萨米尔哈姆丹资助这项技术的发展。 E. 大肠杆菌的蛋白质，从尼克狄克逊教授的实验室中，卧龙岗大学，和T7蛋白质，哈佛医学院教授查尔斯理查德森。工作是支持由美国国立卫生研究院（GM077248 AMvO）和简棺材尔兹基金会（JJL）。

执行单分子的复制实验之前，一些材料需要提前做好准备。 1。 DNA复制模板复制反应的底物是生物素标记，尾M13的滚动一圈，准备使用标准的分子生物学技术1,2。 材料：M13mp18单链DNA，生物素的尾巴寡核苷酸引物（5&#39; -生物素- T的36 AATTCGTAAT CATGGTCATAGCTGTTTCCT - 3“），T7 DNA聚合酶，加 ​​热块，苯酚/异戊醇/氯仿<ol><li>加入10倍，超过TBS缓冲液（330牛尾巴寡，33纳米M13）的寡核苷酸，退火的生物素标记的尾巴寡核苷酸的M13。 </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，以正确的引物退火。 </li><li>催芽M13的64纳米T7 DNA聚合酶和T7复制缓冲区含有dNTPs和MgCl 2的混合物。 </li><li>孵育反应在37 ° 12分钟，并与100毫米EDTA淬火。 </li><li>净化填充产品的DNA，酚/氯仿/异戊醇提取到TE缓冲液透析或其他合适的存储缓冲区，并确定DNA浓度（返回提取每个恢复催芽M13的任何残余的有机相）。 </li><li>一个模板制备的迭代可以很容易地使纳摩尔浓度模板的几毫升，足够几百到成千上万的单分子实验。 </li></ol> 2。官能盖玻片用于连接的DNA玻璃盖玻片，玻璃是先与氨基硅烷，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1,3。 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。超声30分钟重复洗。 </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。再次用丙酮冲洗罐。 </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30分钟的烤箱在110℃烘烤干燥的盖玻片。 </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>干在真空状态下的盖玻片和存储。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>这些材料应提前，然后使用每个实验。要启动每单分子实验，开始组装流室。 3。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1,4。 材料：双面胶带，刀片，与孔管，快干环氧石英片，官能盖玻片（见上文），链解决方案（1毫克/毫升的PBS 25μL），油管，封闭液（20毫米三pH值7.5，2毫米EDTA，50毫米氯化钠，0.2 mg / mL的BSA，0.005％Tween - 20的） <ol><li>首先在干燥器中放置封闭液20-25毫升，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。离开这个孵育20分钟，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ð磁带的石英片的大小相匹配。马克磁带上的油管用铅笔绘制孔，使流室的轮廓，可以在磁带上（1毫克/毫升）位置。 </li><li>使周围的孔2毫米宽的矩形。这将作为流动通道，所以一定要管的入口和出口直边和周围留有余地。如果需要，多渠道或不同大小都可以使用。 </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>削减的进风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。放置在某种支持暂停不易附着在接下来的步骤流细胞管。 </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，只有一面是官能;要小心不要把盖玻片。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> 4。单分子复制实验 材料：编写流室，SYTOX橙，TIRF显微镜，532 nm激光，CCD摄像头，图像采集软件的计算机，滚环DNA底物，复制蛋白<ol><li>流动池已被封锁后，一切准备就绪后开始的单分子实验。 </li><li>以流动细胞和它放在显微镜舞台上。