Name: real-time tracking of dna fragment separation by smartphone
Uploaded: 2017-06-01T15:00:00.000+00:00
Duration: 6 min 58 s
Description: Watch this Scientific Journal Video about Real-time Tracking of DNA Fragment Separation by Smartphone at JoVE.com

我们非常感谢中国国家自然科学基金（21205078）和中国高等教育博士研究基金（No.20123120110002）的支持。这项工作得到了中国国家重点研究发展计划（2016YFB1102303），中国国家基础研究计划（973计划，2015CB352001）和国家自然科学基金（61378060）的部分支持。

SGE芯片的基本设计<ol><li>使用任何透明塑料，如聚甲基丙烯酸甲酯（PMMA）或聚碳酸酯。 注:SGE芯片如图1B所示 。 SGE芯片由用于TBE缓冲器的圆柱形孔，用于DNA分离的通道和沿着电极孔嵌入的两条通道组成。 </li><li>使用激光雕刻机在PMMA块中制造SGE通道阵列。 注意:芯片的几何参数取决于要分离的DNA的大小。例如，如果DNA片段小于1,000bp，则SGE芯片的最佳信道参数为2.5mm×4.0mm×90mm（宽×深×长）。 </li><li>基于SGE设计，制造梳子以在芯片中创建用于加载DNA样品的孔。 </li></ol> 2.琼脂糖凝胶的制备<ol><li>通过混合10×TBE（1×TBE =89mM Tris，89mM硼酸和2mM EDTA; pH 8.4）和蒸馏水以1:19的比例。 </li><li>制备1.0％琼脂糖凝胶，分离100-bp DNA梯。 注意:0.5x TBE缓冲液中琼脂糖的浓度取决于要分离的DNA片段的大小。 </li><li>将0.1 g琼脂糖放入烧瓶中，加入10 mL 0.5x TBE缓冲液。 注意:制备溶液的体积应小于烧瓶容量的1/3。 </li><li>用防腐膜密封烧瓶，然后在微波（培养基，1.0分钟）中加热琼脂糖/缓冲液混合物。在将烧瓶放入微波炉之前，在爆炸的情况下，在防腐膜中形成一些孔。 </li><li>将2.7mL熔融的琼脂糖溶液倒入SGE芯片的4个通道。将梳子放入琼脂糖溶液中以形成孔。 注意:3分钟后，熔化的琼脂糖溶液将在室温下冷却至凝胶状态。 </li><li>从g去除梳子并加入0.9mL 0.5x TBE缓冲液以覆盖凝胶。 </li></ol> 3.在SGE芯片中运行电泳<ol><li>打开LED光源，并在光源上方放置一条425至505 nm的带通滤光片。 </li><li>使用涡旋器混合14.4μL的100bp DNA梯和1.6μL的SYBR Green。 </li><li>在SGE芯片的琼脂糖凝胶的每个孔中加入4μL混合物（ 图2 ）。 </li><li>将电极放入SGE芯片的两条通道。 </li><li>在SGE芯片上方放置550至700 nm带通滤波器。 </li><li>打开电源。将电压设置为180 V（20 V / cm）。打开智能手机记录DNA片段分离过程（ 图3 ）。 </li><li>电泳10分钟后，关掉电源和LED灯。 </li><li>清洁SGE芯片。 </li></ol>

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没有宣布利益冲突。

琼脂糖凝胶电泳广泛用于分离DNA，RNA和蛋白质。这项工作提出了一种替代传统凝胶电泳方法的新方法。结果表明，在这样一个小的组装装置中，50,100和1,000bp的DNA梯子可以很好地分离。该方法的优点在于不仅可以将化学消耗少的核酸分开，还可以记录分离过程。尽管DNA片段在图2中看起来很宽，但可以通过优化SGE芯片的参数和琼脂糖凝胶的浓度来改善结果。此外，如果在芯片?...

