首页> 外文OA文献 >A general method for manipulating DNA sequences from any organism with optical tweezers
【2h】

A general method for manipulating DNA sequences from any organism with optical tweezers

机译:用光镊操作任何生物的DNA序列的一般方法

摘要

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 ∼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.
机译:对单个DNA分子的机械操作可以提供有关DNA特性和蛋白质-DNA相互作用的新颖信息。在这里,我们描述并描述了一种有用的方法,可以用光镊从任何生物中操纵所需的DNA序列。使用标记的引物通过PCR从基因组或克隆的DNA中产生分子,并将分子束缚在两个光学捕获的微球之间。我们证明人,昆虫,植物,细菌和病毒的序列范围约为10至40千个碱基对。力-延伸测量表明,这些构建体显示出均匀的弹性性质,与目标序列的预期轮廓长度一致。介绍了用于制备和操作这些分子的详细方案,并且绑定效率的特征是DNA浓度,离子强度和pH的函数。附着强度的特征在于测量解开时间与施加力的关系。还描述了使用氨基-羧基键的另一种更强的附着方法,该方法可实现可靠的DNA过度拉伸。

著录项

相似文献

  • 外文文献
  • 中文文献
  • 专利

客服邮箱:kefu@zhangqiaokeyan.com

京公网安备:11010802029741号 ICP备案号:京ICP备15016152号-6 六维联合信息科技 (北京) 有限公司©版权所有
  • 客服微信

  • 服务号