中国国立自然科学財団（No 21205078）と中国高等教育博士号プログラム（No.20123120110002）の支援を感謝します。この研究は、中国の国家主要研究開発計画（2016YFB1102303）、中国の国家基礎研究計画（​​973プログラム2015CB352001）、および中国国立自然科学財団（61378060）によって部分的に支持された。

1. SGEチップの基本設計<ol><li>ポリメタクリル酸メチル（PMMA）やポリカーボネートなどの透明プラスチックを使用してください。 注：SGEチップは図1Bに示されています。 SGEチップは、TBEバッファー用の円筒状の穴と、DNA分離用のチャンネルと、電極用の穴に沿って埋め込まれた2つのレーンとからなる。 </li><li>レーザー彫刻機を使用して、PMMAブロック内のSGEチャネルのアレイを製作します。 注：チップの幾何学的パラメータは、分離されるべきDNAのサイズに依存する。例えば、DNAフラグメントが1,000bpよりも小さい場合、SGEチップの最適なチャネルパラメータは、2.5mm×4.0mm×90mm（幅×奥行き×長さ）である。 </li><li> SGEデザインに基づいて、DNAサンプルをロードするためのチップ内にウェルを作成するために櫛を作製する。 </li></ol> 2.アガロースゲルの調製<ol><li> 10×TBE（1×TBE =89mMのトリス、89mMのホウ酸、および2mMのEDTA; pH 8.4）および蒸留水を1:19の比で含む。 </li><li> 100-bp DNAラダーを分離するために1.0％アガロースゲルを調製する。 注：0.5×TBE緩衝液中のアガロースの濃度は、分離すべきDNA断片のサイズに依存する。 </li><li>フラスコに0.1gのアガロースを入れ、0.5x TBE緩衝液10mLを加える。 備考：調製した溶液の容量は、フラスコの容量の1/3未満でなければならない。 </li><li>フラスコを保存用フィルムでシールし、次いで、アガロース/緩衝液混合物をマイクロ波（培地、1.0分）中で加熱する。フラスコをマイクロ波に入れる前に、爆発の場合には防腐膜に穴をあけてください。 </li><li>融解したアガロース溶液2.7 mLをSGEチップの4チャンネルに注ぎます。櫛をアガロース溶液に入れてウェルを作成します。 注：融解したアガロース溶液は、室温で3分後にゲル状態に冷却される。 </li><li> gから櫛を取り除くゲルを覆うため0.9mLの0.5×TBE緩衝液を添加する。 </li></ol> 3. SGEチップにおける電気泳動の実行<ol><li> LED光源を点灯させ、425〜505 nmのバンドパスフィルターを光源の上に置きます。 </li><li>ボルテックス装置を用いて14.4μLの100 bp DNAラダーと1.6μLのSYBR Greenを混合する。 </li><li> 4μLの混合液をSGEチップのアガロースゲルの各ウェルにロードします（ 図2 ）。 </li><li>電極をSGEチップの2つのレーンに入れます。 </li><li> SGEチップの上に550〜700 nmのバンドパスフィルタを配置します。 </li><li>電源を入れます。電圧は180V（20V / cm）に設定してください。スマートフォンをオンにしてDNA断片分離プロセスを記録します（ 図3 ）。 </li><li> 10分後に電気泳動が終了したら、電源とLEDライトをオフにします。 </li><li> SGEチップを清掃してください。 </li></ol>

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利害の衝突は宣言されていない。

アガロースゲル電気泳動は、DNA、RNAおよびタンパク質の分離に広く用いられている。この研究は、伝統的なゲル電気泳動プロトコルを置き換える新しい方法を提案している。結果は、50,100,1000bpのDNAラダーが、このように小型の組立装置で良好に分離できることを示している。この方法の大きな利点は、化学的消費の少ない核酸を分離できるだけでなく、分離プロセスも記録できることである...

