Name: qpcrtag analysis - a high throughput, real time pcr assay for sc2.0 genotyping
Uploaded: 2015-05-25T15:00:00.000+00:00
Duration: 7 min
Description: Watch this Scientific Journal Video about qPCRTag Analysis - A High Throughput, Real Time PCR Assay for Sc2.0 Genotyping at JoVE.com

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><tbody><tr><td colspan="4">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 (0.5 mm)</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 colspan="4">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>1.5 ml 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 colspan="4">Template and primer dispensing</td></tr><tr><td>PCRTag primers</td><td>IDT</td><td>custom</td><td>premixed forward and reverse, 50 &amp;mu;M 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 2.5 nl 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 ~2 mm thick washers</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 colspan="4">Real Time PCR</td></tr><tr><td>LightCycler 1536</td><td>Roche</td><td></td><td>requires 220 V 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.bdigitaluss.remotexs.co/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.bdigitaluss.remotexs.co/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

Universidad San Sebastian

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JoVE Journal

Biology

17.0K 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

High Throughput MicroRNA Profiling: Optimized Multiplex qRT-PCR at Nanoliter Scale on the Fluidigm Dynamic ArrayTM IFCs

The broad involvement of miRNAs in critical processes underlying development, tissue homoeostasis and disease has led to a surging interest among the research and pharmaceutical communities. To study miRNAs, it is essential that the quantification of microRNA levels is accurate and robust. By comparing wild-type to small RNA deficient mouse embryonic stem cells (mESC), we revealed a lack of accuracy and robustness in previous published multiplex qRT-PCR techniques. Here, we describe an optimized method, including purifying away excessive primers from previous multiplex steps before singleplex real time detection, which dramatically increases the accuracy and robustness of the technique. Furthermore, we explain how performing the technique on a microfluidic chip at nanoliter volumes significantly reduces reagent costs and permits time effective high throughput miRNA expression profiling.

Here we describe an optimized multiplex reverse transcriptase quantitative PCR (qRT-PCR) protocol in combination with a microfluidic platform as a cost and time effective high-throughput screening tool for microRNA (miRNA) expression levels, especially when working with limited amounts of sample.

高通量微RNA分析：优化复纳升秤定量RT - PCR Fluidig​​m公司动态ArrayTM国际金融中心

The broad involvement of miRNAs in critical processes underlying development, tissue homoeostasis and disease has led to a surging interest among the ...

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Single-cell Gene Expression Using Multiplex RT-qPCR to Characterize Heterogeneity of Rare Lymphoid Populations

Gene expression heterogeneity is an interesting feature to investigate in lymphoid populations. Gene expression in these cells varies during cell activation, stress, or stimulation. Single-cell multiplex gene expression enables the simultaneous assessment of tens of genes1,2,3. At the single-cell level, multiplex gene expression determines population heterogeneity4,5. It allows for the distinction of population heterogeneity by determining both the probable mix of diverse precursor stages among mature cells and also the diversity of cell responses to stimuli.
Innate lymphoid cells (ILC) have been recently described as a population of innate effectors of the immune response6,7. In this protocol, cell heterogeneity of the ILC hepatic compartment is investigated during homeostasis.
Currently, the most widely used technique to assess gene expression is RT-qPCR. This method measures gene expression only one gene at a time. Additionally, this method cannot estimate heterogeneity of gene expression, since multiple cells are needed for one test. This leads to the measurement of the average gene expression of the population. When assessing large numbers of genes, RT-qPCR becomes a time-, reagent-, and sample-consuming method. Hence, the trade-offs limit the number of genes or cell populations that can be evaluated, increasing the risk of missing the global picture.
This manuscript describes how single-cell multiplex RT-qPCR can be used to overcome these limitations. This technique has benefited from recent microfluidics technological advances1,2. Reactions occurring in multiplex RT-qPCR chips do not exceed the nanoliter-level. Hence, single-cell gene expression, as well as simultaneous multiple gene expression, can be performed in a reagent-, sample-, and cost-effective manner. It is possible to test cell gene signature heterogeneity at the clonal level between cell subsets within a population at different developmental stages or under different conditions4,5. Working on rare populations with large numbers of conditions at the single-cell level is no longer a restriction.

This protocol describes how to assess the expression of a large array of genes at the clonal level. Single-cell RT-qPCR produces highly reliable results with a strong sensitivity for hundreds of samples and genes.

单细胞基因表达利用多重RT-qPCR的表征罕见的淋巴人群的异质性

Gene expression heterogeneity is an interesting feature to investigate in lymphoid populations. Gene expression in these cells varies during cell ...

