The authors acknowledge Liyi Xu for providing MDA reagents and protocols. We also thank David Feldman and Navpreet Ranu for discussions relating to this work. This work was supported in part by a Burroughs Wellcome Career Award at the Scientific Interface to PCB.

注意:制造微流体装置是没有必要的该测定中，作为该协议的液滴可以与现有的商业液滴决策者23,29来形成。 1.使液滴形成微流体装置<ol><li>制备母模的通道与SU-8万事达制作协议先前31所概述的，但有一个液滴发生器掩模图案 32。 </li><li>编造使用软光刻32,33的技术，PDMS设备。 </li></ol> 2.准备反应预混散装滴读数 注意:该协议可以用于量化使用多种类型的扩增反应的核酸。作为一个例子，所需的数是Lambda DNA浓度为MDA试剂给出。试剂细节中列出的具体试剂的表中。模板的液滴内的分布取决于sufficien吨混合样品。 <ol><li>获得或净化Phi29 DNA聚合酶。 <ol><li>净化Phi29如前所述7，或购买来自供应商。用于示出实验的Phi29进行纯化。 </li></ol></li><li>制备出10 ml的变性缓冲液的由65毫米的KOH，1.65毫摩尔EDTA，14毫DTT。 </li><li>制备出10 ml的中和缓冲液的由65毫米的HCl，0.21摩尔Tris-CL pH为7.0，和9毫三Cl pH8.0中。 </li><li>准备两个标准，一种无模板对照（NTC）如无核酸酶水，和一种高模板浓度（约4微克/微升是Lambda DNA）。变性3.3微升每个标准与定量PCR管3.3微升变性缓冲液。在室温下孵育3分钟。淬火每个和缓冲液3.3微升。 </li><li>变性3.3微升与定量PCR管3.3微升变性缓冲液感兴趣的模板。在室温下孵育3分钟。淬火与3.3微升的中和缓冲。 </li><li>准备每个样品11微升主混合物在冰上。的2倍的MDA反应主混合物由2个双链DNA的（双链DNA）结合染料，2倍的qPCR参照染料，50μM随机寡聚，2毫克/毫升牛血清白蛋白，2×Phi29 DNA聚合酶反应缓冲液，4.8毫的dNTPs，无核酸酶水和40微克/毫升Phi29 DNA聚合酶。添加Phi29 DNA聚合酶最后，以确保聚合酶不会遇到的pH水平要比7.5.Mix更高或更低的井。 </li><li>可选:对于减少误判，暴露主结构用UV光形成液滴34之前。 <ol><li>准备10微升每个样品预混合物在冰上0.5 ml或1.5毫升透明管。预混由55微米的随机寡，2.2毫克/毫升BSA，1.1倍Phi29 DNA聚合酶反应缓冲液，5.3毫dNTPs和无核酸酶水。 </li><li>将管中的水在冰上如所述34和暴露于UV光（254 nm）到一个ACCU5.7Ĵmulated剂量/厘米2。 </li><li>双链DNA结合染料至1x，参考染料为1x，并且Phi29添加到40微克/毫升的预混合物。拌匀。 </li></ol></li><li>结合10微升变性的模板或标准和10μl主混合物。拌匀。 </li></ol> 3.液滴形成注意:液滴可以是静电和剪切应力非常敏感。脱去衣服，可能会产生静电，并形成液滴之前自己接地。处理乳液的管子从管的顶部，从乳液尽可能远。吸管乳剂非常慢，优选具有宽孔移液管尖端。当从移液管尖端的分配，观看在顶端侧的乳液，以确定移液速度。 <ol><li>形成液滴。对于所示的实验中，微流体装置具有进油口和两个水入口用流聚焦结用于产生液滴。用两个syriNGE泵通过微流体芯片来控制的55微升油与表面活性剂和20微升反应混合物的流速，以产生20000 1 NL液滴。 </li><li>注意:可以生产具有许多类型的油与表面活性剂的液滴，但该组合物会大大在高温和随着时间的推移影响液滴的稳定性。一种可能的组合是氟化油HFE 7500与阴离子表面活性剂19,35。 </li><li>产生液滴任何尺寸的任何方法在13,36，但是在液滴种群规模可变性将影响散装荧光，因此，测定的精确度。对于所示的实验中，微滴是单分散的1 NL的体积。 </li><li>收集所有的微滴和油的进PCR管。对于每个样品，等份加入30μl的油进入新鲜PCR管具有光学帽，并转移20微升的液滴上的油的顶部。一贯卷保证试验是作为交流策展越好。 </li><li>关闭管帽太紧，松盖将使中石油蒸发。 </li></ol> 4.恒温扩增<ol><li>使用PCR热循环仪，定量PCR热循环仪，或热板，孵育样品在30℃下7小时，和灭活1分钟在75℃。液滴的管可以留在用铝箔纸覆盖在这点上几个小时，一台冰箱。 </li></ol> 5.数据采集与分析<ol><li>紧接着反应失活，测量染料的荧光水平使用qPCR的热循环的所有样本。最佳的过滤器设置将取决于染料的激发和发射光谱。对于这个例子，使用492纳米516纳米滤光器对所述双链DNA结合染料设置，而585纳米-610nm的过滤器的参考染料设定。其它荧光测量技术可用于量化荧光。 </li><li>对于每个样品，通过划分双链DNA结合染料的荧光强度参考染料的荧光强度。减去背景荧光，或无模板对照的标准化荧光，从所有样品。 </li><li>创建使用从标准的校正的荧光测量值的线性标准曲线。 NTC的标准表示荧光为0％荧光滴的样品（"全部阴性"），而高浓度的模板荧光测量是用100％的荧光微滴的样品的预期荧光（"所有阳性"）。使用相应的标准曲线，预测基于它们散装荧光阳性和阴性的液滴的中间比率。 </li></ol> 6.产液滴的证明性原理测量<ol><li>使荧光灯（正）的液滴:准备一个PCR反应混合物由0.1纳克是Lambda DNA，1×双链DNA结合染料，1x引用染料，1.5单位Taq DNA聚合酶，10毫摩尔Tris-盐酸，50mM的氯化钾，1.5米的中号的MgCl 2，0.2 10mM的dNTP，和0.5μM引物。