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>

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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=Comprehensive+human+genome+amplification+using+multiple+displacement+amplification.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+detection+of+single-nucleotide+and+copy-number+variations+of+a+single+human+cell.">Genome-wide detection of single-nucleotide and copy-number variations of a single human cell.</a> Science. 338 (6114), 1622-1626 (2012).</li><li>Abate, A. R., Weitz, D. 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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=G.M.+Softlithographie.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decontamination+of+MDA+reagents+for+single+cell+whole+genome+amplification.">Decontamination of MDA reagents for single cell whole genome amplification.</a> PLoS One. 6 (10), e26161 (2011).</li><li>Hindson, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=System+for+hot-start+amplification+via+a+multiple+emulsion.">System for hot-start amplification via a multiple emulsion.</a> US patent. , (2014).</li><li>Utada, A. 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=Monodisperse+double+emulsions+generated+from+a+microcapillary+device.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Picoinjection+enables+digital+detection+of+RNA+with+droplet+rt-PCR.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mathematical+analysis+of+copy+number+variation+in+a+DNA+sample+using+digital+PCR+on+a+nanofluidic+device.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&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.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Geometrically+mediated+breakup+of+drops+in+microfluidic+devices.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+large-scale+integration+on+a+chip+for+mass+production+of+monodisperse+droplets+and+particles.">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. 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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 border="0" cellpadding="0" cellspacing="0">
	<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 50C and quenched on ice prior to dilution</td>
		</tr>
		<tr>
			<td>Randomized MDA oligos</td>
			<td>Integrated DNA Technologies</td>
			<td></td>
			<td>5&#39;-NNNNN*N-3&#39;</td>
		</tr>
		<tr>
			<td>PCR primers</td>
			<td>Integrated DNA Technologies</td>
			<td></td>
			<td>5&#8217;-CGGCAAACGGGAATGAAACGCC-3&#8217; and 5&#8217;-TGCGGCAAAGACAGCAACGG-3&#8217;</td>
		</tr>
		<tr>
			<td>UltraPure Nuclease-free water</td>
			<td>Life Technologies</td>
			<td>10977-015</td>
			<td>UV-treated for 30m 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 (25uM 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

8.2K 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

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 ...

科研

生物学

Electricity-Free, Sequential Nucleic Acid and Protein Isolation

Traditional and emerging pathogens such as Enterohemorrhagic Escherichia coli (EHEC), Yersinia pestis, or prion-based diseases are of significant concern for governments, industries and medical professionals worldwide. For example, EHECs, combined with Shigella, are responsible for the deaths of approximately 325,000 children each year and are particularly prevalent in the developing world where laboratory-based identification, common in the United States, is unavailable 1. The development and distribution of low cost, field-based, point-of-care tools to aid in the rapid identification and/or diagnosis of pathogens or disease markers could dramatically alter disease progression and patient prognosis. We have developed a tool to isolate nucleic acids and proteins from a sample by solid-phase extraction (SPE) without electricity or associated laboratory equipment 2. The isolated macromolecules can be used for diagnosis either in a forward lab or using field-based point-of-care platforms. Importantly, this method provides for the direct comparison of nucleic acid and protein data from an un-split sample, offering a confidence through corroboration of genomic and proteomic analysis.
Our isolation tool utilizes the industry standard for solid-phase nucleic acid isolation, the BOOM technology, which isolates nucleic acids from a chaotropic salt solution, usually guanidine isothiocyanate, through binding to silica-based particles or filters 3. CUBRC's proprietary solid-phase extraction chemistry is used to purify protein from chaotropic salt solutions, in this case, from the waste or flow-thru following nucleic acid isolation4.
By packaging well-characterized chemistries into a small, inexpensive and simple platform, we have generated a portable system for nucleic acid and protein extraction that can be performed under a variety of conditions. The isolated nucleic acids are stable and can be transported to a position where power is available for PCR amplification while the protein content can immediately be analyzed by hand held or other immunological-based assays. The rapid identification of disease markers in the field could significantly alter the patient's outcome by directing the proper course of treatment at an earlier stage of disease progression. The tool and method described are suitable for use with virtually any infectious agent and offer the user the redundancy of multi-macromolecule type analyses while simultaneously reducing their logistical burden.

A tool and chemistries are described to sequentially isolate nucleic acids followed by proteins from a sample without the need for electricity. The tool consists of a sorbent held within a transfer pipette while the isolation chemistries are based on solid-phase extraction principles. The isolated macromolecules can be analyzed by immuno-based and PCR-based assays.

