Name: direct observation of enzymes replicating dna using a single-molecule dna stretching assay
Uploaded: 2010-03-23T17:30:00
Description: Watch this Scientific Journal Video about Direct Observation of Enzymes Replicating DNA Using a Single-molecule DNA Stretching Assay at JoVE.com

Paul Blainey, Jong-Bong Lee ve Samir Hamdan uzanan DNA testinin geliştirilmesi yardımıyla oldu. Bu çalışma, Charles Richardson (GM54397) ve Antoine van Oijen (GM077248) Sağlık hibe Ulusal Sağlık Enstitüleri tarafından desteklenmektedir.

1. DNA replikasyonu Şablonu Reaksiyon için DNA replikasyon çatal oluşturmak için oligonükleotidler tavlama tarafından değiştirilmiş Lineerleştirilmiş λ DNA. Ayrıca, biotin λ DNA iplikçik sonuna kadar bağlı olduğu ve bir digoxigenin benzer parçaları aynı iplikçik 1 diğer ucuna takılı. Malzemeler: bakteriyofaj λ DNA Oligonükleotidler: biotinlenmiş çatal kol (A: 5&#39;-biotin-AAAAAAAAAAAAAAAAGAGTACTGTACGATCTAGC...

<ol>
	<li>Lee, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+primase+acts+as+a+molecular+brake+in+DNA+replication.">DNA primase acts as a molecular brake in DNA replication.</a> Nature. 439, 621-624 (2006).</li><li>Tanner, N. A., van Oijen, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-molecule+observation+of+prokaryotic+DNA+replication.">Single-molecule observation of prokaryotic DNA replication.</a> Methods Mol Biol. 521, 397-410 (2009).</li><li>Sofia, S. J., Premnath, V. V., Merrill, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Poly(ethylene+oxide)+Grafted+to+Silicon+Surfaces:+Grafting+Density+and+Protein+Adsorption.">Poly(ethylene oxide) Grafted to Silicon Surfaces: Grafting Density and Protein Adsorption.</a> Macromolecules. 31, 5059-5070 (1998).</li><li>Tanner, N. A., Loparo, J. J., van Oijen, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+single-molecule+DNA+replication+with+fluorescence+microscopy.">Visualizing single-molecule DNA replication with fluorescence microscopy.</a> J Vis Exp. , (2009).</li><li>Hamdan, S. M., Loparo, J. J., Takahashi, M., Richardson, C. C., van Oijen, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+DNA+replication+loops+reveal+temporal+control+of+lagging-strand+synthesis.">Dynamics of DNA replication loops reveal temporal control of lagging-strand synthesis.</a> Nature. 457, 336-339 (2009).</li><li>van Oijen, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-molecule+kinetics+of+lambda+exonuclease+reveal+base+dependence+and+dynamic+disorder.">Single-molecule kinetics of lambda exonuclease reveal base dependence and dynamic disorder.</a> Science. 301, 1235-1238 (2003).</li></ol>

Laminer akış boncuklar üzerinde uyguladığı sürtünme kuvveti çoğaltma proteinler enzimatik faaliyetlerin etkisi olmadığından emin olmak için çok önemlidir. Örneğin, 0.012 ml / dk akış oranına karşılık gelen 3 pN gücü önde gelen iplikçik DNA replikasyon etkilemez. Ancak enzimatik faaliyetleri koordine DNA sentezi sırasında yer alan etkilemez ve bu nedenle 1.5 pN azaltılmalıdır. Sürtünme kuvveti kolayca akış hızını veya akış kanalı 5 genişliği değiştirerek kontrol e...