热循环该混合物的30倍（95℃，20秒，55℃，20秒，72℃1分钟），然后形成液滴，如上所述。 </li><li>使非荧光（负）的液滴:准备一个PCR反应混合物由1×双链DNA结合染料的，1x引用染料，1.5单位Taq DNA聚合酶，10毫摩尔Tris-盐酸，50mM的氯化钾，1.5mM的MgCl 2的，为0.2mM的dNTP，和0.5μM的引物。热循环该混合物的30倍（95℃，20秒，55℃，20秒，72℃1分钟），然后形成液滴。 </li><li>形成预混合液滴的比率<ol><li>分发20微升氟化油的成定量PCR管，覆盖有光学帽以防止蒸发。 </li><li>轻轻移取10微升充分混合液滴的期望正:负比对石油的顶部。在示出的结果，正面:负液滴比为1（所有阳性），0.8，0.5，0.2，0.1，0.01，0.001 0.0001，和0（所有阴性ative）。 </li></ol></li><li>测量染料的荧光水平使用qPCR的热循环的所有样本。此协议中，使用492毫微米-516 nm的滤波器的双链DNA结合染料设置，而585纳米-610nm的过滤器的参考染料设定。 <ol><li>规范化双链DNA结合使用参比染料荧光染料的荧光。由"一切积极"样品的荧光进一步规范。 </li></ol></li><li>创建使用从"一切积极"和"消极全部"样本归一化荧光测量线性标准曲线。使用相应的标准曲线，预测基于它们散装荧光阳性和阴性的液滴的中间比率。 </li><li>重复实验的每个比（n = 3）与独立液滴形成并混合每个重复。 </li></ol> 7.验证性原则MDA实验<ol><li>测量的是Lambda DNA原液中的浓度与一个规范trophotometer。 <ol><li>计算需要每滴平均10模板分子的模板浓度。对于这个协议，假设液滴体积为1 NL和1纳克LAMBDA DNA中含有约1.9×10 7份。因此，每滴10模板将每NL 10份，或每微升526 FG LAMBDA DNA。该模板将被摊薄〜6倍的液滴时形成，因此初始模板最高浓度为3.2微克/微升。 </li></ol></li><li>串行稀释LAMBDA DNA以3.2微克/μL变性缓冲液和等量和缓冲液。为0.1的稀释因子，结合10微升样品与45微升变性缓冲液，并在室温下孵育3分钟。加入45为1L和缓冲液的淬火。 </li><li>如步骤7.2中所述，使样品的其余部分的实验进一步稀释样品。在反应的初始模板浓度示分别为3.2皮克，320 FG，160 FG，32 FG，16 FG，3.2 FG，和每微升1.6 FG。 注意:mastermix增设后的最终模板浓度分别为526 FG，52.6 FG，26.3 FG，5.26 FG，2.63 FG，526 Ag，以及263每微升股份公司。 10，1，0.5％，0.1，0.05，0.01，和0.005预期LAMBDA每液滴份，分别。 </li><li>生成并如步骤2.5-5.3中所述分析来自每个样品液滴。 </li><li>获得使用数码相机中所示的荧光图像（图2,3），其安装到一个落射荧光显微镜用在RT 10x物镜。收购查看每个样品12场。正确的荧光图像通过用荧光滑动而得到的背景图像。 </li><li>使用含有室成像37的乳化油将蒸发快。 </li><li>分析使用ImageJ的宏（补充代码文件）单个液滴的强度。 <ol><li>选择最分离非荧光滴在N阈值荧光强度TC图像从高浓度的模板图像的荧光液滴。对于每个未知样品，计算的液滴的馏分高于阈值的荧光强度。 </li></ol></li></ol> 8. PCR和MDA散装反应<ol><li>制备PCR反应。 <ol><li>串联稀释的模板DNA为0.1的稀释因子。每个稀释，结合10微升模板与45微升变性缓冲液，并在室温下孵育3分钟。合并稀释45微升和缓冲液的。 </li><li>准备20微升每个样品PCR反应体系的。的1.1倍的PCR反应主混合物由60单位/微升Taq DNA聚合酶，11毫摩尔Tris盐酸，55MM氯化钾，1.65毫氯化镁 ，0.22 10mM的dNTP，1.1倍的双链DNA结合染料，1.1倍参照染料，和550纳米每个底漆。 </li><li>结合2微升稀释的模板和20微升反应体系的。 注意:最终模板浓度吨他表示PCR反应是50 PG，PG 5，500 FG，50 FG，5 FG，500 Ag和0克LAMBDA每微升的DNA，并一式三份进行。 </li><li>在定量PCR机热循环PCR反应与以下程序。运行的95℃的40个循环，30秒，57℃，20秒，72℃30秒2分钟，然后在72℃进行2分钟，并保持在4℃。 </li><li>规范化双链DNA结合染料的荧光用参比染料荧光之前，进一步的分析。 </li></ol></li><li>准备MDA反应。 <ol><li>如步骤8.1.1中所述稀释样品。 </li><li>准备每个样品11微升主混合物在冰上。的2倍的MDA反应主混合物由2个双链DNA结合染料，2倍参照染料，50μM随机寡聚，2毫克/毫升牛血清白蛋白，2×phi29 DNA聚合酶反应缓冲液，4.8毫的dNTPs，无核酸酶水，和40微克/毫升Phi29 DNA聚合酶。 </li><li>所述Phi29 DNA聚合酶添加至40微克/ ml的持续确保聚合酶ðOES不会遇到的pH水平远高于7.5或更低。拌匀。 </li><li>变性稀释模板3.3μL与定量PCR管3.3微升变性缓冲液。在室温下孵育3分钟。加入3.3微升和缓冲液的。 </li><li>结合10μL的变性模板和10μl主混合物。拌匀。 </li><li>运行定量PCR机MDA反应与以下程序。 </li><li>持在30℃4小时，测量荧光每7.5分钟。 </li><li>灭活在75℃下1分钟。 </li><li>规范化双链DNA结合使用参比染料荧光染料的荧光。由最大荧光每个样品进一步正常化。 </li></ol></li></ol>

