Nanopores pp 287-311 | Cite as

Third Generation DNA Sequencing with a Nanopore

  • Gregory Timp
  • Utkur Mirsaidov
  • Winston Timp
  • Jiwook Shim
  • Deqiang Wang
  • Valentin Dimitrov
  • Jan Scrimgeour
  • Chunchen Lin
  • Jeffrey Comer
  • Anthony H. Ho
  • Xueqing Zou
  • Aleksei Aksimentiev
  • Klaus Schulten


With the advent of Next-Generation-Sequencing (NGS) technologies, an enormous volume of DNA sequencing data can be generated at low cost, placing genomic science within the grasp of everyday medicine. However, mired in this voluminous data, a new problem has emerged: the assembly of the genome from the short reads. In this chapter we examine the prospects for sequencing DNA using a synthetic nanopore. Nanopore sequencing has the potential for very long reads, reducing the computational burden posed by alignment and genome assembly, while at the same time eliminating logistically challenging and error-prone amplification and library formation due to its exquisite single molecule sensitivity. On the other hand, long high fidelity reads demand stringent control over both the DNA configuration in the pore and the translocation kinetics. We examine the prospects for satisfying these specifications with a synthetic nanopore.


Sequencing DNA Trapping DNA Comparison between biological and synthetic nanopores Molecular dynamics simulations Hydrodynamic focusing λ-DNA 



We gratefully acknowledge numerous contributions and our close collaboration with Jiunn Heng, Chuen Ho and Greg Sigalov. This work was funded by grants from National Institutes of Health [R01 HG003713A, PHS 5 P41-RR05969], the Large Resource Allocation Committee [MCA05S028], the Petroleum Research Fund (48352-G6), and the National Science Foundation [TH 2008–01040 ANTC, PHY-0822613 and DMR-0955959].


