拉伸实验的DNA的发展援助，钟奉李保布莱尼和萨米尔哈姆丹。这项工作是由美国国立卫生赠款查尔斯理查德森（GM54397），安托万车Oijen（GM077248）研究院的支持。

1。 DNA复制模板 DNA的反应是一个线性的λDNA寡核苷酸退火形成一个复制叉的修改。此外，生物素连接的λDNA链的结束，一个地高辛基团连接到同一条 1的另一端。 材料：λ噬菌体的DNA，寡核苷酸：生物素化的叉臂（答：5&#39; -生物素- AAAAAAAAAAAAAAAAGAGTACTGTACGATCTAGCATCAATCACAGGGTCAGGTTCGTTATTGTCCAACTTGCTGTCC - 3&#39;），λ互补叉臂（B：5&#39; - GGGCGGCGACCTGGACAGCAAGTTG GACAATCTCGTTCTATCACTAATTCACTAATGCAGGGAGGATTTCAGATATGGCA - 3&#39;），叉底漆（C：5&#39; TGCCATATCTGAAATCCTCCCTGC - 3&#39;），λ互补的地高辛结束（D：5&#39; - AGGTCG CCGCCCAAAAAAAAAAAA地高辛- 3&#39;），T4 DNA连接酶，T4多聚核苷酸激酶，热块。 <ol><li>磷酸寡核苷酸的B和D的5&#39;末端用T4多聚核苷酸激酶。 </li><li>退火寡核苷酸在一个步骤中，A，B和C的λDNA加入一个寡核苷酸A和B超过10倍，和一个超过100倍，在T4 DNA连接酶缓冲液的寡核苷酸彗星。 </li><li>加热至65 ° C在加热块的混合物。加热后，打开热封锁，并允许缓慢冷却到适当的退火寡核苷酸。 </li><li>加入T4 DNA连接酶的混合物，在室温下孵育2小时。 T4 DNA连接酶催化寡核苷酸A和B，和λDNA的正确加入。 </li><li>最后，退火的寡核苷酸D到DNA的复制模板的加入与尊重，在T4 DNA连接酶缓冲液为L的DNA寡核苷酸ð超过100倍。 </li><li>在45 ° C下30分钟的加热块，冷却到室温慢慢转动加热块孵育的混合物。 </li><li>最后DNA的复制模板是现在使用的准备。我们在浓度为1.4 nm的模板存储在4 ° C的几个星期。 </li></ol> 2。珠珠为地高辛与特异性Fab片段与官能。然后它们可以附着到DNA的复制模板2。 材料：对甲苯磺酰激活珠，α地高辛Fab片段，缓冲：0.1 M硼酸pH值9.5，缓冲液B：0.1 M PBS pH值7.4，0.1％W / V牛血清白蛋白，缓冲液C：0.2 M的Tris - HCl pH值8.5（25 ° C）0.1％W / V BSA，磁选机，肩。 <ol><li>重悬珠原液，并转移到Eppendorf管中的一个小等分。放入磁选机和孵化槽管，直到解决方案清除，然后取出上清液吹打。 </li><li>加入缓冲液A，这将激活抗体耦合对甲苯磺酰组。轻轻吹打混合，并删除与磁选机的缓冲区。 </li><li>再次添加缓冲液A中的Fab片段和悬浮珠，调匀，孵化16-24小时，在37 ° C使用肩。孵化后，单独珠使用的磁选机，并删除缓冲区。 </li><li>在4 ° C 5分钟，加入缓冲液B孵育，洗珠。删除缓冲区使用磁选。重复洗两次。 </li><li>新增了4个小时的缓冲液C在37 ° C孵育块游离甲苯磺酰组。独立珠使用磁性分离器，并删除缓冲区。 </li><li>使用的缓冲液B和等分两次清洗。珠可以被保存在4 ° C为1年或更长的时间没有质量的重大损失（我们的库存解决方案包含1 - 2X10 9珠/毫升）。 </li></ol> 3。官能盖玻片为了让玻璃盖玻片DNA附件，玻璃是第一个与氨基硅烷，然后再加以生物素化的聚乙二醇分子官能。这种涂层有助于减少和DNA复制蛋白的非特异性相互作用与表面3,4。 