<ol><li>Sykes, P. J., Neoh, S. H., Brisco, M. J., Hughes, E., Condon, J., Morley, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quantitation+of+targets+for+PCR+by+use+of+limiting+dilution%5BTitle%5D)+AND+%22Biotechniques%22%5BJournal%5D)">Quantitation of targets for PCR by use of limiting dilution.</a> Biotechniques. 13 (3), 444-449 (1992).</li>
<li>Kalinina, O., Lebedeva, I., Brown, J., Silver, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Nanoliter+scale+PCR+with+TaqMan+detection%5BTitle%5D)+AND+%22Nucleic+Acids+Res%22%5BJournal%5D)">Nanoliter scale PCR with TaqMan detection.</a> Nucleic Acids Res. 25 (10), 1999-2004 (1997).</li>
<li>Vogelstein, B., Kinzler, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Digital+PCR%5BTitle%5D)+AND+%22Proc.+Natl.+Acad.+Sci.+U.S.A.%22%5BJournal%5D)">Digital PCR.</a> Proc. Natl. Acad. Sci. U.S.A.. 96 (16), 9236-9241 (1999).</li>
<li>Day, E., Dear, P. H., McCaughan, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Digital+PCR+strategies+in+the+development+and+analysis+of+molecular+biomarkers+for+personalized+medicine%5BTitle%5D)+AND+%22Methods%22%5BJournal%5D)">Digital PCR strategies in the development and analysis of molecular biomarkers for personalized medicine.</a> Methods. 59 (1), 101-107 (2013).</li>
<li>Tatusova, T., Ciufo, S., Fedorov, B., O'Neill, K., Tolstoy, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((RefSeq+microbial+genomes+database%3A+new+representation+and+annotation+strategy%5BTitle%5D)+AND+%22Nucleic+Acids+Res%22%5BJournal%5D)">RefSeq microbial genomes database: new representation and annotation strategy.</a> Nucleic Acids Res. 42 (Database issue), D553-D559 (2014).</li>
<li>Blainey, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+future+is+now%3A+single-cell+genomics+of+bacteria+and+archaea%5BTitle%5D)+AND+%22FEMS+Microbiol.+Rev%22%5BJournal%5D)">The future is now: single-cell genomics of bacteria and archaea.</a> FEMS Microbiol. Rev. 37 (3), 407-427 (2013).</li>
<li>Blainey, P. C., Quake, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Digital+MDA+for+enumeration+of+total+nucleic+acid+contamination%5BTitle%5D)+AND+%22Nucleic+Acids+Res%22%5BJournal%5D)">Digital MDA for enumeration of total nucleic acid contamination.</a> Nucleic Acids Res. 39 (4), e19(2011).</li>
<li>Blainey, P. C., Quake, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dissecting+genomic+diversity%2C+one+cell+at+a+time%5BTitle%5D)+AND+%22Nat.+Methods%22%5BJournal%5D)">Dissecting genomic diversity, one cell at a time.</a> Nat. Methods. 11 (1), 19-21 (2014).</li>
<li>Binga, E. K., Lasken, R. S., Neufeld, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Something+from+%28almost%29+nothing%3A+the+impact+of+multiple+displacement+amplification+on+microbial+ecology%5BTitle%5D)+AND+%22ISME+J%22%5BJournal%5D)">Something from (almost) nothing: the impact of multiple displacement amplification on microbial ecology.</a> ISME J. 2 (3), 233-241 (2008).</li>
<li><a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Method+of+the+Year+2013%5BTitle%5D)+AND+%22Nature+Methods%22%5BJournal%5D)">Method of the Year 2013.</a> Nature Methods. 11 (1), 1(2014).</li>
<li>Ottesen, E. A., Hong, J. W., Quake, S. R., Leadbetter, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microfluidic+digital+PCR+enables+multigene+analysis+of+individual+environmental+bacteria%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Microfluidic digital PCR enables multigene analysis of individual environmental bacteria.</a> Science. 314 (5804), 1464-1467 (2006).</li>
<li>OpenArray Technology Overview. , Life Technologies. <a target="_blank" href="http://www.lifetechnologies.com/us/en/home/life-science/pcr/real-time-pcr/real-time-openarray/open-array-technology.html">http://www.lifetechnologies.com/us/en/home/life-science/pcr/real-time-pcr/real-time-openarray/open-array-technology.html</a> (2014).</li>
<li>Shen, F., Du, W., Kreutz, J. E., Fok, A., Ismagilov, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Digital+PCR+on+a+SlipChip.%5BTitle%5D)+AND+%22Lab+Chip%22%5BJournal%5D)">Digital PCR on a SlipChip.</a> Lab Chip. 10 (20), 2666-2672 (2010).</li>
<li>Heyries, K. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Megapixel+digital+PCR%5BTitle%5D)+AND+%22Nat.+Methods%22%5BJournal%5D)">Megapixel digital PCR.</a> Nat. Methods. 8 (8), 649-651 (2011).</li>
<li>Dressman, D., Yan, H., Traverso, G., Kinzler, K. W., Vogelstein, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Transforming+single+DNA+molecules+into+fluorescent+magnetic+particles+for+detection+and+enumeration+of+genetic+variations%5BTitle%5D)+AND+%22Proc.+Natl.+Acad.+Sci.+U.S.A%22%5BJournal%5D)">Transforming single DNA molecules into fluorescent magnetic particles for detection and enumeration of genetic variations.</a> Proc. Natl. Acad. Sci. U.S.A. 100 (15), 8817-8822 (2003).</li>
<li>Kiss, M. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High-throughput+quantitative+polymerase+chain+reaction+in+picoliter+droplets%5BTitle%5D)+AND+%22Anal.+Chem%22%5BJournal%5D)">High-throughput quantitative polymerase chain reaction in picoliter droplets.</a> Anal. Chem. 80 (23), 8975-8981 (2008).</li>
<li>Baker, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Digital+PCR+hits+its+stride%5BTitle%5D)+AND+%22Nat.+Methods%22%5BJournal%5D)">Digital PCR hits its stride.</a> Nat. Methods. 9 (6), 3(2012).</li>
<li>Marcus, J. S., Anderson, W. F., Quake, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Parallel+picoliter+rt-PCR+assays+using+microfluidics%5BTitle%5D)+AND+%22Anal.+Chem%22%5BJournal%5D)">Parallel picoliter rt-PCR assays using microfluidics.</a> Anal. Chem. 78 (3), 956-958 (2006).</li>
<li>Lee, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Synchronized+reinjection+and+coalescence+of+droplets+in+microfluidics%5BTitle%5D)+AND+%22Lab+Chip%22%5BJournal%5D)">Synchronized reinjection and coalescence of droplets in microfluidics.</a> Lab Chip. 14 (3), 509-513 (2014).</li>
<li>Rhee, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pressure+stabilizer+for+reproducible+picoinjection+in+droplet+microfluidic+systems%5BTitle%5D)+AND+%22Lab+Chip%22%5BJournal%5D)">Pressure stabilizer for reproducible picoinjection in droplet microfluidic systems.</a> Lab Chip. 14 (23), 4533-4539 (2014).</li>
<li>Hatch, A. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((1-Million+droplet+array+with+wide-field+fluorescence+imaging+for+digital+PCR%5BTitle%5D)+AND+%22Lab+Chip%22%5BJournal%5D)">1-Million droplet array with wide-field fluorescence imaging for digital PCR.</a> Lab Chip. 11 (22), 3838-3845 (2011).</li>
