我们承认，化学，地质和生物科学部，办公室基础能源科学，科学办公室，美国能源部（奖DE-FG02-91ER20021）和国家科学基金会（MCB 0948584）（FB）的支持。我们感谢编辑手稿女士卡伦鸟的。

1。 EMS处理 拟南芥种子的诱变诱变剂乙基甲烷磺酸盐（EMS），3,4，这将导致基因组的C-T的变化的C / G / A突变5-7使用。 <ol><li>权衡0.8Ğ拟南芥种子携带的细胞器荧光标记（特别是，在这项研究中，ssGFPHDEL（信号序列的GFP-HDEL四肽）已作为ER标记）（〜40,000种子）。 </li><li>在50毫升的猎鹰管转移的种子，然后加入25毫升蒸馏水。 </li><li>添加0.2％（V / V）乙基甲烷磺酸盐。 </li><li> 16小时孵育关于nutating低速搅拌机。 </li><li>吸液和丢弃到一个瓶含1.0 M氢氧化钠灭活EMS。 </li><li>含种子，靠近猎鹰管加入25毫升水，倒置洗种子的5倍;等到所有的种子纷纷落户，然后吸水和丢弃到1.0米娜<html>俄亥俄州瓶。 </li><li>重复的高达10倍的洗涤步骤。 </li><li>最后一次洗涤后，重新挂起的种子，在少量的水。 </li><li>进行种子消毒，加入25毫升10％的漂白剂，用力摇动30秒。让种子定居的底部，再倒入漂白剂和25毫升无菌水冲洗。倒入无菌水，加入25毫升70％的乙醇。摇管为30秒。让种子定居的底部。倒掉乙醇，25毫升无菌水冲洗。重复两次用无菌水冲洗，然后倒入塑料的Petri菜含有3 MM滤纸上的种子，让引擎盖下干燥。在4°C保存2天。 </li><li>板半，MS phytagel（Murashige和斯库格中期的一半浓度），150毫米的培养皿（〜250-300板种子）的种子。 </li><li> 2周的货币供应量M1种子生长板，然后移植到土壤中。 </li><li>收集个别M1植株M2的种子，产生M2的行（1000独立线）。 </li></ol>ove_title"&gt; 2。共焦M2和M3种群的筛选在本节中，我们描述了与前面所述的8共聚焦荧光显微镜观察幼苗。 <ol><li> 60从每个M2线的种子成长为7至10天，在塑料培养皿，半质谱phytagel;同一板块也增长了5苗EMS-未处理（对照组）。 </li><li>五到十年子叶安装背面朝着镜头（40X）在显微镜幻灯片和盖玻片包围。 </li><li>每个子叶观察，从皮质到内侧区域的荧光下，任何改变的细胞器标记的亚细胞分布。 </li><li>阳性植株移植到土壤中，其种子的收集和筛选，再次确认在M3代的突变体。 </li><li>移除污染背景突变，通过回交至少三次到野生型的基因组可能合作ntaining所需的细胞器荧光标记。 <ol><li>从母株，用细剪刀或镊子删除角果成熟，开放的鲜花。 </li><li>取下芽太小的分生组织。 </li><li>插入一对钳打开一个花蕾，并删除所有花药之间的花瓣和萼片尖端。 </li><li>用一个钳子从父亲的工厂采取一个开放的成熟花和擦上的阉割植物的耻辱的花药。 </li></ol></li></ol> 3。映射本节描述基本上是如何映射隐性突变，从9 Borevitz修改的协议，这是更快，如果比传统的映射方法10,11。这种方法使用的检测能力在一个单一的检测12多个单一功能多态性（SFPS）的高密度寡核苷酸阵列。使用Affymetrix拟南芥ATH1基因芯片阵列是可能的分析约24,000个基因。楼2个人突变池相比，野生型F 2分离群体在同一采集植物池。然后，突变，在突变型等位基因突变池丰富的地区将被映射，因此在同一地区，野生型池将导致野生型的父母等位基因13丰富。 <ol><li>合子突变哥伦比亚（M3）的使用Qiagen公司的DNeasy植物Mini试剂盒提取基因组DNA（3微克）和Illumina的Genome Analyzer的II（GA二）14测序提交。 </li><li>纯合子突变哥伦比亚（M3）兰茨贝格万寿菊杂交产生作图群体。 </li><li> 70显示异常的表型和F2植株与野生型的表型相同数量高达100 F2群体进行映射。 </li><li>收集每个植物叶盘使用打孔（0.60毫米）。 "叶盘应收集相同年龄的叶片，以确保有类似的DNA。可处理的样品基因组提取，单独或团体。在这种情况下，它可以收集每个Eppendorf管5-10样本，以便在年底将有更少的Eppendorf管从基因组DNA提取。 </li><li> MasterPure植物叶片DNA纯化试剂盒（震中）用于提取基因组DNA。从每个样品中获得的基因组DNA进行了量化了的NanoDrop。 </li><li>从每个样品相同数量的基因组DNA，然后放在一起共有300毫微克（约30μL）植物的DNA，添加60μL的2.5倍随机引物溶液125毫米的Tris-HCl（pH值6.8），12.5毫米氯化镁，25毫米2 - 巯基乙醇，750微克/毫升的寡核苷酸引物（随机八聚体）]（Bioprime套件）和42μL水（终体积为132微升）。 </li><li>在变性的DNA&gt; 95℃，5至10分钟。 </li><li>冰酷。 </li><li>每个denatu红色的DNA加入15μL的10倍[1毫米素-14-dCTP，1毫米，2毫米的dATP dCTP，三磷酸2毫米，2毫米dTTP在10毫米的Tris-HCl（pH值7.5），1毫米乙二胺四乙酸二钠与生物素dCTP] dNTPs浓度混合（Bioprime包），并完成与3μL的Klenow聚合酶（Bioprime包）。 </li><li>在25°C孵育过夜</li><li>翌日，添加15μL的3个M醋酸钠和400μL的冷100％的乙醇;组合。 </li><li>在-80°C孵育1小时，然后旋转15分钟20,500 XG，除去上清液，用500μL的冷乙醇75％。 </li><li> 20500 XG旋转10分钟。 </li><li>在37°C干燥的DNA颗粒为10分钟和100μL水重悬。 </li><li>使用5μL的凝胶上（ 图2）的产量和质量检查。 </li><li>发送95μL标记野生型和突变基因芯片拟南芥ATH1基因阵列杂交反应。 </li><li>阵列扫描和分析获得的。CEL文件使用R软件（ <a href="http://www.r-project.org/">http://www.r-project.o</a>RG /）。 </li><li>安装R软件，然后打开该程序，并粘贴以下字符串: 源（" <a href="http://bioconductor.org/biocLite.R">http://bioconductor.org/biocLite.R</a> "） biocLite（） 然后按回车键，这将安 ​​装标准的Bioconductor封装（ <a href="http://www.bioconductor.org">http://www.bioconductor.org</a> ）的。 </li><li>在新的文件夹在桌面上，从以下网站（ <a href="http://www.naturalvariation.org/methods">http://www.naturalvariation.org/methods</a> ）文件下载: readcel.R SFP.R Map.R ath1V5.RData ColLerCEL.zip 解压缩文件ColLerCEL.zip应，并粘贴到新文件夹中生成的文件。 </li><li>重命名从你的基因芯片实验获得的数据到wildtype.CEL和mutant.CEL。 </li><li>现在复制的基因芯片数据（wildtype.CEL和mutant.CEL）到您的文件夹。 </li><li>打开R，适用于Mac）:点击"杂项"，然后点击"更改工作目录。" B）邮编:点击"文件"，然后"更改目录" </li><li>适用于Mac）:选择该文件夹包含的文件。单击"工作区"，"负荷工作区文件"为PC和选择ath1V5.RData，B）:选择包含上述文件的文件夹，单击"文件"，然后选择"载入工作区"，并选择ath1V5.RData。 </li><li>开放readCEL.R使用记事本和整个文本复制到R </li><li>开放SFP.R使用记事本和整个文本复制到R </li><li>开放Map.R使用记事本和整个文本复制到R </li><li>在控制台窗口中，你会看到以下消息: X染色体限制MB YZ 在这个环节上粘贴在的TAIR搜索基因 3 <a href="http://arabidopsis.org/servlets/sv?action=download&amp;chr=x&amp;start=y&amp;end=z">http://arabidopsis.org/servlets/sv?action=download&amp;chr=x&amp;start=y</a>和结束= Z </li><li>复制并粘贴到您的互联网浏览器的链接。将出现一个窗口，并要求打开或保存文件，你应该选择保存。选择一个文件名和附加扩展名为"。XLS"，然后单击"保存"。 </li><li>打开文件"。XLS"在这里你可以找到您突变的坐标（结果显示在图3表示）。 </li><li>读取组装Illumina的映射区域内确定具体的环境管理体系过渡之间的单核苷酸多态性的突变基因。补充转化突变体与野生型基因，使用标准的程序15（S）。 </li></ol> 4。代表结果 图1显示了识别突变体的分泌途径，利用共聚焦显微镜检查所使用的方法。 图2显示了典型的标记基因组DNA阵列杂交准备。在图3中 ，一个典型的后分析基因芯片的ATH1拟南芥基因组阵列得到的数据，提出预期的结果。 <img alt="图1" src="/files/ftp_upload/3809/3809fig1.jpg" /> 图1。拟南芥表达ssGFPHDEL（ER标记）（1）转基因植物种植EMS的治疗（2）产生足够的种子。与EMS处理的种子，然后播种产生（3）货币供应量M1植物。每个货币供应量M1厂代表了不同的线路和种子收集从他们每个人分开。 M2种子被镀上半MS基板（4），然后通过共聚焦显微镜（5）在ER形态缺陷筛选。在筛选中，我们发现，保守的野生型ER形态（6），表明雌激素受体的表型缺陷（7）植物，植物。这些植物生长获得M3代和确认型（8）。 Illumina的测序公司Solexa（一）被用于从M3的植物基因组DNA。同一厂也可用于LER-WT杂交获得的F2作图群体（B）。 <img alt="图2" src="/files/ftp_upload/3809/3809fig2.jpg" /> 图2。Bioprime的随机标记反应（5μL100μL）被装上琼脂糖凝胶1％。巷a是从野生型的F2植株池，车道B从一个突变的F2植株池。 （标记是自NEB 1 kb的DNA阶梯N3232）。 <img alt="图3" src="/files/ftp_upload/3809/3809fig3.jpg" /> 图3。数字代表COL-0突变基因芯片拟南芥ATH1基因组阵列杂交映射的例子。在这个例子中的突变位于1号染色体，由竖线分隔。单杠代表进行检测的阈值。