材料：盖玻片，染色罐，3 -氨丙基甲氧基PEG5k - NHS酯，生物素，PEG5k NHSester，丙酮，1M氢氧化钾，乙醇，烤箱，浴sonicator <ol><li>彻底清洁玻片，放置在染色罐和灌装的罐子，用乙醇。超声30分钟，冲洗出来的罐子和填充用1 M KOH。重复这两个洗。 </li><li>第一官能反应，所有的水的痕迹需要被删除。填写的罐子，用丙酮和10分钟的超声。空，并填写再次用丙酮。 </li><li>准备的硅烷丙酮试剂的2％V / V解决方案。添加到染色罐，搅拌2分钟。这种反应夫妇烷氧基氨基硅烷在玻璃集团，留下了对未来的耦合步骤的反应胺。淬火的大量过剩与水的反应。 </li><li>他们在110烘烤干燥的盖玻片°，C为30分钟的烤箱。 </li><li>准备一个50:1的甲氧基：在100毫米碳酸氢钠 3 pH值8.2生物素聚乙二醇的混合物。旨在为约0.2％W / V的生物素化的聚乙二醇。 </li><li>吸取100μL，到干硅烷盖玻片和另一盖玻片上顶部的PEG混合物。其中包括一个玻璃盖玻片间隔将使分离的盖玻片。 </li><li>在3小时的PEG解决方案孵育盖玻片，然后单独每对盖玻片，用清水洗净广泛。要注意保持官能盖玻片的一面朝上只有一次方将涂用PEG。 </li><li>干燥的空气和下真空干燥器中储存的盖玻片。至少一个月的表面保持稳定，使盖玻片数十可以在一个批次，并根据需要使用。 </li></ol> 4。流室准备这项实验是使用官能盖玻片，双面胶带，石英片和管构造一个简单的流室。一个流室，准备为每个单分子实验2,4。 材料：双面胶带，刀片，与孔管，快干环氧石英片，官能盖玻片，链解决方案（1毫克/毫升的PBS 25μL），油管，封闭液（5X：20毫米的Tris pH值7.5，2毫米EDTA，50 mM氯化钠，0.2 mg / mL的BSA，0.005％Tween - 20的），工作缓冲液（1X：20毫米的Tris - HCl pH值7.5，EDTA的2毫米，50毫米氯化钠）。 <ol><li>开始混合，并把5毫升的封闭液和20毫升工作干燥器中的缓冲区，为后面的步骤中删除任何气泡。 </li><li>采取的PEG -官能盖玻片，PBS稀释的链霉亲和解决方案的125个分布在表面。保留此为20分钟，在室温下孵育，同时准备室的其他部位，使链霉亲和绑定表面biotins。 </li><li>剪下一块双面胶的石英片的大小相匹配。标记的磁带上的油管孔的位置，以便可以在磁带上绘制流室的轮廓。 </li><li>设为3毫米宽的中心通道和矩形包含两个孔。我们使用两个入口和两个出口Y形通道的流量商会。 </li><li>沿着绘制的轮廓切割，直，洗净切块，以确保进入通道没有粘合剂突出。 </li><li>清洁的石英彻底使用丙酮或乙醇删除从以前的流通池建设的粘合剂。 </li><li>找到最佳通道纲要对齐，删除背胶的一面，并小心地将磁带上的石英片。要小心，以正确对齐磁带，入口和出口孔要保持畅通。 </li><li>削减进口和流动池出口的管道长度。它有助于减少约30 °角管，使管会不按对腔底部的平面。将某种支持（如试管架）暂停他们在接下来的步骤流细胞不易附着在管。 </li><li>链霉菌涂层的盖玻片，用水彻底冲洗和干燥的压缩空气。请记住，只有一方是功能化。取出磁带备份的另一边，并将其放置到盖玻片的石英片。 </li><li>盖玻片上轻按推被困在胶粘剂任何空气。这将有助于避免气泡进入流道。 </li><li>用环氧树脂密封室的两侧。插入石英和地方用环氧树脂密封的孔切管。这需要几分钟干。 </li><li>干燥后，开始阻止通过一个管子拉一些脱气封闭液的表面。非常适合一个21号的针管内0.76毫米。冲洗几次，以去除气泡，并允许至少一个半小时的孵化室。 </li></ol> 5。