这项工作是支持部分美国国家科学基金会资助MCB-0718846和美国国防部高级研究计划局合同N66001-12-C-4020（加多宝）。 LAM资金由来自加拿大自然科学和工程研究理事会博士后奖学金。发布这篇文章是由罗氏公司赞助。

1.准备酵母基因组DNA（基因组DNA） <ol><li>制备的酵母的裂解缓冲液9股票包含的50mM Tris pH值8.0，100mM的氯化钠，1％SDS（重量/体积），2％的Triton X-100（V / V），1mM EDTA的pH为8.0。 </li><li>作为起始原料使用〜2×10 6个细胞，其通常对应于〜100微升的饱和培养物对的BY4741 10背景实验室菌株。培养的细胞应该通过离心在1000×g离心收集，在室温下在1.5ml微离心管3分钟。吸出上清液。 </li><li>添加200微升酵母裂解缓冲液和通过移液重悬细胞沉淀。 </li><li>添加〜300微升酸洗涤的玻璃珠，使得珠的水平低于细胞浆液的水平大约1毫米。 </li><li>在通风橱中加入200微升的酚/氯仿/异戊醇（25：24：1）。盖上离心管，并确保没有珠粒被卡住在盖和管之间，因为这将摇动期间导致泄漏（步骤1.6）。翻转3次混合和验证盖密封。 </li><li>摇动该管在使用台式搅拌器室温10分钟，如在Eppendorf 5432。 </li><li>离心20,800 XG在4℃下10分钟。 </li><li>转移75微升的水相，以含有1ml的100％乙醇的一个新的离心管中。反转10次混合。 </li><li>沉淀通过离心该DNA在20800×g离心20分钟，在4℃。 </li><li>吸出上清液没有撞出DNA沉淀。用500μl70％乙醇洗涤沉淀。 </li><li>离心20,800 XG 5分钟，在4℃。 </li><li>吸出上清液，而不撞出DNA沉淀并空气干燥与离心帽打开，直到过量乙醇蒸发后，它通常是约10分钟沉淀。 </li><li>悬浮在75微升的10mM Tris pH值7.4，涡旋混合DNA沉淀。储存样品在-20℃下无限期。使用测量样本中的DNA的浓度台式荧光计，如量子比特，其由RNA区别的DNA。确保的gDNA的终浓度为〜1-2毫微克/微升。 </li><li>使用水和涡流混合制备1:10稀释的基因组DNA。 </li><li>等份加入30μl稀释的gDNA成源板，其是与纳米级声学液体分配器（步骤3）兼容的适当孔。如果使用Labcyte公司回声550，使用384孔聚丙烯板（384PP板）。 注意：当密封适当该板可以在-20℃储存至少一个月。 </li></ol> 2.准备和配给定量PCR主混合物进入1536多孔板<ol><li>准备850微升的qPCR预混，如1536的LightCycler DNA绿法师，在1.5 ml离心管，涡旋混合。离心的qPCR主混合物进行2分钟，在20800×g离心，在室温下，以消除任何气泡。所需的单1536多孔板的定量PCR主混合物的成分如下S： <ol><li>结合170微升酶主混合物（绿站长管1，5×浓缩的），42.5微升SYBR的（绿色硕士管4，20倍浓缩），以及637.5微升水。 </li></ol></li><li>分配500标升/孔成1536多孔板使用散装液体分配器，例如艺术罗宾斯眼镜蛇。 （注意，步骤2.2.1-2.2.4专指眼镜蛇。） <ol><li>创建具有以下设置的分配方案：液体类，水;抽吸量，800微升;分配体积，500 NL;洗时间，20秒。在分配设置中，确定分注速度被设置至49.6毫米/秒，分配偏移为0.25毫米，而过量的吸气液体将被分配回源井。 </li><li>将含有定量PCR主结构的离心管架，它位于甲板位置1.切断离心管的盖子或者干脆把它开A1孔的离心管。通过编辑Aspirat设置“吸好&#39;至A1Ë设置。 </li><li>执行洗涤步骤分配的qPCR预混首先取消选择的吸入和排出步骤程序中设置了步骤2.2.2，然后打&#39;跑&#39;之前。一旦完成，重新选择的吸入和排出之前运行完整的程序来分配的qPCR预混步骤。如果眼镜蛇已经闲置时间少于10分钟的初始洗涤步骤，其用于灌注系统，是不必要的。 </li><li>打“跑”来分配的qPCR预混液，以每口井。 </li></ol></li><li>以及由1536多孔板（30秒，使用一个低速PCR板喷丝）的短暂离心收集的qPCR主混合物在每个孔的底部。 </li></ol> 3.点胶模板DNA和引物到1536多孔板<ol><li>分配5 NL的稀释的gDNA（步骤1.3）转换成使用声液体传输系统，1536多孔板的所需井如回声550。 <ol><li>对于ECHØ550，使用平板格式化软件或交替提供了自定义的Excel电子表格程序转移。确保所选择的水源井的基因组DNA中的节目早已进入你身体已分配的基因组DNA（步骤1.3）的源相匹配。选择源板式&#39;384PP_AQ_BP2&#39;，这表明在384PP板中的液体是没有表面活性剂的缓冲液。 </li></ol></li><li>分配10 NL的引物，预混合正向和反向的各，50μM最终浓度成使用声液体传输系统，1536多孔板的所需井如回声550。 <ol><li>对于液体输送系统中，使用平板格式化软件或交替提供自定义的Excel电子表格程序转移。 注意：这是能够省去引物，使得在1536多孔板的布局相匹配的引物的染色体顺序，所以很容易在视觉上检查实时PCR结果以后（步骤6）。 </li><li>预混合的引物中的板，它与所述声液体分配器兼容。对于回声550，使用Labcyte公司384LDV（低死体积）板，并选择源板型编程时，回声指示液，没有蛋白质一个简单的缓冲区384LDV_AQ_B“。 </li></ol></li></ol> 4.密封和离心1536多孔板<ol><li>使用微板密封器系统，如Plateloc热微孔板封口，使用光学透明的密封是用热封机仪器兼容立即密封1536多孔板。不要触摸密封件的表面，避免污迹。 <ol><li>如果使用的是Plateloc热封口机酶标仪，请使用以下设置：密封时间2.0秒，热封温度162℃，气压82磅​​。该仪器大约需要5分钟升温，因此应提前开启。 </li><li>如果使用的是Plateloc热封口机微孔板，一个adapteR必须是用于提高1536的多孔板在仪器上的阶段的高度，以保证良好的密封。 注意：虽然优选购买舞台板为Plateloc热微孔板封口，一个替代的解决方案是插入四个可用〜2毫米厚的标准垫圈在任何五金店在弯道以提高1536多孔板的高度。 </li></ol></li><li>立即离心密封1536多孔板以2,000×g离心3分钟，在室温下，收集所有的试剂在每个孔的底部。 </li></ol> 5.实时定量PCR <ol><li>程序1536的qPCR仪器，如LightCycler的1536，与两步扩增法，随后通过熔融曲线分析。使用LightCycler的1536软件中设置了一个程序，如下所示： <ol><li>预培养：95℃，保持1分钟，升温速率4.8℃/秒，无重大收购。 </li><li>二步扩增：中[95℃的30个循环，没有保持，斜坡率4.8℃/秒，不采集; 64℃，30秒的保持，升温速率2.5℃/秒，单次采集。 </li><li>熔解曲线：95℃，10秒，升温速率4.8℃/秒，不采集; 60℃，1分钟，升温速率2.5℃/秒，没有收购; 97℃，升温速度0.1℃/秒，5并购/°C（连续）。 </li><li>酷：40℃，30秒，升温速率2.5℃/秒，无重大收购。 </li></ol></li><li>在运行定义选项卡中设置的检测格式“绿色嵌入染料”。 </li><li>坐落在运行定义选项卡的移液控制为“主控”。这个特性作为内部对照，并且检测的qPCR主混合物在1536多孔板，这是有用的，以确定推定的假阴性的每个孔的存在。 </li><li>将制备步骤2-4到1536定量PCR仪1536多孔板和运行程序。 </li></ol> 6.数据分析<ol><li> PERFORM在qPCR的软件内的数据的目视检查。 注：1536软件允许扩增的可视化和熔化曲线，以及一个热图显示的呼叫（正，负，无效，N / A; 图2），所述交叉点的值（滑动色阶为阳性或灰色为负; 图3），或者一个端点荧光（EPF）值（滑动色阶）。 </li><li>从1536软件的形式.txt文件（成绩表）或.xml文件能够与原始数据或从仪器处理离线计算数据导出数据。 注：专有自动化交叉点（CP）调用算法内置于1536软件考虑了形状和扩增曲线的斜率而确定的正/负1和Cp以高置信度值的呼叫，在低子微升反应体积以相对低的荧光值。 </li><li>如果DNA的LightCycler绿色法师被使用d代表定量PCR，验证所有井通过“主控”。故障指示以及未收到的qPCR预混和丢失的数据点应被视为一个潜在的假阴性。验证所有井通过了“主控”。故障指示以及未接受DNA mastermix并考虑丢失的数据点的潜在假阴性。 </li></ol>

