Name: genomic transformation of the picoeukaryote ostreococcus tauri
Uploaded: 2012-07-13T13:00:00
Duration: 10 min 45 s
Description: Watch this Scientific Journal Video about Genomic Transformation of the Picoeukaryote Ostreococcus tauri at JoVE.com

SynthSys 1由BBSRC和EPSRC的奖D019621资助结合系统生物学中心。欧盟FP6的卓越“海洋生物功能基因”的拨款，以FYB网络已资助的遗传转化方法的发展。

1。藻类材料的制备<ol><li> ostreococcus细胞在人工海水（ASW）的培养。海盐（通常约40克每升）溶解在去离子水的盐度30个百分点，作为衡量使用盐度米。富集培养基11,12，微量金属元素和维生素的添加，如表1-3中所述。媒体过滤灭菌，然后通过0.22μm的过滤器。 </li><li>维修，应变OTTH95 Ostreococcus细胞分培养无菌稀释新鲜反潜每7天在1/100光与月光蓝过滤器安装在植物生长培养箱不断生长。光的强度应接近为20μmol米-2 S -1，温度保持在20°C细胞并不需要不断搅拌，但动摇一次，每2至3天，以防止聚合。 </li><li>对于每个改造，50毫升的细胞需要在一个单元格的nsity的20-30×10 6 mL -1时 ，应达 ​​到5-7天，继子培养。可确定近似细胞密度和axeny的的最小的X40倍率要么使用流式细胞仪或血球。 </li></ol> 2。电<ol><li>准备改造的DNA。对于每一个转变，5微克纯，线性化的质粒DNA在浓度为1μg/μL无菌去离子水。为了得到这种DNA，作者建议使用Qiagen公司的MIDI准备套件，尽管其他方法可能同样出色工作。消化酶减少所用的载体的骨干，但不是在转基因或选择基因产品。进一步净化和集中所产生的线性DNA，乙醇沉淀和悬浮的产品在适当的音量高品质的无菌去离子水。 </li><li>准备改造为每个含有5微克DNA的离心管。一个contro没有DNA l是必要的，每个细胞线进行改造。这些管子置于冰上，再加上每个转换为2毫米电试管。 </li><li>准备2.2毫升悬浮缓冲每改造。山梨糖醇溶解到1 M在DDH 2 O的解决方案，添加0.1％聚醚酸F68和过滤消毒。 </li><li>嵌段酸F68，加入终浓度为0.1％的细胞，颗粒8000XĞ10分钟在10°C，在50 ml管锥底。吸取了立即在1毫升的悬浮液悬浮细胞，转移到离心管。 10分钟，在10°C在8000 XG降速，迅速工作，再次重复此清洗步骤。 </li><li>用切尖，悬浮每个最终颗粒的悬浮液在40μL。加入40μL重悬细胞，每管上冰的线性化的DNA，轻轻混匀，转移到电试管。 </li><li>放在electrop在试管祭文机。将设置更改为6千伏，600厘米-1Ω，25μF的。 Electroporate的细胞。 </li><li>孵育10分钟的试管在室温electoporated的细胞，并利用这段时间来准备组织培养瓶中。标记并添加30毫升新鲜反潜每个。取1毫升，每个烧瓶，并轻轻将它添加到相应的试管。孵育2分钟，轻轻地取出反潜，现在含有细胞球，轻轻地，慢慢地吸取到的反潜直接在培养瓶中。 </li><li>细胞通常保持在一个球。请注意，不要动摇或扰乱聚合的细胞，在这一刻。让细胞在孵化器中恢复1-2小时，然后通过摇动烧瓶的悬浮。在这个时候，细胞悬浮自由和无团块应该是可见的。离开文化的孵化器在一夜之间恢复。 </li></ol> 3。列入板细胞在半固体培养基<ol><l我&gt;第二天，高压灭菌器在2.1％低熔点琼脂糖DDH 2瓶在含搅拌棒的解决方案。保持在65-90°C熔化在一个更大的烧杯中含有水加热磁力搅拌器。