Reccius Christian H, Stavis Samuel M, Mannion John T, Walker Larry P, Craighead H G
School of Applied and Engineering Physics, Cornell University, Ithaca, New York 14853, USA.
Biophys J. 2008 Jul;95(1):273-86. doi: 10.1529/biophysj.107.121020. Epub 2008 Mar 13.
A method is presented to rapidly and precisely measure the conformation, length, speed, and fluorescence intensity of single DNA molecules constrained by a nanochannel. DNA molecules were driven electrophoretically from a nanoslit into a nanochannel to confine and dynamically elongate them beyond their equilibrium length for repeated detection via laser-induced fluorescence spectroscopy. A single-molecule analysis algorithm was developed to analytically model bursts of fluorescence and determine the folding conformation of each stretched molecule. This technique achieved a molecular length resolution of 114 nm and an analysis time of around 20 ms per molecule, which enabled the sensitive investigation of several aspects of the physical behavior of DNA in a nanochannel. lambda-bacteriophage DNA was used to study the dependence of stretching on the applied device bias, the effect of conformation on speed, and the amount of DNA fragmentation in the device. A mixture of lambda-bacteriophage with the fragments of its own HindIII digest, a standard DNA ladder, was sized by length as well as by fluorescence intensity, which also allowed the characterization of DNA speed in a nanochannel as a function of length over two and a half orders of magnitude.
本文提出了一种快速精确测量受纳米通道约束的单个DNA分子的构象、长度、速度和荧光强度的方法。DNA分子通过电泳从纳米狭缝进入纳米通道，以限制并动态拉长它们，使其超出平衡长度，以便通过激光诱导荧光光谱进行重复检测。开发了一种单分子分析算法，用于对荧光爆发进行分析建模，并确定每个拉伸分子的折叠构象。该技术实现了114 nm的分子长度分辨率，每个分子的分析时间约为20 ms，从而能够对纳米通道中DNA物理行为的多个方面进行灵敏研究。使用λ噬菌体DNA研究拉伸对施加的器件偏压的依赖性、构象对速度的影响以及器件中DNA片段化的程度。将λ噬菌体与其自身HindIII消化片段的混合物（一种标准DNA梯）按长度和荧光强度进行大小测定，这也使得能够表征纳米通道中DNA速度随长度在两个半数量级上的变化。