  1. 1.
    Lander ES, Linton LM, Birren B, et al (2001) Initial sequencing and analysis of the human genome. Nature, 409(6822), 860–921.CrossRefGoogle Scholar
  2. 2.
    Venter JC, Adams MD, Myers E, Li PW, Mural RJ, et al (2001) The sequence of human genome. Science 291, 1304–1351.CrossRefGoogle Scholar
  3. 3.
    Mardis ER (2008) The impact of next-generation sequencing technology on genetics,” Trends Genetics 24(3), 133–141.CrossRefGoogle Scholar
  4. 4.
    Metzker ML (2010) Sequencing technologies—the next generation. Nature Rev. Genetics 11, 31–46.CrossRefGoogle Scholar
  5. 5.
    Eid J, Fehr A, Gray J, Luong K, Lyle J, et al (2009) Real-time DNA sequencing from single polymerase molecules. Science 323, 133–138.CrossRefGoogle Scholar
  6. 6.
    Pop M, and Salzberg SL (2008) Bioinformatics Challenges in New sequencing Technologies,” Trends in Genetics 24(3), 142149.CrossRefGoogle Scholar
  7. 7.
    Chaisson M, et al (2004) Fragment assembly with short reads. Bioinformatics 20, 2067–2074.CrossRefGoogle Scholar
  8. 8.
    Whiteford N, et al (2005) An analysis of the feasibility of short read sequencing. Nucleic Acids Res. 33, e171.CrossRefGoogle Scholar
  9. 9.
    Voelkerding KV, Dames SA, and Durtschi JD (2009) Next-generation sequencing: from basic research to diagnostics. Clin. Chem. 55(4), 64158.CrossRefGoogle Scholar
  10. 10.
    Anker P, Mulcahy H, Chen XQ, and Stroun M (1999) Detection of circulating tumour DNA in the blood (plasma/serum) of cancer patients. Cancer and Metastasis Reviews 18(1), 65–73.CrossRefGoogle Scholar
  11. 11.
    Branton D, Deamer DW, Marziali A, Bayley H, et al (2008) The potential and challenges of nanopore sequencing. Nature biotechnology 26(10), 1146–1153.CrossRefGoogle Scholar
  12. 12.
    Akeson M, Branton D, Kasianowicz JJ, Brandin E, and Deamer DW (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.CrossRefGoogle Scholar
  13. 13.
    Chen P,et al (2004) Probing single DNA molecule transport using fabricated nanopores. Nano Lett. 4(11), 2293–2298.CrossRefGoogle Scholar
  14. 14.
    Heng JB, et al (2004) Sizing DNA using a nanometer-diameter pore. Biophys. J. 87(4), 2905–2911.CrossRefGoogle Scholar
  15. 15.
    Kasianowicz JJ, Brandin E, Branton D, and Deamer DW (1996) Characterization of individual polynucleotide molecules using a membrane channel. Proc. Natl. Acad. Sci. U.S.A. 93(24), 13770–13773.CrossRefGoogle Scholar
  16. 16.
    Li JL, Gershow M, Stein D, Brandin E, and Golovchenko JA (2003) DNA molecules and configurations in a solid-state nanopore microscope. Nat. Mater. 2(9), 611–615.CrossRefGoogle Scholar
  17. 17.
    Meller A, Nivon L, Brandin E, Golovchenko JA, and Branton D (2000) Rapid nanopore discrimination between single polynucleotide molecules. Proc. Natl. Acad. Sci. U.S.A. 97(3), 1079–1084.CrossRefGoogle Scholar
  18. 18.
    Storm AJ, Chen JH, Zandbergen HW, and Dekker C (2005) Translocation of double-strand DNA through a silicon oxide nanopore. Phys. Rev. E 71(5), 10.Google Scholar
  19. 19.
    Clarke J, et al (2009) Continuous base identification for single-molecule nanopore DNA sequencing. Nat. Nanotechnol doi:  10.1038/NNANO.2009.12.Google Scholar
  20. 20.
    Cockroft S, Chu J, Amorin M, Bayley H, and Ghadiri M (2008) A single-molecule nanopore device detects DNA polymerase activity with single-nucleotide resolution. J. Am. Chem. Soc. 130(3), 818.CrossRefGoogle Scholar
  21. 21.
    Mirsaidov U, Comer J, Dimitrov V, Aksimentiev A, and Timp G (2010) Slowing the Translocation of Double-Stranded DNA Using a Nanopore Smaller than the Double Helix. Nanotechnology 21, 395501.CrossRefGoogle Scholar
  22. 22.
    Stoddart D, Heron AJ, Mikhailova E, Maglia G, and Bayley H (2009) Single-nucleotide discrimination in immobilized DNA oligonucleotides with a biological nanopore. Proceedings of National Academy of Sciences 106(19), 7702–7707.CrossRefGoogle Scholar
  23. 23.
    Likharev KK (1999) Single-Electron Devices and Their Applications. Proc. IEEE 87, 606–632.CrossRefGoogle Scholar
  24. 24.