保持阶段剪辑室的地方，并确保流道直沿Y轴定位。 </li><li>将流动池出口管注射泵使用较大直径的连接管或针。入口放置在封闭液和注射器，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> 25日下午在1毫升封闭液稀释股DNA为模板。在中等流量室的DNA，让良好的表面覆盖的流入。 0.025毫升/分钟的30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>一旦DNA中，洗出多余的DNA，用封闭液。至少有200卷流细胞洗涤除去所有的游离DNA。 </li><li>开始脱气复制缓冲区。对于的大肠杆菌大肠杆菌的复制实验，在37 ° C，以减少在激烈的流动腔泡沫的风险。 </li><li>打开激光和摄像机。冷却CCD需要达到的温度才可以使用。 </li><li>如果执行37 ° C实验，打开加热器。要确定的目标是在与流通池接触，否则将散热片后。 </li><li>洗衣机和脱气后的反应可以有所准备。混合核苷酸，数码地面电视服务，并在复制缓冲区SYTOX。然后添加蛋白质，轻轻混匀。 </li><li>反应混合物流入流通池。这种流动的速度，需要足够的伸展双链。对于一个宽2毫米，高100微米流道0.04 mL / min的作品。 </li><li>允许混合物进入会议厅，并开始成像的时候。一个好的技巧是将视野通道一侧，并专注于不干胶材料。这将确保你聚焦表面附近。 </li><li>调整激光功率，显微镜的重点和TIRF角度。保持低功耗，以避免任何染色的DNA photocleavage。几分钟取决于DNA的复制率在1-5帧/秒的收购。如果需要，重复，以获得更长的的复制轨迹或移动到一个新的领域看到更多的分子。 </li><li>反应后的混合物几乎是空白，它是一个好主意，流更多的缓冲区YTOX看到其他领域的观点。以多个不同的复制分子形象提供processivity决心的统计资料。 </li></ol> 5。代表性的成果积极复制分子很容易被视为增长染色DNA。在我们的实验中，流淌的M13基板时25给出了一个60X，125微米× 125的观点微米领域（图1）100-1000分子。执行复制实验中使用的大肠杆菌大肠杆菌复制体蛋白在37 ° C（详情请参阅材料），每5-50复制分子的视野。当量浓度的T7复制体，在基板上更有效地启动，我们观察到在一个领域复制分子的70％。这些条件会产生一个产品的密度如此之高，单个分子难以解决和分析，这样的T7的实验是在较低的蛋白浓度进行。 数据分析：要获得率数据，只需情节的DNA与时间的终点，并计算斜率（图2 ）。 processivity，确定总长度的DNA。这些数字都将需要转换为碱基对。在进行实验之前，你应该知道在适当的放大倍率的相机的像素大小。碱基的转换，简直是一个很好的估计基础上的DNA晶体的长度，2.9 BP /纳米转换。但是，层流不会完全伸展的DNA，因此应当由执行到一个已知长度的DNA，如48502 BPλDNA，用生物素标记的寡核苷酸，它附着到流通池和测量SYTOX染色长度的DNA长度校准复制实验所用的流量。确定的碱基对/像素数量将允许精确计算的复制率和processivities。以大量的图片和电影，将提供大量的产品分子，使密谋率和processivity分布（图2） 1。 <img src="/files/ftp_upload/1529/1529fig1.jpg" alt="图1" /> 图1：（从编号1）。）卡通的滚环复制检测。 （SA，链）。领先的链合成的延伸的尾部连接圈和表面。尾巴转换为双链DNA的滞后链聚合酶和层流捉襟见肘。 B）SYTOX Orange和532 nm激发的范例领域。小荧光点的系绳，非复制的基板。请注意产品的极端的长度和每场的产品（每个流动池包含5000等领域）的大量。 <img src="/files/ftp_upload/1529/1529fig2.jpg" alt="图2" /> 图2：（改编自文献1）。A，B）典型的复制分子Kymographs T7（A） 和 E 大肠杆菌 （二）实验。 C）a）和b）的分子端点轨迹绘制随时间变化。轨迹都配有线性回归获得复制率。所示的例子是99.4 BP s - 1和467.1 BP s - 1时。 D）复制产品的长度分布，配合单指数衰减，获得processivity：25.3 ± 1.7 KBP（T7），85.3 ± 6.1 KBP（大肠杆菌）。 E）的单分子轨道的速率分布，配合单高斯。方式：75.9 ± 4.8 BP s - 1时（T7），535.5 ± 39 BP s - 1时（ 大肠杆菌 ）。