<li>Illumina. , Technology. <a target="_blank" href="http://www.illumina.com/technology.html">http://www.illumina.com/technology.html</a> (2014).</li>
<li>Droplet digital PCR applications guide. , Bio-Rad. Available from: 
 <a target="_blank" href="http://www.bio-rad.com/en-us/category/digital-pcr">http://www.bio-rad.com/en-us/category/digital-pcr</a> (2014).</li>
<li>Hindson, B. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High-throughput+droplet+digital+PCR+system+for+absolute+quantitation+of+DNA+copy+number%5BTitle%5D)+AND+%22Anal.+Chem%22%5BJournal%5D)">High-throughput droplet digital PCR system for absolute quantitation of DNA copy number.</a> Anal. Chem. 83 (22), 8604-8610 (2011).</li>
<li>Guo, M. T., Rotem, A., Heyman, J. A., Weitz, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Droplet+microfluidics+for+high-throughput+biological+assays%5BTitle%5D)+AND+%22Lab+Chip%22%5BJournal%5D)">Droplet microfluidics for high-throughput biological assays.</a> Lab Chip. 12 (12), 2146-2155 (2012).</li>
<li>Lage, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Whole+genome+analysis+of+genetic+alterations+in+small+DNA+samples+using+hyperbranched+strand+displacement+amplification+and+array-CGH%5BTitle%5D)+AND+%22Genome+Res%22%5BJournal%5D)">Whole genome analysis of genetic alterations in small DNA samples using hyperbranched strand displacement amplification and array-CGH.</a> Genome Res. 13 (2), 294-307 (2003).</li>
<li>Dean, F. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comprehensive+human+genome+amplification+using+multiple+displacement+amplification%5BTitle%5D)+AND+%22Proc.+Natl.+Acad.+Sci.+U.S.A%22%5BJournal%5D)">Comprehensive human genome amplification using multiple displacement amplification.</a> Proc. Natl. Acad. Sci. U.S.A. 99 (8), 5261-5266 (2002).</li>
<li>Zong, C., Lu, S., Chapman, A. R., Xie, X. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genome-wide+detection+of+single-nucleotide+and+copy-number+variations+of+a+single+human+cell%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Genome-wide detection of single-nucleotide and copy-number variations of a single human cell.</a> Science. 338 (6114), 1622-1626 (2012).</li>
<li>Mitos Dropix Systems. , Dolomite. <a target="_blank" href="http://www.dolomite-microfluidics.com/webshop/mitos_dropix_system">http://www.dolomite-microfluidics.com/webshop/mitos_dropix_system</a> (2014).</li>
<li>Abate, A. R., Weitz, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Syringe-vacuum+microfluidics%3A+A+portable+technique+to+create+monodisperse+emulsions%5BTitle%5D)+AND+%22Biomicrofluidics%22%5BJournal%5D)">Syringe-vacuum microfluidics: A portable technique to create monodisperse emulsions.</a> Biomicrofluidics. 5, 14107(2011).</li>
<li>Eyer, K., Kuhn, P., Stratz, S., Dittrich, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+microfluidic+chip+for+the+versatile+chemical+analysis+of+single+cells%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">A microfluidic chip for the versatile chemical analysis of single cells.</a> J Vis Exp. (80), e50618(2013).</li>
<li>Fainman, Y. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aFainman%2C+Y+%22Optofluidics+%3A+fundamentals%2C+devices%2C+and+applications%22">Optofluidics : fundamentals, devices, and applications</a>. , McGraw-Hill. (2010).</li>
<li>Xia, Y. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((G.M.+Softlithographie%5BTitle%5D)+AND+%22Angew+Chem%22%5BJournal%5D)">G.M. Softlithographie.</a> Angew Chem. 110 (5), 26(1998).</li>
<li>Woyke, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Decontamination+of+MDA+reagents+for+single+cell+whole+genome+amplification%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Decontamination of MDA reagents for single cell whole genome amplification.</a> PLoS One. 6 (10), e26161(2011).</li>
<li>System for hot-start amplification via a multiple emulsion. US patent. , (2014).</li>
<li>Utada, A. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Monodisperse+double+emulsions+generated+from+a+microcapillary+device%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Monodisperse double emulsions generated from a microcapillary device.</a> Science. 308 (5721), 537-541 (2005).</li>
<li>Eastburn, D. J., Sciambi, A., Abate, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Picoinjection+enables+digital+detection+of+RNA+with+droplet+rt-PCR%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Picoinjection enables digital detection of RNA with droplet rt-PCR.</a> PLoS One. 8 (4), e62961(2013).</li>
<li>Dube, S., Qin, J., Ramakrishnan, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mathematical+analysis+of+copy+number+variation+in+a+DNA+sample+using+digital+PCR+on+a+nanofluidic+device%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Mathematical analysis of copy number variation in a DNA sample using digital PCR on a nanofluidic device.</a> PLoS One. 3 (8), e2876(2008).</li>
<li>Shen, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Multiplexed+quantification+of+nucleic+acids+with+large+dynamic+range+using+multivolume+digital+RT-PCR+on+a+rotational+SlipChip+tested+with+HIV+and+hepatitis+C+viral+load%5BTitle%5D)+AND+%22J+Am.+Chem.+Soc%22%5BJournal%5D)">Multiplexed quantification of nucleic acids with large dynamic range using multivolume digital RT-PCR on a rotational SlipChip tested with HIV and hepatitis C viral load.</a> J Am. Chem. Soc. 133 (44), 17705-17712 (2011).</li>
<li>Link, D. R., Anna, S. L., Weitz, D. A., Stone, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Geometrically+mediated+breakup+of+drops+in+microfluidic+devices%5BTitle%5D)+AND+%22Phys.+Rev.+Lett%22%5BJournal%5D)">Geometrically mediated breakup of drops in microfluidic devices.</a> Phys. Rev. Lett. 92 (5), 054503(2004).</li>
<li>Nisisako, T., Torii, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microfluidic+large-scale+integration+on+a+chip+for+mass+production+of+monodisperse+droplets+and+particles%5BTitle%5D)+AND+%22Lab+Chip%22%5BJournal%5D)">Microfluidic large-scale integration on a chip for mass production of monodisperse droplets and particles.</a> Lab Chip. 8 (2), 287-293 (2008).</li>
<li>Romanowsky, M. B., Abate, A. R., Rotem, A., Holtze, C., Weitz, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High+throughput+production+of+single+core+double+emulsions+in+a+parallelized+microfluidic+device%5BTitle%5D)+AND+%22Lab+Chip%22%5BJournal%5D)">High throughput production of single core double emulsions in a parallelized microfluidic device.</a> Lab Chip. 12 (4), 802-807 (2012).</li>
</ol>