<ol>
	<li>Marti, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+missense+mutation+in+the+vacuolar+protein+GOLD36+causes+organizational+defects+in+the+ER+and+aberrant+protein+trafficking+in+the+plant+secretory+pathway.">A missense mutation in the vacuolar protein GOLD36 causes organizational defects in the ER and aberrant protein trafficking in the plant secretory pathway.</a> The Plant journal : for cell and molecular biology. 63, 901-913 (2010).</li><li>Stefano, G., Renna, L., Moss, T., McNew, J., Brandizzi, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arabidopsis+the+spatial+and+dynamic+organization+of+the+endoplasmic+reticulum+and+Golgi+apparatus+is+influenced+by+the+integrity+of+the+c-terminal+domain+of+RHD3,+a+non-essential+GTPase.">Arabidopsis the spatial and dynamic organization of the endoplasmic reticulum and Golgi apparatus is influenced by the integrity of the c-terminal domain of RHD3, a non-essential GTPase.</a> The Plant Journal. , (2011).</li><li>Kim, Y., Schumaker, K. S., Zhu, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EMS+mutagenesis+of+Arabidopsis.">EMS mutagenesis of Arabidopsis.</a> Methods in molecular biology. 323, 101-103 (2006).</li><li>Maple, J., Moller, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutagenesis+in+Arabidopsis.">Mutagenesis in Arabidopsis.</a> Methods in molecular biology. 362, 197-206 (2007).</li><li>Greene, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spectrum+of+chemically+induced+mutations+from+a+large-scale+reverse-genetic+screen+in+Arabidopsis.">Spectrum of chemically induced mutations from a large-scale reverse-genetic screen in Arabidopsis.</a> Genetics. 164, 731-740 (2003).</li><li>Krieg, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ethyl+methanesulfonate-induced+reversion+of+bacteriophage+T4rII+mutants.">Ethyl methanesulfonate-induced reversion of bacteriophage T4rII mutants.</a> Genetics. 48, 561-580 (1963).</li><li>Kovalchuk, I., Kovalchuk, O., Hohn, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+variation+of+the+somatic+mutation+frequency+in+transgenic+plants.">Genome-wide variation of the somatic mutation frequency in transgenic plants.</a> The EMBO journal. 19, 4431-4438 (2000).</li><li>Boulaflous, A., Faso, C., Brandizzi, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deciphering+the+Golgi+apparatus:+from+imaging+to+genes.">Deciphering the Golgi apparatus: from imaging to genes.</a> Traffic. 9, 1613-1617 (2008).</li><li>Borevitz, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genotyping+and+mapping+with+high-density+oligonucleotide+arrays.">Genotyping and mapping with high-density oligonucleotide arrays.</a> Methods in molecular biology. 323, 137-145 (2006).</li><li>Konieczny, A., Ausubel, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+procedure+for+mapping+Arabidopsis+mutations+using+co-dominant+ecotype-specific+PCR-based+markers.">A procedure for mapping Arabidopsis mutations using co-dominant ecotype-specific PCR-based markers.</a> The Plant journal : for cell and molecular biology. 4, 403-410 (1993).</li><li>Bell, C. J., Ecker, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assignment+of+30+microsatellite+loci+to+the+linkage+map+of+Arabidopsis.">Assignment of 30 microsatellite loci to the linkage map of Arabidopsis.</a> Genomics. 19, 137-144 (1994).</li><li>Hazen, S. P., Kay, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+arrays+are+not+just+for+measuring+gene+expression.">Gene arrays are not just for measuring gene expression.</a> Trends in Plant Science. 8, 413-416 (2003).</li><li>Hazen, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+array+mapping+of+circadian+clock+and+developmental+mutations+in+Arabidopsis.">Rapid array mapping of circadian clock and developmental mutations in Arabidopsis.</a> Plant Physiology. 138, 990-997 (2005).</li><li>Bentley, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accurate+whole+human+genome+sequencing+using+reversible+terminator+chemistry.">Accurate whole human genome sequencing using reversible terminator chemistry.</a> Nature. 456, 53-59 (2008).</li><li>Weigel, D., Glazebrook, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arabidopsis.">Arabidopsis.</a> A Laboratory Manual. , (2002).</li><li>Voelkerding, K. V., Dames, S., Durtschi, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Next+generation+sequencing+for+clinical+diagnostics-principles+and+application+to+targeted+resequencing+for+hypertrophic+cardiomyopathy:+a+paper+from+the+2009+William+Beaumont+Hospital+Symposium+on+Molecular+Pathology.">Next generation sequencing for clinical diagnostics-principles and application to targeted resequencing for hypertrophic cardiomyopathy: a paper from the 2009 William Beaumont Hospital Symposium on Molecular Pathology.</a> J. Mol. Diagn. 12, 539-551 (2010).</li></ol>

