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

Lo sviluppo del DNA che si estende test è stato aiutato da Paolo Blainey, Lee Jong-Bong, e Samir Hamdan. Questo lavoro è supportato dal National Institutes of Health per borse di Charles Richardson (GM54397), e Antoine van Oijen (GM077248).

1. DNA replica Template DNA per la reazione è un DNA λ linearizzato modificato dalla ricottura di oligonucleotidi per formare una forcella di replica. Inoltre, la biotina è fissata alla fine di un filamento del DNA λ, e un residuo di digossigenina è collegata all&#39;altra estremità del filo stesso 1. Materiali: batteriofago λ DNA, Oligonucleotidi: braccio forcella biotinilato (A: 5&#39;-biotina-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>

E &#39;importante assicurare che la forza di trascinamento esercitata dal flusso laminare perle non influenza le attività enzimatiche delle proteine ​​di replica. Per esempio, un 3 pN forza che corrisponde ad una portata di 0,012 ml / min non influenza la replicazione del filamento di DNA di primo piano. Si pregiudica tuttavia l&#39;attività enzimatiche che avvengono durante la sintesi del DNA coordinato, e deve quindi essere ridotta a 1,5 pN. La forza di trascinamento può essere facilmente controllato modificand...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<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

Descriviamo un metodo per osservare la replicazione in tempo reale di singole molecole di DNA mediate da proteine ​​del sistema di replica batteriofago. Linearizzato λ DNA è modificato in modo da avere un biotina alla fine di un filo, e un residuo di digossigenina sull&#39;altra estremità del filo stesso. La fine biotinilato è collegato a un coprioggetto di vetro funzionalizzati e la fine digoxigeninated ad una piccola biglia. L&#39;assemblaggio di questi DNA-tallone attacchi sulla superficie di una cellula di flusso consente un flusso laminare da applicare a esercitare una forza di resistenza sul tallone. Di conseguenza, il DNA è allungata vicina e parallela alla superficie del vetrino con una forza che è determinata dalla velocità di flusso (Figura 1). La lunghezza del DNA viene misurata attraverso il monitoraggio della posizione del tallone. Differenze di lunghezza tra il DNA a singolo e doppio filamento sono utilizzati per ottenere informazioni in tempo reale l&#39;attività delle proteine ​​replica al bivio. Misurare la posizione del tallone consente precisa determinazione dei tassi e processivities del DNA di svolgimento e di polimerizzazione (Figura 2).

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

Descriviamo un metodo per osservare la replicazione in tempo reale di singole molecole di DNA mediate da proteine ​​del sistema di replica batteriofago.

Osservazione diretta del DNA Enzimi Replica utilizzando un singolo-molecola di DNA Stretching Assay

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

Research

JoVE Journal

Biology

Visualizing Single-molecule DNA Replication with Fluorescence Microscopy

Visualizzazione singola molecola di DNA replica con microscopia a fluorescenza

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

Ricerca

Biologia

DNA Stable-Isotope Probing (DNA-SIP)

DNA Stable-Isotope Probing DNA-SIP

DNA Stabile-isotopica Probing (DNA-SIP)

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

Primer-Free Selezione aptamero utilizzando una libreria di DNA casuale

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

Generazione di DNA / RNA ibrido nel DNA genomico da trasformazione utilizzando RNA contenenti oligonucleotidi

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

Preparazione di DNA-reticolato poliacrilammide idrogel

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

Combinando singola molecola di manipolazione e di imaging per lo studio delle interazioni proteina-DNA

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

In tempo reale Imaging del singolo Engineered RNA trascritti in cellule viventi Utilizzando Ratiometric bimolecolari Beacons

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

Studiare Looping DNA a singola molecola FRET

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

Ridotto gravità ambiente hardware Dimostrazioni di un prototipo miniaturizzato Citofluorimetro e Companion Microfluidic tecnologia di miscelazione

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

A Proteoliposome-Based Efflux Assay per determinare le proprietà singola molecola di Cl<sup&gt; -</sup&gt; Canali e trasportatori

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 ...