单分子复制实验一旦DNA模板和官能珠已准备，并流室已准备就绪，可以进行单分子实验。 材料：准备流室，封闭液（5X：20毫米的Tris - HCl pH值7.5，EDTA的2毫米，50毫米氯化钠，1 mg / mL的BSA，0.025％Tween - 20的），工作缓冲液（1X：20毫米的Tris - HCl pH值7.5，带或不带10MM氯化镁2（1X T7复制缓冲区：40毫米的Tris - HCl pH值7.5，50毫米的K -谷氨酸，2毫米EDTA，100微克/ ml的BSA，10毫米，2毫米EDTA，50毫米氯化钠）数码地面电视，600μMdNTPs），复制的蛋白质，倒与10倍的目标，CCD摄像头，永久的稀土磁铁，注射泵，空气弹簧，光纤照明，计算机图像采集软件光学显微镜。 <ol><li>流后的细胞已被封锁，这是准备开始实验。 </li><li>以流动细胞和它放在显微镜舞台上。保持在舞台剪辑室确保流道与CCD相机对准定位。 </li><li>流通池出口管连接空气弹簧，使用较大直径的连接管或针。使用空气弹簧是稳定的任何流量的违规行为。一个50毫升的塑料管是充满水45毫升。管的盖子是用环氧树脂密封。三孔刺穿盖子，入管插入管件。用环氧，然后密封管的盖子，防止空气进入或逃避任何。空气弹簧是连接注射泵，流动池出口管。 </li><li>放置在封闭液的流动池入口管和注射器，在管中删除任何空气，回拉。一个出水口管轻轻一抖，将帮助被困在流动池清除任何空气气泡。 </li><li>一旦所有的气泡已被删除，阻止一个入口用针管，到1800年的弯曲，并用胶布管的出水口管。 </li><li>至晚上10时，在1毫升脱气封闭液稀释股DNA为模板。在适度的流量室的流量，让良好的表面覆盖的DNA（0.01-0.05毫升/分钟20分钟，效果很好）。基于多少DNA分子表面上每一批盖玻片，或有多少人所需的实验，这可以是多种多样的。 </li><li> 2 - 4 × 6珠/毫升1毫升脱气封闭液稀释的股票珠的解决方案。 0.01-0.05毫升/分钟，15分钟进入会议厅时的流量。 </li><li>一旦加入珠，转流，并允许腔达到平衡。然后关闭这两个插座管。管封闭室没有任何的变化会引起压力波动，将对珠和剪切系绳的DNA分子上的强大力量。 </li><li>座是用来装载用针珠的入口管，并开始清洗流通池，用封闭液脱气1X解决方案，在工作缓冲区的第二个入口。在0.01-0.05毫升/分钟的速度广泛清洗。搅拌手动攻丝，以消除非特异性停留在表面（的任何珠<a href="https://www.jove.com/files/ftp_upload/1689/Tapping.avi">的</a>阶段攻）。 </li><li>一旦自由珠有足够的删除，关闭流和允许腔，达到平衡。关闭打开的出口管，进气口水库和开关蛋白质溶液，慢慢地打开插座，以避免快速压力变化</li><li>您可以检查，如果是拴珠通过改变流动方向（DNA <a href="https://www.jove.com/files/ftp_upload/1689/DNA.avi">的DNA</a> ） 。 </li><li>现在，可以进行酶反应。领先链的合成实验GP4 GP5聚合酶脱气T7复制缓冲区不包含MgCl 2的 （作为一个复杂的，其processivity因素硫氧还蛋白纯化）的解旋酶和20nm的解决方案的20nm的解决方案。 </li><li>在中等流量的流量，让高效的DNA结合的蛋白质溶液进入会议厅。我们使用0.037毫升/分钟，8分钟，然后0.012毫升/分钟7分钟。 </li><li>脱气没有MgCl 2的 T7复制缓冲区的清洗室，删除免费的蛋白质。 0.037毫升/分钟3-10分钟的速度运行良好。 </li><li>洗涤后，流T7 MgCl 2的复制缓冲区，并开始数据采集。