Name: stretching short sequences of dna with constant force axial optical tweezers
Uploaded: 2011-10-13T13:00:00
Description: Watch this Scientific Journal Video about Stretching Short Sequences of DNA with Constant Force Axial Optical Tweezers at JoVE.com

我们感谢陈懿范医生帮助的轴向光镊和他的拉伸数据，这份手稿的贡献。这项工作是由美国国家科学基金会授予的PHY-0957293，FOCUS授予PHY-0114336主办。

1。镊子设置<ol><li>从1064 nm的激光的光束被分成两个正交的偏振光束。一个用来操纵所述生物分子，而另一种是用于校准的目的（参见图2）。 </li><li>操纵光束的强度的控制由一个声光偏转器（AOD），而每束光的位置和焦点被独立地控制由光束转向反射镜和光学望远镜，分别。 </li><li>的光束，然后再结合另一偏振分束器和最后一组的伸缩光学稍微过量填充背面的显微镜物镜的孔径进一步空调。用...

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传统的光学镊子依赖于模拟或电脑控制的反馈上的折光性对象施加一个恒定的力。这些有源反馈系统有困难的其中的突然变化在延长的试样发生的条件下进行，例如，从结合的蛋白质的DNA或一个分子马达沿长丝的快速步进。最近已开发用于施加恒定力的各种无源方法。涉及一种这样的方法，用来解决碱基对组成的RNA聚合酶的步进分辨率的光学势高斯激光束12的线性区域内工作。我们已经适...

<table border="1"><tbody><tr><th>试剂/设备</th><th>公司</th><th>目录号</th><th>评论</th></tr><tr><td> Nd：YVO4激光器</td><td>光谱物理</td><td> T40-Z-106C </td><td></td></tr><tr><td>声光 导流板</td><td> IntraAction </td><td> DTD-274HA6 </td><td></td></tr><tr><td>显微物镜</td><td>奥林巴斯</td><td> PlanApo </td><td> 60X，NA 1.4 </td></tr><tr><td>压电阶段</td><td>疯狂的城市实验室</td><td>纳米LP100 </td><td> XYZ阶段</td></tr><tr><td> CCD相机</td><td> PixeLink </td><td> PL-A741 </td><td></td></tr><tr><td>光电探测器</td><td>光电 技术</td><td> ET-3020 </td><td></td></tr><tr><td>聚苯乙烯珠</td><td> Spherotech </td><td> SVP-08-10 </td><td> 800纳米，链霉亲和素 涂</td></tr><tr><td>抗地高辛</td><td>罗氏公司</TD&gt; <td> 11333089001 </td><td>从羊</td></tr><tr><td>引物</td><td> MWG操纵子</td><td>自定义的寡核苷酸</td><td>一个引物：生物素 其他：地高辛</td></tr><tr><td> PCR试剂</td><td>新英格兰 生物实验室</td><td> TAQ聚合酶， 的dNTPs </td><td></td></tr><tr><td>盖玻片</td><td> Fisher Scientific则</td><td></td><td></td></tr><tr><td>其他化学品 缓冲</td><td> Fisher Scientific则</td><td></td><td></td></tr></tbody></table>补充材料 A.液力摩擦系数用于确定的微球的水动力的摩擦系数的表面附近，1可以使用下面的膨胀5,10： <img src="/files/ftp_upload/3405/3405eq1.jpg" alt="式（1）" />以下简写Ĥ作为介绍： <img src="/files/ftp_upload/3405/3405eq2.jpg" alt="公式2" />的摩擦系数的定义中的流体的粘度η的微球的半径，与微球的中心位于距离η的表面之上。的总和收敛合理扩大到约10条款。 B.刚度标定的轴向位置的影响陷阱刚度涉及校准校准的精度，从而增加与距表面的距离的增加，和实际的轴向位置的陷阱实验使用之间的权衡。在一般情况下，陷阱被校准，从高于实际的实验条件下的表面，这是在约800-1000纳米。 C.改进的蠕虫状链（WLC）模型力拓曲线可以适应修改后的WLC米ODEL占体积排阻效应在零光力如下： <img src="/files/ftp_upload/3405/3405eq3.jpg" alt="式（3）" /> 其中 ，F opt是光学的力中，x o是一个配合的参数的零力延伸中，x opt是下的延伸力，l是长度的DNA的轮廓，和升* p是第二个适合的“有效的参数为“持效期长。f WLC是通常WLC模型11 <img src="/files/ftp_upload/3405/3405eq4.jpg" alt="式（4）" /> 其中ε是相对的DNA延伸。