<ol>
	<li>Annaluru, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Total+synthesis+of+a+functional+designer+eukaryotic+chromosome.">Total synthesis of a functional designer eukaryotic chromosome.</a> Science. 344 (6179), 55-58 (2014).</li><li>Dymond, J. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthetic+chromosome+arms+function+in+yeast+and+generate+phenotypic+diversity+by+design.">Synthetic chromosome arms function in yeast and generate phenotypic diversity by design.</a> Nature. 477 (7365), 471-476 (2011).</li><li>Goffeau, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Life+with+6000+genes.">Life with 6000 genes.</a> Science. 274 (5287), 546-547 (1996).</li><li>Dymond, J., Boeke, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Saccharomyces+cerevisiae+SCRaMbLE+system+and+genome+minimization.">The Saccharomyces cerevisiae SCRaMbLE system and genome minimization.</a> Bioeng Bugs. 3 (3), 168-171 (2012).</li><li>Richardson, S. M., Nunley, P. W., Yarrington, R. M., Boeke, J. D., Bader, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GeneDesign+3.0+is+an+updated+synthetic+biology+toolkit.">GeneDesign 3.0 is an updated synthetic biology toolkit.</a> Nucleic Acids Res. 38 (8), 2603-2606 (2010).</li><li>Richardson, S. M., Liu, S., Boeke, J. D., Bader, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design-A-Gene+with+GeneDesign.">Design-A-Gene with GeneDesign.</a> Methods Mol Biol. 852, 235-247 (2012).</li><li>Lajoie, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probing+the+limits+of+genetic+recoding+in+essential+genes.">Probing the limits of genetic recoding in essential genes.</a> Science. 342 (6156), 361-363 (2013).</li><li>Jovicevic, D., Blount, B. A., Ellis, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Total+synthesis+of+a+eukaryotic+chromosome:+Redesigning+and+SCRaMbLE-ing+yeast.">Total synthesis of a eukaryotic chromosome: Redesigning and SCRaMbLE-ing yeast.</a> Bioessays. 36 (9), 855-860 (2014).</li><li>Dymond, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+genomic+DNA+from+Saccharomyces+cerevisiae.">Preparation of genomic DNA from Saccharomyces cerevisiae.</a> Methods Enzymol. 529, 153-160 (2013).</li><li>Brachmann, C. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Designer+deletion+strains+derived+from+Saccharomyces+cerevisiae+S288C:+a+useful+set+of+strains+and+plasmids+for+PCR-mediated+gene+disruption+and+other+applications.">Designer deletion strains derived from Saccharomyces cerevisiae S288C: a useful set of strains and plasmids for PCR-mediated gene disruption and other applications.</a> Yeast. 14 (2), 115-132 (1998).</li></ol>