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Adapting 3' Rapid Amplification of CDNA Ends to Map Transcripts in Cancer

Maturation of eukaryotic mRNAs involves 3&#39; end formation, which involves the addition of a poly(A) tail. In order to map the 3&#39; end of a gene, the traditional method of choice is 3&#39; rapid amplification of cDNA ends (3&#39; RACE). Protocols for 3&#39; RACE require the careful design and selection of nested primers within the 3&#39; untranslated region (3&#39; UTR) of the target gene of interest. However, with a few modifications the protocol can be used to include the entire 3&#39; UTR and sequences within the open reading frame (ORF), providing a more comprehensive picture of the relationship between the ORF and the 3&#39; UTR. This is in addition to identification of the polyadenylation signal (PAS), as well as the cleavage and polyadenylation site provided by conventional 3&#39; RACE. Expanded 3&#39; RACE can detect unusual 3&#39; UTRs, including gene fusions within the 3&#39; UTR, and the sequence information can be used to predict potential miRNA binding sites as well as AU rich destabilizing elements that may affect the stability of the transcript.

The two different 3&#39; rapid amplification of cDNA ends (3&#39; RACE) protocols described here make use of two different DNA polymerases to map sequences that include a segment of the open reading frame (ORF), the stop codon, and the entire 3&#39; UTR of a transcript using RNA obtained from different cancer cell lines.

3 ' 快速扩增 CDNA 末端, 以图记录癌症

Maturation of eukaryotic mRNAs involves 3&#39; end formation, which involves the addition of a poly(A) tail. In order to map the 3&#39; end of a gene, the ...

Candidate Gene Testing in Clinical Cohort Studies with Multiplexed Genotyping and Mass Spectrometry

Complex diseases are often underpinned by multiple common genetic variants that contribute to disease susceptibility. Here, we describe a cost-effective tag single nucleotide polymorphism (SNP) approach using a multiplexed genotyping assay with mass spectrometry, to investigate gene pathway associations in clinical cohorts. We investigate the food allergy candidate locus Interleukin13 (IL13) as an example. This method efficiently maximizes the coverage by taking advantage of shared linkage disequilibrium (LD) within a region. Selected LD SNPs are then designed into a multiplexed assay, enabling up to 40 different SNPs to be analyzed simultaneously, boosting cost-effectiveness. Polymerase chain reaction (PCR) is used to amplify the target loci, followed by single nucleotide extension, and the amplicons are then measured using matrix-assisted laser desorption/ionization-time of flight(MALDI-TOF) mass spectrometry. The raw output is analyzed with the genotype calling software, using stringent quality control definitions and cut-offs, and high probability genotypes are determined and output for data analysis.

Identification of genetic variants contributing to complex human disease allows us to identify novel mechanisms. Here, we demonstrate a multiplex genotyping approach to candidate genes or gene pathway analysis that maximizes the coverage at low cost and is amenable to cohort-based studies.

应用多重基因分型和质谱法进行临床队列研究的候选基因检测

Complex diseases are often underpinned by multiple common genetic variants that contribute to disease susceptibility. Here, we describe a cost-effective ...

Oncogenic Gene Fusion Detection Using Anchored Multiplex Polymerase Chain Reaction Followed by Next Generation Sequencing

Gene fusions frequently contribute to the oncogenic phenotype of many different types of cancer. Additionally, the presence of certain fusions in samples from cancer patients often directly influences diagnosis, prognosis, and/or therapy selection. As a result, the accurate detection of gene fusions has become a critical component of clinical management for many disease types. Until recently, clinical gene fusion detection was predominantly accomplished through the use of single-gene assays. However, the ever-growing list of gene fusions with clinical significance has created a need for assessing fusion status of multiple genes simultaneously. Next generation sequencing (NGS)-based testing has met this demand through the ability to sequence nucleic acid in massively parallel fashion. Multiple NGS-based approaches that employ different strategies for gene target enrichment are now available for use in clinical molecular diagnostics, each with its own strengths and weaknesses. This article describes the use of anchored multiplex PCR (AMP)-based target enrichment and library preparation followed by NGS to assess for gene fusions in clinical solid tumor specimens. AMP is unique among amplicon-based enrichment approaches in that it identifies gene fusions regardless of the identity of the fusion partner. Detailed here are both the wet-bench and data analysis steps that ensure accurate gene fusion detection from clinical samples.

This article details the use of an anchored multiplex polymerase chain reaction-based library preparation kit followed by next-generation sequencing to assess for oncogenic gene fusions in clinical solid tumor samples. Both wet-bench and data analysis steps are described.

使用锚定多路聚合酶链反应的致癌基因融合检测,随后进行下一代测序

Gene fusions frequently contribute to the oncogenic phenotype of many different types of cancer. Additionally, the presence of certain fusions in samples ...