对于每个转换准备8培养皿直径（55毫米）和8×15毫升管各含9毫升反潜加上所需的选择。如果使用Nourseothricin或G418的，用2毫克/毫升。 </li><li>从孵化器收集的转化细胞。在无菌flowhood工作，加1毫升煮沸的低熔点琼脂糖管9毫升。关闭管，由相混合。再加入0.5毫升的新鲜转化细胞，迅速拌匀，倒入盘。重复这个过程中，与其余7支试管，然后进行下变换瓶。 </li><li>离开板打开了一个小时的流罩，琼脂糖设置。然后关闭板块和它们传送到大广场的培养皿中，将举行各4板。密封方形板机智Ĥ封口膜。请注意该板块将不会设置完全，因此，凝胶是非常脆弱的。不打破凝胶处理板时，应小心。将所有方板的孵化器。 </li></ol> 4。转化殖民地的选择<ol><li>菌落出现后10至21天的转换板，而不是模拟转换板。挑选菌落停产关闭提示使用200微升移液器。只需选择自由的殖民地，并吸出绿色的殖民地。照顾不包括任何细胞从邻近的殖民地。 </li><li>转移细胞至2毫升的液体介质中含有的选择，在24孔板。当，使用Nourseothricin，使用1.75毫克/毫升。混合上下吹打起来。选择24-50％转化菌落。封口膜转移到孵化器密封板。 </li><li>一个星期后，转移100μl每口井的2毫升新鲜反潜选择在24孔板和成长为additionaL 7天。幸存的细胞株可用于进一步的研究。稳定整合到基因组和插入号码应使用PCR和Southern分析。可以为这些目的使用任何规模下的植物DNA的提取标准方法获得基因组DNA。 </li></ol> 5。代表结果细胞分裂后7天增长达到1/100毫升2500万个细胞的密度。导致细胞与相应的设置电时间常数的10至14毫秒。当细胞被转移到培养基培养瓶中，细胞球形成。当一个小时后轻轻动摇，应该很容易悬浮细胞。 0.2％低熔点琼脂1毫升细胞转化纳入应导致在一个一致的，但只有半固态凝胶。殖民地后，应该会出现10至21天。当线性化的DNA转化5微克以上使用的协议，每50-100殖民地改造板是典型的预期，与阴性对照板。 〜80％回升殖民地积极选择在液体培养基中的抗生素耐药性，并在随后的研究中使用。 <table border="1"><tbody><tr><td></td><td> 终浓度 </td><td> 原液 </td><td> 1公升体积 </td></tr><tr><td> NANO 3 </td><td> 8.83×10 -4 M时 </td><td> 75 g / L的DH 2 O的 </td><td> 1毫升</td></tr><tr><td> NH 4 CL </td><td> 3.63×10 -5 M时 </td><td> 2.68克/ L，DH 2 O的 </td><td> 1毫升</td></tr><tr><td> β-甘油</td><td> 1×10 -5 M时 </td><td> 2.16克/ L，DH 2 O的 </td><td> 1毫升</td></tr><tr><td> H 2 SEO 3 </td><td> 1×10 -8 M </td><td> 1.29 mg / L的DH 2 O的 </td><td> 1毫升</td></tr><tr><td> Tris碱（pH值7.2） </td><td> 1×10 -3 M </td><td> 121.1克/ L，DH 2 O的 </td><td> 1毫升</td></tr><tr><td> ķ微量金属解决方案</td><td></td><td>表2 </td><td> 1毫升</td></tr><tr><td> f / 2的维生素解决方案</td><td></td><td>表3 </td><td> 0.5毫升</td></tr></tbody></table> 表1。 野生稻生长的培养基成分金牛 11，12。以总量1公升的人工海水盐度30个百分点，表明添加上述股票解决方案的组成部分。过滤，消毒，在一个星期内的介质和使用。