Çin Ulusal Doğal Bilim Vakfı (No: 21205078) ve Çin Yüksek Öğrenim Doktora Programı Araştırma Fonu (No: 20123120110002) arasındaki desteği memnuniyetle karşılıyoruz. Bu çalışma, Çin Ulusal Temel Araştırma ve Geliştirme Programı (2016YFB1102303), Çin Ulusal Temel Araştırma Programı (973Program 2015CB352001) ve Çin Ulusal Doğa Bilimleri Vakfı (61378060) tarafından kısmen desteklendi.

1. SGE Chip&#39;in Temel Tasarımı <ol><li> Polimetilmetakrilat (PMMA) veya polikarbonat gibi şeffaf bir plastik kullanın. Not: SGE yongası Şekil 1B&#39;de gösterilmiştir. SGE yongası, TBE tamponu için silindirik delikler, DNA ayrılması için kanallar ve elektrot için delikler boyunca gömülü iki şeritten oluşur. </li><li> Bir lazer gravür makinesi kullanarak PMMA bloğundaki dizileri SGE kanalları oluşturun. Not: Çipin geometrik parametreleri, ayrılması gereken DNA&#39;nın boyutuna bağlıdır. Örneğin, DNA fragmanı 1,000 bp&#39;den küçükse, SGE çipi için optimal kanal parametreleri 2,5 mm x 4,0 mm x 90 mm (genişlik x derinlik x uzunluk) şeklindedir. </li><li> SGE tasarımına dayanarak, DNA örneğini yüklemek için yonga kuyuları oluşturmak için tarakları imal edin. </li></ol> 2. Agaroz Jelinin Hazırlanması <ol><li> 0.5 × TBE&#39;yi 10 × TBE (1 x TBE =89 mM Tris, 89 mM borik asit ve 2 mM EDTA; PH 8.4) ve damıtılmış suyun 1:19 oranında çözünmesi. </li><li> 100 bp&#39;lik DNA merdivenlerinin ayrılması için% 1.0 agaroz jeli hazırlayın. Not: 0.5x TBE tamponundaki agaroz konsantrasyonu, ayrılması gereken DNA fragmanlarının boyutlarına bağlıdır. </li><li> Bir balona 0.1 g agaroz yerleştirin ve 10 mL 0.5x TBE tampon ekleyin. Not: Hazırlanan çözeltinin hacmi şişenin kapasitesinin 1 / 3&#39;ünden daha az olmalıdır. </li><li> Şişeyi koruyucu film ile sızdırın ve ardından agaroz / tampon karışımını bir mikrodalga fırında (orta, 1.0 dakika) ısıtın. Şişeyi mikrodalga fırına koymadan önce, bir patlama durumunda koruyucunun filminde bir miktar delik açın. </li><li> 2.7 mL eritilmiş agaroz çözeltisini 4 kanal SGE çipine boşaltın. Kuyu oluşturmak için tarağı agaroz çözeltisine yerleştirin. Not: Erimiş agaroz çözeltisi, oda sıcaklığında 3 dakika sonra jel haline dönecektir. </li><li> Tarağı taraktan çıkarınJel&#39;i örtmek için 0.9 mL 0.5x TBE tampon ilave edin. </li></ol> 3. Elektroforezin SGE Chip&#39;de Çalıştırılması <ol><li> LED ışık kaynağını açın ve ışık kaynağının üzerinde 425 ila 505 nm bant geçiren filtre yerleştirin. </li><li> Bir vorteks kullanılarak, 14.4 uL&#39;lik 100 bp&#39;lik bir DNA merdiveni ve 1.6 uL SYBR Yeşil&#39;ü karıştırın. </li><li> SGE çipi içindeki agaroz jelinin her oyuğuna 4 μL karışım yükleyin ( Şekil 2 ). </li><li> Elektrodu SGE yongasının iki şeridine koyun. </li><li> SGE yongasının üzerinde 550 ila 700 nm bant geçiren filtre yerleştirin. </li><li> Güç kaynağını açın. Elektrik voltajını 180 V (20 V / cm) olarak ayarlayın. DNA fragmanı ayırma işlemini kaydetmek için akıllı telefonu açın ( Şekil 3 ). </li><li> Elektroforez 10 dakika sonra bittiğinde, güç kaynağını ve LED ışığını kapatın. </li><li> SGE yongasını temizleyin. </li></ol>

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Çıkar çatışması ilan edilmez.