使用0.012毫升/分钟，相应的3 PN本议定书中所描述的条件下，DNA的系绳上的拖曳力的流量。 </li><li>放置一个以上的流动细胞稀土永磁前成像技术来防止珠和表面之间的非特异性相互作用。 </li><li>查看10倍的目标和CCD相机领域。聚焦使用拴珠。 </li><li>在10度附近的显微镜显微镜阶段平行之间的入射角光纤照明的位置。暗场照明是用来增加在珠成像的对比度。 </li><li> 20〜30分钟在一个缓慢的帧速率（通常为2-4赫兹）获取数据。 </li></ol> 6。代表性的成果在一个成功的实验，你应该同时能观察到超过100珠<a href="https://www.jove.com/files/ftp_upload/1689/Leading_strand_synthesis.avi">（领先链</a>合成） 。实验产生无数的痕迹，显示领先的双链DNA的合成。 数据分析： 为了测量率和平仓的DNA和聚合processivities，珠精确的位置必须确定。您可以通过它与2维高斯函数拟合这些职位。珠跟踪使用跟踪软件（例如DiaTrack）在每个帧的位置，轨迹，然后可以提取。现在您已经准备密谋珠的位置作为时间的函数，使用任何图形，使可视化轨迹ftware（如原产地）。要获得率数据，配合线性回归图和计算斜率。 processivity，确定总长度的DNA从开始到结束一个缩短事件（图3）。这些数字都将需要被转换成碱基对。珠运动转换成碱基对，首先你需要一个拉长的λDNA分子的长度来衡量反应流量。以来的λDNA的长度是已知的（48502 BP），可以校准实验力的碱基对/像素数。为了提高准确度，减去利息珠系绳酶不改变基准跟踪的痕迹。结合所有单次测量和积率和processivity分布。使用高斯和单指数函数，分别为（图3）数据拟合。 <img src="/files/ftp_upload/1689/1689fig1.jpg" alt="图1" /> 图1单分子实验装置。 （一）双面λDNA（48.5 KB）连接流细胞表面通过一个使用生物素 - 亲和相互作用链5&#39;端和3&#39;同一条连接到珠使用地高辛抗地高辛的相互作用。在对面的珠年底形成一个引物的复制叉允许加载的聚合酶。 （二）珠DNA集会被拉长使用的缓冲层流和成像采用宽视场光学显微镜。跟踪随着时间的推移珠的位置，同时保持一个恒定的拉伸力，可监控的DNA结构的长度。 （三）扩展配置文件的单链DNA（实心圆）和双链DNA（开放圆圈）在低力量。虚线显示了充分的DS -λ的DNA，16.3微米的晶体长度。 ssDNA和dsDNA抗体之间的长度约3 PN的力量相差较大，允许通过监测DNA长度变化的一个直接观察SS -和dsDNA之间的转换。大量的DNA耦合珠的同步可视化，允许在一个实验中的许多个人replisomes的研究。 <img src="/files/ftp_upload/1689/1689fig2.jpg" alt="图2" /> 图2噬菌体T7复制蛋白：DNA聚合酶（GP5和硫氧还复杂）和DNA解旋酶（GP4）合成DNA脱氧核糖核酸和镁的存在，导致钢绞线。这个过程是伴随着缩短到卷取DNA滞后链。双链DNA转换成单链DNA的变化珠的位置。测量珠的位置，可以精确测定DNA复制的速度和processivity。 <img src="/files/ftp_upload/1689/1689fig3.jpg" alt="图3" /> 图3例如，从一个单分子的领先链的合成实验典型的数据。 DNA复制Processivity等于总长度的DNA从开始到结束一个缩短事件。 DNA复制的速度是通过拟合与线性回归的情节和计算斜坡。插图显示率和processivity分布，从测量数据相结合获得。数据进行拟合，分别用高斯和单指数函数。

