Abstract
In the past decade, there have been extensive studies aimed at exploring the potential of protein nanopores to sequence single strands of DNA using resistive pulse sensing. The high speed of DNA electrophoretically driven through these pores (∼l μs/base) necessitates high bandwidth measurements, which prevent resolution of the picoampere differences in blockage current resulting from different nucleotides. Here, we describe a procedure for the immobilization of DNA in the α-hemolysin protein nanopore which enables low-noise, high-precision measurements capable of resolving subpicoampere differences in blockage current associated with differences in the sequence and structure of the DNA.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Braha O et al (2000) Simultaneous stochastic sensing of divalent metal ions. Nat Biotechnol 18(9):1005–1007
Li J et al (2001) Ion-beam sculpting at nanometre length scales. Nature 412(6843):166–169
Gu LQ et al (1999) Stochastic sensing of organic analytes by a pore-forming protein containing a molecular adapter. Nature 398:686–690
Rhee M, Burns MA (2006) Nanopore sequencing technology: research trends and applications. Trends Biotechnol 24(12):580–586
Meller A et al (2000) Rapid nanopore discrimination between single polynucleotide molecules. Proc Natl Acad Sci U S A 97(3):1079–1084
Akeson M et al (1999) Microsecond time-scale discrimination among polycytidylic acid, polyadenylic acid, and polyuridylic acid as homopolymers or as segments within single RNA molecules. Biophys J 77(6):3227–3233
Fologea D et al (2005) Slowing DNA translocation in a solid-state nanopore. Nano Lett 5(9):1734–1737
Song LZ et al (1996) Structure of staphylococcal alpha-hemolysin, a heptameric transmembrane pore. Science 274(5294):1859–1866
Sauer-Budge AF et al (2003) Unzipping kinetics of double-stranded DNA in a nanopore. Phys Rev Lett 90(23):238101
Ashkenasy N et al (2005) Recognizing a single base in an individual DNA strand: a step toward DNA sequencing in nanopores. Angew Chem Int Ed 44(9):1401–1404
Vercoutere W et al (2001) Rapid discrimination among individual DNA hairpin molecules at single-nucleotide resolution using an ion channel (vol 19, pg 248, 2001). Nat Biotechnol 19(7):681–681
Henrickson SE et al (2000) Driven DNA transport into an asymmetric nanometer-scale pore. Phys Rev Lett 85(14):3057–3060
Nakane J, Wiggin M, Marziali A (2004) A nanosensor for transmembrane capture and identification of single nucleic acid molecules (vol 87, pg 615, 2004). Biophys J 87(5):3618–3618
Cockroft SL et al (2008) A single-molecule nanopore device detects DNA polymerase activity with single-nucleotide resolution. J Am Chem Soc 130(3):818–820
Purnell RF, Mehta KK, Schmidt JJ (2008) Nucleotide identification and orientation discrimination of DNA homopolymers immobilized in a protein nanopore. Nano Lett 8(9):3029–3034
Purnell RF, Schmidt JJ (2009) Discrimination of single base substitutions in a DNA strand immobilized in a biological nanopore. ACS Nano 3(9):2533–2538
Stoddart D et al (2009) Single-nucleotide discrimination in immobilized DNA oligonucleotides with a biological nanopore. Proc Natl Acad Sci 106(19):7702–7707
Menestrina G (1986) Ionic channels formed by staphylococcus-aureus alpha-toxin: voltage-dependent inhibition by divalent and trivalent cations. J Membrane Biol 90(2):177–190
Bezrukov SM, Kasianowicz JJ (1993) Current noise reveals protonation kinetics and number of ionizable sites in an open protein ion channel. Phys Rev Lett 70(15): 2352–2355
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2012 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Purnell, R., Schmidt, J. (2012). Measurements of DNA Immobilized in the Alpha-Hemolysin Nanopore. In: Gracheva, M. (eds) Nanopore-Based Technology. Methods in Molecular Biology, vol 870. Humana Press. https://doi.org/10.1007/978-1-61779-773-6_3
Download citation
DOI: https://doi.org/10.1007/978-1-61779-773-6_3
Published:
Publisher Name: Humana Press
Print ISBN: 978-1-61779-772-9
Online ISBN: 978-1-61779-773-6
eBook Packages: Springer Protocols