我们感谢博士T. Helleday和大肠杆菌彼得曼博士与纤维的技术，以及有益的讨论实验室成员的帮助。 WN是由一名高级国际研究，国际癌症研究协会奖学金和科学研究的补助金（N301 165935）波兰国家委员会的支持。

在这里介绍的方法是使用下列出版物:Schwab等，EMBO J.，29（4）:806 - 18 5。 1。 DT40细胞培养 材料:胎牛血清（FBS），，鸡血清，青霉素/链霉素，2 -巯基乙醇的RPMI <ol><li>准备细胞培养液中添加7％FBS，3％的鸡血清，1个青霉素/链霉素和10微米2 - 巯基乙醇的RPMI培养基。 </li><li> DT40细胞生长在无菌细胞培养瓶中，在38 ° C和5％CO 2在培养箱。拆分单元格，每天密度5 × 10 5细胞/ ml 6。 </li></ol> 2， 在体内的标签 材料:IDU，CldU <ol><li>准备核苷酸类似物的股票解决方案:解散IDU在中等至2.5毫米至5毫米和CldU。热两种解决方案简要地60 ° C和旋涡，直到核苷酸类似物UES完全溶解。 </li><li> IDU添加到终浓度为25微米成倍增长DT40细胞和混合细胞悬液。细胞孵育20分钟，38 ° C和5％CO 2 。 </li><li>第一个标签的潜伏期后，添加CldU到终浓度为250微米，并视为在上一步中所描述的细胞。 </li><li>与冰冷的PBS清洗细胞，在浓度7.5 × 10 5细胞/ ml的冷PBS重悬。标记的细胞置于冰上。 </li></ol>标签过程仅仅是一个建议，可以进行修改，以解决具体问题。实验设计的更多信息，请参阅讨论部分。 3。细胞裂解和DNA的蔓延 材料:载玻片，纤维裂解液<ol><li>准备纤维裂解液:在200毫米的Tris - HCl，pH 7.5的50 mM的EDTA，0.5％SDS </li><liSPOT 2μL的细胞悬液到载玻片的一端。空气干燥5分钟或直到下降的体积大大减少，但不干燥。 </li><li>吸取7μL的细胞悬液上裂解液，轻轻搅动枪头混合的解决方案。孵育细胞裂解进行2分钟。 </li><li> 15 °倾斜的幻灯片，让纤维传播沿着幻灯片。 </li><li>一旦光纤解决方案已经达到底部的幻灯片，水平放置风干。干燥后，薄，不透明的线应沿幻灯片可见。此时，应开始拉伸纤维染色线将不可见，再后用铅笔标​​记。此标记稍后将有助于找到的纤维在显微镜下。 </li></ol> 4。免疫荧光染色 材料:甲醇，醋酸，盐酸，5％BSA的PBS，染色缸，抗BrdU抗体（鼠），抗BrdU抗体（鼠），羊抗鼠Cy3标记抗体，羊抗鼠的Alexa Fluor 488抗体，Vectashield安装介质，盖玻片，指甲油<ol><li>沉浸在甲醇/醋酸（3:1）在染色缸和10分钟的孵育幻灯片。 </li><li>在蒸馏水中H 2 O的幻灯片，然后在2.5 M盐酸浸泡80分钟洗净。 </li><li> 5分钟，洗净幻灯片在PBS的3倍。 </li><li>从染色缸中删除的幻灯片，并用纸巾收集多余的PBS。水平放置幻灯片和吸管5％BSA的每张幻灯片上。轻轻盖上盖玻片的幻灯片，传播的BSA均匀地分布在幻灯片和孵育20分钟。 </li><li>淡化在5％BSA在以下浓度的主要抗体:1​​:25抗BrdU（鼠标）和1:400抗BrdU（大鼠）。 </li><li>向下移动载玻片盖玻片，轻轻将其删除。不要使用武力，如果盖玻片坚持幻灯片。在PBS直到盖玻片变得松散一个幻灯片可水化D可以轻松删除。用纸巾收集多余的BSA和横向放置幻灯片。吸取50μL抗体溶液到每个幻灯片。盖上盖玻片，再次传播的抗体溶液均匀地分布在幻灯片和在2个小时的湿盒孵育。 </li><li>取出的盖玻片后，洗的幻灯片，在PBS三次5分钟</li><li>以下浓度稀释在5％BSA的二级抗体:1:500羊抗小鼠Cy3和1:400羊抗鼠的Alexa Fluor 488。 </li><li>应用50μL二次抗体的主要抗体所述的解决方案。防止光线和孵化1小时的幻灯片。 </li><li>取出的盖玻片后，5分钟洗幻灯片在PBS的3倍。 </li><li>添加到每个幻灯片下降Vectashield安装介质和应用盖玻片。轻压盖玻片，并围绕它用纸巾去除多余的液体。与透明的指甲城邦密封盖玻片H和让他们干。存放于-20 ° C的幻灯片</li></ol> 5。图像采集 材料:荧光显微镜，相机放置浸油下降到接近铅笔标记的幻灯片，并开始定位的纤维。通常情况下，有一个主要的纤维束，但这些纤维太纠结，以后不能分析。离主束纤维明确由对方（图2）分离领域。我们将选择的图片，只用一个颜色通道，以避免偏见。然后，我们将采取约10每个时间点，浓度或细胞株的照片。然而，图片的数量取决于多少纤维可以计算每一张图片上。沿着采取不同的图片幻灯片，幻灯片的一个区域可能无法提供有代表性的纤维长度或复制结构。 6。数据分析材料:图片分析程序，例如:ImageJ <a href="http://rsbweb.nih.gov/ij/" target="_blank">（</a> http://rsbweb.nih.gov/ij/ ） 导入的图片分析程序图片。测量长度的纤维束和/或计数不同的复制结构（图3）。只算纤维清晰可见，不扩展到画面边缘。我们通常约100纤维长度测量和计数150-200复制结构，我们将至少三次重复个别实验。 7。代表性的成果: 新复制的DNA可以可视化的抗体标记的核苷酸类似物线。在我们的实验正在进行叉代表相邻的红色和绿色信号（图3）。双标协议还允许我们定义的复制结构的四个主要类别:1）新的启动事件可以divid<html>到发射的起源，而细胞培养与第一个标签，并在第二个标签的潜伏期发射的起源。前者包括邻近的绿 - 红 - 绿信号，后者只有一条绿线。 2）终止事件表现为毗邻的红，绿，红色信号。 3）穿插的起源是在过密的起源基因组中的网站。这类网站包括连续的起源和终止信号。 4）根据实验设计，停顿/倒塌叉可以定义为一个红色的7只信号或一条红线很短的绿道8,9。 这里所描述的条件，野生型DT40细胞中有一个叉速度平均为0.4微米/分钟。我们可以探测到约63％正在进行的叉子，10％起源，16％陷入僵局叉（红色大片），8％终端和3％穿插纤维。 <html>余吕杏茜1"/&gt; 。图1（a）左侧的卡通描绘的DNA标记程序的第一步:加入IDU指数增长DT40细胞，导致容易被纳入到新合成的领先和落后的DNA链的核苷酸类似物。这可以通过使用特定的抗体进行可视化，并在显微镜下观察时，会出现一条红线。随后，同样的过程重复与CldU上图的右侧所示。第二个核苷酸类似物的潜伏期后，积极叉将作为毗邻的红，绿道可见。纤维平均长度是核苷酸类似物孵育时间成正比。 （二）从裂解DT40细胞的DNA是由重力拉伸到载玻片和注册成立的核苷酸类似物是利用特异性抗体和荧光显微镜观察。 igure 2"/&gt; 图2代表的荧光图像显示了从野生型DT40随后与IDU和CldU标记，每次20分钟的细胞纤维。纤维大多是彼此分开，因此可以很容易地分析。白条代表10微米。 <img src="/files/ftp_upload/3255/3255fig3.jpg" alt="图3" /> 图3双标纤维技术允许一个区分不同的复制结构; 1 -积极复制叉，2 -新网站的DNA复制（发射新的起源），3 -叉终止（两个汇聚成叉），4 -穿插纤维间隔紧密的起源（站点）和5 - 停滞叉（红色信号）。