<ol>
	<li>Lee, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+primase+acts+as+a+molecular+brake+in+DNA+replication.">DNA primase acts as a molecular brake in DNA replication.</a> Nature. 439, 621-624 (2006).</li><li>Tanner, N. A., van Oijen, A. M. <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+observation+of+prokaryotic+DNA+replication.">Single-molecule observation of prokaryotic DNA replication.</a> Methods Mol Biol. 521, 397-410 (2009).</li><li>Sofia, S. J., Premnath, V. V., Merrill, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Poly(ethylene+oxide)+Grafted+to+Silicon+Surfaces:+Grafting+Density+and+Protein+Adsorption.">Poly(ethylene oxide) Grafted to Silicon Surfaces: Grafting Density and Protein Adsorption.</a> Macromolecules. 31, 5059-5070 (1998).</li><li>Tanner, N. A., Loparo, J. J., van Oijen, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+single-molecule+DNA+replication+with+fluorescence+microscopy.">Visualizing single-molecule DNA replication with fluorescence microscopy.</a> J Vis Exp. , (2009).</li><li>Hamdan, S. M., Loparo, J. J., Takahashi, M., Richardson, C. C., van Oijen, A. M. <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+DNA+replication+loops+reveal+temporal+control+of+lagging-strand+synthesis.">Dynamics of DNA replication loops reveal temporal control of lagging-strand synthesis.</a> Nature. 457, 336-339 (2009).</li><li>van Oijen, A. M. <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+kinetics+of+lambda+exonuclease+reveal+base+dependence+and+dynamic+disorder.">Single-molecule kinetics of lambda exonuclease reveal base dependence and dynamic disorder.</a> Science. 301, 1235-1238 (2003).</li></ol>

