Name: the visual colorimetric detection of multi-nucleotide polymorphisms on a pneumatic droplet manipulation platform
Uploaded: 2016-09-27T14:00:00.000+00:00
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Description: Watch this Scientific Journal Video about The Visual Colorimetric Detection of Multi-nucleotide Polymorphisms on a Pneumatic Droplet Manipulation Platform at JoVE.com

Ministry of Science and Technology of Taiwan provided financial support of this research under contracts MOST-103-2221-E-002 -097 -MY3.

1.检测方法MNP 注意:本节介绍了一种基于金纳米粒子的杂交介导的生长检测MNP的过程。 <ol><li>制备探针DNA（5&#39;-硫醇GAGCTGGTGGCGTAGGCAAG-3&#39;）的浓度为100μM的溶液。 </li><li>准备探针DNA修饰的金纳米粒子（金纳米粒子探针）的颗粒。21 注意:此处所用的量取决于探针DNA修饰金纳米粒子（金纳米粒子探针的）颗粒的要求。它因此是依赖于要进行的实验次数。我们通常超额准备金纳米粒子探测器粒子作为备用。在这些步骤中使用的体积是可调的和灵活的。 <ol><li>在浓度为1 OD /毫升（100μM，在步骤1.1中制备）添加探针DNA金纳米颗粒的溶液（13纳米，17纳米）。 </li><li>带来十二烷基硫酸钠的最终浓度（NAC 12 H 25 SO 4，SDS）和磷酸盐缓冲液（PBS）中，以0.01％和0.01男，respectivel年。 </li><li> 20分钟后，带来的氯化钠（NaCl）浓度为0.05M的用NaCl（2M）和PBS（0.01M），同时维持在0.01％SDS的溶液中，并孵育20分钟。 </li><li>增加的0.1M的增量NaCl浓度至1M在20分钟的时间间隔的终浓度。 </li><li>与在23℃下在涡旋混合器上振摇孵育过夜。摇动速度不只要DNA样品和金纳米粒子探针成为杂交影响实验。 </li><li>离心金纳米颗粒在7000×g离心30秒，并去除上清。 </li></ol></li><li>在25纳米（金纳米粒子探针溶液）的浓度添加金纳米粒子探针粒子到去离子水（DI水）。 </li><li>制备的靶DNA样品（完全互补:5&#39;-CTTGCCTACGCCACCAGCTC-3&#39;;三碱基对不匹配:5&#39;-CTTGCCTAC TTT ACCAGCTC-3&#39;; 6个碱基对的错配:5&#39;-CTTGCCT TTTTTT CCAGCTC-3&#39;）的浓度0.06，0.11，0.17，0.20，0.30，0.50微米，分别。 </li><li>添加金纳米粒子探针溶液（6微升，25纳米）到DNA样品（350微升）用NaCl（14μM）。 </li><li>摇DNA样品和金纳米粒子探针的混合物用涡旋混合器在23℃下5分钟进行DNA杂交。 </li><li>对于金纳米粒子的生长，添加NH 2 OH（6微升，400毫摩尔）和氯金酸4（6微升，25.4毫摩尔）该溶液中（DNA样品和金纳米粒子探针的混合物）。