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

Agradecemos o apoio da Fundação Nacional de Ciências Naturais da China (nº 21205078) e do Fundo de Pesquisa para o Programa de Doutorado em Educação Superior da China (No.20123120110002). Este trabalho foi parcialmente apoiado pelo Programa Nacional de Pesquisa e Desenvolvimento Chave da China (2016YFB1102303), o Programa Nacional de Pesquisa Básica da China (973Program, 2015CB352001) e a Fundação Nacional de Ciências Naturais da China (61378060).

1. Projeto básico do chip SGE <ol><li> Use qualquer plástico transparente, como polimetilmetacrilato (PMMA) ou policarbonato. Nota: O chip SGE está demonstrado na Figura 1B . O chip SGE consiste em furos cilíndricos para o tampão TBE, canais para separação de DNA e duas pistas incorporadas ao longo dos orifícios para o eletrodo. </li><li> Fabricação de matrizes de canais SGE no bloco PMMA usando uma máquina de gravura a laser. Nota: Os parâmetros geométricos do chip dependem do tamanho do DNA a ser separado. Por exemplo, os parâmetros de canal ótimos para o chip SGE são de 2,5 mm x 4,0 mm x 90 mm (largura x profundidade x comprimento) se o fragmento de DNA for menor que 1.000 pb. </li><li> Com base no design SGE, fabrica pentes para criar os poços no chip para carregar a amostra de DNA. </li></ol> 2. Preparação do Gel de Agarose <ol><li> Prepare 0,5 × TBE misturando 10 × TBE (1 × TBE =Tris 89 mM, ácido bórico 89 mM e EDTA 2 mM; PH 8,4) e água destilada numa proporção 1:19. </li><li> Preparar 1,0% de gel de agarose para a separação de escadas de DNA de 100 pb. Nota: A concentração de agarose em tampão TBE 0,5x depende dos tamanhos dos fragmentos de DNA a serem separados. </li><li> Colocar 0,1 g de agarose num balão e adicionar 10 mL de tampão TBE 0,5x. Nota: O volume da solução preparada deve ser inferior a 1/3 da capacidade do balão. </li><li> Selar o balão com película conservante e depois aquecer a mistura de agarose / tampão em um microondas (meio, 1,0 min). Antes de colocar o balão no microondas, faça alguns orifícios no filme de conservação em caso de explosão. </li><li> Despeje 2,7 mL de solução de agarose derretida em 4 canais do chip SGE. Coloque o pente na solução de agarose para criar os poços. Nota: A solução de agarose derretida arrefecerá para o estado de gel à temperatura ambiente 3 minutos depois. </li><li> Remova o pente do gE adicionar 0,9 mL de tampão TBE 0,5x para cobrir o gel. </li></ol> 3. Correndo a eletroforese no chip SGE <ol><li> Ligue a fonte de luz LED e coloque um filtro de passagem de banda de 425 a 505 nm acima da fonte de luz. </li><li> Misture 14,4 μL de uma escala de DNA de 100 pb e 1,6 μL de SYBR Green usando um vortexer. </li><li> Carregar 4 μL de mistura em cada poço do gel de agarose no chip SGE ( Figura 2 ). </li><li> Coloque o eletrodo nas duas faixas do chip SGE. </li><li> Coloque um filtro de passagem de banda de 550 a 700 nm acima do chip SGE. </li><li> Ligue a fonte de alimentação. Ajuste a tensão elétrica para 180 V (20 V / cm). Ligue o smartphone para gravar o processo de separação de fragmentos de DNA ( Figura 3 ). </li><li> Quando a eletroforese terminar 10 minutos depois, desligue a fonte de energia e a luz LED. </li><li> Limpe o chip SGE. </li></ol>

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Não são declarados conflitos de interesse.

A eletroforese em gel de agarose é amplamente empregada para a separação de DNA, ARN e proteína. Este trabalho propõe um novo método para substituir o protocolo tradicional de eletroforese em gel. Os resultados demonstram que as escadas de DNA de 50, 100 e 1000 pb podem ser separadas bem em um dispositivo tão pequeno montado. A grande vantagem deste método é que não só pode separar os ácidos nucleicos com pouco consumo químico, mas também pode registrar o processo de separação. Embora os fragmentos de DN...