汇集的维生素解决方案以外的所有化合物的股票可以添加6毫升这一解决方案，为每公升介质。 <table border="1"><tbody><tr><td></td><td> 终浓度 </td><td> 原液 </td><td> 1公升金额 </td></tr><tr><td>娜2 </sUB&gt; EDTA·2H 2 O </td><td> 1×10 -4 M时 </td><td></td><td> 41.6克</td></tr><tr><td>三氯化铁3•6H 2 O </td><td> 1×10 -5 M时 </td><td></td><td> 3.15克</td></tr><tr><td>的Na 2 MOO 4•2H 2 O的 </td><td> 1×10 -8 M </td><td> 6.3 g / L的DH 2 O的 </td><td> 1毫升</td></tr><tr><td>一水硫酸锌4•7H 2 </td><td> 1×10 -9 M </td><td> 22.0克/ L，DH 2 O的 </td><td> 1毫升</td></tr><tr><td> CoCl 2•6H 2 O </td><td> 1×10 -9 M </td><td> 10.0 g / L的DH 2 O的 </td><td> 1毫升</td></tr><tr><td> MnCl 2·4H 2 O的 </td><td> 1×10 -8 M </td><td> 180.0克/ L，DH 2 O的 </td><td> 1毫升</td></tr><tr><td>硫酸铜4·5H 2 O </td><td> 1×10 -8 M </td><td> 9.8克/大号DH <sub&gt; 2 O的</td><td> 1毫升</td></tr></tbody></table> 表2。微量金属溶液11,12。弥补共1升，在DH 2 O的热溶解。等分存储解决方案，并冻结。最终浓度指最终的介质中，微量金属溶液。 <table border="1"><tbody><tr><td></td><td> 终浓度 </td><td> 原液 </td><td> 1公升金额 </td></tr><tr><td>维生素B 12 </td><td> 1×10 -10米 </td><td> 1 g / L的DH 2 O的 </td><td> 1毫升</td></tr><tr><td>素</td><td> 1×10 -9 M </td><td> 0.1 g / L的DH 2 O的 </td><td> 10毫升</td></tr><tr><td>硫胺素•盐酸</td><td> 1×10-7 M </td><td></td><td> 200毫克</td></tr></tbody></table> 表3。 F / 2维生素解决方案11。化妆到1公升，过滤，消毒，分装，并冻结在DH 2 O的总。最终浓度指最终介质，而不是维生素解决方案。 <img alt="图1" src="/files/ftp_upload/4074/4074fig1.jpg" /> 图1。的转型过程中的图形概览。基因改造电Ostreococcus金牛的过程示意图。 <img alt="图2" src="/files/ftp_upload/4074/4074fig2.jpg" /> 图2。文化增长。细胞分培养无菌稀释新鲜反潜的1/100，每7天，并不断与月光蓝过滤器安装在植物生长培养箱，种植。光的强度应为20μmol接近米-2 s -1及温度保持在20°C细胞并不需要不断搅拌，但有s哈肯每2至3天，以防止聚合。 <img alt="图3" src="/files/ftp_upload/4074/4074fig3.jpg" /> 图3。集落形成于0.2％低熔点琼脂糖。传输4板，一个大广场的培养皿中，用封口膜密封（左上）。已经形成的殖民地时，只需选择免费（理想锥形）殖民地（右上）和板与P200移液器吸出来。应该有阴性对照组（右下）没有殖民地。