The Broad Institute may move to file a patent application that includes aspects of this work.

数字测定是强大的方法，使检测稀有细胞的计数和个体的核酸分子组成。但是，数字实验仍然不是常规分析实验室应用，部分原因是由于与市售的方法相关联的专门设备的成本。在这里，我们描述端点数字测定用于定量核酸与使用标准的实时定量PCR机器的简化模拟读出。这种方法是快速建立，和读出比液滴计数方法简单得多。 我们的分析将丧失独立液滴测量的单分子（单滴?...

数字测定核酸定量（数字PCR），1-4和基本顺序（顺序）的强烈影响生命科学和医学。数字分析提供分子计数定量在绝对规模（而不是相对于对照），提供高灵敏度，在整个实验中实现浅显的比较，重要的是，使含可比数据5（表1）大型数据库的建设。 在过去的15年中，全基因组扩增（WGA）出现旁边的PCR作为用于核酸扩增的通用工具。像的PCR，WGA为通过放大分钟样品的元素最多可以很容易地检测或用于随后的分析像碱基测序的水平分析和制备应用中有用。不像PCR，WGA不是特定于特定的DNA位点，而允许在样品中的所有序列，包括未知序列的扩增。PCR和WGA之间的这种根本差异使得互补彼此的方法和引起在他们的应用程序不同的挑战。 高产的WGA反应6的使基因组DNA的单分子7，单个细胞8和其它低生物质的样品9，用于量化或进一步分析例程扩增。与WGA化学相关的主要挑战是它的极端敏感性污染物和整个个体模板分子6不均匀放大。但是，WGA逐渐流行为单细胞测序已成为"杀手级应用"WGA技术10，和模板的定量通过WGA是在应用程序7中许多领域的重要。 仪器的数字核酸量化已在各种阀联结且无气门的微流体形式上的先前已经描述TS 11-14包括液滴基试验15,16（ 表2）。然而，商用的微流体系统用于数字分析需要专门的设备以进行反应的设置和产物检测17。自阀微流体是灵活的，但需要精密微细加工和气动控制系统18。虽然这是相对简单的，使单分散的微滴乳化为基础的数字测定19,20，数字读出在技术上是麻烦的，需要无论大型宽视场成像（类似于流行下一代测序技术）21,22或高速的基于流滴检测23-25。理想情况下，数字检测将是从设置简单到读出，减少了需要复杂的仪器，并允许大量样品被迅速地读出。在这里，我们描述了用于数字液滴测定读出一个简化的方法，该方法使用常规的实时PCR我nstrument测量液滴为基础的数字检测散装荧光。 虽然新的方法可以应用到数字PCR测定法，它是用于该模拟的实时扩增分析（即提供优良的效果进行PCR）是有问题的数字的WGA实验特别有利的。 WGA通常应用于样本与模板分子的异质在序列，长度和基本内容。这些不同的模板分子扩增以不同速率6，迫使使用基准（"标准"）具有匹配特性的样品。通常情况下，没有这样的标准是可用的，或传入样本的特性是未知的。输入质量，分子的输入数，两个，或既不的组合- WGA化学的输入材料和长度依赖性属性的异质26还通过创建歧义什么被定量复杂化的结果的解释。 Finally，序列 - 非特异性呈现定量多重置换扩增（MDA）的更多的污染比定量PCR敏感，因为任何序列的污染物分子有一个潜在的干扰。微流体数字分析解决污染通过分离模板分子和减少的反应体积，使得更少的污染物被采样。 在这里，我们使用了流行的等温WGA的方法，MDA 27。值得注意的是，其他几个WGA化学品，包括PicoPlex和MALBAC 28关键取决于最初的等温链置换步骤。等温步骤加剧施加模拟实时测定法进行定量的WGA的挑战。 WGA既不能在安装过程中被完全防止，从而导致不希望的可变预放大，也不离散（"循环"）的方式，其中异构分子的复制可以被驱动到完成并停止之前，下一个周期的PCR像11 </suP&gt;（ 图1）。对于WGA数字测定形式可容纳典型的反应中的设置过程，由于每个分子为枚举在测定端点的偏析（因此扩增前不影响结果）原始读出装置的精度将是基本上独立变化的扩增效率。 该试验依赖于产生油具有均匀体积的液滴，作为每滴的信号电平在测定端点将取决于液滴体积上，我们不希望结果的一致性取决于跨越液滴尺寸的分布平均化。制造单分散的液滴是现在的标准程序（约3500的单分散液滴论文已自2013出版），但要求的微流体的仪器25。事实上，六年多的公司已经开发出依赖于生产这样的液滴独立的商业产品，和液滴制作微流控芯片市售23,29。在这项研究中，我们使用了内部（见协议）生产的定制微流体装置。注射泵来驱动流过器件也可商购的，但可替换地，可以用一个一次性注射器的真空驱动的流动被取代，以降低成本30。

<table><tbody><tr><td>Evagreen Dye</td><td>Biotium</td><td>31000</td><td></td></tr><tr><td>Bovine serum albumin</td><td>New England Biotechnologies</td><td>B9000S</td><td></td></tr><tr><td>Phi29 DNA Polymerase Reaction Buffer</td><td>New England Biotechnologies</td><td>B0269S</td><td>Vortex periodically until aggregate dissolves</td></tr><tr><td>Lambda DNA</td><td>New England Biotechnologies</td><td>N3011S</td><td>Heated to 50 &lt;sup&gt;o&lt;/sup&gt;C and quenched on ice prior to dilution</td></tr><tr><td>Randomized MDA oligos</td><td>Integrated DNA Technologies</td><td></td><td>5&#039;-NNNNN*N-3&#039;</td></tr><tr><td>PCR primers</td><td>Integrated DNA Technologies</td><td></td><td>5&amp;rsquo;-CGGCAAACGGGAATGAAACGCC-3&amp;rsquo; and 5&amp;rsquo;-TGCGGCAAAGACAGCAACGG-3&amp;rsquo;</td></tr><tr><td>UltraPure Nuclease-free water</td><td>Life Technologies</td><td>10977-015</td><td>UV-treated for 30 min in Stratalinker 2400 before use (optional)</td></tr><tr><td>ROX reference dye</td><td>Life Technologies</td><td>12223-012</td><td></td></tr><tr><td>PCR optical strip caps</td><td>Life Technologies</td><td>4323032</td><td></td></tr><tr><td>PCR tubes</td><td>Agilent Technologies</td><td>401428</td><td></td></tr><tr><td>dNTP (25 micromolar each)</td><td>Agilent Technologies</td><td>200415</td><td></td></tr><tr><td>Thermocycler - Mx3000P qPCR system</td><td>Agilent Technologies</td><td>401403</td><td></td></tr><tr><td>Barrier pipette tips</td><td>VWR</td><td>89003-046</td><td></td></tr><tr><td>Bio-rad oil for evagreen</td><td>Bio-rad</td><td>186-4005</td><td></td></tr><tr><td>JumpStart Taq ReadyMix</td><td>Sigma-Aldrich</td><td>P2893-100RXN</td><td></td></tr></tbody></table>