重要的是要确保对珠层流曳力不影响酶活性的复制蛋白。举例来说，一个3 PN的力量，对应到0.012毫升/分钟的流速不影响复制的DNA领先链。但它的影响，采取协调DNA合成过程中酶的活性，因此将减少到1.5 PN。拖曳力可以很容易地控制，通过改变流速或流道 5的宽度。 DNA拉伸实验采用双和单链DNA之间的长度差异。在实验中所描述这里的单链DNA双链DNA转换作为一个领先的?...

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		<th>Type</th>
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		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Bacteriophage &lambda; DNA</td>
<td>&nbsp;</td>
<td>New England Biolabs</td>
<td>N3011L</td>
<td>&nbsp;</td>
<tr>
<td>DNA Oligonucleotides</td>
<td>&nbsp;</td>
<td>Integrated DNA Technologies</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>T4 DNA Ligase</td>
<td>&nbsp;</td>
<td>New England Biolabs</td>
<td>M0202L</td>
<td>&nbsp;</td>
<tr>
<td>T4 Polynucleotide Kinase</td>
<td>&nbsp;</td>
<td>New England Biolabs</td>
<td>M0201L</td>
<td>&nbsp;</td>
<tr>
<td>&alpha;-digoxigenin Fab</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>11214667001</td>
<td>&nbsp;</td>
<tr>
<td>Tosyl Activated Beads</td>
<td>&nbsp;</td>
<td>Dynal/Invitrogen</td>
<td>142-03</td>
<td>&nbsp;</td>
<tr>
<td>Magnetic Separator</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>Dynal MPC</td>
<td>&nbsp;</td>
<tr>
<td>3-aminopropyl-triethoxysilane</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A3648</td>
<td>Other aminosilanes can be used or mixed with non-amine reactive silanes for sparser surfaces</td>
</tr>
<tr>
<td>Succinimidyl propionate PEG</td>
<td>&nbsp;</td>
<td>Nektar</td>
<td>&nbsp;</td>
<td>Similar PEGs can be purchased from Nanocs, CreativePEGWorks, etc.</td>
</tr>
<tr>
<td>Biotin-PEG-NHS</td>
<td>&nbsp;</td>
<td>Nektar</td>
<td>&nbsp;</td>
<td>Similar PEGs can be purchased from Nanocs, CreativePEGWorks, etc.</td>
</tr>
<tr>
<td>Double-sided tape</td>
<td>&nbsp;</td>
<td>Grace BioLabs</td>
<td>SA-S-1L</td>
<td>100 &mu;m thickness</td>
</tr>
<tr>
<td>Quartz slide</td>
<td>&nbsp;</td>
<td>Technical Glass</td>
<td>20 mm (W)x 50 mm (L)x 1mm (H)</td>
<td>Size to fit on coverslips. Drill holes with diamond-tip drill bits (DiamondBurs.net)</td>
</tr>
<tr>
<td>Polyethylene tubing</td>
<td>&nbsp;</td>
<td>Becton Dickinson</td>
<td>427416</td>
<td>0.76 mm ID, 1.22 OD 
Other size tubing can be substituted.</td>
</tr>
<tr>
<td>Streptavidin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>S4762</td>
<td>Make 1 mg/mL solution, 25 &mu;L aliquots in PBS pH 7.3</td>
</tr>
<tr>
<td>Deoxyribonucleotide triphosphate solution mix</td>
<td>&nbsp;</td>
<td>New England Biolabs</td>
<td>N0447</td>
<td>&nbsp;</td>
<tr>
<td>Inverted Optical Microscope with 10X Objective</td>
<td>&nbsp;</td>
<td>Olympus</td>
<td>Olympus IX-51</td>
<td>&nbsp;</td>
<tr>
<td>Permament rare-earth magnet</td>
<td>&nbsp;</td>
<td>National Imports</td>
<td>www.rare-earth-magnets.com</td>
<td>&nbsp;</td>
<tr>
<td>CCD Camera</td>
<td>&nbsp;</td>
<td>QImaging</td>
<td>Rolera-XR Fast 139</td>
<td>&nbsp;</td>
<tr>
<td>Syringe Pump</td>
<td>&nbsp;</td>
<td>Harvard Apparatus</td>
<td>11 Plus</td>
<td>Operate in refill mode to facilitate solution changes</td>
</tr>
<tr>
<td>Fiber Illuminator</td>
<td>&nbsp;</td>
<td>Thorlabs Inc.</td>
<td>OSL1</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