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没有利益冲突的声明。

细胞状态和文化axeny是至关重要的转型过程中。虽然细胞通常保持在12小时光照周期，12小时黑暗，被发现在这些条件下生长的细胞转化效率不足。在重复制粒/再悬浮步骤，细胞总是容易悬浮。如果细胞聚集，或粘液样物质存在，它是更好的再次从头开始的过程。通常情况下，50殖民地，可以预计在每个转换板，与阴性对照板没有。转化效率低的情况下，应改​​变培养条件，以确保细胞在最佳状态。影响转化效率的变量，包括在实验室的环境温度（应该接近到20°C）和处理速度（制粒细胞[2.3]恢复[2.7]程序应采取〜1小时）。同样重要的是，所有的解决方案重新取得了新的前每个转换。列入对于板块中，低熔点琼脂糖的浓度是至关重要的。Ostreococcus细胞不会生长在高琼脂糖浓度，并会在低浓度扩散。 三个转化载体Ostreococcus已发表 ​​过6，预计将产生更多的载体，短期内公布。现有的矢量吕克6罐允许结合的启动子的选择与使用的C-末端的萤火虫荧光素酶标记允许更快的转基因株系的选择加听话的表达模式，而进行的Potox载体6强诱导13启动子从的Ostreococcus采取高亲和磷转运（HAPT）感兴趣的基因表达的基因。 Potox吕克载体派生从Potox，携带一个额外的过度线的间接选择可以使用的荧光素酶标记<s高达&gt; 14。这些载体成功的选择标记Nourseothricin的的和G148。然而，Ostreococcus细胞对多种抗生素耐药，并选择转基因Ostreococcus细胞所必需的化合物的浓度高于传统模型系统（2毫克/毫升）。 虽然过程中出现的效率低下，在此过程所需的细胞和质粒DNA的大量不构成重大障碍。每产生抗性选择标记的线，是需要单独进行分析检查，整个结构的DNA相结合，是一个较大的限制。我们观察到的例子，在选择标记的成功表达不符合利益的实际基因插入;即部分的集成可以发生。由此可见，随机插入到基因组也意味着，有没有使用此方法插入网站的控制，方法B同源重组的层次正在开发。然而，该方法这里描述的是，我们所知，目前只有特点的方法来产生转基因Ostreococcus细胞。 正如金牛Ostreococcus最近才被作为实验模式生物，大多数这些细胞的方法很可能被越来越多的研究涉及社会发展和细化。该协议旨在帮助人发起与Ostreococcus工作，但绝不声称来形容，有了解这微藻基因改造。作者的信任，读者将这个协议能够适应自己的需求小的差异，在孵化器（光水平或质量）和其他参数会存在，并有可能在每一个人的实验室进行了优化，获得此需要的健康文化协议。这里描述后掌握的技术，可以构造向量产生发光，荧光或其他标记的融合线，在控制的范围助剂。这个令人振奋的新的实验平台，将是有益的探讨如何降低复杂性，在药理方法非常方便3真核生物解决复杂的生化问题。