<ol>
	<li>Caldwell, R. B., Fiedler, P., Schoetz, U., Buerstedde, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+function+analysis+using+the+chicken+B-cell+line+DT40.">Gene function analysis using the chicken B-cell line DT40.</a> Methods. Mol. Biol. 408, 193-210 (2007).</li><li>Petes, T. D., Williamson, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fiber+autoradiography+of+replicating+yeast+DNA.">Fiber autoradiography of replicating yeast DNA.</a> Exp. Cell. Res. 95, 103-110 (1975).</li><li>Takeuchi, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+frequency+of+initiation+sites+of+DNA+replication+in+Werner's+syndrome+cells.">Altered frequency of initiation sites of DNA replication in Werner's syndrome cells.</a> Hum. Genet. 60, 365-368 (1982).</li><li>Jackson, D. A., Pombo, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replicon+clusters+are+stable+units+of+chromosome+structure:+evidence+that+nuclear+organization+contributes+to+the+efficient+activation+and+propagation+of+S+phase+in+human+cells.">Replicon clusters are stable units of chromosome structure: evidence that nuclear organization contributes to the efficient activation and propagation of S phase in human cells.</a> J. Cell. Biol. 140, 1285-1295 (1998).</li><li>Schwab, R. A., Blackford, A. N., Niedzwiedz, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ATR+activation+and+replication+fork+restart+are+defective+in+FANCM-deficient+cells.">ATR activation and replication fork restart are defective in FANCM-deficient cells.</a> EMBO. J. 29, 806-818 (2010).</li><li>Saribasak, H., Arakawa, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basic+cell+culture+conditions.">Basic cell culture conditions.</a> Subcell. Biochem. 40, 345-346 (2006).</li><li>Conti, C., Seiler, J. A., Pommier, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mammalian+DNA+replication+elongation+checkpoint:+implication+of+Chk1+and+relationship+with+origin+firing+as+determined+by+single+DNA+molecule+and+single+cell+analyses.">The mammalian DNA replication elongation checkpoint: implication of Chk1 and relationship with origin firing as determined by single DNA molecule and single cell analyses.</a> Cell. Cycle. 6, 2760-2767 (2007).</li><li>Petermann, E., Orta, M. L., Issaeva, N., Schultz, N., Helleday, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydroxyurea-stalled+replication+forks+become+progressively+inactivated+and+require+two+different+RAD51-mediated+pathways+for+restart+and+repair.">Hydroxyurea-stalled replication forks become progressively inactivated and require two different RAD51-mediated pathways for restart and repair.</a> Mol. Cell. 37, 492-502 (2010).</li><li>Edmunds, C. E., Simpson, L. J., Sale, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PCNA+ubiquitination+and+REV1+define+temporally+distinct+mechanisms+for+controlling+translesion+synthesis+in+the+avian+cell+line+DT40.">PCNA ubiquitination and REV1 define temporally distinct mechanisms for controlling translesion synthesis in the avian cell line DT40.</a> Mol. Cell. 30, 519-529 (2008).</li></ol>