免电力，顺序的核酸和蛋白质的分离

Traditional and emerging pathogens such as Enterohemorrhagic Escherichia coli (EHEC), Yersinia pestis, or prion-based diseases are of significant concern ...

Analyzing and Building Nucleic Acid Structures with 3DNA

The 3DNA software package is a popular and versatile bioinformatics tool with capabilities to analyze, construct, and visualize three-dimensional nucleic acid structures. This article presents detailed protocols for a subset of new and popular features available in 3DNA, applicable to both individual structures and ensembles of related structures. Protocol 1 lists the set of instructions needed to download and install the software. This is followed, in Protocol 2, by the analysis of a nucleic acid structure, including the assignment of base pairs and the determination of rigid-body parameters that describe the structure and, in Protocol 3, by a description of the reconstruction of an atomic model of a structure from its rigid-body parameters. The most recent version of 3DNA, version 2.1, has new features for the analysis and manipulation of ensembles of structures, such as those deduced from nuclear magnetic resonance (NMR) measurements and molecular dynamic (MD) simulations; these features are presented in Protocols 4 and 5. In addition to the 3DNA stand-alone software package, the w3DNA web server, located at <a href="http://w3dna.rutgers.edu" target="_blank">http://w3dna.rutgers.edu</a>, provides a user-friendly interface to selected features of the software. Protocol 6 demonstrates a novel feature of the site for building models of long DNA molecules decorated with bound proteins at user-specified locations.

The 3DNA software package is a popular and versatile bioinformatics tool with capabilities to analyze, construct, and visualize three-dimensional nucleic acid structures. This article presents detailed protocols for a subset of new and popular features available in 3DNA, applicable to both individual structures and ensembles of related structures.

核酸结构分析及建设3DNA

The 3DNA software package is a popular and versatile bioinformatics tool with capabilities to analyze, construct, and visualize three-dimensional nucleic ...

FtsZ Polymerization Assays: Simple Protocols and Considerations

During bacterial cell division, the essential protein FtsZ assembles in the middle of the cell to form the so-called Z-ring. FtsZ polymerizes into long filaments in the presence of GTP in vitro, and polymerization is regulated by several accessory proteins. FtsZ polymerization has been extensively studied in vitro using basic methods including light scattering, sedimentation, GTP hydrolysis assays and electron microscopy. Buffer conditions influence both the polymerization properties of FtsZ, and the ability of FtsZ to interact with regulatory proteins. Here, we describe protocols for FtsZ polymerization studies and validate conditions and controls using Escherichia coli and Bacillus subtilis FtsZ as model proteins. A low speed sedimentation assay is introduced that allows the study of the interaction of FtsZ with proteins that bundle or tubulate FtsZ polymers. An improved GTPase assay protocol is described that allows testing of GTP hydrolysis over time using various conditions in a 96-well plate setup, with standardized incubation times that abolish variation in color development in the phosphate detection reaction. The preparation of samples for light scattering studies and electron microscopy is described. Several buffers are used to establish suitable buffer pH and salt concentration for FtsZ polymerization studies. A high concentration of KCl is the best for most of the experiments. Our methods provide a starting point for the in vitro characterization of FtsZ, not only from E. coli and B. subtilis but from any other bacterium. As such, the methods can be used for studies of the interaction of FtsZ with regulatory proteins or the testing of antibacterial drugs which may affect FtsZ polymerization.

Polymerization of FtsZ is essential for bacterial cell division. In this report, we detail simple protocols to monitor FtsZ polymerization activity and discuss the influence of buffer composition. The protocols can be used to study the interaction of FtsZ with regulatory proteins or antibacterial drugs that affect FtsZ polymerization.

FtsZ的聚合反应检测：简单的协议和注意事项

During bacterial cell division, the essential protein FtsZ assembles in the middle of the cell to form the so-called Z-ring. FtsZ polymerizes into long ...

