Conformation, Length, and Speed Measurements of Electrodynamically Stretched DNA in Nanochannels

Abstract

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.

Probing transient protein-mediated DNA linkages using nanoconfinement

“…However, the configuration was such that the folded segment trailed the molecule through the channel. That in stark contrast to loops at high driving pressure, 75 and in particular loops in the absence of T4 DNA ligase, where we see almost exclusively folded segments leading the molecule. The only explanation for this asymmetry is the formation of transient loops in solution.…”

DNA methylation profiling in nanochannels

“…37 The capability for automated high-throughput mapping has been demonstrated. 38 While nanochannel mapping appears similar to stretching through molecular combing, it differs in that the stretched state represents the equilibrium configuration of the molecule such that the molecule can fluctuate around that equilibrium configuration. In contrast, fiber-FISH and molecular combing lock the nucleic acid in a single non-equilibrium configuration.…”

Monte Carlo simulation of chromatin fibre confined in a nanochannel

“…Thus, a comparison cannot be carried out in the present paper. Nevertheless, many results on the DNA stretching by the nanoconfinement have been reported [19][20][21]. Generally, the stretching of DNA in a channel is in combination with other strategies, i.e., oscillatory shear flow [19], electrophoresis technique [20] and shear flow stretching by fixing one end of DNA [21].…”