<html>板状凝胶电泳（SGE）是DNA片段分离最有效的方法1,2,3,4,5 ，因此被认为是生物化学和生物学分析中的通用工具6,7,8。然而，许多实验表明，SGE受到以下四个问题的限制:（1）分离需要很多小时甚至几天; （2）化学品消耗量非常高; （3）需要复杂的装置（ 例如， 2D电泳池，电泳电源和凝胶成像系统）; （4）实验完成后，凝胶成像系统只能观察分离的DNA片段。此外，SGE 9中通常使用的溴化乙锭（EtBr）ass ="xref"&gt; 10，是致突变性和致癌性11,12 。因此，在装载含有EtBr的凝胶时，应始终佩戴手套。 与SGE相比，毛细管电泳（CE）具有许多优点，如自动操作，分离时间短，消耗量少等优点，13，14，15，16，17。然而，CE仪器相当昂贵。因此，为了克服这些限制，已经开发了一种用于分离DNA的系统（ 图1 ）。这样的系统不仅可以大大降低化学消耗，还可以节省SGE实验时间（&lt;8分钟），而且可以通过智能手机对琼脂糖凝胶中的DNA分离过程进行实时跟踪。按照本协议中描述的程序，学生们可以可以设计和制造SGE芯片，在芯片中制备琼脂糖凝胶，用智能手机设置简单的SGE系统，并将DNA迁移过程记录在琼脂糖凝胶中。

<table><tbody><tr><td>10&amp;times;TBE</td><td>Beijing Solarbio Science &amp;amp; Technology Co., Ltd.</td><td>T1051</td><td></td></tr><tr><td>50 bp DNA ladder</td><td>Takara Bio Inc.</td><td>3421A</td><td></td></tr><tr><td>100 bp DNA ladder</td><td>Takara Bio Inc.</td><td>3422A</td><td></td></tr><tr><td>1 kbp DNA ladder</td><td>Takara Bio Inc.</td><td>3426A</td><td></td></tr><tr><td>SYBR GREEN</td><td>Takara Bio Inc.</td><td>5760A</td><td></td></tr><tr><td>Agarose</td><td>Sigma-Aldrich Corporate</td><td>V900510</td><td></td></tr></tbody></table>

real-time tracking of dna fragment separation by smartphone

板状凝胶电泳（SGE）是分离DNA片段的最常用方法;因此，它广泛应用于生物学等领域。然而，传统的SGE协议相当乏味，实验需要很长时间。此外，SGE实验中的化学消耗量非常高。这项工作提出了一种基于SGE芯片分离DNA片段的简单方法。该芯片由雕刻机制成。两个塑料片用于光信号的激发和发射波长。通过智能手机收集DNA条带的荧光信号。为了验证该方法，分离出50,100和1,000bp DNA梯。结果表明，使用该方法可以在12分钟内解析出小于5,000bp的DNA梯度，分辨率高，表明它是传统SGE方法的理想替代品。

<html> 图4 ， 图5和图6代表了在50,100和1,000bp DNA梯级的凝胶电泳之后的典型结果。实验后，DNA片段分离良好。此外，在SGE芯片的4个通道中分离相同的样品，显示出相同尺寸的DNA片段在每个实验中移动相同的距离。 DNA梯度的分离性能可以通过分析迁移距离与DNA大小对数的关系进行评估;因此，SGE可用于计算未知DNA...

Watch this Scientific Journal Video about Real-time Tracking of DNA Fragment Separation by Smartphone at JoVE.com

Real-time Tracking of DNA Fragment Separation by Smartphone

通过智能手机实时跟踪DNA片段分离

传统的平板凝胶电泳（SGE）实验需要复杂的装置和高的化学消耗。这项工作提出了一个协议，描述了一种在短时间内分离DNA片段的低成本方法。

Slab gel electrophoresis (SGE) is the most common method for the separation of DNA fragments; thus, it is broadly applied to the field of biology and ...

real-time-tracking-of-dna-fragment-separation-by-smartphone

Universidad San Sebastian

Research

JoVE Journal

Biochemistry

14.4K 次观看.  University of Shanghai for Science and Technology. 传统的平板凝胶电泳（SGE）实验需要复杂的装置和高的化学消耗。这项工作提出了一个协议，描述了一种在短时间内分离DNA片段的低成本方法。 

视频: Real-time Tracking of DNA Fragment Separation by Smartphone

Detection of Copy Number Alterations Using Single Cell Sequencing

Detection of genomic changes at single cell resolution is important for characterizing genetic heterogeneity and evolution in normal tissues, cancers, and microbial populations. Traditional methods for assessing genetic heterogeneity have been limited by low resolution, low sensitivity, and/or low specificity. Single cell sequencing has emerged as a powerful tool for detecting genetic heterogeneity with high resolution, high sensitivity and, when appropriately analyzed, high specificity. Here we provide a protocol for the isolation, whole genome amplification, sequencing, and analysis of single cells. Our approach allows for the reliable identification of megabase-scale copy number variants in single cells. However, aspects of this protocol can also be applied to investigate other types of genetic alterations in single cells.

