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J Biomol Tech. 2010 September; 21(3 Suppl): S2.
PMCID: PMC2918088

The Prospects for Sequencing a Single Molecule of Double-Stranded DNA in a Nanopore With a Diameter Smaller than the Double Helix

D. Wang,1 U. Mirsaidov,1 W. Timp,2 J. Comer,1 and A. Aksimentiev1
1Beckman Institute, University of Illinois, Urbana, IL, United States;
2School of Medicine, Johns Hopkins University, Baltimore, MD, United States;
3Stinson-Remick Laboratory, Notre Dame University, South Bend, IN, United States

Abstract

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Single molecule DNA sequencing using a nanopore represents the logical end-of-the-line in development of sequencing technology, which extracts the maximum information with minmal material and pre-processing. Nanopore sequencing relies on the electrical signal that develops when DNA, immersed in electrolyte, translocates across a membrane through the pore. If each nucleobase has an electrical signature, then ostensibly a nanopore could be used to analyze the DNA chemical structure in a single, long read. The main problems with nanopore sequencing stem from the lack of control over the translocation kinetics and the molecule configuration in the pore. The molecular configuration determines how the ions passing through the pore come into contact with the nucleotides, while the kinetics affect the time in which the same nucleotides are held in the constriction as data is acquired. We describe a method that addresses these two problems by trapping a single molecule of double-stranded DNA (dsDNA) in a nanopore, smaller in diameter than the doulbe helix, in a solid-state membrane. We show that it is possible to trap dsDNA in a nanopore <3nm in diameter by applying a voltage larger than the stretching threshold and forcing the molecule to translocate through the pore. According to molecular dynamics (MD) simulations, this leaves the dsDNA stretched in the pore constriction with the base-pairs tilted, while the B-form canonical structure is preserved outside the pore. If the voltage is then rapidly switched to a value below the threshold during the translocation, the dsDNA becomes trapped in a harmonic potential in this configuration, which facilitates a read. If the time the molecule spends in the trap is commensurate with the bandwidth, it is possible to distinguish signatures of the base-pairs by simply measuring the pore current. This possibility is demonstrated by MD and by trapping biotinylated dsDNA bound to streptavidin.


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