Conformation, length, and speed measurements of electrodynamically stretched DNA in nanochannels

Christian H. Reccius, Samuel M. Stavis, John T. Mannion, Larry P. Walker, H. G. Craighead

Research output: Contribution to journalArticlepeer-review

73 Scopus citations

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. λ-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 λ- 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.

Original languageEnglish (US)
Pages (from-to)273-286
Number of pages14
JournalBiophysical Journal
Volume95
Issue number1
DOIs
StatePublished - Jul 1 2008

ASJC Scopus subject areas

  • Biophysics

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