<ol>
	<li>Tanner, N. A., 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=Real-time+single-molecule+observation+of+rolling-circle+DNA+replication.">Real-time single-molecule observation of rolling-circle DNA replication.</a> Nucleic Acids Res. 37, e27 (2009).</li><li>Tabor, S., Huber, H. E., Richardson, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Escherichia+coli+thioredoxin+confers+processivity+on+the+DNA+polymerase+activity+of+the+gene+5+protein+of+bacteriophage+t7.">Escherichia coli thioredoxin confers processivity on the DNA polymerase activity of the gene 5 protein of bacteriophage t7.</a> J Biol Chem. 262, 16212-16223 (1987).</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., van Oijen, A. M., Vengrova, S., Dalgaard, J. Z. <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> DNA replication: Methods and protocols. 521, 397-410 (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>Heller, R. C., Marians, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replication+fork+reactivation+downstream+of+a+blocked+nascent+leading+strand.">Replication fork reactivation downstream of a blocked nascent leading strand.</a> Nature. 439, 557-562 (2006).</li></ol>

一个关键控制研究的污点，SYTOX橙的效果，感兴趣的复制蛋白。一个简单的方法做，这是执行中所述的流通池，但SYTOX遗漏的复制实验。反应混合物通过室流出后，添加SYTOX缓冲区，染色的DNA，并检查复制的分子长度分布。另外，标准的散装反应，可以用来检查任何SYTOX复制速度和效率的影响。 这里所描述的实验中只使用一个单一的流道。这是可以改变的，很容易通过建立多渠道的流量商会或采用硅橡胶或类似的微流体装置。增加频道数量，大大有利于蛋白质的浓度，突变体，或抑制剂分子的筛选和增加了复制数据收集的速度。 如上所述，我们执行的E.大肠杆菌复制实验在37 ° C使用一个自制的铝流细胞加热器，电阻加热元件（墨盒热水器）和可变电源。这使良好的温度稳定性和避免购买一个客观的加热器。要校准加热器，我们只需在石英流通池的顶部中心钻了一个洞，流道插入热电偶和流动是正常的缓冲区。在增加电压的流通池缓冲区的温度测量，可以实现精确的加热。

T7启动复制 ：40毫米的Tris pH值7.5，50毫米钾谷氨酸，10毫米氯化镁，100微克/毫升BSA，5毫米二硫苏糖醇，600μMdNTPs，ATP的300微米，300微米的CTP，和15纳米SYTOX橙之前，立即加入使用。蛋白质补充：5纳米GP4（六聚体），聚合酶（1:1 GP5：硫氧还蛋白）40纳米，360纳米gp2.5 1,5。 大肠杆菌复制：立即使用前的50 mM HEPES pH值7.9，12毫米醋酸镁，氯化钾80毫米，100微克/毫升BSA 10毫米二硫苏糖醇，dNTPs 40微米，200μMrNTPs，15纳米SYTOX橙补充。蛋白补充说：αεθ，15纳米30纳米DnaB（六聚体）30纳米，180纳米DNAC（单体），τ2γ1δδ&#39;χψ或τ3δδ&#39;χψ，β（二聚体）30纳米，300纳米DnaG，250纳米单边带（四聚体），20纳米PRIA，40纳米PriB，320纳米PriC，480纳米DNAT 1,6

visualizing single-molecule dna replication with fluorescence microscopy

我们描述了一个简单的基于荧光显微镜实时的方法，在单分子水平上观察DNA的复制。一个圆形，分叉的DNA为模板，连接到一个官能的玻璃盖玻片和广泛复制后，引进复制的蛋白质和核苷酸（图1）。增长的产品双链DNA（dsDNA）的扩展与层流，通过使用嵌入染料的可视化。测量实时日益增长的DNA末端的位置，允许复制率精确测定（图2）。此外，完成DNA产品的长度报告上的复制processivity。这个实验可以很容易和迅速地执行，只需要一个相当敏感的相机的荧光显微镜。

Watch this Scientific Journal Video about Visualizing Single-molecule DNA Replication with Fluorescence Microscopy at JoVE.com

Visualizing Single-molecule DNA Replication with Fluorescence Microscopy

此协议表明一个简单的可视化实时个人replisomes的DNA复制的单分子荧光显微镜技术。

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 ...

visualizing-single-molecule-dna-replication-with-fluorescence

Research

JoVE Journal

Biology

22.3K 次观看.  Harvard Medical School. 此协议表明一个简单的可视化实时个人replisomes的DNA复制的单分子荧光显微镜技术。

