수 있고, biotins이 양단 태그 것이 가능하다. </li><li> 0.5 M EDTA, pH가 8의 5 μl를 추가하여 반응을 중지합니다. </li><li> 4 ° C에서 튜브를 유지합니다. </li></ol></li><li> DNA 리가 제를 사용하여 접착 종단 DNA의 바이오 티 닐화 참고 : 끈적 엔드 DNA이다 사용 λ DNA (48.5 KBP). &lt;올&gt; <li> T4 DNA 리가 아제의 1 μL, 10 × 내고 버퍼의 5 μl를 25 ng의 1 μl를 추가 / λ 파지 DNA μL, 50 μL의 최종 부피를 만들기 위해 물을 43 μl를 추가합니다. </li><li> 4 ° C에서 튜브를 유지합니다. </li></ol></li></ol> 2. 기능화 표면 유도체 화 주 :.도 1에 도시 된 바와 같이 유리 표면에 DNA 분자의 말단 밧줄하기 위해, 차 아민기를 바이오틴 PEG 코팅 하였다 커버 슬립에 실란 화되고 이는 PEG 화 과정은 단일 분자 DNA 이미징에 중요한 그 때문에 크게 표면에 원하지 않는 분자의 부착에 의해 생성 된 랜덤 노이즈를 감소시킬 수있다. <ol><li> 피라냐 청소 참고 : 피라냐 솔루션 따라서는 적절한 안전 지침에 따라주의하여 취급해야한다, 유기 물질과 격렬하게 반응한다. <ol><li> 장소는 폴리 테트라 플루오로 에틸렌 (PTFE) 랙 된 커버, 및 B을 길게Y PTFE 길이 스레드 인감 테이프, 랙에 표면의 가장자리 반에 반. 래핑 후, 세정 과정에서 랙을 처리 테이프의 긴 조각 (~ 5cm)를 떠난다. </li><li> SO H 2 O 2의 4, 150 ml의 흄 후드에서 피라 솔루션을 만들기 위해 H 2의 350 ml의 1 L의 유리 비커를 입력합니다. 2 시간 동안 피라냐 용액에 선반을 두십시오. </li><li> 비커에서의 pH까지 탈 이온수로 철저하게 빈 비커에서 피라 솔루션 및 린스 된 커버는 중립에 도달한다. 산도 종이 스트립을 사용합니다. </li><li> 30 분 동안 비이커에서 물을 함유 커버 슬립의 랙 초음파 처리. 비커에서 불과 아미노 실란 화하기 전에 빈 물. 유리 기판 청소 및 유도체 화하는 초음파 전력 75 W를 사용합니다. </li></ol></li><li> 유리 표면에 Aminosilanization <ol><li> N 2 ㎖를 추가 - [3- (트리 메 톡시 실릴) 프로필] 에틸렌 디아민, 200 mL의 빙초산 10 ㎖aminosilanization 용액을 제조 깨끗한 폴리 프로필렌 용기에 메틸 알콜. </li><li> 폴리 프로필렌 용기에 세척 된 커버를 놓습니다. 30 분 동안 100 rpm으로 실온에서 그들을 흔들어. </li><li> W. 적어도 30 분 동안 100 rpm으로 실온에서 그들을 흔들어 75에서 15 분 동안 유도 된 커버 슬립으로 비이커를 초음파 처리. </li><li> 에틸 알코올로 두 번 비커에서 솔루션을 비우기, 메틸 알코올로 한번 신중 된 커버를 세척합니다. 저장 에틸 알콜 된 커버와 2 주 내에서 사용합니다. </li></ol></li><li> 커버 슬립의 PEG 화 <ol><li> 0.1 M 중탄산 나트륨 10 ㎖ (의 NaHCO3)을 만든다. 0.22 μm의 주사기 필터 솔루션 필터. </li><li> 비오틴-PEG 숙신 카보네이트 (비오틴-PEG-SC)과의 NaHCO3 350 μL에 PEG 숙신 발레 레이트 80 mg의 (MPEG-SVG)의 2 mg을 녹이고. 가벼운 보호 튜브를 사용합니다. </li><li> 1 적극적으로 튜브를 와동0 초 원심 분리는 1 분 거품을 제거하는 10,000 × g에서. </li><li> 에틸 알콜로 세척 한 다음 아세톤 유리 슬라이드를 헹군다. 공기 슬라이드가 완전히 건조 할 수 있습니다. </li><li> 깨끗한 유리 슬라이드에 PEG 용액의 방울 (50 μl를) 놓습니다. 