金纳米粒子的生长需要完成大约30秒。颜色的变化稳定保持1个多小时。 注意:用氯金酸皮肤接触可能会产生严重的毒性作用。使用前请咨询材料安全数据表（MSDS）。请戴手套，并使用通风橱使用氯金酸4时。 </li></ol> 2.气动操纵液滴平台的制造注:基于PDMS滴操控平台由两部分组成:一名PDMS膜（100微米）用超疏水性表面和空气沟道层（5毫米）。没有的MEMS工艺中，常见的加工过程被用于制造该装置，其中包括计算机数字控制（CNC）微机械加工制造的模具中，PDMS铸造和复制的微流体部件的快速原型设计，并激光微加工用于制造超疏水表面（见图3）。 <ol><li>使用CNC机床配备有钻头（0.5毫米）到产生PMMA基主模具（ 见图3）具有微结构。使用钻头7位毫米/秒的进给速度和旋转26000 rpm的速率。使用鼓风机和DI水以除去PMMA废料和清洁主模具的表面上。 </li><li>制备的PDMS基底和在比10的固化剂的混合物:1。脱气干燥器内的混合物30分钟，或直到所有的气泡都被删除。 </li><li>倒入PDMS进入次È母模，并在烘箱中烘烤3小时的PDMS在60℃，得到的气室的逆微结构（PDMS膜）和空气通道（PDMS层）。 </li><li>取下母模（用手）的PDMS层。冲PDMS层的空气吸入口上的孔。 </li><li>放PDMS膜，与PDMS层和玻璃基板到氧等离子体处理系统的室中。氧等离子体处理后，一起在电炉上（90℃）10分钟粘合PDMS膜，与PDMS层和玻璃基板。 </li><li>把复合芯片进入激光切割机（PDMS膜面朝上）。导入.dwg文件来定义超疏水区（15毫米×12毫米）。参见图3。直接与二氧化碳激光雕刻机的PDMS膜的PDMS膜（功率12 W，扫描速度106.68毫米/秒）创建超疏水区域。 </li><li>清洁用DI水的超疏水性表面洗远表面上的碳化残留物。 </li><li>通过电磁阀连接空气吸入口和真空泵（ 见图4）。电磁阀到计算机和控制连接与数字I / O USB模块。 </li></ol> 3.气动平台的MNP的检测操作注:这部分描述的操作以色度和迅速查明气动液滴操纵平台上的MNP（ 见图5）。所有步骤取在23℃（环境温度）的地方，相对湿度85％。 <ol><li>放置靶DNA样品的液滴（10微升，0.5μM）上的充气液滴操纵平台的超疏水性区域。 </li><li>放置金纳米粒子探针的液滴（10微升，25纳米）上的充气液滴操纵平台的超疏水性区域。 </li><li>控制吸气使PDMS膜的偏转用的电磁阀和一个SImple程序（ 例如，Labview的）。提供在PDMS膜使得液滴相互碰撞和聚结在沿液滴（含有靶DNA和金纳米粒子探针分别）的迁移路径各空气室的空气吸入。使用-80千帕（表压）的真空泵的工作压力。 <ol><li>可替代地，只要手动作为微滴充分混合控制空气吸入。用手，连接或断开真空泵和空气室的入口与在测试的早期阶段的管。 </li></ol></li><li>控制吸气滚动聚结的液滴前进和后退用电磁阀的控制系统，以提高混合效率为3分钟。靶DNA与金纳米粒子探针变得很好地混合，在3分钟内进行杂交。使用的驱动频率和5赫兹和-80千帕（表压）的工作压力，分别。 </li><li>放置装有NH 2 OH（2微升，400毫摩尔）和氯金酸的液滴4（2微升，25.4毫摩尔）上的充气液滴操纵平台的超疏水性区域。 </li><li>控制吸气（-80千帕表压），以让液滴彼此和聚结发生碰撞。则控制吸气（5赫兹和-80千帕表压），以让所述聚结的液滴滚向前和向后以提高混合效率，持续1分钟。 </li><li>测量和记录用肉眼聚结液滴的颜色。 </li></ol>