我们什么都没有透露。

在这里，我们描述了共聚焦显微镜为基础的筛选为内膜突变体鉴定。这种方法可以很容易地扩展到其他细胞的细胞器，为特定的荧光蛋白标记。基于屏幕的鉴定表明，无论是在目标器官或细胞器，不应该包含标记荧光标记的异常分布的突变。 ，这些突变体分别代表人群中受到损害的器官的能力subcompartimentalize标记或突变体不能正常转运细胞器之间的标记。在筛选中，我们注意到，一些突变表明，?...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td> ethylmethane磺酸盐</td><td>西格玛</td><td> M0880 </td></tr><tr><td>氢氧化钠</td><td>托德·贝克</td><td> 3722-05 </td></tr><tr><td> murashige斯库格培养基的瓦特gamborg维生素</td><td>植物技术的laboratorie </td><td> M404 </td></tr><tr><td> phytagel </td><td>西格玛</td><td> P8169-1千克</td></tr><tr><td>的RNeasy迷你套件</td><td> Qiagen公司</td><td> 74104 </td></tr><tr><td>大师纯植物叶片DNA纯化试剂盒</td><td>震中</td><td> MPP92100 </td></tr><tr><td> bioprime的DNA标签制度</td><td> Invitrogen公司</td><td> 18094-011 </td></tr><tr><td>酒精200证明</td><td> decan实验室公司。 </td><td> 2716 </td></tr><tr><td>醋酸钠</td><td>托德·贝克</td><td></td></tr><tr><td>基因芯片拟南芥ATH1基因组阵列</td><td> Affymetrix公司</td><td> 900385 </td></tr><tr><td>猎鹰管50毫升</td><td>康宁</td><td> 430290 </td></tr><tr><td> Eppendorf管1.5毫升</td><td></td><td></td></tr><tr><td>滤波纸90毫米</td><td>瓦特曼</td><td> 1001090 </td></tr><tr><td>分析天平</td><td>梅特勒 - 托利多AB54-S的</td><td> NA </td></tr><tr><td> nutating（波）振动筛</td><td> heidolph普力马1040 </td><td> NA </td></tr><tr><td>离心分离</td><td> Eppendorf公司5417 - R </td><td> NA </td></tr></tbody></table>

fluorescence-microscopy screening and next-generation sequencing: useful tools for the identification of genes involved in organelle integrity