视频: Visualizing Single-molecule DNA Replication with Fluorescence Microscopy

Visualizing Single Molecular Complexes In Vivo Using Advanced Fluorescence Microscopy

Full insight into the mechanisms of living cells can be achieved only by investigating the key processes that elicit and direct events at a cellular level. To date the shear complexity of biological systems has caused precise single-molecule experimentation to be far too demanding, instead focusing on studies of single systems using relatively crude bulk ensemble-average measurements. However, many important processes occur in the living cell at the level of just one or a few molecules; ensemble measurements generally mask the stochastic and heterogeneous nature of these events. Here, using advanced optical microscopy and analytical image analysis tools we demonstrate how to monitor proteins within a single living bacterial cell to a precision of single molecules and how we can observe dynamics within molecular complexes in functioning biological machines. The techniques are directly relevant physiologically. They are minimally-perturbative and non-invasive to the biological sample under study and are fully attuned for investigations in living material, features not readily available to other single-molecule approaches of biophysics. In addition, the biological specimens studied all produce fluorescently-tagged protein at levels which are almost identical to the unmodified cell strains ("genomic encoding"), as opposed to the more common but less ideal approach for generating significantly more protein than would occur naturally ('plasmid expression'). Thus, the actual biological samples which will be investigated are significantly closer to the natural organisms, and therefore the observations more relevant to real physiological processes.

Visualizing Single Molecular Complexes In Vivo Using Advanced Fluorescence Microscopy

Here we demonstrate the protocols for performing single-molecule fluorescence microscopy on living bacterial cells to enable functional molecular complexes to be detected, tracked and quantified.

可视化单分子络合物在体内使用高级荧光显微

Full insight into the mechanisms of living cells can be achieved only by investigating the key processes that elicit and direct events at a cellular ...

科研

生物学

Automated System for Single Molecule Fluorescence Measurements of Surface-immobilized Biomolecules

Fluorescence Resonance Energy Transfer (FRET) microscopy has been widely used to study the structure and dynamics of molecules of biological interest, such as nucleic acids and proteins. Single molecule FRET (sm-FRET) measurements on immobilized molecules permit long observations of the system -effectively until both dyes photobleach- resulting in time-traces that report on biomolecular dynamics with a broad range of timescales from milliseconds to minutes. To facilitate the acquisition of large number of traces for statistical analyses, the process must be automated and the sample environment should be tightly controlled over the entire measurement time (~12 hours). This is accomplished using an automated scanning confocal microscope that allows the interrogation of thousands of single molecules overnight, and a microfluidic cell that permits the controlled exchange of buffer, with restricted oxygen content and maintains a constant temperature throughout the entire measuring period. Here we show how to assemble the microfluidic device and how to activate its surface for DNA immobilization. Then we explain how to prepare a buffer to maximize the photostability and lifetime of the fluorophores. Finally, we show the steps involved in preparing the setup for the automated acquisition of time-resolved single molecule FRET traces of DNA molecules.

In this article we describe how we obtain FRET traces from individual DNA molecules immobilized to a surface using an automated scanning confocal microscope.

表面固定生物分子的单分子荧光检测的自动化系统

Fluorescence Resonance Energy Transfer (FRET) microscopy has been widely used to study the structure and dynamics of molecules of biological interest, ...

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.

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. ...

Time-lapse Imaging of Mitosis After siRNA Transfection

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and cellular content. Hallmark events of mitosis, such as chromosome movement, can be readily tracked on an individual cell basis using time-lapse fluorescence microscopy of mammalian cell lines expressing specific GFP-tagged proteins. In combination with RNAi-based depletion, this can be a powerful method for pinpointing the stage(s) of mitosis where defects occur after levels of a particular protein have been lowered. In this protocol, we present a basic method for assessing the effect of depleting a potential mitotic regulatory protein on the timing of mitosis. Cells are transfected with siRNA, placed in a stage-top incubation chamber, and imaged using an automated fluorescence microscope. We describe how to use software to set up a time-lapse experiment, how to process the image sequences to make either still-image montages or movies, and how to quantify and analyze the timing of mitotic stages using a cell-line expressing mCherry-tagged histone H2B. Finally, we discuss important considerations for designing a time-lapse experiment. This strategy is complementary to other approaches and offers the advantages of 1) sensitivity to changes in kinetics that might not be observed when looking at cells as a population and 2) analysis of mitosis without the need to synchronize the cell cycle using drug treatments. The visual information from such imaging experiments not only allows the sub-stages of mitosis to be assessed, but can also provide unexpected insight that would not be apparent from cell cycle analysis by FACS.

