Our aim is to develop a DNA sequencing device based upon the blockade of ionic current in the transmembrane protein pore a-hemolysin (aHL). Compared to other proposed nanopore-based approaches, the protein pore current blockade (PPCB) method is arguably the simplest, both conceptually and technologically, but recently has been passed over in favor of more complex methods. Recent electronic readout and bilayer lipid membrane advances by the proposers have greatly alleviated prior signal-to-noise ratio and robustness issues. New experimental data and an accurate Stochastic Model for DNA Motion (SMDM) within a-HL now indicate threshold feasibility for sequencing by the PPCB method. Inspection of calculated SMDM responses to known input DNA sequences shows that the principal issue for sequencing via the PPCB method is random variance in the order the bases pass through the pore, leading to three quantifiable sources of error. This program will make specific structural and measurement parameter modifications to the present apparatus to reduce each type of error and to produce a minimal overall sequencing error. The final apparatus will be evaluated by sequencing up to 30 Mbases of natural DNA.

Public Health Relevance

This project offers a path to low cost DNA sequencing. Determining the DNA sequence is useful in basic research studying fundamental biological processes, as well as in applied fields such as diagnostic or forensic research. The advent of DNA sequencing has significantly accelerated biological research and discovery, but current methods are complicated and expensive, thus limiting their applicability. Simple, inexpensive DNA sequencing offers the capability to apply the benefits of the process to everyday medical and forensic applications.

National Institute of Health (NIH)
National Human Genome Research Institute (NHGRI)
Research Project (R01)
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Special Emphasis Panel (ZHG1-HGR-N (M1))
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Schloss, Jeffery
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Electronic Biosciences, Inc.
San Diego
United States
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De Biase, Pablo M; Ervin, Eric N; Pal, Prithwish et al. (2016) What controls open-pore and residual currents in the first sensing zone of alpha-hemolysin nanopore? Combined experimental and theoretical study. Nanoscale 8:11571-9
De Biase, Pablo M; Markosyan, Suren; Noskov, Sergei (2015) BROMOC suite: Monte Carlo/Brownian dynamics suite for studies of ion permeation and DNA transport in biological and artificial pores with effective potentials. J Comput Chem 36:264-71
Ervin, Eric N; Barrall, Geoffrey A; Pal, Prithwish et al. (2014) Creating a Single Sensing Zone within an Alpha-Hemolysin Pore Via Site Directed Mutagenesis. Bionanoscience 4:78-84
De Biase, Pablo M; Markosyan, Suren; Noskov, Sergei (2014) Microsecond simulations of DNA and ion transport in nanopores with novel ion-ion and ion-nucleotides effective potentials. J Comput Chem 35:711-21
Wang, Guihua; Wang, Liang; Han, Yujing et al. (2014) Nanopore detection of copper ions using a polyhistidine probe. Biosens Bioelectron 53:453-8
Markosyan, Suren; De Biase, Pablo M; Czapla, Luke et al. (2014) Effect of confinement on DNA, solvent and counterion dynamics in a model biological nanopore. Nanoscale 6:9006-16
Wolna, Anna H; Fleming, Aaron M; An, Na et al. (2013) Electrical Current Signatures of DNA Base Modifications in Single Molecules Immobilized in the ?-Hemolysin Ion Channel. Isr J Chem 53:417-430
Wang, Guihua; Zhao, Qitao; Kang, Xiaofeng et al. (2013) Probing mercury(II)-DNA interactions by nanopore stochastic sensing. J Phys Chem B 117:4763-9
Jin, Qian; Fleming, Aaron M; Burrows, Cynthia J et al. (2012) Unzipping kinetics of duplex DNA containing oxidized lesions in an *-hemolysin nanopore. J Am Chem Soc 134:11006-11
An, Na; White, Henry S; Burrows, Cynthia J (2012) Modulation of the current signatures of DNA abasic site adducts in the ýý-hemolysin ion channel. Chem Commun (Camb) 48:11410-2

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