<html>スラブゲル電気泳動（SGE）は、DNA断片分離1,2,3,4,5に最も有効な方法であり、生化学的および生物学的分析における多目的ツールであると考えられている6,7,8。しかし、多くの実験では、SGEが以下の4つの問題によって制限されていることが示されている。（1）分離に数時間および数日かかる。 （2）化学消費量が非常に高い。 （3）複雑な装置（ 例えば、 2D電気泳動セル、電気泳動電源、およびゲルイメージングシステム）を必要とする。 （4）ゲルイメージングシステムは、実験が終了したときにのみ、分離されたDNA断片を観察することができる。さらに、SGE9で一般的に使用されている臭化エチジウム（EtBr）は、突然変異原性であり、発癌性である11,12 。従って、EtBrを含むゲルを手渡すときは常に手袋を着用しなければならない。 キャピラリー電気泳動（CE）には、自動操作、短時間の分離、低消費など、SGEと比較して13,14,15,16,17の利点があります。しかし、CE機器は非常に高価です。従って、これらの制限を克服するために、DNAの分離のためのシステムが開発されている（ 図1 ）。このようなシステムは、化学物質の消費を大幅に削減し、SGE実験時間（8分未満）を節約するだけでなく、スマートフォンによるアガロースゲル中のDNA分離プロセスのリアルタイム追跡も行うことができる。このプロトコルに記載されている手順に従って、SGEチップの設計と製作、チップ内でのアガロースゲルの調製、スマートフォンによるシンプルなSGEシステムのセットアップ、アガロースゲルへのDNAマイグレーションプロセスの記録が可能です。

<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

スラブゲル電気泳動（SGE）は、DNAフラグメントの分離のための最も一般的な方法である;生物学などの分野に広く応用されている。しかし、従来のSGEプロトコルは非常に面倒であり、実験には長い時間がかかります。さらに、SGE実験における化学消費は非常に高い。この研究は、SGEチップに基づくDNA断片の分離のための簡単な方法を提案する。チップは彫刻機で作られています。 2つのプラスチックシートが、光信号の励起波長および発光波長に対して使用される。 DNAバンドの蛍光シグナルはスマートフォンで収集されます。この方法を検証するために、50,100および1,000bpのDNAラダーを分離した。この結果は、この方法を使用する場合、従来のSGE法の理想的な代替物であることを示しており、5,000bpより小さいDNAラダーが12分以内に高分解能で分離できることを示している。

<html> 図4 、 図5および図6は、 50,100および1,000bpのDNAラダーのゲル電気泳動後の典型的な結果を表す。実験後、DNA断片は十分に分離されていた。さらに、同じサンプルをSGEチップの4チャンネルで分離したところ、同じサイズのDNA断片が各実験において同じ距離を移動することが示された。 DNAラダーの分離?...

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

スマートフォンによるDNA断片分離のリアルタイム追跡

伝統的なスラブゲル電気泳動（SGE）実験は、複雑な装置および高い化学消費を必要とする。この研究は、短い時間枠内でDNA断片を分離する低コストの方法を記載したプロトコールを提示する。

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

Research

JoVE Journal

Biochemistry

14.5K ビュー.  University of Shanghai for Science and Technology. 伝統的なスラブゲル電気泳動（SGE）実験は、複雑な装置および高い化学消費を必要とする。この研究は、短い時間枠内でDNA断片を分離する低コストの方法を記載したプロトコールを提示する。 

ビデオ: Real-time Tracking of DNA Fragment Separation by Smartphone

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.

In-gelハイブリダイゼーションを用いたテロメア長の定量化のための末端制限制限断片の解析

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

がん研究

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.

シンプルで堅牢、かつ高スループットの単一分子 DNA 分子の輸送を研究するためのストレッチ法の実装を流れ

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 を介した DNA 鎖交換反応のリアルタイム観察

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.

D1S80ローカスを用いたDNAフィンガープリントのラボクラスへの応用

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

遺伝学

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.

制限エンドヌクレアーゼ媒介性DNA切断の1分子滞留時間解析

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

Bradford Assayを使用してタンパク質を定量する分析デバイスとしてのスマートフォン

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.

著者スポットライト:スマートフォンを使用したBradford Assayによるタンパク質定量への代替アプローチ(英語) 

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:スマートフォンカメラによるタンパク質定量(英語)

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>ssDNA-RPAトラクト成長によるCMG媒介DNA巻き戻しの観察

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>著者スポットライト:真核生物のDNA複製のダイナミクスを解明する:単一分子の可視化による

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.

CMGヘリカーゼによるDNA巻き戻しの1分子リアルタイム可視化

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

スマートフォンによるDNA断片分離のリアルタイム追跡

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RGBradford:スマートフォンカメラによるタンパク質定量(英語)

RGBradford:スマートフォンカメラによるタンパク質定量(英語)

RGBradford:スマートフォンカメラによるタンパク質定量(英語)

CMGヘリカーゼによるDNA巻き戻しの1分子リアルタイム可視化

CMGヘリカーゼによるDNA巻き戻しの1分子リアルタイム可視化