没有利益冲突的声明。

我们描述了一种方法，允许参数的影响在单分子水平的S期DNA合成进行定量分析。在过去的十多年中，不同版本的DNA纤维fluorography技术开发的可视化的活细胞内的个体复制叉的运动。在所有这些技术的关键参数是用于实现提供良好的分离DNA纤维的玻璃表面上的DNA最好的伸展的程序。实现这一目标的一个关键步骤是显微镜幻灯片，以及溶解时间的细胞悬液的孵化时间。这些参数依赖于一个特定的实验室的温度和气流，并应凭经验确定。 DNA纤维实验的实践部分通常是在一天之内完成。然而，随后的图像采集和分析是更费时，需要实践。 标签协议可以广告艾普特解答各种科学问题:使用在第二个脉冲标记的干扰与复制代理显示器叉伸长/复制强调拖延。在这种情况下，第一个标签不需要洗第二个核苷酸是添加过量。另外，阻碍复制的药物，可以组合使用，或只是除了第一个标签后。这就要求在第一个标签，并应用到删除前的第二个核苷酸类似物药物。此过程允许一个确定的各种DNA修复因子的复制叉稳定性/复制压力的条件下（即叉拖延和/或折叠）复苏的重要性。值得注意的是，一个更详细的分析，DNA复制计划的详情，可以使用DNA的梳理和荧光原位杂交相结合的一种替代方法。然而，这在技术上更有挑战性的，需要昂贵sive设备。 能够定性和定量分析DNA复制的资料调查，在DNA复制本身的缺陷以及抑制基因组不稳定与复制叉相关的途径，提供了一个必不可少的工具。毫无疑问，这个实验将进一步帮助摆脱复制介导的DNA修复机制，使正常细胞响应/适应环境的变化以及癌细胞如何利用这些机制，以抵御针对复制叉的药物的毒性的光。

<table border="1"><tbody><tr><th>试剂名称</th><th>公司</th><th>目录编号</th><th>评论（可选） </th></tr><tr><td>胎牛血清金（FBS） </td><td>临时机场管理局</td><td> A15 - 151 </td><td></td></tr><tr><td>鸡血清</td><td>西格玛</td><td> C5405 </td><td></td></tr><tr><td>青霉素/链霉素</td><td>临时机场管理局</td><td> P11 - 010 </td><td></td></tr><tr><td> RPMI 1640培养</td><td> Gibco公司</td><td> 21875 </td><td></td></tr><tr><td> 5 - 碘-2&#39; - 脱氧尿苷（IDU） </td><td> Sigma - Aldrich公司</td><td> I7125 </td><td></td></tr><tr><td> 5 - 氯-2&#39; - 脱氧尿苷（CldU） </td><td>西格玛</td><td> C6891 </td><td></td></tr><tr><td>显微镜载玻片SuperFrost </td><td> VWR </td><td> 631-0910 </td><td></td></tr><tr><td>盖玻片1号</td><td>科学实验室suppplies </td><td> MIC3234 </td><td></td></tr><tr><td> Vectashield安装介质</td><td> H - 1000矢量实验室</td><td> H - 1000 </td><td></td></tr><tr><td>抗BrdU抗体（鼠） </td><td> BD公司</td><td> 347580 </td><td></td></tr><tr><td>抗BrdU抗体（鼠） </td><td> Abcam公司</td><td> ab6326 </td><td></td></tr><tr><td>羊抗小鼠Cy3标记</td><td>西格玛</td><td> C2181 </td><td></td></tr><tr><td>山羊抗大鼠的Alexa Fluor 488抗体</td><td> Invitrogen公司</td><td> A110060 </td><td></td></tr><tr><td></td><td></td><td></td><td></td></tr></tbody></table>

visualization of dna replication in the vertebrate model system dt40 using the dna fiber technique