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The authors declare that they have no competing financial interests.

在这个协议中，一个简单的比色方法检测MNP可以在浓度范围为0.11-0.50微米的微离心管中实现。此外，所提出MNP检测方法上的充气液滴操作平台，该平台具有用于DNA筛查和其它生物医学应用的高电位下进行。在实践中，样品DNA浓度的可检测范围取决于的操作平台的混合效率。以确保该聚结的液滴充分混合，在该协议中的关键步骤是依赖于聚结液滴的大小吸气（压力和频率）的控制。完全互补的样本D...

单核苷酸多态性（SNP），这是在DNA序列的单碱基对差异，是最常见的遗传变异之一。目前的研究报告，单核苷酸多态性与疾病风险，药效和通过影响基因功能的个人副作用有关。1,2-最近的研究还表明，双或多点突变（多核苷酸多态性）引起特定疾病和个体在疾病的影响差异。3,4核苷酸多态性的检测是因此病预检势在必行。为快速检测序列特异性寡核苷酸的简单而有效的方法，在过去的二十年是高度发达的1,5电流的方法来识别DNA突变通常涉及程序，包括探针固定，荧光标记，凝胶电泳等 ，6,7但这些方法一般需要很长的分析过程，expen西伯设备，训练有素的技术人员，以及样品和试剂的消耗显著。 与表面积与体积和独特的物理化学性质的大比率的纳米粒子是作为用于特定的生物标记物的高灵敏度和廉价的检测平台的理想材料。金纳米颗粒（金纳米粒子）被广泛用于DNA检测的，因为它们的大能力与寡核苷酸探针进行修改。8-10 SNP检测技术是使用金纳米粒子还开发11-13在这项工作中，我们采用了一种新颖的比色方法检测多核苷酸多态性（MNP）通过14金纳米粒子探针的DNA杂交介导的生长，这种简单的和快速的探测方法是基于这样的理论缀合到单链DNA（ssDNA）或双链DNA（dsDNA）的的不同长度金纳米粒子影响金纳米粒子的生长的大小和形状（参见图1）。15的DNA检测的这种方法设有试剂的消耗量小，小试验的持续时间（几分钟），并且没有热控制一个简单的过程，是前瞻性适用于临床诊断和国内医学筛查。 几个微流体系统以检测DNA序列已被开发; 16这些微流体系统中，从传统的实验方案演化而来，需要更少的块的大型设备，并简化了实验方案，以提高灵敏度，检测极限和特异性的DNA生物传感器。在微流体系统的DNA检测方法仍需要，但是，信号放大和荧光读取器的随后的处理的仪器如PCR（聚合酶链式反应）机对单个读出，以确定异构SNP。17,18开发一个简单的无随后的处理平台，以直接读出的多核苷酸多态性的结果是非常需要的。比起很好用的，传统的，封闭的微流体系统，开放式表面微流体装置有为地提供一些优势，比如一个明确的光路，一个简单的方法来访问样本，直接无障碍环境并没有容易形成气穴或界面阻塞通道19我们以前的工作引入开放表面的液滴操作（ 见图2）。20简单气动平台在此平台上，液滴可同时运输和使用抽吸力从驱动能量而不干扰操纵，其中有一个在生物和化学的应用潜力巨大。因此，该气动平台利用结合使用金纳米粒子探针的DNA杂交介导的生长的概念比色方法执行DNA样品的操纵对MNP的检测。 本文介绍的协议描述多核苷酸多态性的一个简单的目视检测气动操纵液滴平台上的开放面。这项工作证实，多核苷酸多态性是用肉眼可检测的;建议的气动平台，适用于生物和化学应用。

<table><tbody><tr><td>PDMS</td><td>Dow Corning</td><td>SYLGARD 184</td><td></td></tr><tr><td>benchtop engravers</td><td>Roland DG</td><td>EGX-400</td><td></td></tr><tr><td>laser cutting machine</td><td>Universal Laser Systems, Inc.</td><td>VLS 3.50</td><td></td></tr><tr><td>Oxygen plasma treatment system</td><td>Femto Science Inc. Korea</td><td>CUTE-MPR</td><td></td></tr><tr><td>solenoid valve</td><td></td><td></td><td>home built</td></tr><tr><td>vacuum pump</td><td>ULVAC KIKO, Inc.</td><td>DA-30D</td><td></td></tr><tr><td>13-nm AuNP solution</td><td>TAN Bead Inc., Taiwan</td><td>NG-13</td><td></td></tr><tr><td>DNA (with 5&#039;-end labeled thiol)</td><td>MDBio, Inc., Taiwan</td><td></td><td></td></tr><tr><td>phosphate buffered saline (PBS)</td><td>UniRegion&amp;nbsp;Bio-Tech,. Taiwan</td><td>PBS001-1L</td><td></td></tr><tr><td>sodium dodecyl sulfate (SDS)</td><td>J. T. baker</td><td>4095-04</td><td></td></tr><tr><td>Hydroxylamine solution (NH&lt;sub&gt;2&lt;/sub&gt;OH)</td><td>Sigma-Aldrich</td><td>467804</td><td></td></tr><tr><td>Chloroauric acid (HAuCl&lt;sub&gt;4&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>G4022</td><td></td></tr><tr><td>sodium chloride (NaCl)</td><td></td><td></td><td></td></tr><tr><td>vortex mixer</td><td>Digisystem Laboratory Instruments Inc.</td><td>VM-2000</td><td></td></tr><tr><td>centrifuge</td><td>Hermle Labortechnik GmbH.</td><td>Z 216 MK</td><td></td></tr></tbody></table>