Agaroz jel elektroforezi, DNA, RNA ve proteinin ayrılması için yaygın olarak kullanılmaktadır. Bu çalışma, geleneksel jel elektroforez protokolünün yerini alacak yeni bir yöntem önermektedir. Sonuçlar, 50, 100 ve 1.000 bp&#39;lik DNA merdivenlerinin böyle küçük bir şekilde monte edilmiş bir cihazda iyi ayrılabildiğini göstermektedir. Bu yöntemin en büyük avantajı, nükleik asitlerin az miktarda kimyasal tüketimi ile ayrılabilmesinin yanı sıra ayırma işlemini de kaydedebilmesidir. DNA fra...

<html> Slab jel elektroforezi (SGE), DNA fragman ayırma 1 , 2 , 3 , 4 , 5 için en etkili yöntemdir ve bu nedenle biyokimyasal ve biyolojik analizlerde 6 , 7 , 8 çok yönlü bir araç olarak kabul edilmektedir. Bununla birlikte, birçok deney, SGE&#39;nin aşağıdaki dört problemle sınırlandığını göstermektedir: (1) ayrımlar birçok saat, hatta günler sürmektedir; (2) kimyasal tüketim çok yüksek; (3) karmaşık bir aparat gerektirir ( örn. 2D elektroforez hücresi, elektroforez güç kaynağı ve jel görüntüleme sistemi); (4) jel görüntüleme sistemi, deney sona erdikten sonra yalnızca ayrılmış DNA parçalarını gözlemleyebilir. Ayrıca, SGE 9&#39;da yaygın olarak kullanılan etidyum bromür (EtBr)Ass = &quot;xref&quot;&gt; 10, mutajenik ve kanserojen 11,12 . Bu nedenle, EtBr içeren jelleri verirken eldivenler giyilmelidir. Kapiler elektroforez (CE), otomatik çalışma, kısa ayırma süresi ve daha düşük tüketim gibi SGE&#39;ye kıyasla 13 , 14 , 15 , 16 , 17 birçok avantaja sahiptir. Bununla birlikte, CE cihazı oldukça pahalıdır. Bu nedenle, bu kısıtlamaların üstesinden gelmek için DNA&#39;nın ayrılması için bir sistem geliştirilmiştir ( Şekil 1 ). Böyle bir sistem, kimyasal tüketimini büyük ölüde azaltamaz ve SGE deney süresinden (&lt;8 dak) tasarruf sağlayamaz, aynı zamanda akıllıca agaroz jelinde DNA ayırma işleminin gerçek zamanlı izlenmesini de gerçekleştirebilir. Bu protokolde açıklanan prosedürleri izleyerek, öğrencilerSGE çipini tasarlayıp imal edip, agaroz jeli çipte hazırlayabilir, akıllı bir telefonla basit bir SGE sistemi kurabilir ve DNA migrasyon sürecini agaroz jelinde kaydedebilirim.

<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

Slab jel elektroforezi (SGE), DNA fragmanlarının ayrılması için en yaygın yöntemdir; Bu nedenle, biyoloji ve diğer alanlara genel olarak uygulanır. Bununla birlikte, geleneksel SGE protokolü oldukça sıkıcıdır ve deneyi uzun zaman almaktadır. Üstelik, SGE deneylerindeki kimyasal tüketim çok yüksektir. Bu çalışma, bir SGE çipine dayalı DNA fragmanlarının ayrılması için basit bir yöntem önermektedir. Çip bir gravür makinesi ile yapılır. Optik sinyalin uyarma ve emisyon dalga boyları için iki plastik levha kullanılır. DNA bantlarının floresan sinyali akıllı telefon ile toplanır. Bu yöntemi doğrulamak için 50, 100 ve 1.000 bp&#39;lik DNA merdivenleri ayrılmıştır. Sonuçlar, 5.000 bp&#39;den daha küçük bir DNA merdiveninin 12 dakika içinde çözünebileceğini ve bu yöntemi kullanırken yüksek özünürlükle çözülebildiğini gösteriyor, bu da bunun geleneksel SGE yönteminin ideal bir yerini belirttiğini gösteriyor.