Single cell sequencing is an increasingly popular and accessible tool for addressing genomic changes at high resolution. We provide a protocol that uses single cell sequencing to identify copy number alterations in single cells.

单细胞测序拷贝数改变的检测

Detection of genomic changes at single cell resolution is important for characterizing genetic heterogeneity and evolution in normal tissues, cancers, and ...

科研

遗传学

Modified Terminal Restriction Fragment Analysis for Quantifying Telomere Length Using In-gel Hybridization

There are several different techniques for measuring telomere length, each with their own advantages and disadvantages. The traditional approach, Telomere Restriction Fragment (TRF) analysis, utilizes a DNA hybridization technique whereby genomic DNA samples are digested with restriction enzymes, leaving behind telomere DNA repeats and some sub-telomeric DNA. These are separated by agarose gel electrophoresis, transferred to a filter membrane and hybridized to oligonucleotide probes tagged with either chemiluminescence or radioactivity to visualize telomere restriction fragments. This approach, while requiring a larger quantity of DNA than other techniques such as PCR, can measure the telomere length distribution of a population of cells and allows measurement expressed in absolute kilobases. This manuscript demonstrates a modified DNA hybridization procedure for determining telomere length. Genomic DNA is first digested with restriction enzymes (that do not cut telomeres) and separated by agarose gel electrophoresis. The gel is then dried and the DNA is denatured and hybridized in situ to a radiolabeled oligonucleotide probe. This in situ hybridization avoids loss of telomere DNA and improves signal intensity. Following hybridization, the gels are imaged utilizing phosphor screens and the telomere length is quantified using a graphing program. This procedure was developed by the laboratories of Drs. Woodring Wright and Jerry Shay at the University of Texas Southwestern1,2. Here, we present a detailed description of this procedure, with some modifications.

In this article, a detailed protocol for quantifying telomere length using a modified terminal restriction fragment analysis is discussed that provides fast and efficient direct measurement of telomere length. This technique can be applied to a variety of cell sources of DNA for quantifying telomere length.

使用凝胶内杂交来定量端粒长度的修饰末端限制片段分析

There are several different techniques for measuring telomere length, each with their own advantages and disadvantages. The traditional approach, Telomere ...

癌症研究

A Simple, Robust, and High Throughput Single Molecule Flow Stretching Assay Implementation for Studying Transport of Molecules Along DNA

We describe a simple, robust and high throughput single molecule flow-stretching assay for studying 1D diffusion of molecules along DNA. In this assay, glass coverslips are functionalized in a one-step reaction with silane-PEG-biotin. Flow cells are constructed by sandwiching an adhesive tape with pre-cut channels between a functionalized coverslip and a PDMS slab containing inlet and outlet holes. Multiple channels are integrated into one flow cell and the flow of reagents into each channel can be fully automated, which significantly increases the assay throughput and reduces hands-on time per assay. Inside each channel, biotin-&#955;-DNAs are immobilized on the surface and a laminar flow is applied to flow-stretch the DNAs. The DNA molecules are stretched to &#62;80% of their contour length and serve as spatially extended templates for studying the binding and transport activity of fluorescently labeled molecules. The trajectories of single molecules are tracked by time-lapse Total Internal Reflection Fluorescence (TIRF) imaging. Raw images are analyzed using streamlined custom single particle tracking software to automatically identify trajectories of single molecules diffusing along DNA and estimate their 1D diffusion constants.

This protocol demonstrates a simple, robust and high throughput single molecule flow-stretching assay for studying one-dimensional (1D) diffusion of molecules along DNA.

一种简单、健壮且高通量的单分子流动拉伸试验研究分子沿 DNA 运移的方法

We describe a simple, robust and high throughput single molecule flow-stretching assay for studying 1D diffusion of molecules along DNA. In this assay, ...