本协议描述了拟南芥幼苗的荧光显微镜为基础的筛选，并介绍如何将隐性突变，改变了一个特定的标记分泌途径中的荧光标记的亚细胞分布。 拟南芥是一种强大的生物遗传研究模型，因为它的基因组大小，一代的时间，并节约王国之间的分子机制。作为一种方法来映射替代传统的方法，基于分子标记的突变基因分型阵列是有利的，因为它是相对较快，可能允许在一个非常短的时间框架的几个突变的映射。这种方法允许蛋白质的鉴定，可以影响任何在植物细胞器的完整性。在这里，作为一个例子，我们提出了一个重要的内质网（ER）的完整性的地图基因的屏幕。然而，我们的做法，可以很容易地扩展到其他植物细胞器（例如，见1,2），因此代表了对理解其他亚细胞结构的分子基础的重要一步。

Watch this Scientific Journal Video about Fluorescence-microscopy Screening and Next-generation Sequencing: Useful Tools for the Identification of Genes Involved in Organelle Integrity at JoVE.com

Fluorescence-microscopy Screening and Next-generation Sequencing: Useful Tools for the Identification of Genes Involved in Organelle Integrity

在细胞生物学的一个基本任务是定义机制的基础，使真核细胞的细胞器的身份。在这里，我们提出了一个方法，以确定该基因的植物细胞器，利用荧光显微镜和下一代测序工具的形态和功能的完整性负责。

荧光显微镜筛选和新一代测序:参与细胞器完整性的基因鉴定的有用工具

This protocol describes a fluorescence microscope-based screening of Arabidopsis seedlings and describes how to map recessive mutations that alter the ...

fluorescence-microscopy-screening-next-generation-sequencing-useful

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

12.2K 次观看.  Michigan State University. 在细胞生物学的一个基本任务是定义机制的基础，使真核细胞的细胞器的身份。在这里，我们提出了一个方法，以确定该基因的植物细胞器，利用荧光显微镜和下一代测序工具的形态和功能的完整性负责。

视频: Fluorescence-microscopy Screening and Next-generation Sequencing: Useful Tools for the Identification of Genes Involved in Organelle Integrity

Using RNA-mediated Interference Feeding Strategy to Screen for Genes Involved in Body Size Regulation in the Nematode C. elegans

Double-strand RNA-mediated interference (RNAi) is an effective strategy to knock down target gene expression1-3. It has been applied to many model systems including plants, invertebrates and vertebrates. There are various methods to achieve RNAi in vivo4,5. For example, the target gene may be transformed into an RNAi vector, and then either permanently or transiently transformed into cell lines or primary cells to achieve gene knockdown effects; alternatively synthesized double-strand oligonucleotides from specific target genes (RNAi oligos) may be transiently transformed into cell lines or primary cells to silence target genes; or synthesized double-strand RNA molecules may be microinjected into an organism. Since the nematode C. elegans uses bacteria as a food source, feeding the animals with bacteria expressing double-strand RNA against target genes provides a viable strategy6. Here we present an RNAi feeding method to score body size phenotype. Body size in C. elegans is regulated primarily by the TGF- &beta; - like ligand DBL-1, so this assay is appropriate for identification of TGF-&beta; signaling components7. We used different strains including two RNAi hypersensitive strains to repeat the RNAi feeding experiments. Our results showed that rrf-3 strain gave us the best expected RNAi phenotype. The method is easy to perform, reproducible, and easily quantified. Furthermore, our protocol minimizes the use of specialized equipment, so it is suitable for smaller laboratories or those at predominantly undergraduate institutions.

Using RNA-mediated Interference Feeding Strategy to Screen for Genes Involved in Body Size Regulation in the Nematode C. elegans

We demonstrate how to use the RNAi feeding technique to knock down target genes and score body size phenotype in C. elegans. This method could be used for a large scale screen to identify potential genetic components of interest, such as those involved in body size regulation by DBL-1/TGF-&beta; signaling.