LAM，NAP，果酱和加多宝都没有透露。 RC和AL是由罗氏公司生命科学就业，美国和合作发展与LAM应用为原则，实验证明的一部分。

结合实时定量PCR检测到PCRTag基因分型检测对于Sc2.0项目的一个重要的发展，因为它使显著更高的吞吐量。先前的工作流中指定2.5μl的反应在384孔PCR板，1.5小时的热循环的运行时间，琼脂糖凝胶电泳和凝胶的人工注释。 这里介绍的工作流程，叫qPCRTag分析，克服几大瓶颈。首先，一个qPCRTag运行凝结4×384孔板成可以处理的，从开始到结束（板设置加上运行时间），在大约一小时一次实验。它是显著地注意到，每孔的试剂成本qPCRTag分析看齐与较低吞吐量琼脂糖凝胶为基础的方法。然而，通过绕过凝胶电泳和人工注释，所述qPCRTag工作流得到大幅节省时间和劳力，这是主要的优点。重要的是qPCRTag工作流程连接自动化tirely兼容。 一个主要关注与使用实时检测作为PCRTag测定的输出是假阳性和假阴性率。由于PCRTag引物原本不是设计用于在实时PCR使用，预期是，并非所有的引物对将适合于与该输出的使用。为此，它验证所有PCRTag引物对的功能和特异性排除不在前面的实时为基础的检测工作的子集是重要的。例如，3号染色体PCRTag误报和漏报是由和-大可再现的实时PCR数据（ 图2和3），并且这些可排除进一步的分析。此外，已知的失败的引物对（通过凝胶电泳评估，并在图S6和的Annaluru 等人 1 的S7示出）也可以被排除。这是很容易实现的简单exclu鼎设置故障引物对时了声传输协议。 最喜欢的高通量分析，我们打算使用实时PCRTag检测作为主屏幕，以确定值得进一步验证下游转化。随后，将金标准二次筛选将保持与凝胶电泳作为读PCRTag分析。除了PCRTag分析的应用Sc2.0，结合国家的最先进的纳米液体处理系统具有高吞吐量的实时PCR技术能快速和自动分析和有可能影响到很多领域。例如，该工作流程可以适用于高通量库筛选，感染性疾病的诊断，微生物分析，和尖端的基因组编辑办法试图同时修改多个基因座。

