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

拉伸实验的DNA的发展援助，钟奉李保布莱尼和萨米尔哈姆丹。这项工作是由美国国立卫生赠款查尔斯理查德森（GM54397），安托万车Oijen（GM077248）研究院的支持。

1。 DNA复制模板 DNA的反应是一个线性的λDNA寡核苷酸退火形成一个复制叉的修改。此外，生物素连接的λDNA链的结束，一个地高辛基团连接到同一条 1的另一端。 材料：λ噬菌体的DNA，寡核苷酸：生物素化的叉臂（答：5&#39; -生物素- AAAAAAAAAAAAAAAAGAGTACTGTACGATCTAGCATCAATCACAGGGTCAGGTTCGTTATTGTCCAACTTGCTGTCC - 3&#39;），λ互补叉臂（B：5&#39; - GGGCGGCGACCTGGA...

<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>

重要的是要确保对珠层流曳力不影响酶活性的复制蛋白。举例来说，一个3 PN的力量，对应到0.012毫升/分钟的流速不影响复制的DNA领先链。但它的影响，采取协调DNA合成过程中酶的活性，因此将减少到1.5 PN。拖曳力可以很容易地控制，通过改变流速或流道 5的宽度。 DNA拉伸实验采用双和单链DNA之间的长度差异。在实验中所描述这里的单链DNA双链DNA转换作为一个领先的?...

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		<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

我们描述了一个观察单个DNA分子介导的蛋白质的噬菌体复制系统的实时复制的方法。线性λDNA被修改，有一个链上的生物素，地高辛基团上的同一条电缆的另一端。生物素标记的一端连接到一个官能的玻璃盖玻片和digoxigeninated结束的小珠。一个流细胞表面上这些DNA珠系绳大会允许应用层流珠施加的阻力。因此，被拉伸的DNA靠近和平行的流量（图1）确定力在盖玻片表面。测量长度的DNA监测珠的位置。单双链DNA之间的长度差异，利用获得的实时信息，在交岔路口的复制蛋白的活性。测量珠的位置可以精确测定平仓DNA和聚合（图2）的速率和processivities。

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

我们描述了一个观察单个DNA分子介导的蛋白质的噬菌体复制系统的实时复制的方法。

使用单分子DNA拉伸含量直接观察DNA复制的酶

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

可视化的单分子荧光显微镜下的DNA复制

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

科研

生物学

DNA Stable-Isotope Probing (DNA-SIP)

DNA Stable-Isotope Probing DNA-SIP

DNA的稳定同位素探测（DNA的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

引物的适体的选择，使用一个随机DNA文库

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

改造使用含RNA寡核苷酸基因组DNA的RNA / DNA杂交一代

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交联聚丙烯酰胺水凝胶的制备

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

结合单分子操纵和成像蛋白-DNA相互作用的研究

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

单工程的RNA转录物的活细胞实时成像采用比例式双分子信标

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

研究DNA环接了单分子荧光共振能量转移

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

样机小型化流式细胞仪及配套的混合微流控技术降低重力环境硬件演示

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

一个脂蛋白体为基础的外排检测，以确定氯的单分子性质<sup&gt; -</sup&gt;通道和转运

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 ...