<table border="1"><tbody><tr><td colspan="4"> 培养基成分 </td></tr><tr><td> 化工 </td><td colspan="2" > 目录编号 </td><td> 供应商 </td></tr><tr><td>即时海洋海盐</td><td colspan="3">从<a href="http://amazon.co.uk" target="_blank">amazon.co.uk</a>或<a href="http://rocketaquatics.co.uk" target="_blank">rocketaquatics.co.uk</a> </td></tr><tr><td> NANO 3 </td><td colspan="2"> S5506-250G </td><td>西格玛</td></tr><tr><td> NH 4 CL </td><td colspan="2"> A9434-500G </td><td>西格玛</td></tr><tr><td>甘油磷酸钠盐水合物</td><td colspan="2"> G9422-100G </td><td>西格玛</td></tr><tr><td> H 2 SEO 3 </td><td colspan="2"> 84920-250G </td><td>西格玛</td></tr><tr><td>超纯净的TRIS </td><td colspan="2"> 15504-020 </td><td> Invitrogen公司</td></tr><tr><td>娜2 EDTA·2H 2 O </td><td colspan="2"> BPE120-500G </td><td> Fisher Scientific则</td></tr><tr><td>三氯化铁3•6H 2 O </td><td colspan="2"> 157740 </td><td>西格玛</td></tr><tr><td>的Na 2 MOO 4•2H 2 O的 </td><td colspan="2"> 31439-100G-R的</td><td>西格玛</td></tr><tr><td>一水硫酸锌4•7H 2 </td><td colspan="2"> Z0251 </td><td>西格玛</td></tr><tr><td> CoCl 2•6H 2 O </td><td colspan="2"> 31277 </td><td>西格玛</td></tr><tr><td> MnCl 2·4H 2 O的 </td><td colspan="2"> 203734-5G </td><td>西格玛</td></tr><tr><td>硫酸铜4·5H 2 O </td><td colspan="2"> C8027 </td><td>西格玛</td></tr><tr><td> 维生素 B12（氰钴胺） </td><td colspan="2"> V2876 </td><td>西格玛</td></tr><tr><td>素</td><td colspan="2"> 47868 </td><td>西格玛</td></tr><tr><td>硫胺素•盐酸</td><td colspan="2"> T4625 </td><td>西格玛</td></tr><tr><td>氨苄青霉素</td><td colspan="2"> A9518-25G </td><td>西格玛</td></tr><tr><td>新霉素</td><td colspan="2"> N6386 </td><td>西格玛</td></tr><tr><td>卡那霉素</td><td colspan="2"> K4000。 </td><td>西格玛</td></tr><tr><td colspan="4"> 消耗品培养 </td></tr><tr><td colspan="2"> 项目 </td><td> 目录编号 </td><td> 供应商 </td></tr><tr><td colspan="2">组织培养瓶中的T25悬浮浇注通道排出的文化</td><td> 83.1810.502 </td><td> starstedt </td></tr><tr><td colspan="2">组织培养瓶中的T75悬浮浇注通道排出的文化</td><td> 83.1813.502 </td><td> Starstedţ </td></tr><tr><td colspan="2">组织培养瓶T175悬浮浇注通道排出的文化</td><td> 83.1812.502 </td><td> starstedt </td></tr><tr><td colspan="2"> TPP的瓶顶过滤器，1L，完整</td><td> 99950T </td><td>拿生命科学</td></tr><tr><td colspan="2"> TPP的瓶顶过滤器，500毫升， </td><td> 99500T </td><td>拿生命科学</td></tr><tr><td colspan="2"> TPP的瓶顶过滤器，250毫升， </td><td> 99250T </td><td>拿生命科学</td></tr><tr><td colspan="2"> TPP的瓶顶过滤器，为150mL， </td><td> 99150T </td><td>拿生命科学</td></tr><tr><td colspan="2">盐度计的海洋水族馆</td><td> DSG-10 </td><td> daeyoon </td></tr></tbody></table><table border="1"><tbody><tr><td colspan="3" > 转型的化学品 </td></tr><tr><td> 化工 </td><td> 目录数</td><td> 供应商 </td></tr><tr><td> D-山梨醇</td><td> S6021-1KG </td><td>西格玛</td></tr><tr><td>聚醚F-68的解决方案</td><td> P5556-100ML </td><td>西格玛</td></tr><tr><td> ClonNat </td><td> 51000 </td><td>沃纳</td></tr><tr><td>低熔点琼脂糖</td><td> 16520-050 </td><td> Invitrogen公司</td></tr><tr><td colspan="3"> 耗材转型 </td></tr><tr><td> 项目 </td><td> 目录编号 </td><td> 供应商 </td></tr><tr><td>组织培养瓶中的T25悬浮浇注通道排出的文化</td><td> 83.1810.502 </td><td> starstedt </td></tr><tr><td>组织培养瓶中的T75悬浮浇注通道排出的文化</td><td> 83.1813.502 </td><td> starstedt </td></tr><tr><td>组织培养瓶T175暂停铜ltures发泄浇注通道</td><td> 83.1812.502 </td><td> starstedt </td></tr><tr><td>格雷纳离心管50毫升</td><td> 227261 </td><td>全球生物多样性展望</td></tr><tr><td>基因脉冲/ MicroPulser比色皿，0.2厘米的缝隙，50 </td><td> 165-2086 </td><td> BIO-RAD </td></tr><tr><td>培养皿一轮PS 3通风口H14.2Φ55 </td><td> 391-0895 </td><td> VWR国际</td></tr><tr><td>四个通风口聚苯乙烯照射明确120毫米无菌培养皿平方米</td><td> FB51788 </td><td> Fisher Scientific则</td></tr><tr><td>月光蓝灯过滤器</td><td> 183 </td><td>李照明</td></tr></tbody></table>