维护稳定复制叉是分裂的细胞保存的可行性和预防疾病的最重要的。涉及的流程，不仅确保在面对内源性和外源性DNA损伤的忠实的基因组的重复，又要防止基因组不稳定性，一个公认的肿瘤发展的致病因素。 在这里，我们描述了一个简单的和具有成本效益的荧光显微镜为基础的方法，可视化在禽流感的B细胞DT40的DNA复制。该细胞系提供了强大的的工具，以调查在 1脊椎动物细胞的反向遗传学蛋白质在体内的功能。缺乏一种特定的基因在DT40细胞的DNA纤维fluorography允许一个解释这种基因产品在DNA复制和基因组稳定性的功能。依靠传统的方法来分析，在脊椎动物细胞复制叉动态测量人口脉冲标记细胞DNA合成的整体失业率。这是一个定量方法和定性分析影响DNA合成的参数，不允许。相反，应遵循积极叉的运动速度可以直接使用DNA纤维技术 2-4时。在这种方法中，新生的DNA标记在体内卤化核苷酸的掺入（图1A） 。随后，个别纤维拉伸到载玻片上，并标记的DNA复制大片特异性抗体染色，荧光显微镜（图1B）可视化。开始复制以及叉的方向性是由两个不同修改的类似物的连续使用。此外，双标记的方法允许参数的定量分析，在S期DNA合成的影响，即复制结构，如正在进行的和停滞的叉，复制起始密度以及叉终端。最后，实验过程中可以完成海关在一天之内，只需要一般的实验室设备和荧光显微镜。

Watch this Scientific Journal Video about Visualization of DNA Replication in the Vertebrate Model System DT40 using the DNA Fiber Technique at JoVE.com

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

DT40，模型脊椎动物的遗传系统，提供了强大的工具来分析蛋白质的功能。在这里，我们描述了一个简单的方法，允许在DT40细胞在单分子水平的S期DNA合成的影响参数的定性分析。

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 ...

visualization-dna-replication-vertebrate-model-system-dt40-using-dna

Research

JoVE Journal

Biology

39.7K 次观看.  University of Oxford. DT40，模型脊椎动物的遗传系统，提供了强大的工具来分析蛋白质的功能。在这里，我们描述了一个简单的方法，允许在DT40细胞在单分子水平的S期DNA合成的影响参数的定性分析。

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

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

Genetic Studies of Human DNA Repair Proteins Using Yeast as a Model System 

Understanding the roles of human DNA repair proteins in genetic pathways is a formidable challenge to many researchers. Genetic studies in mammalian systems have been limited due to the lack of readily available tools including defined mutant genetic cell lines, regulatory expression systems, and appropriate selectable markers. To circumvent these difficulties, model genetic systems in lower eukaryotes have become an attractive choice for the study of functionally conserved DNA repair proteins and pathways. We have developed a model yeast system to study the poorly defined genetic functions of the Werner syndrome helicase-nuclease (WRN) in nucleic acid metabolism. Cellular phenotypes associated with defined genetic mutant backgrounds can be investigated to clarify the cellular and molecular functions of WRN through its catalytic activities and protein interactions. The human WRN gene and associated variants, cloned into DNA plasmids for expression in yeast, can be placed under the control of a regulatory plasmid element. The expression construct can then be transformed into the appropriate yeast mutant background, and genetic function assayed by a variety of methodologies. Using this approach, we determined that WRN, like its related RecQ family members BLM and Sgs1, operates in a Top3-dependent pathway that is likely to be important for genomic stability. This is described in our recent publication [1] at <a href="http://www.impactaging.com">www.impactaging.com</a>. Detailed methods of specific assays for genetic complementation studies in yeast are provided in this paper.

Genetic Studies of Human DNA Repair Proteins Using Yeast as a Model System

Genetic studies in yeast can be employed to investigate the molecular and cellular functions of human genes in cellular DNA metabolism. Methods are described for the genetic characterization of the human WRN gene product defective in the premature aging disorder Werner syndrome in functionally conserved pathways using yeast as a tractable model system.

人类DNA修复蛋白的遗传研究利用酵母作为模型系统

Understanding the roles of human DNA repair proteins in genetic pathways is a formidable challenge to many researchers.  Genetic studies in mammalian ...