Here we describe a basic protocol to image and quantify the mitotic timing of live mammalian tissue culture cells after siRNA transfection.

有丝分裂的siRNA转染后的时间推移成像

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and ...

Cell Electrofusion Visualized with Fluorescence Microscopy

Cell electrofusion is a safe, non-viral and non-chemical method that can be used for preparing hybrid cells for human therapy. Electrofusion involves application of short high-voltage electric pulses to cells that are in close contact. Application of short, high-voltage electric pulses causes destabilization of cell plasma membranes. Destabilized membranes are more permeable for different molecules and also prone to fusion with any neighboring destabilized membranes. Electrofusion is thus a convenient method to achieve a non-specific fusion of very different cells in vitro. In order to obtain fusion, cell membranes, destabilized by electric field, must be in a close contact to allow merging of their lipid bilayers and consequently their cytoplasm. In this video, we demonstrate efficient electrofusion of cells in vitro by means of modified adherence method. In this method, cells are allowed to attach only slightly to the surface of the well, so that medium can be exchanged and cells still preserve their spherical shape. Fusion visualization is assessed by pre-labeling of the cytoplasm of cells with different fluorescent cell tracker dyes; half of the cells are labeled with orange CMRA and the other half with green CMFDA. Fusion yield is determined as the number of dually fluorescent cells divided with the number of all cells multiplied by two.

In this video we demonstrate efficient electrofusion of cells in vitro by means of modified adherence method using electroporation and the subsequent detection of fused cells visualization with fluorescence microscopy.

荧光显微镜下细胞电融合的可视化

Cell electrofusion is a safe, non-viral and non-chemical method that can be used for preparing hybrid cells for human therapy. Electrofusion involves ...

Monitoring Plasmid Replication in Live Mammalian Cells over Multiple Generations by Fluorescence Microscopy

Few naturally-occurring plasmids are maintained in mammalian cells. Among these are genomes of gamma-herpesviruses, including Epstein-Barr virus (EBV) and Kaposi's Sarcoma-associated herpesvirus (KSHV), which cause multiple human malignancies 1-3. These two genomes are replicated in a licensed manner, each using a single viral protein and cellular replication machinery, and are passed to daughter cells during cell division despite their lacking traditional centromeres 4-8.
Much work has been done to characterize the replications of these plasmid genomes using methods such as Southern blotting and fluorescence in situ hybridization (FISH). These methods are limited, though. Quantitative PCR and Southern blots provide information about the average number of plasmids per cell in a population of cells. FISH is a single-cell assay that reveals both the average number and the distribution of plasmids per cell in the population of cells but is static, allowing no information about the parent or progeny of the examined cell.
Here, we describe a method for visualizing plasmids in live cells. This method is based on the binding of a fluorescently tagged lactose repressor protein to multiple sites in the plasmid of interest 9. The DNA of interest is engineered to include approximately 250 tandem repeats of the lactose operator (LacO) sequence. LacO is specifically bound by the lactose repressor protein (LacI), which can be fused to a fluorescent protein. The fusion protein can either be expressed from the engineered plasmid or introduced by a retroviral vector. In this way, the DNA molecules are fluorescently tagged and therefore become visible via fluorescence microscopy. The fusion protein is blocked from binding the plasmid DNA by culturing cells in the presence of IPTG until the plasmids are ready to be viewed.
This system allows the plasmids to be monitored in living cells through several generations, revealing properties of their synthesis and partitioning to daughter cells. Ideal cells are adherent, easily transfected, and have large nuclei. This technique has been used to determine that 84% of EBV-derived plasmids are synthesized each generation and 88% of the newly synthesized plasmids partition faithfully to daughter cells in HeLa cells. Pairs of these EBV plasmids were seen to be tethered to or associated with sister chromatids after their synthesis in S-phase until they were seen to separate as the sister chromatids separated in Anaphase10. The method is currently being used to study replication of KSHV genomes in HeLa cells and SLK cells. HeLa cells are immortalized human epithelial cells, and SLK cells are immortalized human endothelial cells. Though SLK cells were originally derived from a KSHV lesion, neither the HeLa nor SLK cell line naturally harbors KSHV genomes11. In addition to studying viral replication, this visualization technique can be used to investigate the effects of the addition, removal, or mutation of various DNA sequence elements on synthesis, localization, and partitioning of other recombinant plasmid DNAs.