genomic transformation of the picoeukaryote ostreococcus tauri

阻碍植物科学的快速进步的共同问题，包括细胞，组织和整个生物体的复杂性，尤其是高层次的基因冗余影响在高等​​植物中的简单的遗传学研究。新模型的有机体Ostreococcus金牛是迄今已知最小的自由生活真核生物，拥有大大降低的基因组大小和细胞复杂1,2，表现的只是其中的大多数细胞器（线粒体，叶绿体，高尔基栈）每存在细胞，只有约8000个基因的基因组。此外，结合unicellularity和易于文化提供了一个平台，适合化学生物学方法。最近，Ostreococcus已成功地采用了以研究昼夜报时3-6的基本机制。从这个模型有机体的结果，这不仅影响植物科学，但也哺乳动物生物学7。这个例子强调如何在一个快速实验从简单的真核生物的绿色系可以加速在更复杂的生物体产生可验证的假设使用技术上是可行的，只有在这个背景降低复杂性的方法研究。在任何模式物种基因组和基因修改的可能性的知识是必不可少的工具。转录基因组1，8，9蛋白质组学这一物种的信息是免费提供的，而以前报道的方法6,10到基因改造Ostreococcus的被称为世界各地的一些实验室。 在这篇文章中，实验方法，以基因改造与过度新型模式生物电建设中概述的细节，以及列入在低百分比琼脂糖转化细胞的方法，允许选择转换线从原产单细胞。继成功Ostreoco应用日益增长的兴趣在Ostreococcus昼夜研究ccus到，可以预计从不同的研究领域内，外植物科学，包括生物技术领域。研究人员从一个保守的生化途径的生物学和医学科学的范围广泛，可考虑追求在Ostreococcus的研究，从更大的模式物种的基因组和生物体的复杂性。

Watch this Scientific Journal Video about Genomic Transformation of the Picoeukaryote Ostreococcus tauri at JoVE.com

Genomic Transformation of the Picoeukaryote Ostreococcus tauri

本文介绍了单细胞海藻的遗传转化<em&gt; Ostreococcus金牛</em&gt;电。这是真核有机体的有效模式，为高等植物平台，possesing基因和细胞的复杂性大大降低，并随时服从细胞培养和化学生物学。

基因改造的Picoeukaryote<em&gt; Ostreococcus金牛</em

Common problems hindering rapid progress in Plant Sciences include cellular, tissue and whole organism complexity, and notably the high level of genomic ...

genomic-transformation-of-the-picoeukaryote-ostreococcus-tauri

Research

JoVE Journal

Biology

Genomic Transformation of the Picoeukaryote Ostreococcus tauri

Isolation of Genomic DNA from Mouse Tails

从鼠尾基因组DNA提取

科研

生物学

Large Insert Environmental Genomic Library Production

The vast majority of microbes in nature currently remain inaccessible to traditional cultivation methods. Over the past decade, culture-independent environmental genomic (i.e. metagenomic) approaches have emerged, enabling researchers to bridge this cultivation gap by capturing the genetic content of indigenous microbial communities directly from the environment. To this end, genomic DNA libraries are constructed using standard albeit artful laboratory cloning techniques. Here we describe the construction of a large insert environmental genomic fosmid library with DNA derived from the vertical depth continuum of a seasonally hypoxic fjord. This protocol is directly linked to a series of connected protocols including coastal marine water sampling [1], large volume filtration of microbial biomass [2] and a DNA extraction and purification protocol [3]. At the outset, high quality genomic DNA is end-repaired with the creation of 5 -phosphorylated blunt ends. End-repaired DNA is subjected to pulsed-field gel electrophoresis (PFGE) for size selection and gel extraction is performed to recover DNA fragments between 30 and 60 thousand base pairs (Kb) in length. Size selected DNA is purified away from the PFGE gel matrix and ligated to the phosphatase-treated blunt-end fosmid CopyControl vector pCC1 (EPICENTRE <a href="http://www.epibio.com/item.asp?ID=385">http://www.epibio.com/item.asp?ID=385</a>). Linear concatemers of pCC1 and insert DNA are subsequently headfull packaged into phage particles by lambda terminase, with subsequent infection of phage-resistant E. coli cells. Successfully transduced clones are recovered on LB agar plates under antibiotic selection and archived in 384-well plate format using an automated colony picking robot (Qpix2, GENETIX). The current protocol draws from various sources including the CopyControl Fosmid Library Production Kit from EPICENTRE and the published works of multiple research groups [4-7]. Each step is presented with best practice in mind. Whenever possible we highlight subtleties in execution to improve overall quality and efficiency of library production. The whole process of fosmid library production and automated colony picking takes at least 7-10 days as there are many incubation steps included. However, there are several stopping points possible which are mentioned within the protocol.