Sc2.0，或合成的酵母基因组计划（ <a href="http://www.syntheticyeast.org" target="_blank">www.syntheticyeast.org</a> ），设置了设计和建造的完全合成的真核基因组中的目标。使用酿酒酵母 3为起点的高度策划的基因组序列，每个十六线性染色体已经被重新设计，以满足一组指定维持细胞的健身，提高基因组稳定性，以及增加的遗传灵活性设计原则。例如，去稳定化元素，如重复从Sc2.0染色体中删除。的TAG终止密码子的所有实例都重新编码到TAA到“释放”的密码子在用于引入一个非遗传编码氨基酸的最终劳损。另外一种可诱导系统演变，争夺，由酶Cre-lox系统启用，允许前所未有的能力，以产生衍生的基因组结构新颖4。 <p class=“jove_content”&gt;在Sc2.0基因组的另一个主要设计元素是引入PCRTags，可以作为DNA水印，使合成的和野生型DNA跟踪。 PCRTags是合成染色体的ORF短，重新编码段;而PCRTag序列在合成的和野生型染色体之间的DNA含量差异，所编码的蛋白质在氨基酸序列上，因此，据推测，功能相同。 PCRTag序列被专门设计为引物的结合位点，以促进选择性扩增（ 图1A）。 PCRTag设计是使用了“最不同的”算法GeneDesign 5,6-，屈服重新编码是通常〜60％，比之间58℃和60℃，并在天然序列（最小33％）与熔化温度不同合成序列200-500个碱基对2之间扩增长度。重新编码是不是第100bp的各ORF的在允许范围内，因为这些区域是已知的，以具有密码子使用的7方面特殊偏好。总之，这些设计规则偏向几乎所有PCRTags的在一组的PCR条件，由此合成的和野生型PCRTag引物对完全结合并扩增合成的和天然的DNA，分别为（ 图1B）的高性能。 <img alt="图1" src="/files/ftp_upload/52941/52941fig1.jpg" /> 图1：PCRTag示意图 （A） 的 PCRTags被重新编码的基因上Sc2.0染色体的开放阅读框（ORF）中的序列。 （B）合成器（SYN）和野生型（WT）PCRTag引物完全结合并扩增合成的和野生型基因组DNA（gDNA的），分别。这里展示的是染色体6的左侧臂的〜30 kb的片段的分析，检测使用任一WT或半synVIL2的gDNA为模板13 PCRTag引物对。在许多情况下，一个单一的ORF编码多于一个PCRTag。 PCRTag扩增子的存在/不存在通过琼脂糖凝胶电泳评估。 PCRTag扩增子的尺寸范围从200基点，至500基点。越快迁移物种在面板的底部是引物二聚体。 PCRTag分析已被证明是在Sc2.0染色体的组装的重要工具。在典型的实验30-50 kb的合成DNA的编码20-30 PCRTags，转化到酵母细胞，以取代相应的野生型DNA 1,2,8。 PCRTag分析然后被用于鉴定编码合成但不与野生型PCRTags跨越DNA的片段的转化体，或所谓的“赢家”。它通常需要测试多个转化体，以确定“获胜者”，所以吞吐量和PCRTag分析的成本是重要的考虑因素。目前2 Sc2.0染色体已经完成（synIII 1和synIXR 2），代表Sc2.0基因组的10％以内，中高音霍夫多于其余染色体的一半目前正在进行合成和装配。 PCRTag分析的规模需要对此项目的快速超越运行凝胶和手动得分合成DNA和不存在下的野生型DNA的存在的能力。 为了提高我们已开发了使用实时PCR来规避利用琼脂糖凝胶电泳的工作流的PCRTag测定的吞吐量。工作流利用一个散装液体分配器的分配的qPCR mastermix成1536多孔板，纳米级的声学液体分配器传送模板DNA和引物的各孔中，一个1536的qPCR热循环仪，使我们能够小型化的反应，以500 NL和最大限度地提高吞吐量。此外分析可以实现自动化。这种类型的高通量基因分型的协议应该推广到任何需要的许多克隆的分析，在多个位点的项目。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Yeast gDNA prep</td>
		</tr>
		<tr>
			<td>yeast gDNA</td>
			<td>custom</td>
			<td>custom</td>
			<td>template for real time PCR</td>
		</tr>
		<tr>
			<td>Acid-washed glass beads (0.5mm)</td>
			<td>Sigma</td>
			<td>G8772</td>
			<td>yeast cell lysis</td>
		</tr>
		<tr>
			<td>Ultrapure Phenol:Chloroform:Isoamyl Alcohol (25:24:1, v/v)</td>
			<td>Invitrogen</td>
			<td>15593-031</td>
			<td>yeast cell lysis</td>
		</tr>
		<tr>
			<td>5432 Mixer</td>
			<td>Eppendorf</td>
			<td>5432</td>
			<td>yeast cell lysis</td>
		</tr>
		<tr>
			<td>Microcentrifuge 5417R</td>
			<td>Eppendorf</td>
			<td>22621807</td>
			<td>yeast cell lysis</td>
		</tr>
		<tr>
			<td>Qubit 3.0 Fluorometer</td>
			<td>Life Technologies</td>
			<td>Q33216</td>
			<td>gDNA quantification</td>
		</tr>
		<tr>
			<td>Qubit dsDNA BR Assay Kit</td>
			<td>Life Technologies</td>
			<td>Q32850</td>
			<td>gDNA quantification</td>
		</tr>
		<tr>
			<td>Labcyte 384PP plate</td>
			<td>Labcyte</td>
			<td>P-05525</td>
			<td>gDNA source plate</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>DNA Green Mastermix prep and dispensing</td>
		</tr>
		<tr>
			<td>LightCycler 1536 DNA Green</td>
			<td>Roche</td>
			<td>5573092001</td>
			<td>real time PCR mastermix</td>
		</tr>
		<tr>
			<td>1.5mL Microfuge Tube Holder</td>
			<td>ARI</td>
			<td>EST BD060314-1</td>
			<td>microfuge tube holder for deck of Cobra</td>
		</tr>
		<tr>
			<td>LightCycler 1536 Multiwell Plate</td>
			<td>Roche</td>
			<td>5358639001</td>
			<td></td>
		</tr>
		<tr>
			<td>Cobra liquid handling system</td>
			<td>Art Robbins Instruments</td>
			<td>630-1000-10</td>
			<td>dispense qPCR master mix into 1536 plate</td>
		</tr>
		<tr>
			<td>PCR Plate Spinner</td>
			<td>VWR</td>
			<td>89184-608</td>
			<td>cenrifugation of 1536 plate</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Template and primer dispensing</td>
		</tr>
		<tr>
			<td>PCRTag primers</td>
			<td>IDT</td>
			<td>custom</td>
			<td>premixed forward and reverse, [50uM] each</td>
		</tr>
		<tr>
			<td>TempPlate pierceable sealing foil, sterile</td>
			<td>USA Scientific</td>
			<td>2923-0110</td>
			<td>temporary seal for PCRTag primer plates</td>
		</tr>
		<tr>
			<td>Labcyte LDV 384 well plate</td>
			<td>Labcyte</td>
			<td>LP-0200</td>
			<td>pre-mixed primer source plate</td>
		</tr>
		<tr>
			<td>Echo 550 Liquid Handler</td>
			<td>Labcyte</td>
			<td></td>
			<td>transfer 2.5nL drops of primer and template DNA into 1536 plate</td>
		</tr>
		<tr>
			<td>Plateloc Thermal Microplate Sealer</td>
			<td>Agilent</td>
			<td>G5402-90001</td>
			<td>heat seal for 1536 plate prior to LC1536 run</td>
		</tr>
		<tr>
			<td>Clear Permanent Seal</td>
			<td>Agilent</td>
			<td>24212-001</td>
			<td>optically clear heat seal for LC1536 multiwell plate</td>
		</tr>
		<tr>
			<td>PlateLoc Roche/LightCycler 1536 Plate Stage</td>
			<td>Agilent</td>
			<td>G5402-20008</td>
			<td>can substitute ~2mm thick washers&#160;</td>
		</tr>
		<tr>
			<td>Sorvall Legend XTR</td>
			<td>Thermo Scientfic</td>
			<td>75-004-521</td>
			<td>centrifuge heat sealed 1536 plate</td>
		</tr>
		<tr>
			<td>Industrial Air Compressor</td>
			<td>Jun Air</td>
			<td>1795011</td>
			<td>to run the Echo and Heat Sealer</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Real Time PCR</td>
		</tr>
		<tr>
			<td>LightCycler 1536</td>
			<td>Roche</td>
			<td></td>
			<td>requires 220V outlet</td>
		</tr>
	</tbody>
</table>