the visual colorimetric detection of multi-nucleotide polymorphisms on a pneumatic droplet manipulation platform

是在一个开放的表面上液滴的气动操纵平台进行一个简单直观的方法来检测多核苷酸多态性（MNP）。这种方法比色检测DNA的基础上金纳米粒子探测器（探头金纳米粒子）的杂交介导的生长。该金纳米粒子的生长尺寸和结构通过与探针杂交的DNA样品的数量支配。根据纳米颗粒的具体大小 - 和形状依赖性的光学特性，错配的在样品DNA片段与探针的数目是能够被区分。试验是分别通过含有试剂和DNA样品的液滴中进行，被运送并与基于PDMS柔性超疏水性膜的受控气动吸入混合气的平台上。液滴可以同时并准确传递一个开放表面上所提出的气动平台，是没有副作用EFF高度的生物相容性液滴内的DNA样品的等。两个提出的方法相结合，该多核苷酸多态性可在视线气动液滴操纵平台上被检测;没有额外的仪器是必需的。从平台到最终结果上安装的液滴的步骤需要不到5分钟，比用现有方法少得多。此外，该组合MNP检测方法仅需要10微升，在每个操作中，这比一个宏系统的显着更小的样品体积。

在这项工作中，三种DNA样品使用检测的简单且新颖的方法通过金纳米粒子探针的DNA杂交介导的生长测试。探针DNA和三种DNA样本的序列，具体而言，CDNA（完全互补的探针DNA），TMDNA（三碱基对的错配的DNA），和SixMDNA（6个碱基对的错配的DNA）的协议步骤1中列出。此处所测试的DNA样品的探针的错配是无论在DNA样品的中间段。 图6示出了用于在不同的浓度的DNA样品的...

Watch this Scientific Journal Video about The Visual Colorimetric Detection of Multi-nucleotide Polymorphisms on a Pneumatic Droplet Manipulation Platform at JoVE.com

The Visual Colorimetric Detection of Multi-nucleotide Polymorphisms on a Pneumatic Droplet Manipulation Platform

这项工作提出了一个简单直观的方法来检测气动操纵液滴平台的多核苷酸多态性。与所提出的方法中，在整个实验，包括液滴操作和多核苷酸多态性检测，可以接近23°C不先进仪器的帮助下进行。

在气动操纵液滴平台多核苷酸多态性的视觉比色法检测

A simple and visual method to detect multi-nucleotide polymorphism (MNP) was performed on a pneumatic droplet manipulation platform on an open surface. ...

the-visual-colorimetric-detection-multi-nucleotide-polymorphisms-on

Research

JoVE Journal

Genetics

7.6K 次观看.  National Taiwan University. 这项工作提出了一个简单直观的方法来检测气动操纵液滴平台的多核苷酸多态性。与所提出的方法中，在整个实验，包括液滴操作和多核苷酸多态性检测，可以接近23°C不先进仪器的帮助下进行。 

视频: The Visual Colorimetric Detection of Multi-nucleotide Polymorphisms on a Pneumatic Droplet Manipulation Platform