癌症研究

Cerebrospinal Fluid MicroRNA Profiling Using Quantitative Real Time PCR

MicroRNAs (miRNAs) constitute a potent layer of gene regulation by guiding RISC to target sites located on mRNAs and, consequently, by modulating their translational repression. Changes in miRNA expression have been shown to be involved in the development of all major complex diseases. Furthermore, recent findings showed that miRNAs can be secreted to the extracellular environment and enter the bloodstream and other body fluids where they can circulate with high stability. The function of such circulating miRNAs remains largely elusive, but systematic high throughput approaches, such as miRNA profiling arrays, have lead to the identification of miRNA signatures in several pathological conditions, including neurodegenerative disorders and several types of cancers. In this context, the identification of miRNA expression profile in the cerebrospinal fluid, as reported in our recent study, makes miRNAs attractive candidates for biomarker analysis.
There are several tools available for profiling microRNAs, such as microarrays, quantitative real-time PCR (qPCR), and deep sequencing. Here, we describe a sensitive method to profile microRNAs in cerebrospinal fluids by quantitative real-time PCR. We used the Exiqon microRNA ready-to-use PCR human panels I and II V2.R, which allows detection of 742 unique human microRNAs. We performed the arrays in triplicate runs and we processed and analyzed data using the GenEx Professional 5 software.
Using this protocol, we have successfully profiled microRNAs in various types of cell lines and primary cells, CSF, plasma, and formalin-fixed paraffin-embedded tissues.

We describe a protocol of real time PCR to profile microRNAs in the cerebrospinal fluid (CSF). With the exception of RNA extraction protocols, the procedure can be extended to RNA extracted from other body fluids, cultured cells, or tissue specimens.

脑脊液的microRNA分析采用实时定量PCR技术

MicroRNAs (miRNAs) constitute a potent layer of gene regulation by guiding RISC to target sites located on mRNAs and, consequently, by modulating their ...

医学

Simultaneous Quantification of T-Cell Receptor Excision Circles (TRECs) and K-Deleting Recombination Excision Circles (KRECs) by Real-time PCR

T-cell receptor excision circles (TRECs) and K-deleting recombination excision circles (KRECs) are circularized DNA elements formed during recombination process that creates T- and B-cell receptors. Because TRECs and KRECs are unable to replicate, they are diluted after each cell division, and therefore persist in the cell. Their quantity in peripheral blood can be considered as an estimation of thymic and bone marrow output. By combining well established and commonly used TREC assay with a modified version of KREC assay, we have developed a duplex quantitative real-time PCR that allows quantification of both newly-produced T and B lymphocytes in a single assay. The number of TRECs and KRECs are obtained using a standard curve prepared by serially diluting TREC and KREC signal joints cloned in a bacterial plasmid, together with a fragment of T-cell receptor alpha constant gene that serves as reference gene. Results are reported as number of TRECs and KRECs/106 cells or per ml of blood. The quantification of these DNA fragments have been proven useful for monitoring immune reconstitution following bone marrow transplantation in both children and adults, for improved characterization of immune deficiencies, or for better understanding of certain immunomodulating drug activity.

Simultaneous Quantification of T-Cell Receptor Excision Circles TRECs and K-Deleting Recombination Excision Circles KRECs by Real-time PCR

Here, we describe a method for simultaneous quantification of T-cell receptor excision circles (TRECs) and K-deleting recombination excision circles (KRECs). The TREC/KREC assay can be used as marker of thymic and bone marrow output.

T细胞受体删除环同时定量（TRECs）和K-删除重组删除环（KRECs）通过实时PCR

T-cell receptor excision circles (TRECs) and K-deleting recombination excision circles (KRECs) are circularized DNA elements formed during recombination ...

免疫与感染

Infinium Assay for Large-scale SNP Genotyping Applications

Genotyping variants in the human genome has proven to be an efficient method to identify genetic associations with phenotypes. The distribution of variants within families or populations can facilitate identification of the genetic factors of disease. Illumina&#39;s panel of genotyping BeadChips allows investigators to genotype thousands or millions of single nucleotide polymorphisms (SNPs) or to analyze other genomic variants, such as copy number, across a large number of DNA samples. These SNPs can be spread throughout the genome or targeted in specific regions in order to maximize potential discovery. The Infinium assay has been optimized to yield high-quality, accurate results quickly. With proper setup, a single technician can process from a few hundred to over a thousand DNA samples per week, depending on the type of array. This assay guides users through every step, starting with genomic DNA and ending with the scanning of the array. Using propriety reagents, samples are amplified, fragmented, precipitated, resuspended, hybridized to the chip, extended by a single base, stained, and scanned on either an iScan or Hi Scan high-resolution optical imaging system. One overnight step is required to amplify the DNA. The DNA is denatured and isothermally amplified by whole-genome amplification; therefore, no PCR is required. Samples are hybridized to the arrays during a second overnight step. By the third day, the samples are ready to be scanned and analyzed. Amplified DNA may be stockpiled in large quantities, allowing bead arrays to be processed every day of the week, thereby maximizing throughput.