Visualization of Caenorhabditis elegans Cuticular Structures Using the Lipophilic Vital Dye DiI

The cuticle of C. elegans is a highly resistant structure that surrounds the exterior of the animal1-4. The cuticle not only protects the animal from the environment, but also determines body shape and plays a role in motility4-6. Several layers secreted by epidermal cells comprise the cuticle, including an outermost lipid layer7.
Circumferential ridges in the cuticle called annuli pattern the length of the animal and are present during all stages of development8. Alae are longitudinal ridges that are present during specific stages of development, including L1, dauer, and adult stages2,9. Mutations in genes that affect cuticular collagen organization can alter cuticular structure and animal body morphology5,6,10,11. While cuticular imaging using compound microscopy with DIC optics is possible, current methods that highlight cuticular structures include fluorescent transgene expression12, antibody staining13, and electron microscopy1. Labeled wheat germ agglutinin (WGA) has also been used to visualize cuticular glycoproteins, but is limited in resolving finer cuticular structures14. Staining of cuticular surface using fluorescent dye has been observed, but never characterized in detail15. We present a method to visualize cuticle in live C. elegans using the red fluorescent lipophilic dye DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate), which is commonly used in C. elegans to visualize environmentally exposed neurons. This optimized protocol for DiI staining is a simple, robust method for high resolution fluorescent visualization of annuli, alae, vulva, male tail, and hermaphrodite tail spike in C. elegans.

Visualization of Caenorhabditis elegans Cuticular Structures Using the Lipophilic Vital Dye DiI

We present a method to visualize cuticle in live C. elegans using the red fluorescent lipophilic dye DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate), which is commonly used in C. elegans to visualize environmentally exposed neurons. With this optimized protocol, alae and annular cuticular structures are stained by DiI and observed using compound microscopy.

可视化线虫角质层结构的亲脂性的重要染料，直接投资收益

The cuticle of C. elegans is a highly resistant structure that surrounds the exterior of the animal1-4. The cuticle not only protects the animal from the ...

Selective Capture of 5-hydroxymethylcytosine from Genomic DNA

5-methylcytosine (5-mC) constitutes ~2-8% of the total cytosines in human genomic DNA and impacts a broad range of biological functions, including gene expression, maintenance of genome integrity, parental imprinting, X-chromosome inactivation, regulation of development, aging, and cancer1. Recently, the presence of an oxidized 5-mC, 5-hydroxymethylcytosine (5-hmC), was discovered in mammalian cells, in particular in embryonic stem (ES) cells and neuronal cells2-4. 5-hmC is generated by oxidation of 5-mC catalyzed by TET family iron (II)/&alpha;-ketoglutarate-dependent dioxygenases2, 3. 5-hmC is proposed to be involved in the maintenance of embryonic stem (mES) cell, normal hematopoiesis and malignancies, and zygote development2, 5-10. To better understand the function of 5-hmC, a reliable and straightforward sequencing system is essential. Traditional bisulfite sequencing cannot distinguish 5-hmC from 5-mC11. To unravel the biology of 5-hmC, we have developed a highly efficient and selective chemical approach to label and capture 5-hmC, taking advantage of a bacteriophage enzyme that adds a glucose moiety to 5-hmC specifically12.
Here we describe a straightforward two-step procedure for selective chemical labeling of 5-hmC. In the first labeling step, 5-hmC in genomic DNA is labeled with a 6-azide-glucose catalyzed by &beta;-GT, a glucosyltransferase from T4 bacteriophage, in a way that transfers the 6-azide-glucose to 5-hmC from the modified cofactor, UDP-6-N3-Glc (6-N3UDPG). In the second step, biotinylation, a disulfide biotin linker is attached to the azide group by click chemistry. Both steps are highly specific and efficient, leading to complete labeling regardless of the abundance of 5-hmC in genomic regions and giving extremely low background. Following biotinylation of 5-hmC, the 5-hmC-containing DNA fragments are then selectively captured using streptavidin beads in a density-independent manner. The resulting 5-hmC-enriched DNA fragments could be used for downstream analyses, including next-generation sequencing.
Our selective labeling and capture protocol confers high sensitivity, applicable to any source of genomic DNA with variable/diverse 5-hmC abundances. Although the main purpose of this protocol is its downstream application (i.e., next-generation sequencing to map out the 5-hmC distribution in genome), it is compatible with single-molecule, real-time SMRT (DNA) sequencing, which is capable of delivering single-base resolution sequencing of 5-hmC.