使用屏幕在车身尺寸规例中的线虫基因介导的RNA干扰取食策略<em&gt; C.线虫</em

Double-strand RNA-mediated interference (RNAi) is an effective strategy to knock down target gene expression1-3. It has been applied to many model systems ...

科研

生物学

Next-generation Sequencing of 16S Ribosomal RNA Gene Amplicons

One of the major questions in microbial ecology is &#8220;who is there?&#8221; This question can be answered using various tools, but one of the long-lasting gold standards is to sequence 16S ribosomal RNA (rRNA) gene amplicons generated by domain-level PCR reactions amplifying from genomic DNA. Traditionally, this was performed by cloning and Sanger (capillary electrophoresis) sequencing of PCR amplicons. The advent of next-generation sequencing has tremendously simplified and increased the sequencing depth for 16S rRNA gene sequencing. The introduction of benchtop sequencers now allows small labs to perform their 16S rRNA sequencing in-house in a matter of days. Here, an approach for 16S rRNA gene amplicon sequencing using a benchtop next-generation sequencer is detailed. The environmental DNA is first amplified by PCR using primers that contain sequencing adapters and barcodes. They are then coupled to spherical particles via emulsion PCR. The particles are loaded on a disposable chip and the chip is inserted in the sequencing machine after which the sequencing is performed. The sequences are retrieved in fastq format, filtered and the barcodes are used to establish the sample membership of the reads. The filtered and binned reads are then further analyzed using publically available tools. An example analysis where the reads were classified with a taxonomy-finding algorithm within the software package Mothur is given. The method outlined here is simple, inexpensive and straightforward and should help smaller labs to take advantage from the ongoing genomic revolution.

Characterizing microbial community has been a longstanding goal in environmental microbiology. Next-generation sequencing methods now allow for the characterization of microbial communities at an unprecedented depth with minimal cost and labor. We detail here our approach to sequence bacterial 16S ribosomal RNA genes using a benchtop sequencer.

的16S核糖体RNA基因的扩增子的新一代测序

One of the major questions in microbial ecology is &#8220;who is there?&#8221; This question can be answered using various tools, but one of the ...

Targeted DNA Methylation Analysis by Next-generation Sequencing

The role of epigenetic processes in the control of gene expression has been known for a number of years. DNA methylation at cytosine residues is of particular interest for epigenetic studies as it has been demonstrated to be both a long lasting and a dynamic regulator of gene expression. Efforts to examine epigenetic changes in health and disease have been hindered by the lack of high-throughput, quantitatively accurate methods. With the advent and popularization of next-generation sequencing (NGS) technologies, these tools are now being applied to epigenomics in addition to existing genomic and transcriptomic methodologies. For epigenetic investigations of cytosine methylation where regions of interest, such as specific gene promoters or CpG islands, have been identified and there is a need to examine significant numbers of samples with high quantitative accuracy, we have developed a method called Bisulfite Amplicon Sequencing (BSAS). This method combines bisulfite conversion with targeted amplification of regions of interest, transposome-mediated library construction and benchtop NGS. BSAS offers a rapid and efficient method for analysis of up to 10 kb of targeted regions in up to 96 samples at a time that can be performed by most research groups with basic molecular biology skills. The results provide absolute quantitation of cytosine methylation with base specificity. BSAS can be applied to any genomic region from any DNA source. This method is useful for hypothesis testing studies of target regions of interest as well as confirmation of regions identified in genome-wide methylation analyses such as whole genome bisulfite sequencing, reduced representation bisulfite sequencing, and methylated DNA immunoprecipitation sequencing.

Bisulfite amplicon sequencing (BSAS) is a method for quantifying cytosine methylation in targeted genomic regions of interest. This method uses bisulfite conversion paired with PCR amplification of target regions prior to next-generation sequencing to produce absolute quantitation of DNA methylation at a base-specific level.