<html> Şekil 4 , Şekil 5 ve Şekil 6 , 50, 100 ve 1.000 bp DNA merdivenlerinin jel elektroforezinden sonra tipik bir sonucu temsil etmektedir. Deneyden sonra, DNA parçaları iyi ayrılmıştı. Ayrıca, aynı numuneler SGE çipinin 4 kanalında ayrıldı ve aynı boyuttaki DNA fragmanlarının her deneyde aynı mesafeyi hareket ettirdiğini gösterdi. DNA merdiveninin ayırma performansı, göç mesaf...

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

Akıllı Telefon ile DNA Parçalarının Ayırılmasının Gerçek Zamanlı İzlenmesi

Geleneksel kütük jel elektroforezi (SGE) deneyleri karmaşık bir aparat ve yüksek kimyasal tüketim gerektirir. Bu çalışma kısa bir zaman aralığında DNA parçalarını ayırmak için düşük maliyetli bir yöntemi açıklayan bir protokol sunmaktadır.

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 Görüntüleme.  University of Shanghai for Science and Technology.  Geleneksel kütük jel elektroforezi (SGE) deneyleri karmaşık bir aparat ve yüksek kimyasal tüketim gerektirir. Bu çalışma kısa bir zaman aralığında DNA parçalarını ayırmak için düşük maliyetli bir yöntemi açıklayan bir protokol sunmaktadır. 

Video: 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.

Jel Hibridizasyonunu Kullanarak Telomer Uzunluğunu Nicellemek İçin Modifiye Terminal Restriction Fragment Analysis

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

Kanser Araştırmaları

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.

Basit, sağlam ve yüksek üretilen iş tek bir molekül akışı germe tahlil uygulama boyunca DNA moleküllerinin taşıma eğitimi için

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

Biyokimya

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 tarafından aracılı DNA Strand Exchange tepki gerçek zamanlı gözlem

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.

Laboratuvar Sınıflarında D1S80 Locus Kullanılarak DNA Parmak İzi Uygulaması

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

Genetik

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.

Restriksiyon endonükleaz aracılı DNA bölünmesinin tek moleküllü bekleme süresi analizi

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

Bradford Tahlili Kullanarak Proteini Ölçmek için Analitik Bir Cihaz Olarak Akıllı Telefon

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.

Yazar Spotlight: Akıllı Telefon Kullanarak Bradford Tahlili ile Protein Miktar Tayinine Alternatif Bir Yaklaşım 

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: Akıllı Telefon Kamerası ile Protein Kantitasyonu

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 Yolu Büyümesi ile Gözlenen CMG Aracılı DNA Çözülmesi

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>Yazar Spotlight: Tek Moleküllü Görselleştirme Yoluyla Ökaryotik DNA Replikasyonunun Dinamiklerini Çözme

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 Helikaz ile DNA Çözmenin Tek Moleküllü Gerçek Zamanlı Görselleştirilmesi

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

Akıllı Telefon ile DNA Parçalarının Ayırılmasının Gerçek Zamanlı İzlenmesi

Özet

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RGBradford: Akıllı Telefon Kamerası ile Protein Kantitasyonu

RGBradford: Akıllı Telefon Kamerası ile Protein Kantitasyonu

RGBradford: Akıllı Telefon Kamerası ile Protein Kantitasyonu

CMG Helikaz ile DNA Çözmenin Tek Moleküllü Gerçek Zamanlı Görselleştirilmesi

CMG Helikaz ile DNA Çözmenin Tek Moleküllü Gerçek Zamanlı Görselleştirilmesi