simple bulk readout of digital nucleic acid quantification assays

数字测定是强大的方法，使检测稀有细胞和单个核酸分子的计数。但是，数字实验仍然没有常规应用，由于成本，并与商购的方法相关联的特定设备。在这里，我们提出了一个简单的方法，使用传统的实时PCR仪测量液滴为基础的数字检测散装荧光数字墨滴检测读数。 我们表征使用合成的液滴的混合物和液滴数字多重置换扩增（MDA）的测定中的散装读数测定法的性能。定量MDA特别是从数字的反应形式的好处，但我们的新方法适用于任何数字检测。为建立数字测定协议，如数字PCR，该方法用于加快和简化测定读数。 我们的批量读取方法带来partitione的优势ð测定，而不需要专门的读取仪​​表。墨滴计数平台以及需要的标准样品相比，大部分的读出方法的主要限制是减少的动态范围，虽然对于这个标准的要求比以往的实时实验要求不高。定量全基因组扩增（WGA）被用来测​​试在WGA反应污染物是最敏感的方法来检测具有未知序列的DNA片段的存在，从而在不同的应用领域，包括药物质量控制和天体生物学方法巨大潜力。

而常规的本体/实时读数可用于既定量PCR和定量的WGA实验（图1），数字定量分析提供的优点（见表1）。中描述的方法，我们读出的微量流体格式数字分析用一个简单的批量终点测量（ 图2）。虽然这种方法可广泛应用，我们专注于定量WGA（MDA），因为这种方法提出了传统的实时检测特殊的挑战。 要检查我们的实时PCR仪是否能检测出不同的...

Watch this Scientific Journal Video about Simple Bulk Readout of Digital Nucleic Acid Quantification Assays at JoVE.com

Simple Bulk Readout of Digital Nucleic Acid Quantification Assays

We describe an endpoint digital assay for quantifying nucleic acids with a simplified (analog) readout. We measure bulk fluorescence of droplet-based digital assays using a standard qPCR machine rather than specialized instrumentation and confirm our results by microscopy.

数字核酸定量实验分析的简单堆积读数

Digital assays are powerful methods that enable detection of rare cells and counting of individual nucleic acid molecules. However, digital assays are ...

simple-bulk-readout-of-digital-nucleic-acid-quantification-assays

Research

JoVE Journal

Biology

8.3K 次观看.  Broad Institute. We describe an endpoint digital assay for quantifying nucleic acids with a simplified (analog) readout. We measure bulk fluorescence of droplet-based digital assays using a standard qPCR machine rather than specialized instrumentation and confirm our results by microscopy. 

视频: Simple Bulk Readout of Digital Nucleic Acid Quantification Assays

Rapid Colorimetric Assays to Qualitatively Distinguish RNA and DNA in Biomolecular Samples

Biochemical experimentation generally requires accurate knowledge, at an early stage, of the nucleic acid, protein, and other biomolecular components in potentially heterogeneous specimens. Nucleic acids can be detected via several established approaches, including analytical methods that are spectrophotometric (e.g., A260), fluorometric (e.g., binding of fluorescent dyes), or colorimetric (nucleoside-specific chromogenic chemical reactions).1 Though it cannot readily distinguish RNA from DNA, the A260/A280 ratio is commonly employed, as it offers a simple and rapid2 assessment of the relative content of nucleic acid, which absorbs predominantly near 260 nm and protein, which absorbs primarily near 280 nm. Ratios &lt; 0.8 are taken as indicative of 'pure' protein specimens, while pure nucleic acid (NA) is characterized by ratios &gt; 1.53.
However, there are scenarios in which the protein/NA content cannot be as clearly or reliably inferred from simple uv-vis spectrophotometric measurements. For instance, (i) samples may contain one or more proteins which are relatively devoid of the aromatic amino acids responsible for absorption at &asymp;280 nm (Trp, Tyr, Phe), as is the case with some small RNA-binding proteins, and (ii) samples can exhibit intermediate A260/A280 ratios (~0.8 &lt; ~1.5), where the protein/NA content is far less clear and may even reflect some high-affinity association between the protein and NA components. For such scenarios, we describe herein a suite of colorimetric assays to rapidly distinguish RNA, DNA, and reducing sugars in a potentially mixed sample of biomolecules. The methods rely on the differential sensitivity of pentoses and other carbohydrates to Benedict's, Bial's (orcinol), and Dische's (diphenylamine) reagents; the streamlined protocols can be completed in a matter of minutes, without any additional steps of having to isolate the components. The assays can be performed in parallel to differentiate between RNA and DNA, as well as indicate the presence of free reducing sugars such as glucose, fructose, and ribose (Figure 1).

A suite of colorimetric assays is described for rapidly distinguishing protein, RNA, DNA, and reducing sugars in potentially heterogeneous biomolecular samples.

快速比色测定RNA和DNA生物分子样品进行定性区分

Biochemical experimentation generally requires accurate knowledge, at an early stage, of the nucleic acid, protein, and other biomolecular components in ...