Preparation, Imaging, and Quantification of Bacterial Surface Motility Assays

Bacterial surface motility, such as swarming, is commonly examined in the laboratory using plate assays that necessitate specific concentrations of agar and sometimes inclusion of specific nutrients in the growth medium. The preparation of such explicit media and surface growth conditions serves to provide the favorable conditions that allow not just bacterial growth but coordinated motility of bacteria over these surfaces within thin liquid films. Reproducibility of swarm plate and other surface motility plate assays can be a major challenge. Especially for more &#8220;temperate swarmers&#8221; that exhibit motility only within agar ranges of 0.4%-0.8% (wt/vol), minor changes in protocol or laboratory environment can greatly influence swarm assay results. &#8220;Wettability&#8221;, or water content at the liquid-solid-air interface of these plate assays, is often a key variable to be controlled. An additional challenge in assessing swarming is how to quantify observed differences between any two (or more) experiments. Here we detail a versatile two-phase protocol to prepare and image swarm assays. We include guidelines to circumvent the challenges commonly associated with swarm assay media preparation and quantification of data from these assays. We specifically demonstrate our method using bacteria that express fluorescent or bioluminescent genetic reporters like green fluorescent protein (GFP), luciferase (lux operon), or cellular stains to enable time-lapse optical imaging. We further demonstrate the ability of our method to track competing swarming species in the same experiment.

Swarming motility is influenced by physical and environmental factors. We describe a two-phase protocol and guidelines to circumvent the challenges commonly associated with swarm assay preparation and data collection. A macroscopic imaging technique is employed to obtain detailed information on swarm behavior that is not provided by current analysis techniques.

准备，成像和定量的细菌表面运动性测定

Bacterial surface motility, such as swarming, is commonly examined in the laboratory using plate assays that necessitate specific concentrations of agar ...

Efficient Nucleic Acid Extraction and 16S rRNA Gene Sequencing for Bacterial Community Characterization

There is a growing appreciation for the role of microbial communities as critical modulators of human health and disease. High throughput sequencing technologies have allowed for the rapid and efficient characterization of bacterial communities using 16S rRNA gene sequencing from a variety of sources. Although readily available tools for 16S rRNA sequence analysis have standardized computational workflows, sample processing for DNA extraction remains a continued source of variability across studies. Here we describe an efficient, robust, and cost effective method for extracting nucleic acid from swabs. We also delineate downstream methods for 16S rRNA gene sequencing, including generation of sequencing libraries, data quality control, and sequence analysis. The workflow can accommodate multiple samples types, including stool and swabs collected from a variety of anatomical locations and host species. Additionally, recovered DNA and RNA can be separated and used for other applications, including whole genome sequencing or RNA-seq. The method described allows for a common processing approach for multiple sample types and accommodates downstream analysis of genomic, metagenomic and transcriptional information.

We describe an efficient, robust, and cost effective method for extracting nucleic acid from swabs for characterization of bacterial communities using 16S rRNA gene amplicon sequencing. The method allows for a common processing approach for multiple sample types and accommodates a number of downstream analytic processes.

高效的核酸提取和16S rRNA基因序列的细菌群落特征

There is a growing appreciation for the role of microbial communities as critical modulators of human health and disease. High throughput sequencing ...

Filtration Isolation of Nucleic Acids:  A Simple and Rapid DNA Extraction Method

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad to extract cellular DNA from whole blood in less than 2 min. The blood specimen is treated with detergent, mixed briefly and applied by pipet to the separation membrane. The lysate wicks into the blotting pad due to capillary action, capturing the genomic DNA on the surface of the separation membrane. The extracted DNA is retained on the membrane during a simple wash step wherein PCR inhibitors are wicked into the absorbent blotting pad. The membrane containing the entrapped DNA is then added to the PCR reaction without further purification. This simple method does not require laboratory equipment and can be easily implemented with inexpensive laboratory supplies. Here we describe a protocol for highly sensitive detection and quantitation of HIV-1 proviral DNA from 100 &#181;l whole blood as a model for early infant diagnosis of HIV that could readily be adapted to other genetic targets.

We describe here a simple and rapid paper-based DNA extraction method of HIV proviral DNA from whole blood detected by quantitative PCR. This protocol can be extended for use in detecting other genetic markers or using alternative amplification methods.

核酸的分离过滤：一种简单快速的DNA提取方法

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad ...

Magnetic-Activated Cell Sorting Strategies to Isolate and Purify Synovial Fluid-Derived Mesenchymal Stem Cells from a Rabbit Model

Mesenchymal stem cells (MSCs) are the main cell source for cell-based therapy. MSCs from articular cavity synovial fluid could potentially be used for cartilage tissue engineering. MSCs from synovial fluid (SF-MSCs) have been considered promising candidates for articular regeneration, and their potential therapeutic benefit has made them an important research topic of late. SF-MSCs from the knee cavity of the New Zealand white rabbit can be employed as an optimized translational model to assess human regenerative medicine. By means of CD90-based magnetic activated cell sorting (MACS) technologies, this protocol successfully obtains rabbit SF-MSCs (rbSF-MSCs) from this rabbit model and further fully demonstrates the MSC phenotype of these cells by inducing them to differentiate to osteoblasts, adipocytes, and chondrocytes. Therefore, this approach can be applied in cell biology research and tissue engineering using simple equipment and procedures.