Described is a two-step labeling process using &beta;-glucosyltransferase (&beta;-GT) to transfer an azide-glucose to 5-hmC, followed by click chemistry to transfer a biotin linker for easy and density-independent enrichment. This efficient and specific labeling method enables enrichment of 5-hmC with extremely low background and high-throughput epigenomic mapping via next-generation sequencing.

从基因组DNA选择性捕获的5  - 羟甲基

5-methylcytosine (5-mC) constitutes ~2-8% of the total cytosines in human genomic DNA and impacts a broad range of biological functions, including gene ...

Study of the DNA Damage Checkpoint using Xenopus Egg Extracts

On a daily basis, cells are subjected to a variety of endogenous and environmental insults. To combat these insults, cells have evolved DNA damage checkpoint signaling as a surveillance mechanism to sense DNA damage and direct cellular responses to DNA damage. There are several groups of proteins called sensors, transducers and effectors involved in DNA damage checkpoint signaling (Figure 1). In this complex signaling pathway, ATR (ATM and Rad3-related) is one of the major kinases that can respond to DNA damage and replication stress. Activated ATR can phosphorylate its downstream substrates such as Chk1 (Checkpoint kinase 1). Consequently, phosphorylated and activated Chk1 leads to many downstream effects in the DNA damage checkpoint including cell cycle arrest, transcription activation, DNA damage repair, and apoptosis or senescence (Figure 1). When DNA is damaged, failing to activate the DNA damage checkpoint results in unrepaired damage and, subsequently, genomic instability. The study of the DNA damage checkpoint will elucidate how cells maintain genomic integrity and provide a better understanding of how human diseases, such as cancer, develop.
Xenopus laevis egg extracts are emerging as a powerful cell-free extract model system in DNA damage checkpoint research. Low-speed extract (LSE) was initially described by the Masui group1. The addition of demembranated sperm chromatin to LSE results in nuclei formation where DNA is replicated in a semiconservative fashion once per cell cycle.
The ATR/Chk1-mediated checkpoint signaling pathway is triggered by DNA damage or replication stress 2. Two methods are currently used to induce the DNA damage checkpoint: DNA damaging approaches and DNA damage-mimicking structures 3. DNA damage can be induced by ultraviolet (UV) irradiation, &gamma;-irradiation, methyl methanesulfonate (MMS), mitomycin C (MMC), 4-nitroquinoline-1-oxide (4-NQO), or aphidicolin3, 4. MMS is an alkylating agent that inhibits DNA replication and activates the ATR/Chk1-mediated DNA damage checkpoint 4-7. UV irradiation also triggers the ATR/Chk1-dependent DNA damage checkpoint 8. The DNA damage-mimicking structure AT70 is an annealed complex of two oligonucleotides poly-(dA)70 and poly-(dT)70. The AT70 system was developed in Bill Dunphy's laboratory and is widely used to induce ATR/Chk1 checkpoint signaling 9-12.
Here, we describe protocols (1) to prepare cell-free egg extracts (LSE), (2) to treat Xenopus sperm chromatin with two different DNA damaging approaches (MMS and UV), (3) to prepare the DNA damage-mimicking structure AT70, and (4) to trigger the ATR/Chk1-mediated DNA damage checkpoint in LSE with damaged sperm chromatin or a DNA damage-mimicking structure.

Study of the DNA Damage Checkpoint using Xenopus Egg Extracts

Xenopus egg extract is a useful model system to investigate the DNA damage checkpoint. This protocol is for the preparation of Xenopus egg extracts and DNA damage checkpoint inducing reagents. These techniques are adaptable to a variety of DNA damaging approaches in the study of the DNA damage checkpoint signaling.