stretching short sequences of dna with constant force axial optical tweezers

拉伸DNA的轮廓长度小于一个碱基的单分子技术是充满了实验的困难。然而，许多有趣的生物事件，如组蛋白结合的DNA 1,2和蛋白介导的循环，出现该长度的规模。近年来，DNA的机械性能已被证明是在基本的细胞过程中起到显着的作用类似的包装成紧凑的核小体的DNA和染色质的纤维3,4。显然，这是重要，了解短的DNA片段的力学性能。在本文中，我们提供了一个实用指南如何运用眼线改变单分子光学技术，我们已经开发研究的力学行为与轮廓长度短到几百个碱基对的DNA。 伸展短的DNA片段的主要障碍是，传统的光学镊子的设计，一般使用武力中的directioŇ横向的阶段5,6，（参见图1）。在这种几何形状，胎圈和盖玻片之间的角度，其中DNA是拴，变得很陡的亚微米长度的DNA。必须现在可以占的轴向位置，这可以是一个挑战，并且，由于扩展的微球拖动接近盖玻片，空间位阻效应增强。此外，作为微球的不对称的结果，横向扩展，会产生不同水平的转矩由于由于在延长的方向上的反作用力的变化的光陷阱内的微球的旋转。 拉伸亚微米DNA的替代方法，对自己的独特的障碍。比如，双束光阱是有限的拉伸周围的波长，在该DNA的两个陷阱的点之间的干涉效应和从光散射之间的微球体开始构成了显着的问题，。更换一个陷阱用微量最有可​​能遭受类似的挑战。虽然人们可以直接使用的轴向电位伸展DNA，有源反馈方案将需要施加一个恒定的力的带宽，这将是相当有限的，尤其是在低力量。 我们规避这些基本问题直接拉的DNA，从盖玻片上使用恒定的力的轴向光镊7,8。这是通过捕集的胎圈在光学势的线性区域，其中的光学力是恒定的强度可以通过调整激光功率调谐。俘获的线性区域内还用作所有的光学近350nm的在轴向方向上延伸的DNA上的力夹紧。我们同时补偿通过微调整的位置的阶段，这样的热性能和机械漂移盖玻片粘贴至一个参考微球保持在相同的位置和焦点，一个llowing一个几乎是无限的观察期。

Watch this Scientific Journal Video about Stretching Short Sequences of DNA with Constant Force Axial Optical Tweezers at JoVE.com

Stretching Short Sequences of DNA with Constant Force Axial Optical Tweezers

我们说明了使用一个恒定的力的轴向光镊探索短的DNA分子的力学性能。轴向拉伸DNA，我们尽量减少在传统的横向操作所产生的立体障碍和文物，使我们能够研究DNA分子的短〜100 nm之间。

伸展的DNA短序列恒力轴向光镊

Single-molecule techniques for stretching DNA of contour lengths  less than a kilobase are fraught with experimental difficulties. However, many ...

Research

JoVE Journal

Bioengineering

Visualization of Recombinant DNA and Protein Complexes Using Atomic Force Microscopy

重组DNA和蛋白质复合物，用原子力显微镜的可视化

Atomic force microscopy (AFM) allows for the visualizing of individual proteins, DNA molecules, protein-protein complexes, and DNA-protein complexes. On ...

科研

生物工程

Multiplexed Single-molecule Force Proteolysis Measurements Using Magnetic Tweezers

警队复用单分子蛋白水解测量磁性镊子

The  generation and detection of mechanical forces is a ubiquitous aspect of cell physiology,  with direct relevance to cancer metastasis1, atherogenesis2 ...

Constant Pressure-controlled Extrusion Method for the Preparation of Nano-sized Lipid Vesicles

恒压控制的挤压方法制备纳米脂质体

Liposomes are artificially prepared vesicles consisting of natural and synthetic phospholipids that are widely used as a cell membrane mimicking platform ...

Quantifying the Mechanical Properties of the Endothelial Glycocalyx with Atomic Force Microscopy

量化的内皮细胞糖复合物的力学性能，用原子力显微镜

Our  understanding of the interaction of leukocytes and the vessel wall during  leukocyte capture is limited by an incomplete understanding of the ...

Investigating Receptor-ligand Systems of the Cellulosome with AFM-based Single-molecule Force Spectroscopy

调查纤维素酶与基于AFM的单分子力谱受体 - 配体系统

Cellulosomes are discrete multienzyme complexes used by a subset of anaerobic bacteria and fungi to digest lignocellulosic substrates. Assembly of the ...

Quantitative Optical Microscopy: Measurement of Cellular Biophysical Features with a Standard Optical Microscope

定量的光学显微镜：测量细胞生物物理特性与标准光学显微镜

We describe the use of a standard optical microscope to perform quantitative measurements of mass, volume, and density on cellular specimens through a ...

Stretching Micropatterned Cells on a PDMS Membrane

在硅橡胶膜拉伸微图案细胞

Mechanical forces exerted on cells and/or tissues  play a major role in numerous processes. We have developed a device to stretch  cells plated on a ...

Magnetic Tweezers for the Measurement of Twist and Torque

磁性镊子的扭曲和扭矩的测量

Single-molecule techniques make it possible to investigate the behavior of individual biological molecules in solution in real time. These techniques ...

Nanomanipulation of Single RNA Molecules by Optical Tweezers

单个RNA分子的光镊纳米操作

A large portion of the human genome is transcribed but not translated. In this post genomic era, regulatory functions of RNA have been shown to be ...

Double Emulsion Generation Using a Polydimethylsiloxane (PDMS) Co-axial Flow Focus Device

Double Emulsion Generation Using a Polydimethylsiloxane PDMS Co-axial Flow Focus Device

复乳代使用聚二甲基硅氧烷（PDMS）同轴流量聚焦设备

Double emulsions are useful in a number of biological and industrial applications in which it is important to have an aqueous carrier fluid. This paper ...