This article presents a simple and economic protocol for the straightforward isolation and purification of mesenchymal stem cells from New Zealand white rabbit synovial fluid.

从兔模型中分离纯化滑液源性间充质干细胞的磁活化细胞分选策略

Mesenchymal stem cells (MSCs) are the main cell source for cell-based therapy. MSCs from articular cavity synovial fluid could potentially be used for ...

The Lactate Dehydrogenase Sequestration Assay &#8212; A Simple and Reliable Method to Determine Bulk Autophagic Sequestration Activity in Mammalian Cells

Bulk autophagy is characterized by the sequestration of large portions of cytoplasm into double/multi-membrane structures termed autophagosomes. Here a simple protocol to monitor this process is described. Moreover, typical results and experimental validation of the method under autophagy-inducing conditions in various types of cultured mammalian cells are provided. During bulk autophagy, autophagosomes sequester cytosol, and thereby also soluble cytosolic proteins, alongside other autophagic cargo. LDH is a stable and highly abundant, soluble cytosolic enzyme that is non-selectively sequestered into autophagosomes. The amount of LDH sequestration therefore reflects the amount of bulk autophagic sequestration. To efficiently and accurately determine LDH sequestration in cells, we employ an electrodisruption-based fractionation protocol that effectively separates sedimentable from cytosolic LDH, followed by measurement of enzymatic activity in sedimentable fractions versus whole-cell samples. Autophagic sequestration is determined by subtracting the proportion of sedimentable LDH in untreated cells from that in treated cells. The advantage of the LDH sequestration assay is that it gives a quantitative measure of the autophagic sequestration of endogenous cargo, as opposed to other methods that either involve ectopic expression of sequestration probes or semi-quantitative protease protection analyses of autophagy markers or receptors.

The Lactate Dehydrogenase Sequestration Assay &amp;#8212; A Simple and Reliable Method to Determine Bulk Autophagic Sequestration Activity in Mammalian Cells

Here a simple and well-validated protocol for measuring bulk autophagic sequestration activity in mammalian cells is described. The method is based on quantifying the proportion of lactate dehydrogenase (LDH) in sedimentable cell fractions compared to total cellular LDH levels.

乳酸脱氢酶螯合测定--一种简便、可靠的测定哺乳动物细胞中自噬性螯合活性的方法

Bulk autophagy is characterized by the sequestration of large portions of cytoplasm into double/multi-membrane structures termed autophagosomes. Here a ...

Planarian Scrunching as a Quantitative Behavioral Readout for Noxious Stimuli Sensing

Freshwater planarians normally glide smoothly through ciliary propulsion on their ventral side. Certain environmental conditions, however, can induce musculature-driven forms of locomotion: peristalsis or scrunching. While peristalsis results from a ciliary defect, scrunching is independent of cilia function and is a specific response to certain stimuli, including amputation, noxious temperature, extreme pH, and ethanol. Thus, these two musculature-driven gaits are mechanistically distinct. However, they can be difficult to distinguish qualitatively. Here, we provide a protocol for inducing scrunching using various physical and chemical stimuli. We detail the quantitative characterization of scrunching, which can be used to distinguish it from peristalsis and gliding, using freely available software. Since scrunching is a universal planarian gait, albeit with characteristic species-specific differences, this protocol can be broadly applied to all species of planarians, when using appropriate considerations. To demonstrate this, we compare the response of the two most popular planarian species used in behavioral research, Dugesia japonica and Schmidtea mediterranea, to the same set of physical and chemical stimuli. Furthermore, the specificity of scrunching allows this protocol to be used in conjunction with RNA interference and/or pharmacological exposure to dissect the molecular targets and neuronal circuits involved, potentially providing mechanistic insight into important aspects of nociception and neuromuscular communication.

Freshwater planarians exhibit three gaits (gliding, peristalsis, and scrunching) that are distinguishable by quantitative behavioral analysis. We describe a method to induce scrunching using various noxious stimuli, quantification thereof, and distinction from peristalsis and gliding. Using gene knockdown, we demonstrate the specificity of scrunching as a quantitative phenotypic readout.

平面性皱毛作为有毒刺激感的定量行为读出

Freshwater planarians normally glide smoothly through ciliary propulsion on their ventral side. Certain environmental conditions, however, can induce ...