A general method for manipulating DNA sequences from any organism with optical tweezers
A general method for manipulating DNA sequences from any organism with optical tweezers
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DOI：
10.1093/nar/gnj016
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发表时间：
2006-01-01
影响因子：
14.9
通讯作者：
Smith, DE
中科院分区：
文献类型：
--
作者：
Fuller, DN;Gemmen, GJ;Smith, DE
对单个DNA分子的机械操纵可以提供有关DNA性质和蛋白质-DNA相互作用的新信息。在这里，我们描述并描述了一种有用的方法，可以用光学镊子从任何生物体中操作所需的DNA序列。分子是由基因组或克隆的DNA通过使用标记的引物通过聚合酶链式反应产生的，并被拴在两个光学捕获的微球之间。我们证明，人类、昆虫、植物、细菌和病毒的序列从类似到10千到40千个基数都是可以操纵的。力伸展测量表明，这些结构显示出与目标序列的预期轮廓长度一致的弹性性质。介绍了制备和操作这些分子的详细方案，并用DNA浓度、离子强度和pH来表征拴系效率。附着强度的特征是测量作为作用力的函数的解粘时间。还描述了使用氨基-羧基连接的另一种更强的连接方法，该方法允许可靠的DNA过度伸展。
Mechanical manipulation of single DNA molecules can provide novel information about DNA properties and protein-DNA interactions. Here we describe and characterize a useful method for manipulating desired DNA sequences from any organism with optical tweezers. Molecules are produced from either genomic or cloned DNA by PCR using labeled primers and are tethered between two optically trapped microspheres. We demonstrate that human, insect, plant, bacterial and viral sequences ranging from similar to 10 to 40 kilobasepairs can be manipulated. Force-extension measurements show that these constructs exhibit uniform elastic properties in accord with the expected contour lengths for the targeted sequences. Detailed protocols for preparing and manipulating these molecules are presented, and tethering efficiency is characterized as a function of DNA concentration, ionic strength and pH. Attachment strength is characterized by measuring the unbinding time as a function of applied force. An alternative stronger attachment method using an amino-carboxyl linkage, which allows for reliable DNA overstretching, is also described.