有针对性的DNA甲基化分析通过新一代测序

The role of epigenetic processes in the control of gene expression has been known for a number of years. DNA methylation at cytosine residues is of ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

高通量筛选转录后调控基因的化学调节剂

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

Identification of Kinesin-1 Cargos Using Fluorescence Microscopy

Fluorescence microscopy is employed to identify Kinesin-1 cargos. Recently, the heavy chain of Kinesin-1 (KIF5B) was shown to transport the nuclear transcription factor c-MYC for proteosomal degradation in the cytoplasm. The method described here involves the study of a motorless KIF5B mutant for fluorescence microscopy. The wild-type and motorless KIF5B proteins are tagged with the fluorescent protein tdTomato. The wild-type tdTomato-KIF5B appears homogenously in the cytoplasm, while the motorless tdTomato-KIF5B mutant forms aggregates in the cytoplasm. Aggregation of the motorless KIF5B mutant induces aggregation of its cargo c-MYC in the cytoplasm. Hence, this method provides a visual means to identify the cargos of Kinesin-1. A similar strategy can be utilized to identify cargos of other motor proteins.

Here, a protocol is presented to identify Kinesin-1 cargos. A motorless mutant of the Kinesin-1 heavy chain (KIF5B) aggregates in the cytoplasm and induces aggregation of its cargos. Both aggregates are detected under fluorescence microscopy. A similar strategy can be employed to identify cargos of other motor proteins.

使用荧光显微镜驱动蛋白-1的货物的识别

Fluorescence microscopy is employed to identify Kinesin-1 cargos. Recently, the heavy chain of Kinesin-1 (KIF5B) was shown to transport the nuclear ...

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 ...

Interactome-Seq: A Protocol for Domainome Library Construction, Validation and Selection by Phage Display and Next Generation Sequencing

Folding reporters are proteins with easily identifiable phenotypes, such as antibiotic resistance, whose folding and function is compromised when fused to poorly folding proteins or random open reading frames. We have developed a strategy where, by using TEM-1 &#946;-lactamase (the enzyme conferring ampicillin resistance) on a genomic scale, we can select collections of correctly folded protein domains from the coding portion of the DNA of any intronless genome. The protein fragments obtained by this approach, the so called &#34;domainome&#34;, will be well expressed and soluble, making them suitable for structural/functional studies.
By cloning and displaying the &#34;domainome&#34; directly in a phage display system, we have showed that it is possible to select specific protein domains with the desired binding properties (e.g., to other proteins or to antibodies), thus providing essential experimental information for gene annotation or antigen identification.
The identification of the most enriched clones in a selected polyclonal population can be achieved by using novel next-generation sequencing technologies (NGS). For these reasons, we introduce deep sequencing analysis of the library itself and the selection outputs to provide complete information on diversity, abundance and precise mapping of each of the selected fragment. The protocols presented here show the key steps for library construction, characterization, and validation.

The protocols described allow the construction, characterization and selection (against the target of choice) of a &#34;domainome&#34; library made from any DNA source. This is achieved by a research pipeline that combines different technologies: phage display, a folding reporter and next generation sequencing with a web tool for data analysis.

Interactome: Domainome 图书馆建设、噬菌体展示和下一代测序的验证与选择协议

Folding reporters are proteins with easily identifiable phenotypes, such as antibiotic resistance, whose folding and function is compromised when fused to ...

SA-&#946;-Galactosidase-Based Screening Assay for the Identification of Senotherapeutic Drugs

Cell senescence is one of the hallmarks of aging known to negatively influence a healthy lifespan. Drugs able to kill senescent cells specifically in cell culture, termed senolytics, can reduce the senescent cell burden in vivo and extend healthspan. Multiple classes of senolytics have been identified to date including HSP90 inhibitors, Bcl-2 family inhibitors, piperlongumine, a FOXO4 inhibitory peptide and the combination of Dasatinib/Quercetin. Detection of SA-&#946;-Gal at an increased lysosomal pH is one of the best characterized markers for the detection of senescent cells. Live cell measurements of senescence-associated &#946;-galactosidase (SA-&#946;-Gal) activity using the fluorescent substrate C12FDG in combination with the determination of the total cell number using a DNA intercalating Hoechst dye opens the possibility to screen for senotherapeutic drugs that either reduce overall SA-&#946;-Gal activity by killing of senescent cells (senolytics) or by suppressing SA-&#946;-Gal and other phenotypes of senescent cells (senomorphics). Use of a high content fluorescent image acquisition and analysis platform allows for the rapid, high throughput screening of drug libraries for effects on SA-&#946;-Gal, cell morphology and cell number.

Cellular senescence is the key factor in the development of chronic age-related pathologies. Identification of therapeutics that target senescent cells show promise for extending healthy aging. Here, we present a novel assay to screen for the identification of senotherapeutics based on measurement of senescence associated &#946;-Galactosidase activity in single cells.