生物化学

Real-time Observation of the DNA Strand Exchange Reaction Mediated by Rad51

The DNA strand exchange reaction mediated by Rad51 is a critical step of homologous recombination. In this reaction, Rad51 forms a nucleoprotein filament on single-stranded DNA (ssDNA) and captures double-stranded DNA (dsDNA) non-specifically to interrogate it for a homologous sequence. After encountering homology, Rad51 catalyzes DNA strand exchange to mediate pairing of the ssDNA with the complementary strand of the dsDNA. This reaction is highly regulated by numerous accessary proteins in vivo. Although conventional biochemical assays have been successfully employed to examine the role of such accessory protein in vitro, kinetic analysis of intermediate formation and its progression into a final product has proven challenging due to the unstable and transient nature of the reaction intermediates. To observe these reaction steps directly in solution, fluorescence resonance energy transfer (FRET)-based real-time observation systems of this reaction were established. Kinetic analysis of real-time observations shows that the DNA strand exchange reaction mediated by Rad51 obeys a three-step reaction model involving the formation of a three-strand DNA intermediate, maturation of this intermediate, and the release of ssDNA from the mature intermediate. The Swi5-Sfr1 complex, an accessary protein conserved in eukaryotes, strongly enhances the second and third steps of this reaction. The FRET-based assays presented here enable us to uncover the molecular mechanisms through which recombination accessary proteins stimulate the DNA strand exchange activity of Rad51. The primary goal of this protocol is to enhance the repertoire of techniques available to researchers in the field of homologous recombination, particularly those working with proteins from species other than Schizosaccharomyces pombe, so that the evolutionary conservation of the findings presented herein can be determined.

Fluorescence resonance energy transfer-based real-time observation systems of the DNA strand exchange reaction mediated by Rad51 were developed. Using the protocols presented here, we are able to detect the formation of reaction intermediates and their conversion into products, while also analyzing the enzymatic kinetics of the reaction.

rad51 介导的 dna 链交换反应的实时观测

The DNA strand exchange reaction mediated by Rad51 is a critical step of homologous recombination. In this reaction, Rad51 forms a nucleoprotein filament ...

Application of DNA Fingerprinting using the D1S80 Locus in Lab Classes

In biological sciences, DNA fingerprinting has been widely used for paternity testing, forensic applications and phylogenetic studies. Here, we describe a reliable and robust method for genotyping individuals by Variable Number of Tandem Repeat (VNTR) analysis in the context of undergraduate laboratory classes. The human D1S80 VNTR locus is used in this protocol as a highly polymorphic marker based on variation in the number of repetitive sequences.
This simple protocol conveys useful information for teachers and the implementation of DNA fingerprinting in practical laboratory classes. In the presented laboratory exercise, DNA extraction followed by PCR amplification is used to determine genetic variation at the D1S80 VNTR locus. Differences in the fragment size of PCR products are visualized by agarose gel electrophoresis. The fragment sizes and repeat numbers are calculated based on a linear regression of the size and migration distance of a DNA size standard.
Following this guide, students should be able to:
&#8226;&#160; Harvest and extract DNA from buccal mucosa epithelial cells 
&#8226;&#160; Perform a PCR experiment and understand the function of various reaction components 
&#8226;&#160; Analyze the amplicons by agarose gel electrophoresis and interpret the results 
&#8226;&#160; Understand the use of VNTRs in DNA fingerprinting and its application in biological sciences

Application of DNA Fingerprinting using the D1S80 Locus in Lab Classes

Here we describe a simple protocol to generate DNA fingerprinting profiles by amplifying the VNTR locus D1S80 from epithelial cell DNA.

使用 D1S80 轨迹在实验室类中应用 DNA 指纹

In biological sciences, DNA fingerprinting has been widely used for paternity testing, forensic applications and phylogenetic studies. Here, we describe a ...

Single-Molecule Dwell-Time Analysis of Restriction Endonuclease-Mediated DNA Cleavage

This novel total internal reflection fluorescence microscopy-based assay facilitates the simultaneous measurement of the length of the catalytic cycle for hundreds of individual restriction endonuclease (REase) molecules in one experiment. This assay does not require protein labeling and can be carried out with a single imaging channel. In addition, the results of multiple individual experiments can be pooled to generate well-populated dwell-time distributions. Analysis of the resulting dwell-time distributions can help elucidate the DNA cleavage mechanism by revealing the presence of kinetic steps that cannot be directly observed. Example data collected using this assay with the well-studied REase, EcoRV - a dimeric Type IIP restriction endonuclease that cleaves the palindromic sequence GAT&#8595;ATC (where &#8595; is the cut site) - are in agreement with prior studies. These results suggest that there are at least three steps in the pathway to DNA cleavage that is initiated by introducing magnesium after EcoRV binds DNA in its absence, with an average rate of 0.17 s-1 for each step.