<html> A eletroforese em gel de laje (SGE) é o método mais eficaz para a separação dos fragmentos de DNA 1 , 2 , 3 , 4 , 5 e, portanto, é considerada uma ferramenta versátil nas análises bioquímicas e biológicas 6 , 7 , 8 . No entanto, muitas experiências indicam que o SGE é restrito pelos seguintes quatro problemas: (1) as separações demoram muitas horas e até dias; (2) o consumo químico é muito alto; (3) requer um aparelho complicado ( por exemplo, célula de eletroforese 2D, fonte de energia de eletroforese e sistema de imagem em gel); (4) o sistema de imagem em gel só pode observar os fragmentos de DNA separados quando a experiência terminar. Além disso, o brometo de etídio (EtBr), que é comumente usado no SGE 9 ,Ass = &quot;xref&quot;&gt; 10, é mutagênico e cancerígeno 11 , 12 . Assim, sempre devem ser usadas luvas ao entregar géis contendo EtBr. A eletroforese capilar (CE) tem inúmeras vantagens 13 , 14 , 15 , 16 , 17 em comparação com SGE, como operação automática, tempo de separação curto e menor consumo. No entanto, o instrumento CE é bastante caro. Portanto, para superar essas limitações, um sistema foi desenvolvido ( Figura 1 ) para a separação do DNA. Tal sistema não só pode reduzir muito o consumo de produtos químicos e economizar no tempo de experiência de SGE (&lt;8 min), mas também pode realizar rastreamento em tempo real do processo de separação de DNA no gel de agarose pelo smartphone. Seguindo os procedimentos descritos neste protocolo, os estudantes shouPoderia projetar e fabricar o chip SGE, preparar o gel de agarose no chip, configurar um sistema SGE simples com um smartphone e registrar o processo de migração do DNA no gel de agarose.

<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

A eletroforese em gel de laje (SGE) é o método mais comum para a separação de fragmentos de DNA; Portanto, é amplamente aplicado ao campo da biologia e outros. No entanto, o protocolo SGE tradicional é bastante tedioso, e o experimento leva muito tempo. Além disso, o consumo químico em experimentos SGE é muito alto. Este trabalho propõe um método simples para a separação de fragmentos de DNA com base em um chip SGE. O chip é feito por uma máquina de gravura. São utilizadas duas folhas de plástico para os comprimentos de onda de excitação e emissão do sinal óptico. O sinal de fluorescência das bandas de DNA é coletado por smartphone. Para validar este método, as escadas de DNA de 50, 100 e 1000 pb foram separadas. Os resultados demonstram que uma escala de DNA menor que 5.000 pb pode ser resolvida dentro de 12 min e com alta resolução ao usar este método, indicando que é um substituto ideal para o método SGE tradicional.

<html> A Figura 4 , Figura 5 , e a Figura 6 representam um resultado típico após a eletroforese em gel de 50, 100 e 1.000 pb de escadas de DNA. Após o experimento, os fragmentos de DNA estavam bem separados. Além disso, as mesmas amostras foram separadas nos 4 canais do chip SGE, mostrando que fragmentos de DNA do mesmo tamanho movem a mesma distância em cada experimento. O desempenho de separação...

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

Rastreamento em tempo real da separação de fragmentos de DNA por smartphone

As experiências tradicionais de eletroforese em gel de laje (SGE) requerem um aparelho complicado e alto consumo químico. Este trabalho apresenta um protocolo que descreve um método de baixo custo para separar fragmentos de DNA dentro de um prazo curto.

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

14.4K visualizações.  University of Shanghai for Science and Technology.  As experiências tradicionais de eletroforese em gel de laje (SGE) requerem um aparelho complicado e alto consumo químico. Este trabalho apresenta um protocolo que descreve um método de baixo custo para separar fragmentos de DNA dentro de um prazo curto. 

Vídeo: 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.

Detecção de Copy Number Alterações Usando Sequencing única célula

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

Genética

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.

Análise de fragmentos de restrição de terminal modificada para quantificar o comprimento de Telomere usando a hibridação em gel

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

Pesquisa do câncer

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.

Uma molécula simples, robusto e alta taxa de transferência de fluxo alongamento do ensaio implementação para estudar o transporte de moléculas ao longo de 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, ...

Bioquímica

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.

Observação em tempo real da reação de troca de fita de DNA mediada por Rad51

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.

Aplicação de impressão digital de DNA usando o Lócus D1S80 em classes de laboratório

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.

Análise de tempo de permanência de molécula única de clivagem de DNA mediada por endonuclease de restrição

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

Smartphone como um dispositivo analítico para quantificar proteínas usando o ensaio de Bradford

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.

Autor em destaque: uma abordagem alternativa para a quantificação de proteínas pelo ensaio de Bradford usando um smartphone 

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: Quantificação de proteínas com uma câmera de smartphone

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>Desenrolamento de DNA mediado por CMG observado pelo crescimento do trato ssDNA-RPA

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>Autor em destaque: Desvendando a dinâmica da replicação do DNA eucariótico por meio da visualização de uma única molécula

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.

Visualização em tempo real de molécula única do desenrolamento do DNA por 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 ...

Rastreamento em tempo real da separação de fragmentos de DNA por smartphone

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RGBradford: Quantificação de proteínas com uma câmera de smartphone

Visualização em tempo real de molécula única do desenrolamento do DNA por CMG helicase