Construction of a fosmid library with environmental genomic DNA isolated from the vertical depth continuum of a seasonally hypoxic fjord is described. The resulting clone library is picked into 384-well plates and archived for downstream sequencing and functional screening by the application of an automated colony picking system.

大型插入环境基因组文库生产

The vast majority of microbes in nature currently remain inaccessible to traditional cultivation methods. Over the past decade, culture-independent ...

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 ...

Genomic MRI - a Public Resource for Studying Sequence Patterns within Genomic DNA

Non-coding genomic regions in complex eukaryotes, including intergenic areas, introns, and untranslated segments of exons, are profoundly non-random in their nucleotide composition and consist of a complex mosaic of sequence patterns. These patterns include so-called Mid-Range Inhomogeneity (MRI) regions -- sequences 30-10000 nucleotides in length that are enriched by a particular base or combination of bases (e.g. (G+T)-rich, purine-rich, etc.). MRI regions are associated with unusual (non-B-form) DNA structures that are often involved in regulation of gene expression, recombination, and other genetic processes (Fedorova &amp; Fedorov 2010). The existence of a strong fixation bias within MRI regions against mutations that tend to reduce their sequence inhomogeneity additionally supports the functionality and importance of these genomic sequences (Prakash et al. 2009).
Here we demonstrate a freely available Internet resource -- the Genomic MRI program package -- designed for computational analysis of genomic sequences in order to find and characterize various MRI patterns within them (Bechtel et al. 2008). This package also allows generation of randomized sequences with various properties and level of correspondence to the natural input DNA sequences. The main goal of this resource is to facilitate examination of vast regions of non-coding DNA that are still scarcely investigated and await thorough exploration and recognition.

We present a public computational web site for the analysis of genomic sequences. It detects DNA sequence patterns with various non-random nucleotide compositions. This resource also generates randomized sequences with diverse levels of complexity.

磁共振成像基因 - 公共资源为研究基因组DNA序列内模式

Non-coding genomic regions in complex eukaryotes, including intergenic areas, introns, and untranslated segments of exons, are profoundly non-random in ...

Competitive Genomic Screens of Barcoded Yeast Libraries

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily. The tempo of these advances is accelerating, promising greater depth and breadth. In light of these extraordinary advances, the need for fast, parallel methods to define gene function becomes ever more important. Collections of genome-wide deletion mutants in yeasts and E. coli have served as workhorses for functional characterization of gene function, but this approach is not scalable, current gene-deletion approaches require each of the thousands of genes that comprise a genome to be deleted and verified. Only after this work is complete can we pursue high-throughput phenotyping. Over the past decade, our laboratory has refined a portfolio of competitive, miniaturized, high-throughput genome-wide assays that can be performed in parallel. This parallelization is possible because of the inclusion of DNA 'tags', or 'barcodes,' into each mutant, with the barcode serving as a proxy for the mutation and one can measure the barcode abundance to assess mutant fitness. In this study, we seek to fill the gap between DNA sequence and barcoded mutant collections. To accomplish this we introduce a combined transposon disruption-barcoding approach that opens up parallel barcode assays to newly sequenced, but poorly characterized microbes. To illustrate this approach we present a new Candida albicans barcoded disruption collection and describe how both microarray-based and next generation sequencing-based platforms can be used to collect 10,000 - 1,000,000 gene-gene and drug-gene interactions in a single experiment.

We have developed comprehensive, unbiased genome-wide screens to understand gene-drug and gene-environment interactions. Methods for screening these mutant collections are presented.

条形码酵母图书馆竞争力的基因屏幕

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily.  The tempo of these advances is ...

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 ...

Array Comparative Genomic Hybridization (Array CGH) for Detection of Genomic Copy Number Variants

Array CGH for the detection of genomic copy number variants has replaced G-banded karyotype analysis. This paper describes the technology and its application in a clinical diagnostic service laboratory. DNA extracted from a patient&#8217;s sample (blood, saliva or other tissue types) is labeled with a fluorochrome (either cyanine 5 or cyanine 3). A reference DNA sample is labeled with the opposite fluorochrome. There follows a cleanup step to remove unincorporated nucleotides before the labeled DNAs are mixed and resuspended in a hybridization buffer and applied to an array comprising ~60,000 oligonucleotide probes from loci across the genome, with high probe density in clinically important areas. Following hybridization, the arrays are washed, then scanned and the resulting images are analyzed to measure the red and green fluorescence for each probe. Software is used to assess the quality of each probe measurement, calculate the ratio of red to green fluorescence and detect potential copy number variants.