direct observation of enzymes replicating dna using a single-molecule dna stretching assay

我们描述了一个观察单个DNA分子介导的蛋白质的噬菌体复制系统的实时复制的方法。线性λDNA被修改，有一个链上的生物素，地高辛基团上的同一条电缆的另一端。生物素标记的一端连接到一个官能的玻璃盖玻片和digoxigeninated结束的小珠。一个流细胞表面上这些DNA珠系绳大会允许应用层流珠施加的阻力。因此，被拉伸的DNA靠近和平行的流量（图1）确定力在盖玻片表面。测量长度的DNA监测珠的位置。单双链DNA之间的长度差异，利用获得的实时信息，在交岔路口的复制蛋白的活性。测量珠的位置可以精确测定平仓DNA和聚合（图2）的速率和processivities。

Watch this Scientific Journal Video about Direct Observation of Enzymes Replicating DNA Using a Single-molecule DNA Stretching Assay at JoVE.com

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

我们描述了一个观察单个DNA分子介导的蛋白质的噬菌体复制系统的实时复制的方法。

使用单分子DNA拉伸含量直接观察DNA复制的酶

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

direct-observation-enzymes-replicating-dna-using-single-molecule-dna

Research

JoVE Journal

Biology

18.6K 次观看.  Harvard Medical School. 我们描述了一个观察单个DNA分子介导的蛋白质的噬菌体复制系统的实时复制的方法。

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

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.

可视化的单分子荧光显微镜下的DNA复制

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

科研

生物学

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的稳定同位素探测（DNA的SIP）

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

Generation of RNA/DNA Hybrids in Genomic DNA by Transformation using RNA-containing Oligonucleotides

Synthetic short nucleic acid polymers, oligonucleotides (oligos), are the most functional and widespread tools of molecular biology. Oligos can be produced to contain any desired DNA or RNA sequence and can be prepared to include a wide variety of base and sugar modifications. Moreover, oligos can be designed to mimic specific nucleic acid alterations and thus, can serve as important tools to investigate effects of DNA damage and mechanisms of repair. We found that Thermo Scientific Dharmacon RNA-containing oligos with a length between 50 and 80 nucleotides can be particularly suitable to study, in vivo, functions and consequences of chromosomal RNA/DNA hybrids and of ribonucleotides embedded into DNA. RNA/DNA hybrids can readily form during DNA replication, repair and transcription, however, very little is known about the stability of RNA/DNA hybrids in cells and to which extent these hybrids can affect the genetic integrity of cells. RNA-containing oligos, therefore, represent a perfect vector to introduce ribonucleotides into chromosomal DNA and generate RNA/DNA hybrids of chosen length and base composition. Here we present the protocol for the incorporation of ribonucleotides into the genome of the eukaryotic model system yeast /Saccharomyces cerevisiae/. Yet, our lab has utilized Thermo Scientific Dharmacon RNA-containing oligos to generate RNA/DNA hybrids at the chromosomal level in different cell systems, from bacteria to human cells.

This work shows how to form an RNA/DNA hybrid at the chromosomal level and reveal transfer of genetic information from RNA to genomic DNA in yeast cells.

改造使用含RNA寡核苷酸基因组DNA的RNA / DNA杂交一代

Synthetic short nucleic acid polymers, oligonucleotides (oligos), are the most functional and widespread tools of molecular biology. Oligos can be ...

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.

使用可视化的DNA复制DNA纤维的技术，在脊椎动物的模型系统DT40

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

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交联聚丙烯酰胺水凝胶的制备

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

Combining Single-molecule Manipulation and Imaging for the Study of Protein-DNA Interactions

The paper describes the combination of optical tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method offers the opportunity of investigating interactions occurring in solution (thus avoiding problems due to closeby surfaces as in other single molecule methods), controlling the DNA extension and tracking interaction dynamics as a function of both mechanical parameters and DNA sequence. The methods for establishing successful optical trapping and nanometer localization of single molecules are illustrated. We illustrate the experimental conditions allowing the study of interaction of lactose repressor (lacI), labeled with Atto532, with a DNA molecule containing specific target sequences (operators) for LacI binding. The method allows the observation of specific interactions at the operators, as well as one-dimensional diffusion of the protein during the process of target search. The method is broadly applicable to the study of protein-DNA interactions but also to molecular motors, where control of the tension applied to the partner track polymer (for example actin or microtubules) is desirable.

Here we describe the instrumentation and methods for detecting single fluorescently-labeled protein molecules interacting with a single DNA molecule suspended between two optically trapped microspheres.