A protocol is described that uses Illumina&#39;s Infinium assays to perform large-scale genotyping. These assays can reliably genotype millions of SNPs across hundreds of individual DNA samples in three days. Once generated, these genotypes can be used to check for associations with a variety of different diseases or phenotypes.

Infinium检测的大型SNP基因分型应用

Genotyping variants in the human genome has proven to be an efficient method to identify genetic associations with phenotypes. The distribution of ...

生物学

Chip-Based Digital PCR to Detect Rare Transcript Variants Using a Nanofluidic Chip

Source: Molinari, C., et al. <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/57066">Detection of a CDH1 Rare Transcript Variant in Fresh-frozen Gastric Cancer Tissues by Chip-based Digital PCR.</a> J. Vis. Exp. (2018).
This video demonstrates chip-based digital PCR &#8212; a variation of the digital PCR technique that is useful in detecting rare transcript variants. The PCR reaction is partitioned into the chambers of a nanofluidic chip, each of which acts as an independent reaction. The detection of fluorescence signals from the chambers with amplified targets confirms the presence of rare transcript variants in the sample.

基于芯片的数字 PCR 使用纳米流控芯片检测罕见的转录本变体

Source: Molinari, C., et al. Detection of a CDH1 Rare Transcript Variant in Fresh-frozen Gastric Cancer Tissues by Chip-based Digital PCR. J. Vis. Exp. ...

JoVE Encyclopedia of Experiments

Biological Techniques

PCR Techniques

Chip-in-a-Tube-Based Digital PCR for Quantification of Single Nucleotide Variants

Source: Kahyo, T., et al. <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/58199">Digital Polymerase Chain Reaction Assay for the Genetic Variation in a Sporadic Familial Adenomatous Polyposis Patient Using the Chip-in-a-tube Format.</a> J. Vis. Exp. (2018).
This video demonstrates the utility of digital PCR in a chip-in-a-tube format for the detection of single nucleotide variations in DNA samples. A single PCR reaction is partitioned into chambers that act as independent PCR reactions, and the detection of fluorescence signals from the amplified targets in the chambers is used to compute the frequency of the variant allele in the sample.

基于 Chip-in-a-Tube 的数字 PCR，用于定量单核苷酸变异

Source: Kahyo, T., et al. Digital Polymerase Chain Reaction Assay for the Genetic Variation in a Sporadic Familial Adenomatous Polyposis Patient Using the ...

qPCRTag分析 - 高通量，实时定量PCR检测方法的基因分型Sc2.0

摘要

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高通量微RNA分析：优化复纳升秤定量RT - PCR Fluidigm公司动态ArrayTM国际金融中心

单细胞基因表达利用多重RT-qPCR的表征罕见的淋巴人群的异质性

3 ' 快速扩增 CDNA 末端, 以图记录癌症

应用多重基因分型和质谱法进行临床队列研究的候选基因检测

使用锚定多路聚合酶链反应的致癌基因融合检测,随后进行下一代测序

脑脊液的microRNA分析采用实时定量PCR技术

T细胞受体删除环同时定量（TRECs）和K-删除重组删除环（KRECs）通过实时PCR

Infinium检测的大型SNP基因分型应用

基于芯片的数字 PCR 使用纳米流控芯片检测罕见的转录本变体

基于 Chip-in-a-Tube 的数字 PCR，用于定量单核苷酸变异

qPCRTag分析 - 高通量，实时定量PCR检测方法的基因分型Sc2.0

摘要

探索更多视频

此视频中的章节

高通量微RNA分析：优化复纳升秤定量RT - PCR Fluidig​​m公司动态ArrayTM国际金融中心

单细胞基因表达利用多重RT-qPCR的表征罕见的淋巴人群的异质性

3 ' 快速扩增 CDNA 末端, 以图记录癌症

应用多重基因分型和质谱法进行临床队列研究的候选基因检测

使用锚定多路聚合酶链反应的致癌基因融合检测,随后进行下一代测序

脑脊液的microRNA分析采用实时定量PCR技术

T细胞受体删除环同时定量（TRECs）和K-删除重组删除环（KRECs）通过实时PCR

Infinium检测的大型SNP基因分型应用

基于芯片的数字 PCR 使用纳米流控芯片检测罕见的转录本变体

基于 Chip-in-a-Tube 的数字 PCR，用于定量单核苷酸变异

高通量微RNA分析：优化复纳升秤定量RT - PCR Fluidigm公司动态ArrayTM国际金融中心