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		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>Bacteriophage &lambda; DNA</td>
<td>&nbsp;</td>
<td>New England Biolabs</td>
<td>N3011L</td>
<td>&nbsp;</td>
<tr>
<td>DNA Oligonucleotides</td>
<td>&nbsp;</td>
<td>Integrated DNA Technologies</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>T4 DNA Ligase</td>
<td>&nbsp;</td>
<td>New England Biolabs</td>
<td>M0202L</td>
<td>&nbsp;</td>
<tr>
<td>T4 Polynucleotide Kinase</td>
<td>&nbsp;</td>
<td>New England Biolabs</td>
<td>M0201L</td>
<td>&nbsp;</td>
<tr>
<td>&alpha;-digoxigenin Fab</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>11214667001</td>
<td>&nbsp;</td>
<tr>
<td>Tosyl Activated Beads</td>
<td>&nbsp;</td>
<td>Dynal/Invitrogen</td>
<td>142-03</td>
<td>&nbsp;</td>
<tr>
<td>Magnetic Separator</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>Dynal MPC</td>
<td>&nbsp;</td>
<tr>
<td>3-aminopropyl-triethoxysilane</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A3648</td>
<td>Other aminosilanes can be used or mixed with non-amine reactive silanes for sparser surfaces</td>
</tr>
<tr>
<td>Succinimidyl propionate PEG</td>
<td>&nbsp;</td>
<td>Nektar</td>
<td>&nbsp;</td>
<td>Similar PEGs can be purchased from Nanocs, CreativePEGWorks, etc.</td>
</tr>
<tr>
<td>Biotin-PEG-NHS</td>
<td>&nbsp;</td>
<td>Nektar</td>
<td>&nbsp;</td>
<td>Similar PEGs can be purchased from Nanocs, CreativePEGWorks, etc.</td>
</tr>
<tr>
<td>Double-sided tape</td>
<td>&nbsp;</td>
<td>Grace BioLabs</td>
<td>SA-S-1L</td>
<td>100 &mu;m thickness</td>
</tr>
<tr>
<td>Quartz slide</td>
<td>&nbsp;</td>
<td>Technical Glass</td>
<td>20 mm (W)x 50 mm (L)x 1mm (H)</td>
<td>Size to fit on coverslips. Drill holes with diamond-tip drill bits (DiamondBurs.net)</td>
</tr>
<tr>
<td>Polyethylene tubing</td>
<td>&nbsp;</td>
<td>Becton Dickinson</td>
<td>427416</td>
<td>0.76 mm ID, 1.22 OD 
Other size tubing can be substituted.</td>
</tr>
<tr>
<td>Streptavidin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>S4762</td>
<td>Make 1 mg/mL solution, 25 &mu;L aliquots in PBS pH 7.3</td>
</tr>
<tr>
<td>Deoxyribonucleotide triphosphate solution mix</td>
<td>&nbsp;</td>
<td>New England Biolabs</td>
<td>N0447</td>
<td>&nbsp;</td>
<tr>
<td>Inverted Optical Microscope with 10X Objective</td>
<td>&nbsp;</td>
<td>Olympus</td>
<td>Olympus IX-51</td>
<td>&nbsp;</td>
<tr>
<td>Permament rare-earth magnet</td>
<td>&nbsp;</td>
<td>National Imports</td>
<td>www.rare-earth-magnets.com</td>
<td>&nbsp;</td>
<tr>
<td>CCD Camera</td>
<td>&nbsp;</td>
<td>QImaging</td>
<td>Rolera-XR Fast 139</td>
<td>&nbsp;</td>
<tr>
<td>Syringe Pump</td>
<td>&nbsp;</td>
<td>Harvard Apparatus</td>
<td>11 Plus</td>
<td>Operate in refill mode to facilitate solution changes</td>
</tr>
<tr>
<td>Fiber Illuminator</td>
<td>&nbsp;</td>
<td>Thorlabs Inc.</td>
<td>OSL1</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

direct observation of enzymes replicating dna using a single-molecule dna stretching assay

Biz bireysel DNA molekülleri bakteriyofaj çoğaltma sistemi proteinleri aracılığı ile gerçek zamanlı çoğaltma gözlemlemek için bir yöntem açıklanmaktadır. Lineerleştirilmiş λ DNA bir iplikçik sonunda bir biotin, ve aynı iplikli diğer ucunu bir digoxigenin benzer parçaları değiştirilir. Biotinlenmiş sonuna Fonksiyonlu cam lamel ve küçük bir boncuk digoxigeninated sonuna eklenir. Bir akış hücre yüzeyinde bu DNA boncuk hayvan iplerini montaj, laminer akış, boncuk üzerinde sürükleyin kuvvet uygularlar uygulanmasına olanak veriyor. Sonuç olarak, DNA ve paralel akış hızı (Şekil 1) tarafından belirlenen bir kuvvet lamel yüzeye yakın gerilir. DNA uzunluğu boncuk pozisyonunun izlenmesi ile ölçülür. Tek ve çift iplikli DNA arasındaki uzunluk farklılıkları çatal çoğaltma proteinlerin aktivitesini gerçek zamanlı bilgi elde etmek için kullanılmaktadır. Boncuk pozisyon ölçme gevşemek DNA ve polimerizasyon (Şekil 2) oranları ve processivities hassas belirliyor.