科研

化学

Amplification of Near Full-length HIV-1 Proviruses for Next-Generation Sequencing

The Full-Length Individual Proviral Sequencing (FLIPS) assay is an efficient and high-throughput method designed to amplify and sequence single, near full-length (intact and defective), HIV-1 proviruses. FLIPS allows determination of the genetic composition of integrated HIV-1 within a cell population. Through identifying defects within HIV-1 proviral sequences that arise during reverse transcription, such as large internal deletions, deleterious stop codons/hypermutation, frameshift mutations, and mutations/deletions in cis acting elements required for virion maturation, FLIPS can identify integrated proviruses incapable of replication. The FLIPS assay can be utilized to identify HIV-1 proviruses that lack these defects and are&#160;therefore potentially replication-competent. The FLIPS protocol involves: lysis of HIV-1 infected cells, nested PCR of near full-length HIV-1 proviruses (using primers targeted to the HIV-1 5&#39; and 3&#39; LTR), DNA purification and quantification, library preparation for Next-generation Sequencing (NGS), NGS, de novo assembly of proviral contigs, and a simple process of elimination for identifying replication-competent proviruses. FLIPS provides advantages over traditional methods designed to sequence integrated HIV-1 proviruses, such as single-proviral sequencing. FLIPS amplifies and sequences near full-length proviruses enabling replication competency to be determined, and also uses fewer amplification primers, preventing the consequences of primer mismatches. FLIPS is a useful tool for understanding the genetic landscape of integrated HIV-1 proviruses, especially within the latent reservoir, however, its utilization can extend to any application in which the genetic composition of integrated HIV-1 is required.

Full-length individual proviral sequencing (FLIPS) provides an efficient and high-throughput method for the amplification and sequencing of single, near full-length (intact and defective) HIV-1 proviruses and allows for determination of their potential replication-competency. FLIPS overcomes limitations of previous assays designed to sequence the latent HIV-1 reservoir.

用于下一代测序的近全长 HIV-1 Proviruses 的扩增

The Full-Length Individual Proviral Sequencing (FLIPS) assay is an efficient and high-throughput method designed to amplify and sequence single, near ...

遗传学

Circulating MicroRNA Quantification Using DNA-binding Dye Chemistry and Droplet Digital PCR

Circulating (of cell-free) microRNAs (miRNAs) are released from cells into the blood stream. The amount of specific microRNAs in the circulation has been linked to a disease state and has the potential to be used as disease biomarker. A sensitive and accurate method for circulating microRNA quantification using a dye-based chemistry and droplet digital PCR technology has been recently developed. Specifically, using Locked Nucleic Acid (LNA)-based miRNA-specific primers with a green fluorescent DNA-binding dye in a compatible droplet digital PCR system it is possible to obtain the absolute quantification of specific miRNAs. Here, we describe how performing this technique to assess miRNA amount in biological fluids, such as plasma and serum, is both feasible and effective.

A sensitive and accurate method for cell-free microRNAs quantification using a dye-based chemistry and droplet digital PCR technology is described.

微RNA循环使用定量DNA结合染料化学和液滴的数字PCR

Circulating (of cell-free) microRNAs (miRNAs) are released from cells into the blood stream. The amount of specific microRNAs in the circulation has been ...

生物工程

Quantification of dsDNA using the Hitachi F-7000 Fluorescence Spectrophotometer and PicoGreen Dye

Quantification of DNA, especially in small concentrations, is an important task with a wide range of biological applications including standard molecular biology assays such as synthesis and purification of DNA, diagnostic applications such as quantification of DNA amplification products, and detection of DNA molecules in drug preparations. During this video we will demonstrate the capability of the Hitachi F-7000 Fluorescence Spectrophotometer equipped with a Micro Plate Reader accessory to perform dsDNA quantification using Molecular Probes Quant-it PicoGreen dye reagent kit.
The F-7000 Fluorescence Spectrophotometer offers high sensitivity and high speed measurements. It is a highly flexible system capable of measuring fluorescence, luminescence, and phosphorescence. Several measuring modes are available, including wavelength scan, time scan, photometry and 3-D scan measurement. The spectrophotometer has sensitivity in the range of 50 picomoles of fluorescein when using a 300 &#956;L sample volume in the microplate, and is capable of measuring scan speeds of 60,000 nm/minute. It also has a wide dynamic range of up to 5 orders of magnitude which allows for the use of calibration curves over a wide range of concentrations. The optical system uses all reflective optics for maximum energy and sensitivity. The standard wavelength range is 200 to 750 nm, and can be extended to 900 nm when using one of the optional near infrared photomultipliers. The system allows optional temperature control for the plate reader from 5 to 60 degrees Celsius using an optional external temperature controlled liquid circulator. The microplate reader allows for the use of 96 well microplates, and the measuring speed for 96 wells is less than 60 seconds when using the kinetics mode.
Software controls for the F-7000 and Microplate Reader are also highly flexible. Samples may be set in either column or row formats, and any combination of wells may be chosen for sample measurements. This allows for optimal utilization of the microplate. Additionally, the software allows importing micro plate sample configurations created in Excel and saved in comma separated values, or "csv" format. Microplate measuring configurations can be saved and recalled by the software for convenience and increased productivity. Data results can be output to a standard report, to Excel, or to an optional Report Generator Program.

Demonstration of quantification of dsDNA using Molecular Probes PicoGreen dye and Hitachi F-7000 Fluorescence Spectrophotometer equipped with a microplate reader accessory.

双链DNA定量，使用日立F - 7000荧光分光光度计和PicoGreen染料

Quantification of DNA, especially in small concentrations, is an important task with a wide range of biological applications including standard molecular ...

生物学

NanoDrop Microvolume Quantitation of Nucleic Acids

Biomolecular assays are continually being developed that use progressively smaller amounts of material, often precluding the use of conventional cuvette-based instruments for nucleic acid quantitation for those that can perform microvolume quantitation.
The NanoDrop microvolume sample retention system (Thermo Scientific NanoDrop Products) functions by combining fiber optic technology and natural surface tension properties to capture and retain minute amounts of sample independent of traditional containment apparatus such as cuvettes or capillaries. Furthermore, the system employs shorter path lengths, which result in a broad range of nucleic acid concentration measurements, essentially eliminating the need to perform dilutions. Reducing the volume of sample required for spectroscopic analysis also facilitates the inclusion of additional quality control steps throughout many molecular workflows, increasing efficiency and ultimately leading to greater confidence in downstream results.
The need for high-sensitivity fluorescent analysis of limited mass has also emerged with recent experimental advances. Using the same microvolume sample retention technology, fluorescent measurements may be performed with 2 &mu;L of material, allowing fluorescent assays volume requirements to be significantly reduced. Such microreactions of 10 &mu;L or less are now possible using a dedicated microvolume fluorospectrometer.
Two microvolume nucleic acid quantitation protocols will be demonstrated that use integrated sample retention systems as practical alternatives to traditional cuvette-based protocols. First, a direct A260 absorbance method using a microvolume spectrophotometer is described. This is followed by a demonstration of a fluorescence-based method that enables reduced-volume fluorescence reactions with a microvolume fluorospectrometer. These novel techniques enable the assessment of nucleic acid concentrations ranging from 1 pg/ &mu;L to 15,000 ng/ &mu;L with minimal consumption of sample.