모든 거품을 생성하지 않고, 아미노 실란 화 커버 슬립으로 부드럽게 방울을 커버. </li><li> 3 시간 동안 장소 슬라이드 밤새 어둠 속에서, 실온에서 습기 챔버를 잘 평준화합니다. 바닥에 물 챔버로 빈 피펫 팁 홀더를 사용합니다. 튜브에 잔류 PEG 용액을 밀봉하고 4 ℃에서 보관하십시오. </li><li> 탈 이온수로 철저하게 PEG 화 coverslip에 린스. 사용할 때까지 어둡고 건조한 장소에 보관하십시오. </li></ol></li></ol> 3. 유량 용기를 조립 <ol><li> .도 2에서와 같이, 펀칭 아크릴 홀더를 제작 튜브 인서트의 직경이 0.762 mm 인 것을 확인하고 하나의 구멍 입구 인 지정 다른 것은출구 (그림 2). </li><li> 모든 채널에 구멍을 교란하지, 입구와 출구 구멍에 수직으로 정렬 양면 접착 테이프 스트립 아크릴 홀더 (폭 5-6mm) (그림 2)를 놓습니다. 멀티 채널 벽 챔버를 만들기 위해 이러한 테이프 스트립을 사용합니다. 피펫 팁을 사용하여 테이프를 스크럽. </li><li> 인쇄면이 밑으로 가도록 PEG 화면이 흐름 챔버를 만들기 위해 상단에 PEG 화 커버 슬립을 놓습니다. </li><li> 누출되는 모든 솔루션을 방지하기 위해 양면 테이프가 배치되는 지역을 통해 커버 슬립의 상단을 누릅니다. 상단 및 하단 (그림 2 노란색)에서 챔버의 가장자리를 닫습니다 속건 에폭시 접착제를 추가합니다. </li><li> 기밀 주사기에 튜브 (외경, OD, 0.042 &quot;)의 짧은 조각 (2.5 cm)를 연결하고 에폭시 접착제와 공동 인감. </li><li> 주사기에 연결된 튜브와 에폭시 접착제와 공동 밀봉 : 긴 유연한 튜브 (0.03 &quot;OD)을 연결합니다. </li><li> TUBI 채우기NG는 DI 워터와 주사기에 연결. 공기 거품이없는 것을 확인합니다. </li><li> 에폭시 접착제로 밀봉 유량 용기의 구멍에 튜브를 삽입합니다. </li><li> 저수지와 다른 구멍에 노란색 팁 (200 μL 팁)을 놓습니다. </li></ol> 유량 용기에 4 샘플로드 주 : 뉴트라 비딘는 스트렙 아비딘과 같은 다른 단백질로 대체 될 수있다. 이 언급하지 않는 한 모든 반응을 실온에서 수행 될 수있다. 노란색 끝에서 시료 용액을 가지고, 아크릴 홀더 (그림 2b)의 구멍에 솔루션과 팁을 설치합니다. <ol><li> / min로 50 μL로 시린지 펌프의 유량을 설정한다. 로드 아비딘 단백질의 20 μL (T50 용액 25 μg의 / ㎖, 10 mM의 트리스, 50 mM의 염화나트륨, pH가 8), 10 분 동안 유지. </li><li> 로드 (20) 비오틴 올리고 데 옥시 뉴클레오티드의 μL (1 × TE 100 μM), 10 분 동안 유지. 터미널 트랜스퍼가 사용되는 경우,로드를 건너올리고 데 옥시 뉴클레오티드의. </li><li> 부하 (20) 10 μL / 분의 유속으로 유동 챔버로 1 단계의 DNA 용액 μL, 30 분 동안 유지. </li><li> 1 × TE와 유량 용기를 세척하고, FP-DBP (10) (~ 80 nm의) 40 μl를로드합니다. </li><li> 60X 대물 렌즈에 1 × TE에서 염색 분자의 연속적인 흐름과 형광 현미경으로 DNA를 관찰한다. FP (eGFP는) -DBP의 여기에 대한 488 nm의 고체 레이저를 사용합니다. DNA 분자의 전체 신장을 위해 사용되는 DNA의 길이에 따라 서로 다른 유속을 적용한다. 예를 들어, T4 DNA의 166 KBP에 대한 λ DNA의 48.5 KBP, 100 μL / 분 동안 50 μL / 분을 적용합니다. <ol><li> 아크릴 홀더의 재활용, 가정용 세제 (12)에 조립 흐름 챔버를 흡수. 완전히 벗고 손으로 면도날과 마찰와 테이프와 에폭시를 제거합니다. 주사기 바늘 구멍을 방해를 없애기. 더 사용할 때까지 탈 이온수의 소유자를 유지합니다. </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.