Fluorescence detection methods for microfluidic droplet platforms

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that accompany system miniaturisation. Advantages include the ability to efficiently process nano- to femoto- liter volumes of sample, facile integration of functional components, an intrinsic predisposition towards large-scale multiplexing, enhanced analytical throughput, improved control and reduced instrumental footprints.1
In recent years much interest has focussed on the development of droplet-based (or segmented flow) microfluidic systems and their potential as platforms in high-throughput experimentation.2-4 Here water-in-oil emulsions are made to spontaneously form in microfluidic channels as a result of capillary instabilities between the two immiscible phases. Importantly, microdroplets of precisely defined volumes and compositions can be generated at frequencies of several kHz. Furthermore, by encapsulating reagents of interest within isolated compartments separated by a continuous immiscible phase, both sample cross-talk and dispersion (diffusion- and Taylor-based) can be eliminated, which leads to minimal cross-contamination and the ability to time analytical processes with great accuracy. Additionally, since there is no contact between the contents of the droplets and the channel walls (which are wetted by the continuous phase) absorption and loss of reagents on the channel walls is prevented.
Once droplets of this kind have been generated and processed, it is necessary to extract the required analytical information. In this respect the detection method of choice should be rapid, provide high-sensitivity and low limits of detection, be applicable to a range of molecular species, be non-destructive and be able to be integrated with microfluidic devices in a facile manner. To address this need we have developed a suite of experimental tools and protocols that enable the extraction of large amounts of photophysical information from small-volume environments, and are applicable to the analysis of a wide range of physical, chemical and biological parameters. Herein two examples of these methods are presented and applied to the detection of single cells and the mapping of mixing processes inside picoliter-volume droplets. We report the entire experimental process including microfluidic chip fabrication, the optical setup and the process of droplet generation and detection.

Droplet-based microfluidic platforms are promising candidates for high throughput experimentation since they are able to generate picoliter, self-compartmentalized vessels inexpensively at kHz rates. Through integration with fast, sensitive and high resolution fluorescence spectroscopic methods, the large amounts of information generated within these systems can be efficiently extracted, harnessed and utilized.

荧光检测方法，为微流体液滴平台

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that ...

科研

生物工程

Single Droplet Digital Polymerase Chain Reaction for Comprehensive and Simultaneous Detection of Mutations in Hotspot Regions

Droplet digital polymerase chain reaction (ddPCR) is a highly sensitive quantitative polymerase chain reaction (PCR) method based on sample fractionation into thousands of nano-sized water-in-oil individual reactions. Recently, ddPCR has become one of the most accurate and sensitive tools for circulating tumor DNA (ctDNA) detection. One of the major limitations of the standard ddPCR technique is the restricted number of mutations that can be screened per reaction, as specific hydrolysis probes recognizing each possible allelic version are required. An alternative methodology, the drop-off ddPCR, increases throughput, since it requires only a single pair of probes to detect and quantify potentially all genetic alterations in the targeted region. Drop-off ddPCR displays comparable sensitivity to conventional ddPCR assays with the advantage of detecting a greater number of mutations in a single reaction. It is cost-effective, conserves precious sample material, and can also be used as a discovery tool when mutations are not known a priori.

Here, we present a protocol to accurately quantify multiple genetic alterations of a target region in a single reaction using drop-off ddPCR and a unique pair of hydrolysis probes.

单液滴数字聚合酶链反应对热点地区突变的综合和同步检测

Droplet digital polymerase chain reaction (ddPCR) is a highly sensitive quantitative polymerase chain reaction (PCR) method based on sample fractionation ...

遗传学

Digital-Droplet PCR to Detect Indels Mutations in Genetically Modified Anopheline Mosquito Populations

Recent advances in mosquito genomics and genetic engineering technologies have fostered a need for quick and efficient methods for detecting targeted DNA sequence variation on a large scale. Specifically, detecting insertions and deletions (indels) at gene-edited sites generated by CRISPR guide RNA (gRNA)/Cas9-mediated non-homologous end-joining (NHEJ) is important for assessing the fidelity of the mutagenesis and the frequency of unintended changes. We describe here a protocol for digital-droplet PCR (ddPCR) that is well-suited for high-throughput NHEJ analysis. While this method does not produce data that identifies individual sequence variation, it provides a quantitative estimate of the sequence variation within a population. Additionally, with appropriate resources, this protocol can be implemented in a field-site laboratory setting more easily than next-generation or Sanger sequencing. ddPCR also has a faster turn-around time for results than either of those methods, which allows a more quick and complete analysis of genetic variation in wild populations during field trials of genetically-engineered organisms.