A method of observing individual DNA molecules in live cells is described. The technique is based on the binding of a fluorescently tagged lac repressor protein to binding sites engineered into the DNA of interest. This method can be adapted to follow many recombinant DNAs in live cells over time.

在Live哺乳动物细胞中质粒复制多代荧光显微镜监测

Few naturally-occurring plasmids are maintained in mammalian cells.  Among these are genomes of gamma-herpesviruses, including Epstein-Barr virus  (EBV) ...

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 ...

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 ...

Visualizing Stromule Frequency with Fluorescence Microscopy

Stromules, or "stroma-filled tubules", are narrow, tubular extensions from the surface of the chloroplast that are universally observed in plant cells but whose functions remain mysterious. Alongside growing attention on the role of chloroplasts in coordinating plant responses to stress, interest in stromules and their relationship to chloroplast signaling dynamics has increased in recent years, aided by advances in fluorescence microscopy and protein fluorophores that allow for rapid, accurate visualization of stromule dynamics. Here, we provide detailed protocols to assay stromule frequency in the epidermal chloroplasts of Nicotiana benthamiana, an excellent model system for investigating chloroplast stromule biology. We also provide methods for visualizing chloroplast stromules in vitro by extracting chloroplasts from leaves. Finally, we outline sampling strategies and statistical approaches to analyze differences in stromule frequencies in response to stimuli, such as environmental stress, chemical treatments, or gene silencing. Researchers can use these protocols as a starting point to develop new methods for innovative experiments to explore how and why chloroplasts make stromules.

Protocols to investigate the dynamics of chloroplast stromules, the stroma-filled tubules that extend from the surface of chloroplasts, are described.

可视化Stromule频率与荧光显微镜

Stromules, or "stroma-filled tubules", are narrow, tubular extensions from the surface of the chloroplast that are universally observed in plant cells but ...

Method for Labeling Transcripts in Individual Escherichia coli Cells for Single-molecule Fluorescence In Situ Hybridization Experiments

A method is described for labeling individual messenger RNA (mRNA) transcripts in fixed bacteria for use in single-molecule fluorescence in situ hybridization (smFISH) experiments in E. coli. smFISH allows the measurement of cell-to-cell variability in mRNA copy number of genes of interest, as well as the subcellular location of the transcripts. The main steps involved are fixation of the bacterial cell culture, permeabilization of cell membranes, and hybridization of the target transcripts with sets of commercially available short fluorescently-labeled oligonucleotide probes. smFISH can allow the imaging of the transcripts of multiple genes in the same cell, with limitations imposed by the spectral overlap between different fluorescent markers. Following completion of the protocol illustrated below, cells can be readily imaged using a microscope coupled with a camera suitable for low-intensity fluorescence. These images, together with cell contours obtained from segmentation of phase contrast frames, or from cell membrane staining, allow the calculation of the mRNA copy number distribution of a sample of cells using open-source or custom-written software. The labeling method described here can also be applied to image transcripts with stochastic optical reconstruction microscopy (STORM).

Method for Labeling Transcripts in Individual Escherichia coli Cells for Single-molecule Fluorescence In Situ Hybridization Experiments

This manuscript describes a method for labeling individual messenger RNA (mRNA) transcripts with fluorescently-labeled DNA probes, for use in single-molecule fluorescence in situ hybridization (smFISH) experiments in E. coli. smFISH is a visualization method that allows the simultaneous detection, localization, and quantification of single mRNA molecules in fixed individual cells.