qpcrtag analysis - a high throughput, real time pcr assay for sc2.0 genotyping

合成酵母基因组计划（Sc2.0）旨在打造16设计师酵母染色体和它们合并成一个单一的酵母细胞。迄今一种合成染色体，synIII 1，和一种合成染色体臂，synIXR 2，已构建和其在体内的功能验证在没有相应的野生型染色体。 Sc2.0染色体的一个重要的设计特点是引入PCRTags，这是短，重新编码的开放阅读框（ORFs），使合成的染色体分化从它们的野生型对应于序列。 PCRTag引物退火选择性地合成的或野生型染色体和存在/不存在每种类型的DNA可以使用一个简单的PCR测定法进行测试。所述PCRTag测定的标准的读出是通过琼脂糖凝胶电泳来评估存在/不存在的扩增子。然而，有一个平均PCRTag扩增子密度为每1.5 kb和股份公司的〜12 MB enome大小，完成基因组Sc2.0将编码大约8000 PCRTags。以提高吞吐量，我们开发了用于PCRTag基因分型，我们称之为qPCRTag分析实时PCR为基础的检测分析。工作流指定一个1536多孔板500 NL反应，使我们可以测试多达768 PCRTags用在一个实验中合成的和野生型引物对。

我们测试了合成的和野生型染色体3 PCRTag引物对1与来自四个不同的菌株中提取的酵母基因组DNA（gDNA的）。染色体3具有跨越染色体长度186 PCRTag引物对（synIII是〜270 kb和野生型染色体3是〜315 kb的）。为了测试这四个菌株这两套引物中，我们将多孔板分成四个象限，每个类型的gDNA，分配合成PCRTag引物以每一个象限的上半部和野生型的底部一半。基因组DNA从酵母菌株，其中两个编码野生型染色体3（野生型，synIXR 2）萃取，而剩下的两个编码合成染色体3（synIII 1，synIII synIXR）。使用本体液体分配器，接着通过使用回波550的引物的gDNA和PCRTag引物在相同进行排列的qPCR主混合物分配到一个1536多孔板的每个孔多孔板为容易视觉比较的每个象限。的多孔板，然后加热密封用光学透明的密封，并进行实时PCR分析。 在此qPCRTag实验中，我们观察到，在大多数情况下，放大如预期，由此合成引物只扩增的合成DNA， 反之亦然 （ 图2和3）。然而，我们还观察到几个偏离预期的图案，表明假阴性和假阳性的数据集。在该实验中，在井100％，检测主控制，指示散装液体分配器成功地分配mastermix到每一个孔上的板（未显示数据）。这排除了假阴性一个潜在来源。此外，一些染色体3 PCRTags已知失败（在图S6中所示和Annaluru 等人 的S7。1），包括至少2 SYN和1重量引物对;因而这些井可以在每个象限被忽略。真假阴性可以在我们的经验然而源自缺乏模板的gDNA或引物的转移，给出的回声550的正确的校准以及制备的gDNA和所描述的引物，这一直没有误差的主要来源。整体而言，在该实验中，假阴性率为极低为WT的引物与野生型模板（〜2％），尽管对于SYN引物与SYN的DNA（〜8％）稍高。 误报，信号在孔中的检测，其中的SYN引物与WT的gDNA（反之亦然）的混合，可以产生从横扩增或引物二聚体。的确，引物二聚体经常是通过凝胶电泳（ 图1B）可见，代表误差的合理来源。用于定量PCR的应用，熔体曲线的检查可能是有用的，以确定是否不同的物种，如引物二聚体，可能是导致一个信号。此外，香水orming的对照实验，由此引物被分配在不存在模板的DNA可以帮助识别与倾向二聚引物。横扩增可以通过凝胶电泳来观察，特别是如果太多PCR循环被执行，或者如果退火温度太低。我们试图尽量减少误报的数量，由于交叉扩增通过优化这些参数qPCRTag协议。最后，检查每个交叉点（CP）以及值可以帮助识别那些不适合实时为基础的检测引物（ 图3， 如 Bb22，FB22，Bb46，FB46）。整体而言，在该实验中，假阳性率为低的SYN引物与WT模板（〜5％）和更高的WT的引物与模板的SYN（〜10％）。 <img alt="图2" src="/files/ftp_upload/52941/52941fig2.jpg" /> 图2：板式换热地图上显示存在/ ABSENCE呼叫要qPCRTag实验。四种不同类型的基因组DNA（象限分离固体白线）使用合成器（SYN）进行PCRTag分析和野生型（WT）3号染色体PCRTag引物（的虚线白线分开的SYN（顶）和WT（下图）在每个象限中）。 WT和基因组DNA synIXR编码野生型3号染色体，产生放大与WT PCRTag引物。synIII和synIII synIXR基因组DNA编码合成3号染色体，产生扩增SYN PCRTag引物。引物根据其左到右的染色体定位阵列和定位在相同的四个象限进行比较。 <a href="https://www.jove.com/files/ftp_upload/52941/52941fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/52941/52941fig3.jpg" /> 图3：板式换热地图为一个qPCRTag实验显示交叉点（CP）值，这是相同的数据集， 如图2和板布局，因此相同的。 N / A是指“未放大&#39;。 <a href="https://www.jove.com/files/ftp_upload/52941/52941fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about qPCRTag Analysis - A High Throughput, Real Time PCR Assay for Sc2.0 Genotyping at JoVE.com

qPCRTag Analysis - A High Throughput, Real Time PCR Assay for Sc2.0 Genotyping

Designer chromosomes of the Synthetic Yeast Genome project, Sc2.0, can be distinguished from their native counterparts using a PCR-based genotyping assay called PCRTagging, which has a presence/absence endpoint. Here we describe a high-throughput real time PCR detection method for PCRTag genotyping.

qPCRTag分析 - 高通量，实时定量PCR检测方法的基因分型Sc2.0

The Synthetic Yeast Genome Project (Sc2.0) aims to build 16 designer yeast chromosomes and combine them into a single yeast cell. To date one synthetic ...

qpcrtag-analysis-high-throughput-real-time-pcr-assay-for-sc20

Research

JoVE Journal

Biology

16.8K 次观看.  Institute for Systems Genetics. Designer chromosomes of the Synthetic Yeast Genome project, Sc2.0, can be distinguished from their native counterparts using a PCR-based genotyping assay called PCRTagging, which has a presence/absence endpoint. Here we describe a high-throughput real time PCR detection method for PCRTag genotyping.