This protocol provides the steps from DNA extraction to experimental set-up for digital droplet PCR (ddPCR), including analysis for the identification and quantification of non-homologous end-joining (NHEJ) events at target sites following gRNA-induced Cas9 cleavage and DNA repair. Other uses of this method include applications such as polymorphism detection and gene-editing variant verification.

数字液滴PCR检测转基因按蚊种群中的Indels突变

Recent advances in mosquito genomics and genetic engineering technologies have fostered a need for quick and efficient methods for detecting targeted DNA ...

Multicolor Fluorescence Detection for Droplet Microfluidics Using Optical Fibers

Fluorescence assays are the most common readouts used in droplet microfluidics due to their bright signals and fast time response. Applications such as multiplex assays, enzyme evolution, and molecular biology enhanced cell sorting require the detection of two or more colors of fluorescence. Standard multicolor detection systems that couple free space lasers to epifluorescence microscopes are bulky, expensive, and difficult to maintain. In this paper, we describe a scheme to perform multicolor detection by exciting discrete regions of a microfluidic channel with lasers coupled to optical fibers. Emitted light is collected by an optical fiber coupled to a single photodetector. Because the excitation occurs at different spatial locations, the identity of emitted light can be encoded as a temporal shift, eliminating the need for more complicated light filtering schemes. The system has been used to detect droplet populations containing four unique combinations of dyes and to detect sub-nanomolar concentrations of fluorescein.

Multicolor fluorescence detection in droplet microfluidics typically involves bulky and complex epifluorescence microscope-based detection systems. Here we describe a compact and modular multicolor detection scheme that utilizes an array of optical fibers to temporally encode multicolor data collected by a single photodetector.

使用多色荧光检测液滴微流控光纤

Fluorescence assays are the most common readouts used in droplet microfluidics due to their bright signals and fast time response. Applications such as ...

Visual Detection of Multiple Nucleic Acids in a Capillary Array

Multi-target, short time, and resource-affordable methodologies for the detection of multiple nucleic acids in a single, easy to operate test are urgently needed in disease diagnosis, microbial monitoring, genetically modified organism (GMO) detection, and forensic analysis. We have previously described the platform called CALM (Capillary Array-based Loop-mediated isothermal amplification for Multiplex visual detection of nucleic acids). Herein, we describe improved fabrication and performance processes for this platform. Here, we apply a small, ready-to-use cassette assembled by capillary array for multiplex visual detection of nucleic acids. The capillary array is pre-treated into a hydrophobic and hydrophilic pattern before fixing loop-mediated isothermal amplification (LAMP) primer sets in capillaries. After assembly of the loading adaptor, LAMP reaction mixture is loaded and isolated into each capillary, due to capillary force by a single pipetting step. The LAMP reactions are performed in parallel in the capillaries. The results are visually read out by illumination with a hand-held UV flashlight. Using this platform, we demonstrate monitoring of 8 frequently appearing elements and genes in GMO samples with high specificity and sensitivity. In summary, the platform described herein is intended to facilitate the detection of multiple nucleic acids. We believe it will be widely applicable in fields where high-throughput nucleic acid analysis is required.

This protocol describes the fabrication of a small, ready-to-use cassette that can be applied for visual detection of multiple nucleic acids in a single, test that is easy to operate. In this approach, a capillary array was used for multiplex and highly efficient detection of GMO targets.

毛细管阵列中多核酸的视觉检测

Multi-target, short time, and resource-affordable methodologies for the detection of multiple nucleic acids in a single, easy to operate test are urgently ...

Methylation Specific Multiplex Droplet PCR using Polymer Droplet Generator Device for Hematological Diagnostics

A multiplexed droplet PCR (mdPCR) workflow and detailed protocol for determining epigenetic-based white blood cell (WBC) differential count is described, along with a thermoplastic elastomer (TPE) microfluidic droplet generation device. Epigenetic markers are used for WBC&#160;subtyping which is of important prognostic value in different diseases. This is achieved through the quantification of DNA methylation patterns of specific CG-rich regions in the genome (CpG loci). In this paper, bisulfite-treated DNA from peripheral blood mononuclear cells (PBMCs) is encapsulated in droplets with mdPCR reagents including primers and hydrolysis fluorescent probes specific for CpG loci that correlate with WBC sub-populations. The multiplex approach allows for the interrogation of many CpG loci without the need for separate mdPCR reactions, enabling more accurate parametric determination of WBC sub-populations using epigenetic analysis of methylation sites. This precise quantification can be extended to different applications and highlights the benefits for clinical diagnosis and subsequent prognosis.