여기에 우리가 표면에 고정을 위해 비오틴 긴 DNA 분자를 시각화 플랫폼을 제시한다. 우리는 바이오틴 소 혈청 알부민과 아비딘 단백질이 코팅 된 표면에 닿는 DNA 분자에 대한 접근 방식. 6 이전 방법에서보고, 우리는에 닿는 DNA 분자를 얼룩 비스 - 인터 염료에 의한 DNA의 사진 분열의 중요한 문제를 발견 표면. 이러한 지속적 여기 형광체는 여기 광 전력이 광 분열을 방지하기 위해 최소화?...

유리 구슬 표면에 닿는 큰 DNA 분자의 시각화 DNA 기판 1,2- 고분자 물리학 DNA - 단백질 상호 작용, 단백질 역학 조사에 이용되었다. 3,4- 단일 닿는 큰 DNA 분자를위한 플랫폼 구별 몇 갖는다 이점, 다른 DNA의 고정화 방법으로 비교 하였다. (5) 우선, 표면에 닿는 큰 DNA 분자의 결합 부위를 인식하는 DNA 결합 단백질에 대한 중요하다 전단 흐름없이 자연스러운 임의 코일 형태를 갖는다. 둘째, 유동 챔버에서 일련의 효소 반응에 대한 DNA 분자 주위의 화학적 환경을 변경하는 것은 매우 쉽다. 셋째, 미세 유체 전단 흐름 대안 DNA 신장을 사용하여 달성하는 것은 매우 어렵다 전체 윤곽 길이의 100 %까지 연신 분자량 DNA는 표면 고정화 6 및 나노 채널 가둠으로 접근 유도한다.도 7a stretche 완전히D DNA 분자는 게놈지도에서 효소의 움직임을 모니터링하는 데 유용 할 수의 위치 정보를 제공한다. 그럼에도 불구하고, DNA 테 더링 방식은 YOYO-1은 일반적으로 닿는 DNA 분자가 용이 형광 여기 광에 의해 파손되게로서 그 인터 염료의 중요한 단점을 갖는다. 일반적으로, 많은 DNA 분자는 형광 현미경 시각화 형광 염료로 염색 될 수있다. 이를 위해, YOYO-1 또는 다른 TOTO 계 염료는 이중 가닥 DNA를 층간 삽입 할 때 이러한 염료 만 형광 때문에 주로 사용된다. (8) 단, 비스 인터 염료 광 - 유도 DNA의 광 분열로 인해 발생하는 것이 잘 알려져 형광체의 인터. 9 항, 표면에 닿는 형광 염색 DNA 분자는 전단 흐름이 자유롭게 DNA 분자 이동에 힘을 깨고 발휘할 수 있기 때문에 더 약하고. 따라서, 우리는 새로운 D-FP로 DBP 개발표면에 닿는 큰 DNA 분자 이미징 NA 단백질 염색 염료. FP-DBP를 사용하는 이점은 그것이 바인딩하는 DNA 분자의 광 분열이 발생하지 않는다는 것이다. (10) 또한 비스 인터 염료 윤곽 길이를 증가하면서, FP-DBP는 DNA의 윤곽 길이가 증가하지 않는다 약 33 % 정도. 이 비디오 방법은 PEG-비오틴 표면에 큰 DNA 분자를 테 더링에 대한 실험 방법을 소개합니다. (1) 평활 말단 스티커 끝으로 DNA를 테 더링의 서로 다른 접근 방식을 보여줍니다. 따라서, 이러한 착색 방법은 DNA 분자의 모든 유형에 적용 할 수있다. 2 DNA 분자를 늘릴뿐만 아니라 화학적 및 효소를로드하는 전단 흐름을 생성하기 위해 주사기 펌프에 의해 제어 될 수 유량 용기 조립체의 개략적 인 표현을 도시 한 도표 솔루션을 제공합니다. 그림 3은 PEGylat에 닿는 완전히 뻗어 DNA 분자의 현미경 사진을 보여줍니다에드면 (11) 및 FP-DBP로 염색.

<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).

도 1은도 1b도.도 1a는 스티키 종단 DNA 분자가 아비딘 코팅 된 PEG 상에 고정화 상보 오티 닐화 올리고 뉴클레오티드 혼성화 방법을 도시한다. DNA 분자의 말단 구조에 따라 두 개의 서로 다른 DNA 테 더링 방법을 도시의 첨가를 보여준다 바이오틴 어떤 ddNTP 또는 터미널 전이에 의해 무딘 엔드 DNA의 3 &#39;히드 록실 그룹의 dNTP. ...

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.

펩타이드를 결합 형광 단백질 DNA와 표면 닿는 큰 DNA 분자의 시각화

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 조회수.  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. 

비디오: 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.

형광 현미경과 함께 머릿속으로 단일 분자의 DNA 복제

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

연구

생물학

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.

효소 복제 DNA의 직접 관찰 어세을 스트레칭 단일 분자 DNA를 사용하여

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.

DNA 안정 - 동위 원소 (DNA - SIP) 탐색

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 복제의 시각화는 척추 모델 시스템 DT40의 DNA 섬유 기술을 사용하여

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.

대형 Unilamellar Vesicles로 펩타이드의 Translocation 측정

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-가교 폴리 아크릴 아미드 하이드로 겔의 제조

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.

비례 생체 분자 비콘을 사용하여 살아있는 세포의 단일 엔지니어링 RNA 성적 증명서의 실시간 영상

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.

프로토 타입 소형 흐름 사이토와 동반자 미세 유체 혼합 기술의 감소 - 중력 환경 하드웨어 데모

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.

막 단백질의 녹색 형광 단백질 기반의 표현 상영에<em&gt; 대장균</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.

전자 분광 이미징과 결합 miniSOG 태그 DNA 복구 단백질의 시각화 (ESI)

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