Using quantum-dot-labeled DNA and total internal reflection fluorescence microscopy, we can investigate the reaction mechanism of restriction endonucleases while using unlabeled protein. This single-molecule technique allows for massively multiplexed observation of individual protein-DNA interactions, and data can be pooled to generate well-populated dwell-time distributions.

限制性核酸内切酶介导的 DNA 切割的单分子停留时间分析

This novel total internal reflection fluorescence microscopy-based assay facilitates the simultaneous measurement of the length of the catalytic cycle for ...

智能手机作为使用 Bradford 分析定量蛋白质的分析设备

RGBradford: Protein Quantitation with a Smartphone Camera

Protein quantitation is an essential procedure in life sciences research. Amongst several other methods, the Bradford assay is one of the most used. Because of its widespread, the limitations and advantages of the Bradford assay have been exhaustively reported, including several modifications of the original method to improve its performance. One of the alterations of the original method is the use of a smartphone camera as an analytical instrument. Taking advantage of the three forms of the Coomassie Brilliant Blue dye that exist in the conditions of the Bradford assay, this paper describes how to accurately quantify protein in samples using color data extracted from a single picture of a microplate. After performing the assay in a microplate, a picture is taken using a smartphone camera, and RGB color data is extracted from the picture using a free and open-source image analysis software application. Then, the ratio of blue to green intensity (in the RGB scale) of samples with unknown concentrations of protein is used to calculate the protein content based on a standard curve. No significant difference is observed between values calculated using RGB color data and those calculated using conventional absorbance data.

作者聚焦:使用智能手机通过 Bradford 分析进行蛋白质定量的另一种方法 

This paper provides a protocol for protein quantification using the Bradford assay and a smartphone as an analytical device. Protein levels in samples can be quantified using color data extracted from a picture of a microplate taken with a smartphone.

RGBradford:使用智能手机相机进行蛋白质定量

Protein quantitation is an essential procedure in life sciences research. Amongst several other methods, the Bradford assay is one of the most used. ...

<meta charset="utf8"></head><body>通过 ssDNA-RPA 束生长观察到的 CMG 介导的 DNA 解旋

Single-Molecule Real-Time Visualization of DNA Unwinding by CMG Helicase

Faithful genome duplication is essential for preserving the genetic stability of dividing cells. DNA replication is carried out during the S phase by a dynamic complex of proteins termed the replisome. At the heart of the replisome is the&#160;CDC45-MCM2-7-GINS (CMG) helicase, which separates the two strands of the DNA double helix such that DNA polymerases can copy each strand. During genome duplication, replisomes must overcome a plethora of obstacles and challenges. Each of these threatens genome stability, as failure to replicate DNA completely and accurately can lead to mutations, diseases, or cell death. Therefore, it is of great interest to understand how CMG functions in the replisome during both normal replication and replication stress. Here, we describe a total internal reflection fluorescence (TIRF) microscopy assay using recombinant purified proteins, which allows for real-time visualization of surface-tethered stretched DNA molecules by individual CMG complexes. This assay provides a powerful platform to investigate CMG behavior at the single-molecule level, allowing helicase dynamics to be directly observed with real-time control over reaction conditions.

<meta charset="utf8"></head><body>作者聚焦:通过单分子可视化揭示真核 DNA 复制的动力学

This protocol demonstrates performing a single-molecule assay for live visualization of DNA unwinding by CMG helicase. It describes (1) preparing a DNA substrate, (2) purifying fluorescently labeled Drosophila melanogaster CMG helicase, (3) preparing a microfluidic flow cell for total internal reflection fluorescence (TIRF) microscopy, and (4) the single-molecule DNA unwinding assay.

通过 CMG 解旋酶对 DNA 解旋进行单分子实时可视化

Faithful genome duplication is essential for preserving the genetic stability of dividing cells. DNA replication is carried out during the S phase by a ...

通过智能手机实时跟踪DNA片段分离

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限制性核酸内切酶介导的 DNA 切割的单分子停留时间分析

RGBradford:使用智能手机相机进行蛋白质定量

RGBradford:使用智能手机相机进行蛋白质定量

RGBradford:使用智能手机相机进行蛋白质定量

通过 CMG 解旋酶对 DNA 解旋进行单分子实时可视化