SA-β-加拉基沙酶筛查检测,用于识别治疗药物

Cell senescence is one of the hallmarks of aging known to negatively influence a healthy lifespan. Drugs able to kill senescent cells specifically in cell ...

Tick Microbiome Characterization by Next-Generation 16S rRNA Amplicon Sequencing

In recent decades, vector-borne diseases have re-emerged and expanded at alarming rates, causing considerable morbidity and mortality worldwide. Effective and widely available vaccines are lacking for a majority of these diseases, necessitating the development of novel disease mitigation strategies. To this end, a promising avenue of disease control involves targeting the vector microbiome, the community of microbes inhabiting the vector. The vector microbiome plays a pivotal role in pathogen dynamics, and manipulations of the microbiome have led to reduced vector abundance or pathogen transmission for a handful of vector-borne diseases. However, translating these findings into disease control applications requires a thorough understanding of vector microbial ecology, historically limited by insufficient technology in this field. The advent of next-generation sequencing approaches has enabled rapid, highly parallel sequencing of diverse microbial communities. Targeting the highly-conserved 16S rRNA gene has facilitated characterizations of microbes present within vectors under varying ecological and experimental conditions. This technique involves amplification of the 16S rRNA gene, sample barcoding via PCR, loading samples onto a flow cell for sequencing, and bioinformatics approaches to match sequence data with phylogenetic information. Species or genus-level identification for a high number of replicates can typically be achieved through this approach, thus circumventing challenges of low detection, resolution, and output from traditional culturing, microscopy, or histological staining techniques. Therefore, this method is well-suited for characterizing vector microbes under diverse conditions but cannot currently provide information on microbial function, location within the vector, or response to antibiotic treatment. Overall, 16S next-generation sequencing is a powerful technique for better understanding the identity and role of vector microbes in disease dynamics.

Here we present a next-generation sequencing protocol for 16S rRNA sequencing which enables identification and characterization of microbial communities within vectors. This method involves DNA extraction, amplification and barcoding of samples through PCR, sequencing on a flow-cell, and bioinformatics to match sequence data to phylogenetic information.

下一代 16S rRNA 扩增子测序的蜱菌群鉴定

In recent decades, vector-borne diseases have re-emerged and expanded at alarming rates, causing considerable morbidity and mortality worldwide. Effective ...

Microbiota Analysis Using Two-step PCR and Next-generation 16S rRNA Gene Sequencing

The human gut is colonized by trillions of bacteria that support physiologic functions such as food metabolism, energy harvesting, and regulation of the immune system. Perturbation of the healthy gut microbiome has been suggested to play a role in the development of inflammatory diseases, including multiple sclerosis (MS). Environmental and genetic factors can influence the composition of the microbiome; therefore, identification of microbial communities linked with a disease phenotype has become the first step towards defining the microbiome&#8217;s role in health and disease. Use of 16S rRNA metagenomic sequencing for profiling bacterial community has helped in advancing microbiome research. Despite its wide use, there is no uniform protocol for 16S rRNA-based taxonomic profiling analysis. Another limitation is the low resolution of taxonomic assignment due to technical difficulties such as smaller sequencing reads, as well as use of only forward (R1) reads in the final analysis due to low quality of reverse (R2) reads. There is need for a simplified method with high resolution to characterize bacterial diversity in a given biospecimen. Advancements in sequencing technology with the ability to sequence longer reads at high resolution have helped to overcome some of these challenges. Present sequencing technology combined with a publicly available metagenomic analysis pipeline such as R-based Divisive Amplicon Denoising Algorithm-2 (DADA2) has helped advance microbial profiling at high resolution, as DADA2 can assign sequence at the genus and species levels. Described here is a guide for performing bacterial profiling using two-step amplification of the V3-V4 region of the 16S rRNA gene, followed by analysis using freely available analysis tools (i.e., DADA2, Phyloseq, and METAGENassist). It is believed that this simple and complete workflow will serve as an excellent tool for researchers interested in performing microbiome profiling studies.

Described here is a simplified standard operating procedure for microbiome profiling using 16S rRNA metagenomic sequencing and analysis using freely available tools. This protocol will help researchers who are new to the microbiome field as well as those requiring updates on methods to achieve bacterial profiling at a higher resolution.

使用两步PCR和下一代16S rRNA基因测序进行微生物群分析

The human gut is colonized by trillions of bacteria that support physiologic functions such as food metabolism, energy harvesting, and regulation of the ...