The use of NanoDrop microvolume systems as practical and efficient alternatives to traditional nucleic acid quantitation methodology is described through the demonstration of two microvolume nucleic acid quantitation protocols.

NanoDrop微量核酸的定量

Biomolecular assays are continually being developed that use progressively smaller amounts of material, often precluding the use of conventional ...

<meta charset="utf8"></head><body>使用数字液滴 PCR 进行病毒基因组定量的标准化方案

Quantification of Adeno-Associated Viral Genomes in Purified Vector Samples by Digital Droplet Polymerase Chain Reaction

Adeno-associated virus (AAV) is a non-pathogenic virus used as a delivery vehicle to transfer therapeutic genes into patients. Accurate quantification of AAV genome copy number in vector preparations is essential for bioprocess optimization and dosage calculation in both preclinical and clinical studies of AAV-based gene therapy products. Currently, a consensus protocol for AAV viral genome titration is lacking. Here, we present a digital droplet PCR (dd_PCR) protocol for the quantification of viral genomes in purified vector samples. Samples are treated with DNase I to eliminate unpackaged contaminant DNA. DNase-treated samples are then mixed with an appropriate primer-probe set (designed according to the target AAV genome) and PCR reagents, and then loaded into a droplet generator. The prepared droplets are transferred into a PCR plate, where PCR amplification is performed and analyzed. The viral genome titer is calculated based on the concentration (copies/&#181;L), accounting for sample dilutions. A successful measurement shows a clear separation of the positive and negative droplet clouds, has at least 10,000 accepted droplets, shows a value between 10 copies/&#181;L and 10,000 copies/&#181;L, and has a coefficient of variation (CV) between repeats lower than 20%. Reliable viral genome titration will aid in the development of safe and effective AAV-based gene therapy products.

<meta charset="utf8"></head><body>作者聚焦:优化数字微滴 PCR 方法以实现准确的腺相关病毒基因组定量

Precise quantification of adeno-associated virus (AAV) vector genome copies is critical, but a standardized protocol has yet to be established. This protocol describes a validated method for preparing purified AAV samples and conducting digital droplet polymerase chain reaction (dd_PCR) to reliably quantify the viral genome titer.

通过数字液滴聚合酶链反应定量纯化载体样品中的腺相关病毒基因组

Adeno-associated virus (AAV) is a non-pathogenic virus used as a delivery vehicle to transfer therapeutic genes into patients. Accurate quantification of ...

Two-Step Reverse Transcription Droplet Digital PCR Protocols for SARS-CoV-2 Detection and Quantification

Diagnosis of the ongoing SARS-CoV-2 pandemic is a priority for all countries across the globe. Currently, reverse transcription quantitative PCR (RT-qPCR) is the gold standard for SARS-CoV-2 diagnosis as no permanent solution is available. However effective this technique may be, research has emerged showing its limitations in detection and diagnosis especially when it comes to low abundant targets. In contrast, droplet digital PCR (ddPCR), a recent emerging technology with superior advantages over qPCR, has been shown to overcome the challenges of RT-qPCR in diagnosis of SARS-CoV-2 from low abundant target samples. Prospectively, in this article, the capabilities of RT-ddPCR are further expanded by showing steps on how to develop simplex, duplex, triplex probe mix, and quadruplex assays using a two-color detection system. Using primers and probes targeting specific sites of the SARS-CoV-2 genome (N, ORF1ab, RPP30, and RBD2), the development of these assays is shown to be possible. Additionally, step by step detailed protocols, notes, and suggestions on how to improve the assays workflow and analyze data are provided. Adapting this workflow in future works will ensure that the maximum number of targets can be sensitively detected in a small sample significantly improving on cost and sample throughput.

This work summarizes steps on developing different assays for SARS-CoV-2 detection using a two color ddPCR system. The steps are elaborate and notes have been included on how to improve the assays and experiment performance. These assays may be used for multiple SARS-CoV-2 RT-ddPCR applications.

用于SARS-CoV-2检测和定量的两步逆转录液滴数字PCR方案

Diagnosis of the ongoing SARS-CoV-2 pandemic is a priority for all countries across the globe. Currently, reverse transcription quantitative PCR (RT-qPCR) ...

Quantification of Circular RNAs Using Digital Droplet PCR

Digital droplet polymerase chain reaction (dd-PCR) is one of the most sensitive quantification methods; it fractionates the reaction into nearly 20,000 water-in-oil droplets, and the PCR occurs in the individual droplets. The dd-PCR has several advantages over conventional real-time qPCR, including increased accuracy in detecting low-abundance targets, omitting reference genes for quantification, eliminating technical replicates for samples, and showing high resilience to inhibitors in the samples. Recently, dd-PCR has become one of the most popular methods for accurately quantifying target DNA or RNA for gene expression analysis and diagnostics. Circular RNAs (circRNAs) are a large family of recently discovered covalently closed RNA molecules lacking 5' and 3' ends. They have been shown to regulate gene expression by acting as sponges for RNA-binding proteins and microRNAs. Furthermore, circRNAs are secreted into body fluids, and their resistance to exonucleases makes them serve as biomarkers for disease diagnosis. This article aims to show how to perform divergent primer design, RNA extraction, cDNA synthesis, and dd-PCR analysis to accurately quantify specific circular RNA (circRNA) levels in cells. In conclusion, we demonstrate the precise quantification of circRNAs using dd-PCR.

This protocol describes the detailed method of digital droplet PCR (dd-PCR) for precise quantification of circular RNA (circRNA) levels in cells using divergent primers.

使用数字微滴PCR定量环状RNA

Digital droplet polymerase chain reaction (dd-PCR) is one of the most sensitive quantification methods; it fractionates the reaction into nearly 20,000 ...

数字核酸定量实验分析的简单堆积读数

摘要

探索更多视频

快速比色测定RNA和DNA生物分子样品进行定性区分

用于下一代测序的近全长 HIV-1 Proviruses 的扩增

微RNA循环使用定量DNA结合染料化学和液滴的数字PCR

双链DNA定量，使用日立F - 7000荧光分光光度计和PicoGreen染料

NanoDrop微量核酸的定量

通过数字液滴聚合酶链反应定量纯化载体样品中的腺相关病毒基因组

通过数字液滴聚合酶链反应定量纯化载体样品中的腺相关病毒基因组

通过数字液滴聚合酶链反应定量纯化载体样品中的腺相关病毒基因组

用于SARS-CoV-2检测和定量的两步逆转录液滴数字PCR方案

使用数字微滴PCR定量环状RNA