Array Comparative Genomic Hybridization Array CGH for Detection of Genomic Copy Number Variants

Array CGH for the detection of genomic copy number variants has replaced G-banded karyotype analysis. This paper describes the technology and its application in a diagnostic service laboratory.

阵列比较基因组杂交（CGH阵列），用于检测基因拷贝数变异的

Array CGH for the detection of genomic copy number variants has replaced G-banded karyotype analysis. This paper describes the technology and its ...

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 ...

Targeted RNA Sequencing Assay to Characterize Gene Expression and Genomic Alterations

RNA sequencing (RNAseq) is a versatile method that can be utilized to detect and characterize gene expression, mutations, gene fusions, and noncoding RNAs. Standard RNAseq requires 30 - 100 million sequencing reads and can include multiple RNA products such as mRNA and noncoding RNAs. We demonstrate how targeted RNAseq (capture) permits a focused study on selected RNA products using a desktop sequencer. RNAseq capture can characterize unannotated, low, or transiently expressed transcripts that may otherwise be missed using traditional RNAseq methods. Here we describe the extraction of RNA from cell lines, ribosomal RNA depletion, cDNA synthesis, preparation of barcoded libraries, hybridization and capture of targeted transcripts and multiplex sequencing on a desktop sequencer. We also outline the computational analysis pipeline, which includes quality control assessment, alignment, fusion detection, gene expression quantification and identification of single nucleotide variants. This assay allows for targeted transcript sequencing to characterize gene expression, gene fusions, and mutations.

We describe a targeted RNA sequencing-based method that includes preparation of indexed cDNA libraries, hybridization and capture with custom probes and data analysis to interrogate selected transcripts for gene expression, mutations, and gene fusions. Targeted RNAseq permits cost-effective, rapid evaluation of selected transcripts on a desktop sequencer.

有针对性的RNA测序分析表征基因表达和基因组改变

RNA sequencing (RNAseq) is a versatile method that can be utilized to detect and characterize gene expression, mutations, gene fusions, and noncoding ...

Preparing and Injecting Embryos of Culex Mosquitoes to Generate Null Mutations using CRISPR/Cas9

Culex mosquitoes are the major vectors of several diseases that negatively impact human and animal health including West Nile virus and diseases caused by filarial nematodes such as canine heartworm and elephantasis. Recently, CRISPR/Cas9 genome editing has been used to induce site-directed mutations by injecting a Cas9 protein that has been complexed with a guide RNA (gRNA) into freshly laid embryos of several insect species, including mosquitoes that belong to the genera Anopheles and Aedes. Manipulating and injecting Culex mosquitoes is slightly more difficult as these mosquitoes lay their eggs upright in rafts rather than individually like other species of mosquitoes. Here we describe how to design gRNAs, complex them with Cas9 protein, induce female mosquitoes of Culex pipiens to lay eggs, and how to prepare and inject newly laid embryos for microinjection with Cas9/gRNA. We also describe how to rear and screen injected mosquitoes for the desired mutation. The representative results demonstrate that this technique can be used to induce site-directed mutations in the genome of Culex mosquitoes and, with slight modifications, can be used to generate null-mutants in other mosquito species as well.&#160;

Preparing and Injecting Embryos of Culex Mosquitoes to Generate Null Mutations using CRISPR/Cas9

CRISPR/Cas9 is increasingly used to characterize gene function in non-model organisms. This protocol describes how to generate knock-out lines of Culex pipiens, from preparing injection mixes, to obtaining and injecting mosquito embryos, as well as how to rear, cross, and screen injected mosquitoes and their progeny for desired mutations.

准备和注射 Culex 蚊子的胚胎，使用CRISPR/Cas9产生空突变

Culex mosquitoes are the major vectors of several diseases that negatively impact human and animal health including West Nile virus and diseases caused by ...