视频: qPCRTag Analysis - A High Throughput, Real Time PCR Assay for Sc2.0 Genotyping

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

一个神经元和星形胶质细胞共培养检测神经毒性的高含量分析

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

科研

生物学

Quantitative Real-Time PCR using the Thermo Scientific Solaris qPCR Assay

The Solaris qPCR Gene Expression Assay is a novel type of primer/probe set, designed to simplify the qPCR process while maintaining the sensitivity and accuracy of the assay. These primer/probe sets are pre-designed to &gt;98% of the human and mouse genomes and feature significant improvements from previously available technologies. These improvements were made possible by virtue of a novel design algorithm, developed by Thermo Scientific bioinformatics experts.
Several convenient features have been incorporated into the Solaris qPCR Assay to streamline the process of performing quantitative real-time PCR. First, the protocol is similar to commonly employed alternatives, so the methods used during qPCR are likely to be familiar. Second, the master mix is blue, which makes setting the qPCR reactions easier to track. Third, the thermal cycling conditions are the same for all assays (genes), making it possible to run many samples at a time and reducing the potential for error. Finally, the probe and primer sequence information are provided, simplifying the publication process.
Here, we demonstrate how to obtain the appropriate Solaris reagents using the GENEius product search feature found on the ordering web site (www.thermo.com/solaris) and how to use the Solaris reagents for performing qPCR using the standard curve method.

The Solaris qPCR Gene Expression Assays are novel pre-designed qPCR primer/probe combinations designed to simplify the qPCR process without sacrificing the specificity and robustness of the assay.

实时定量PCR使用的Thermo Scientific的Solaris定量PCR检测

The Solaris qPCR Gene Expression Assay is a novel type of primer/probe set, designed to simplify the qPCR process while maintaining the sensitivity and ...

Analysis of Gene Expression in Emerald Ash Borer (Agrilus planipennis) Using Quantitative Real Time-PCR

Emerald ash borer (EAB, Agrilus planipennis) is an exotic invasive pest, which has killed millions of ash trees (Fraxinus spp) in North America. EAB continues to spread rapidly and attacks ash trees of different ages, from saplings to mature trees. However, to date very little or no molecular knowledge exists for EAB. We are interested in deciphering the molecular-based physiological processes at the tissue level that aid EAB in successful colonization of ash trees. In this report we show the effective use of quantitative real-time PCR (qRT-PCR) to ascertain mRNA levels in different larval tissues (including midgut, fat bodies and cuticle) and different developmental stages (including 1st-, 2nd-, 3rd-, 4th-instars, prepupae and adults) of EAB. As an example, a peritrophin gene (herein named, AP-PERI1) is exemplified as the gene of interest and a ribosomal protein (AP-RP1) as the internal control. Peritrophins are important components of the peritrophic membrane/matrix (PM), which is the lining of the insect gut. The PM has diverse functions including digestion and mechanical protection to the midgut epithelium.

Analysis of Gene Expression in Emerald Ash Borer Agrilus planipennis Using Quantitative Real Time-PCR

Quantitative real-time PCR (qRT-PCR) is an effective tool to diagnose mRNA levels in different insect tissues and developmental stages. In this report we show the use of qRT-PCR to ascertain mRNA levels in different larval tissues and developmental stages of the invasive insect species, emerald ash borer.

白蜡窄吉丁的基因表达分析（ Agrilus planipennis）使用实时定量PCR

Emerald ash borer (EAB, Agrilus planipennis) is an exotic invasive pest, which has killed millions of ash trees (Fraxinus spp) in North America. EAB ...

Profiling of Pre-micro RNAs and microRNAs using Quantitative Real-time PCR (qPCR) Arrays

Quantitative real-time PCR (QPCR) has emerged as an accurate and valuable tool in profiling gene expression levels.  One of its many advantages is a lower detection limit compared to other methods of gene expression profiling while using smaller amounts of input for each assay.  Automated qPCR setup has improved this field by allowing for greater reproducibility.  Its convenient and rapid setup allows for high-throughput experiments, enabling the profiling of many different genes simultaneously in each experiment.  This method along with internal plate controls also reduces experimental variables common to other techniques.  
We recently developed a qPCR assay for profiling of pre-microRNAs  (pre-miRNAs) using a set of 186 primer pairs.  MicroRNAs have emerged as a novel class of small, non-coding RNAs with the ability to regulate many mRNA targets at the post-transcriptional level.  These small RNAs are first transcribed by RNA polymerase II as a primary miRNA (pri-miRNA) transcript, which is then cleaved into the precursor miRNA (pre-miRNA).  Pre-miRNAs are exported to the cytoplasm where Dicer cleaves the hairpin loop to yield mature miRNAs.  Increases in miRNA levels can be observed at both the precursor and mature miRNA levels and profiling of both of these forms can be useful.  There are several commercially available assays for mature miRNAs; however, their high cost may deter researchers from this profiling technique.  Here, we discuss a cost-effective, reliable, SYBR-based qPCR method of profiling pre-miRNAs.  Changes in pre-miRNA levels often reflect mature miRNA changes and can be a useful indicator of mature miRNA expression.  However, simultaneous profiling of both pre-miRNAs and mature miRNAs may be optimal as they can contribute nonredundant information and provide insight into microRNA processing.  Furthermore, the technique described here can be expanded to encompass the profiling of other library sets for specific pathways or pathogens.