结合单分子操纵和成像蛋白-DNA相互作用的研究

The paper describes the combination of optical tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method ...

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.

单工程的RNA转录物的活细胞实时成像采用比例式双分子信标

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

Studying DNA Looping by Single-Molecule FRET

Bending of double-stranded DNA (dsDNA) is associated with many important biological processes such as DNA-protein recognition and DNA packaging into nucleosomes. Thermodynamics of dsDNA bending has been studied by a method called cyclization which relies on DNA ligase to covalently join short sticky ends of a dsDNA. However, ligation efficiency can be affected by many factors that are not related to dsDNA looping such as the DNA structure surrounding the joined sticky ends, and ligase can also affect the apparent looping rate through mechanisms such as nonspecific binding. Here, we show how to measure dsDNA looping kinetics without ligase by detecting transient DNA loop formation by FRET (Fluorescence Resonance Energy Transfer). dsDNA molecules are constructed using a simple PCR-based protocol with a FRET pair and a biotin linker. The looping probability density known as the J factor is extracted from the looping rate and the annealing rate between two disconnected sticky ends. By testing two dsDNAs with different intrinsic curvatures, we show that the J factor is sensitive to the intrinsic shape of the dsDNA.

This study presents a detailed experimental procedure to measure looping dynamics of double-stranded DNA using single-molecule Fluorescence Resonance Energy Transfer (FRET). The protocol also describes how to extract the looping probability density called the J factor.

研究DNA环接了单分子荧光共振能量转移

Bending of double-stranded DNA (dsDNA) is associated with many important biological processes such as DNA-protein recognition and DNA packaging into ...

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.

样机小型化流式细胞仪及配套的混合微流控技术降低重力环境硬件演示

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

A Proteoliposome-Based Efflux Assay to Determine Single-molecule Properties of Cl- Channels and Transporters

The last 15 years have been characterized by an explosion in the ability to overexpress and purify membrane proteins from prokaryotic organisms as well as from eukaryotes. This increase has been largely driven by the successful push to obtain structural information on membrane proteins. However, the ability to functionally interrogate these proteins has not advanced at the same rate and is often limited to qualitative assays of limited quantitative value, thereby limiting the mechanistic insights that they can provide. An assay to quantitatively investigate the transport activity of reconstituted Cl- channels or transporters is described. The assay is based on the measure of the efflux rate of Cl- from proteoliposomes following the addition of the K+ ionophore valinomycin to shunt the membrane potential. An ion sensitive electrode is used to follow the time-course of ion efflux from proteoliposomes reconstituted with the desired protein. The method is highly suited for mechanistic studies, as it allows for the quantitative determination of key properties of the reconstituted protein, such as its unitary transport rate, the fraction of active protein and the molecular mass of the functional unit. The assay can also be utilized to determine the effect of small molecule compounds that directly inhibit/activate the reconstituted protein, as well as to test the modulatory effects of the membrane composition or lipid-modifying reagents. Where possible, direct comparison between results obtained using this method were found to be in good agreement with those obtained using electrophysiological approaches. The technique is illustrated using CLC-ec1, a CLC-type H+/Cl- exchanger, as a model system. The efflux assay can be utilized to study any Cl- conducting channel/transporter and, with minimal changes, can be adapted to study any ion-transporting protein.

A Proteoliposome-Based Efflux Assay to Determine Single-molecule Properties of Cl- Channels and Transporters

Proteoliposomes are used to study purified channels and transporters reconstituted in a well-defined biochemical environment. An experimental procedure to measure efflux mediated by these proteins is illustrated. The steps to prepare proteoliposomes, perform the recordings, and analyze data to quantitatively determine the functional properties of the reconstituted protein are described.

一个脂蛋白体为基础的外排检测，以确定氯的单分子性质<sup&gt; -</sup&gt;通道和转运

The last 15 years have been characterized by an explosion in the ability to overexpress and purify membrane proteins from prokaryotic organisms as well as ...