Epigenetic markers are used for white blood cell (WBC) subtyping through the quantification of DNA methylation patterns. This protocol presents a multiplex droplet polymerase chain reaction (mdPCR) method using a thermoplastic elastomer (TPE)-based microfluidic device for droplet generation allowing for precise and multiplex methylation-specific target quantification of WBC differential counts.

甲基化特异性多路复用液滴PCR，使用聚合物液滴发生器设备进行血液诊断

A multiplexed droplet PCR (mdPCR) workflow and detailed protocol for determining epigenetic-based white blood cell (WBC) differential count is described, ...

Simple Bulk Readout of Digital Nucleic Acid Quantification Assays

Digital assays are powerful methods that enable detection of rare cells and counting of individual nucleic acid molecules. However, digital assays are still not routinely applied, due to the cost and specific equipment associated with commercially available methods. Here we present a simplified method for readout of digital droplet assays using a conventional real-time PCR instrument to measure bulk fluorescence of droplet-based digital assays.
We characterize the performance of the bulk readout assay using synthetic droplet mixtures and a droplet digital multiple displacement amplification (MDA) assay. Quantitative MDA particularly benefits from a digital reaction format, but our new method&#160;applies to any digital assay. For established digital assay protocols such as digital PCR, this method serves to speed up and simplify assay readout.
Our bulk readout methodology brings the advantages of partitioned assays without the need for specialized readout instrumentation. The principal limitations of the bulk readout methodology are reduced dynamic range compared with droplet-counting platforms and the need for a standard sample, although the requirements for this standard are less demanding than for a conventional real-time experiment. Quantitative whole genome amplification (WGA) is used to test for contaminants in WGA reactions and is the most sensitive way to detect the presence of DNA fragments with unknown sequences, giving the method great promise in diverse application areas including pharmaceutical quality control and astrobiology.

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 ...

生物学

Digital Droplet Polymerase Chain Reaction for Indel Mutation Detection in Target Genes

Source: Carballar-Lejaraz&#250;, R., et al. <a href="https://app.jove.com/v/62607">Digital-Droplet PCR to Detect Indels Mutations in Genetically Modified Anopheline Mosquito Populations.</a> J. Vis. Exp. (2021).
This video demonstrates the role of digital droplet polymerase chain reaction in identifying indel mutation in a DNA sample. The high precision and sensitivity of ddPCR enable accurate and consistent detection of rare mutations present in a sample, even at low levels. It can identify and quantify indel mutations in different regions of the genome, providing valuable insights into the mechanisms of mutation and the functional consequences of these mutations.&#160;

用于靶基因 Indel 突变检测的数字液滴聚合酶链反应

Source: Carballar-Lejaraz&#250;, R., et al. Digital-Droplet PCR to Detect Indels Mutations in Genetically Modified Anopheline Mosquito Populations. J. ...

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/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 ...

Droplet Digital Polymerase Chain Reaction: A Method to Generate Nanodroplet PCR Reactions for Detecting Rare Tumor Mutations

Source: Bonner, E. R. et al. <a href="https://www.jove.com/video/59721" target="_blank">Detection and Monitoring of Tumor Associated Circulating DNA in Patient Biofluids.</a> J. Vis. Exp. (2019)
In this video, we present a step-by-step procedure to generate emulsified nano-droplets to detect target nucleic acids by digital polymerase chain reaction or dPCR.

液滴数字聚合酶链反应:一种产生纳米液滴 PCR 反应以检测罕见肿瘤突变的方法

Source: Bonner, E. R. et al. Detection and Monitoring of Tumor Associated Circulating DNA in Patient Biofluids. J. Vis. Exp. (2019)
In this video, we ...