Profiling of Pre-micro RNAs and microRNAs using Quantitative Real-time PCR qPCR Arrays

We will demonstrate the setup and analysis of pre-microRNA 96-well arrays for QPCR using a robot as well as by hand with a Thermo Scientific Matrix multichannel pipette.

分析的预微RNA和小分子RNA采用实时定量PCR（定量PCR）阵列

Quantitative real-time PCR (QPCR) has emerged as an accurate and valuable tool in profiling gene expression levels.  One of its many advantages is a lower ...

High-throughput Saccharification Assay for Lignocellulosic Materials

Polysaccharides that make up plant lignocellulosic biomass can be broken down to produce a range of sugars that subsequently can be 
used in establishing a biorefinery. These raw materials would constitute a new industrial platform, which is both sustainable and carbon neutral, to replace 
the current dependency on fossil fuel. The recalcitrance to deconstruction observed in lignocellulosic materials is produced by several intrinsic properties 
of plant cell walls. Crystalline cellulose is embedded in matrix polysaccharides such as xylans and arabinoxylans, and the whole structure is encased by the 
phenolic polymer lignin, that is also difficult to digest 1. In order to improve the digestibility of plant materials we need to discover the main bottlenecks 
for the saccharification of cell walls and also screen mutant and breeding populations to evaluate the variability in saccharification 2. These tasks require 
a high throughput approach and here we present an analytical platform that can perform saccharification analysis in a 96-well plate format. This platform has 
been developed to allow the screening of lignocellulose digestibility of large populations from varied plant species. We have scaled down the reaction volumes 
for gentle pretreatment, partial enzymatic hydrolysis and sugar determination, to allow large numbers to be assessed rapidly in an automated system.
This automated platform works with milligram amounts of biomass, performing ball milling under controlled conditions to reduce the plant 
materials to a standardised particle size in a reproducible manner. Once the samples are ground, the automated formatting robot dispenses specified and recorded 
amounts of material into the corresponding wells of 96 deep well plate (Figure 1). Normally, we dispense the same material into 4 wells to have 4 replicates for 
analysis. Once the plates are filled with the plant material in the desired layout, they are manually moved to a liquid handling station (Figure 2). In this 
station the samples are subjected to a mild pretreatment with either dilute acid or alkaline and incubated at temperatures of up to 90&deg;C. The pretreatment solution 
is subsequently removed and the samples are rinsed with buffer to return them to a suitable pH for hydrolysis. The samples are then incubated with an enzyme
mixture for a variable length of time at 50&deg;C. An aliquot is taken from the hydrolyzate and the reducing sugars are automatically determined by the MBTH 
colorimetric method.

A simple, rapid method for determining the saccharification potential of large numbers of plant biomass samples is described. The automated platform for this analysis involves the preparation of the plant biomass for analysis in 96 well plates and the subsequent performance of pretreatment, hydrolysis and quantification of the sugars released.

木质纤维素材料的高通量检测糖化

Polysaccharides that make up plant lignocellulosic biomass can be broken down to produce a range of sugars that subsequently can be 
used in establishing ...

A PCR-based Genotyping Method to Distinguish Between Wild-type and Ornamental Varieties of Imperata cylindrica

Wild-type I. cylindrica (cogongrass) is one of the top ten worst invasive plants in the world, negatively impacting agricultural and natural resources in 73 different countries throughout Africa, Asia, Europe, New Zealand, Oceania and the Americas1-2. Cogongrass forms rapidly-spreading, monodominant stands that displace a large variety of native plant species and in turn threaten the native animals that depend on the displaced native plant species for forage and shelter. To add to the problem, an ornamental variety [I. cylindrica var. koenigii (Retzius)] is widely marketed under the names of Imperata cylindrica 'Rubra', Red Baron, and Japanese blood grass (JBG). This variety is putatively sterile and noninvasive and is considered a desirable ornamental for its red-colored leaves. However, under the correct conditions, JBG can produce viable seed (Carol Holko, 2009 personal communication) and can revert to a green invasive form that is often indistinguishable from cogongrass as it takes on the distinguishing characteristics of the wild-type invasive variety4 (Figure 1). This makes identification using morphology a difficult task even for well-trained plant taxonomists. Reversion of JBG to an aggressive green phenotype is also not a rare occurrence. Using sequence comparisons of coding and variable regions in both nuclear and chloroplast DNA, we have confirmed that JBG has reverted to the green invasive within the states of Maryland, South Carolina, and Missouri. JBG has been sold and planted in just about every state in the continental U.S. where there is not an active cogongrass infestation. The extent of the revert problem in not well understood because reverted plants are undocumented and often destroyed.
Application of this molecular protocol provides a method to identify JBG reverts and can help keep these varieties from co-occurring and possibly hybridizing. Cogongrass is an obligate outcrosser and, when crossed with a different genotype, can produce viable wind-dispersed seeds that spread cogongrass over wide distances5-7. JBG has a slightly different genotype than cogongrass and may be able to form viable hybrids with cogongrass. To add to the problem, JBG is more cold and shade tolerant than cogongrass8-10, and gene flow between these two varieties is likely to generate hybrids that are more aggressive, shade tolerant, and cold hardy than wild-type cogongrass. While wild-type cogongrass currently infests over 490 million hectares worldwide, in the Southeast U.S. it infests over 500,000 hectares and is capable of occupying most of the U.S. as it rapidly spreads northward due to its broad niche and geographic potential3,7,11. The potential of a genetic crossing is a serious concern for the USDA-APHIS Federal Noxious Week Program. Currently, the USDA-APHIS prohibits JBG in states where there are major cogongrass infestations (e.g., Florida, Alabama, Mississippi). However, preventing the two varieties from combining can prove more difficult as cogongrass and JBG expand their distributions. Furthermore, the distribution of the JBG revert is currently unknown and without the ability to identify these varieties through morphology, some cogongrass infestations may be the result of JBG reverts. Unfortunately, current molecular methods of identification typically rely on AFLP (Amplified Fragment Length Polymorphisms) and DNA sequencing, both of which are time consuming and costly. Here, we present the first cost-effective and reliable PCR-based molecular genotyping method to accurately distinguish between cogongrass and JBG revert.