研究DNA损伤检验点<em&gt;爪蟾</em&gt;卵提取物

On a daily basis, cells are subjected to a variety of endogenous and environmental insults. To combat these insults, cells have evolved DNA damage ...

Robust 3D DNA FISH Using Directly Labeled Probes

3D DNA FISH has become a major tool for analyzing three-dimensional organization of the nucleus, and several variations of the technique have been published. In this article we describe a protocol which has been optimized for robustness, reproducibility, and ease of use. Brightly fluorescent directly labeled probes are generated by nick-translation with amino-allyldUTP followed by chemical coupling of the dye. 3D DNA FISH is performed using a freeze-thaw step for cell permeabilization and a heating step for simultaneous denaturation of probe and nuclear DNA. The protocol is applicable to a range of cell types and a variety of probes (BACs, plasmids, fosmids, or Whole Chromosome Paints) and allows for high-throughput automated imaging. With this method we routinely investigate nuclear localization of up to three chromosomal regions.

We describe a robust and versatile protocol for analyzing nuclear architecture by 3D DNA FISH using directly labeled fluorescent probes.

强大的3D直接标记的探针DNA鱼

3D DNA FISH has become a major tool for  analyzing three-dimensional organization of the nucleus, and several variations  of the technique have been ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

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 ...

Amplification, Next-generation Sequencing, and Genomic DNA Mapping of Retroviral Integration Sites

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of diverse libraries of retroviral integration sites using ligation-mediated PCR (LM-PCR) amplification and next-generation sequencing (NGS), (2) mapping the genomic location of each virus-host junction using BEDTools, and (3) analyzing the data for statistical relevance. Genomic DNA extracted from infected cells is fragmented by digestion with restriction enzymes or by sonication. After suitable DNA end-repair, double-stranded linkers are ligated onto the DNA ends, and semi-nested PCR is conducted using primers complementary to both the long terminal repeat (LTR) end of the virus and the ligated linker DNA. The PCR primers carry sequences required for DNA clustering during NGS, negating the requirement for separate adapter ligation. Quality control (QC) is conducted to assess DNA fragment size distribution and adapter DNA incorporation prior to NGS. Sequence output files are filtered for LTR-containing reads, and the sequences defining the LTR and the linker are cropped away. Trimmed host cell sequences are mapped to a reference genome using BLAT and are filtered for minimally 97% identity to a unique point in the reference genome. Unique integration sites are scrutinized for adjacent nucleotide (nt) sequence and distribution relative to various genomic features. Using this protocol, integration site libraries of high complexity can be constructed from genomic DNA in three days. The entire protocol that encompasses exogenous viral infection of susceptible tissue culture cells to integration site analysis can therefore be conducted in approximately one to two weeks. Recent applications of this technology pertain to longitudinal analysis of integration sites from HIV-infected patients.

We describe a protocol for amplifying retroviral integration sites from the genomic DNA of infected cells, sequencing the amplified virus-host junctions, and then mapping these sequences to a reference genome. We also describe techniques to quantify the distribution of integration sites relative to various genomic annotations using BEDTools.

逆转录病毒整合位点的扩增，新一代测序和基因组DNA制图

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of ...

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).

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.

表面拴DNA大分子的可视化与荧光蛋白的DNA结合肽

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

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

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此视频中的章节

可视化的单分子荧光显微镜下的DNA复制

人类DNA修复蛋白的遗传研究利用酵母作为模型系统

可视化线虫角质层结构的亲脂性的重要染料，直接投资收益

从基因组DNA选择性捕获的5 - 羟甲基

研究DNA损伤检验点

强大的3D直接标记的探针DNA鱼

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

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

逆转录病毒整合位点的扩增，新一代测序和基因组DNA制图

表面拴DNA大分子的可视化与荧光蛋白的DNA结合肽