Watch this Scientific Journal Video about Direct Observation of Enzymes Replicating DNA Using a Single-molecule DNA Stretching Assay at JoVE.com

Direct Observation of Enzymes Replicating DNA Using a Single-molecule DNA Stretching Assay

Biz bireysel DNA molekülleri bakteriyofaj çoğaltma sistemi proteinleri aracılığı ile gerçek zamanlı çoğaltma gözlemlemek için bir yöntem açıklanmaktadır.

Enzimlerin Klonlanması DNA Doğrudan Gözlem Tek bir molekülü DNA Testi Esneme

We describe a method for observing real time replication of individual DNA molecules mediated by proteins of the bacteriophage replication system. ...

Research

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Biology

Visualizing Single-molecule DNA Replication with Fluorescence Microscopy

Floresan Mikroskop ile görselleştirme Tek molekül DNA replikasyonu

We describe a simple fluorescence microscopy-based real-time method for observing DNA replication at the single-molecule level. A circular, forked DNA ...

DNA Stable-Isotope Probing (DNA-SIP)

DNA Stable-Isotope Probing DNA-SIP

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DNA stable-isotope probing (DNA-SIP) is a powerful technique for identifying active microorganisms that assimilate particular carbon substrates and ...

Primer-Free Aptamer Selection Using A Random DNA Library

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Aptamers are highly structured oligonucleotides (DNA or RNA) that can bind to targets with affinities comparable to antibodies 1. They are identified ...

Generation of RNA/DNA Hybrids in Genomic DNA by Transformation using RNA-containing Oligonucleotides

RNA içeren Oligonükleotidler kullanarak Dönüşüm Genomik DNA, RNA / DNA Hibritlerin Üretimi

Synthetic short nucleic acid polymers, oligonucleotides (oligos), are the most functional and widespread tools of molecular biology. Oligos can be ...

Preparation of DNA-crosslinked Polyacrylamide Hydrogels

DNA çapraz bağlı poliakrilamid hidrojellerinin hazırlanışı

Mechanobiology is an emerging scientific area that addresses the critical role of physical cues in directing cell morphology and function. For example, ...

Combining Single-molecule Manipulation and Imaging for the Study of Protein-DNA Interactions

Protein-DNA Etkileşiminin Çalışmaları Tek molekül Manipülasyon ve Görüntüleme birleştiren

The paper describes the combination of optical tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method ...

Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons

Oranlı metrik Bimoleküler Fenerleri kullanma Yaşam Hücrelerde Tek Engineered RNA Transkriptlerinin Gerçek zamanlı Görüntüleme

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the ...

Studying DNA Looping by Single-Molecule FRET

Tek Molekül FRET DNA döngü oluşturma incelenmesi

Bending of double-stranded DNA (dsDNA) is associated with many important biological processes such as DNA-protein recognition and DNA packaging into ...

Reduced-gravity Environment Hardware Demonstrations of a Prototype Miniaturized Flow Cytometer and Companion Microfluidic Mixing Technology

Prototip Minyatür Akış Sitometresi ve Companion mikroakışkan Karıştırma Teknoloji İndirimli-gravite Çevre Donanım Gösterileri

Until recently, astronaut blood samples were collected in-flight, transported to earth on the Space Shuttle, and analyzed in terrestrial laboratories. If ...

A Proteoliposome-Based Efflux Assay to Determine Single-molecule Properties of Cl- Channels and Transporters

A Proteoliposome-Based Efflux Assay to Determine Single-molecule Properties of Cl- Channels and Transporters

Bir Proteoliposome Tabanlı effluks Deney Cl Tek-molekül Properties belirleme<sup&gt; -</sup&gt; Kanallar ve Nakliyeciler

The last 15 years have been characterized by an explosion in the ability to overexpress and purify membrane proteins from prokaryotic organisms as well as ...