A PCR-based Genotyping Method to Distinguish Between Wild-type and Ornamental Varieties of Imperata cylindrica

We provide a cost-effective and rapid molecular genotyping protocol that employs variety-specific PCR primers that target DNA sequence differences within the chloroplast trnL-F spacer region to differentiate between varieties of Imperata cylindrica (cogongrass) that cannot be distinguished by morphology alone. These varieties include the federally listed noxious weed, cogongrass and closely-related, wide-spread ornamental variety, I. cylindrica var. koenigii (Japanese blood grass).

一个基于PCR的基因分型方法来区分野生型和观赏品种白茅</em

Wild-type I. cylindrica (cogongrass) is one of the top ten worst invasive plants in the world, negatively impacting agricultural and natural resources in ...

Ice-Cap: A Method for Growing Arabidopsis and Tomato Plants in 96-well Plates for High-Throughput Genotyping

It is becoming common for plant scientists to develop projects that require the genotyping of large numbers of plants. The first step in any genotyping project is to collect a tissue sample from each individual plant. The traditional approach to this task is to sample plants one-at-a-time. If one wishes to genotype hundreds or thousands of individuals, however, using this strategy results in a significant bottleneck in the genotyping pipeline. The Ice-Cap method that we describe here provides a high-throughput solution to this challenge by allowing one scientist to collect tissue from several thousand seedlings in a single day 1,2. This level of throughput is made possible by the fact that tissue is harvested from plants 96-at-a-time, rather than one-at-a-time.
The Ice-Cap method provides an integrated platform for performing seedling growth, tissue harvest, and DNA extraction. The basis for Ice-Cap is the growth of seedlings in a stacked pair of 96-well plates. The wells of the upper plate contain plugs of agar growth media on which individual seedlings germinate. The roots grow down through the agar media, exit the upper plate through a hole, and pass into a lower plate containing water. To harvest tissue for DNA extraction, the water in the lower plate containing root tissue is rapidly frozen while the seedlings in the upper plate remain at room temperature. The upper plate is then peeled away from the lower plate, yielding one plate with 96 root tissue samples frozen in ice and one plate with 96 viable seedlings. The technique is named "Ice-Cap" because it uses ice to capture the root tissue. The 96-well plate containing the seedlings can then wrapped in foil and transferred to low temperature. This process suspends further growth of the seedlings, but does not affect their viability. Once genotype analysis has been completed, seedlings with the desired genotype can be transferred from the 96-well plate to soil for further propagation. We have demonstrated the utility of the Ice-Cap method using Arabidopsis thaliana, tomato, and rice seedlings. We expect that the method should also be applicable to other species of plants with seeds small enough to fit into the wells of 96-well plates.

Ice-Cap: A Method for Growing Arabidopsis and Tomato Plants in 96-well Plates for High-Throughput Genotyping

The Ice-Cap method allows one to grow plants in 96-well plates and non-destructively harvest root tissue from each seedling. DNA extracted from this root tissue can be used for genotyping reactions. We have found that Ice-Cap works well for Arabidopsis thaliana, tomato, and rice seedlings.

冰帽：为发展方法拟南芥和番茄植株在96孔板高通量的基因分型

It is becoming common for plant scientists to develop projects that require the genotyping of large numbers of plants. The first step in any genotyping ...

Rapid and Efficient Zebrafish Genotyping Using PCR with High-resolution Melt Analysis

Zebrafish is a powerful vertebrate model system for studying development, modeling disease, and performing drug screening. Recently a variety of genetic tools have been introduced, including multiple strategies for inducing mutations and generating transgenic lines. However, large-scale screening is limited by traditional genotyping methods, which are time-consuming and labor-intensive. Here we describe a technique to analyze zebrafish genotypes by PCR combined with high-resolution melting analysis (HRMA).&#160;This approach is rapid, sensitive, and inexpensive, with lower risk of contamination artifacts.&#160;Genotyping by PCR with HRMA can be used for embryos or adult fish, including in high-throughput screening protocols.

PCR combined with high-resolution melt analysis (HRMA) is demonstrated as a rapid and efficient method to genotype zebrafish.

快速，高效的斑马鱼基因分型采用PCR与高分辨率熔体分析

Zebrafish is a powerful vertebrate model system for studying development, modeling disease, and performing drug screening. Recently a variety of genetic ...

A High Throughput MHC II Binding Assay for Quantitative Analysis of Peptide Epitopes

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or design1,2. Here, a peptide-MHC II binding assay is scaled to 384-well format. The scaled down protocol reduces reagent costs by 75% and is higher throughput than previously described 96-well protocols1,3-5. Specifically, the experimental design permits robust and reproducible analysis of up to 15 peptides against one MHC II allele per 384-well ELISA plate. Using a single liquid handling robot, this method allows one researcher to analyze approximately ninety test peptides in triplicate over a range of eight concentrations and four MHC II allele types in less than 48 hr. Others working in the fields of protein deimmunization or vaccine design and development may find the protocol to be useful in facilitating their own work. In particular, the step-by-step instructions and the visual format of JoVE should allow other users to quickly and easily establish this methodology in their own labs.

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or design. &#160;Here, a peptide-MHC II binding assay scaled to 384-well plates is described. This cost effective format should prove useful in the fields of protein deimmunization and vaccine design and development.

高通量MHC II类分子结合分析抗原表位肽的定量分析

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or ...

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 ...