    Lagerqvist J, Zwolak M, and Di Ventra M (2006) Fast DNA sequencing via transverse electronic transport,” Nano Letters 6(4), 779.CrossRefGoogle Scholar
  25. 25.
    Chang SA, He J, Lin LS, et al (2009) Tunnel conductance of Watson-Crick nucleoside-base pairs from telegraph noise. Nanotechnology 20(18), 7.CrossRefGoogle Scholar
  26. 26.
    He J, Lin LS, Liu H, et al (2009) A hydrogen-bonded electron-tunneling circuit reads the base composition of unmodified DNA. Nanotechnology 20(7), 8.CrossRefGoogle Scholar
  27. 27.
    Meunier V, and Krstic PS (2008) Enhancement of the transverse conductance in DNA nucleotides. Journal of Chemical Physics 128(4), 4.CrossRefGoogle Scholar
  28. 28.
    Sigalov G, Comer J, Timp G, et al (2008) Detection of DNA Sequences Using an Alternating Electric Field in a Nanopore Capacitor, Nano Letters 8(1), 56–63.CrossRefGoogle Scholar
  29. 29.
    Fologea D, Uplinger J, Thomas B, McNabb DS and Li JL (2005) Slowing DNA translocation in a solid-state nanopore. Nano Lett. 5, 1734–1737.CrossRefGoogle Scholar
  30. 30.
    Storm AJ, Storm C, Chen JH, Zandbergen H, Joanny JF, and Dekker C (2005) Fast DNA translocation through a solid-state nanopore. Nano Lett. 5, 1193–1197.Google Scholar
  31. 31.
    Soni GV and Meller A (2007) Progress toward ultrafast DNA Sequencing using solid-state nanopores. Clinical Chemistry 53(11), 1996–2001.CrossRefGoogle Scholar
  32. 32.
    Gershow M and Golovchenko JA (2007) Recapturing and trapping single molecules with a solid-state nanopore. Nat. Nanotechnol. 2(12), 775–779.CrossRefGoogle Scholar
  33. 33.
    Timp W, Mirsaidov UM, Wang D, Comer J, Aksimentiev O, and Timp G (2010) Nanopore Sequencing: Electrical Measurements of the Code of Life. IEEE Trans. Nanotechnology 9, 281–294.CrossRefGoogle Scholar
  34. 34.
    Mirsaidov UM, Wang D, Timp W, and Timp G (2010) Molecular Diagnostics for Personal Medicine Using a Nanopore,” WIRES Review Nanomedicine Nanobiotechnology,2, 367–381.CrossRefGoogle Scholar
  35. 35.
    Heng JB, Aksimentiev A, Ho C, Dimitrov V, Sorsch T, Miner J, Mansfield W, Schulten K, and Timp G (2005) Beyond the Gene Chip. Bell Labs Tech. J. 10(3), 5–22.CrossRefGoogle Scholar
  36. 36.
    Cruz-Chu ER, Ritz T, Siwy ZS, Schulten K (2009) Molecular control of ionic conduction in polymer nanopores. Faraday Disc. 143, 47–62.CrossRefGoogle Scholar
  37. 37.
    Ho C, Qiao R, Heng JB, Chatterjee A, Timp R, Aluru NR, and Timp G (2005) Electrolytic transport through a synthetic nanometer-diameter pore. Proceedings of National Academy of Sciences 102(30), 10445–10450.CrossRefGoogle Scholar
  38. 38.
    Venkatesan BM, Dorvel B, Yemenicioglu S, Watkins N, Petrov I, and Bashir R (2009) Highly sensitive, mechanically stable nanopore sensors for DNA analysis Advanced Materials, 21(27), 2771–2776.CrossRefGoogle Scholar
  39. 39.
    Dimitrov V, Aksimentiev A, Schulten K, Heng JB, Sorsch T, et al (2006) Exploring the Prospects for a Nanometer-scale Gene Chip. IEDM Proceedings 169–172.Google Scholar
  40. 40.
    Fischbein MD and Drndić M (2007) Sub-10 nm Device Fabrication in a Transmission Electron Microscope. Nano Lett. 7(5), 1329–1337.CrossRefGoogle Scholar
  41. 41.
    Martin CR, Nishizawa M, Jiarge K, Kang M, and Lee SB (2001) Controlling Ion Transport Selectively in Gold Nanotubule Membranes. Adv. Mater. 13, 13511362.CrossRefGoogle Scholar
  42. 42.
    Li J, Stein D, McMullan C, et al (2001) Ion-beam sculpting at nanometre length scales. Nature 412, 6843, 166–169.CrossRefGoogle Scholar
  43. 43.
    Siwy Z and Fulinski A (2002) Fabrication of a synthetic nanopore ion pump. Physical Review Letters 89(19), 4.CrossRefGoogle Scholar
  44. 44.
    Dimitrov V, Mirsaidov UM, Wang D, Sorsch T, et al (2010) Nanopores in Solid-State Membranes Engineered for Single-Molecule Detection. Nanotechnology 21, 065502.CrossRefGoogle Scholar
  45. 45.
    Storm AJ, Chen JH, Ling XS, et al (2003) Fabrication of solid-state nanopores with single-nanometre precision. Nature Materials 2(8), 537–540.CrossRefGoogle Scholar
  46. 46.
    Nair PR and Alam MA (2006) Performance limits of nanobiosensors. Appl. Phys. Lett. 88, 233120.CrossRefGoogle Scholar
  47. 47.
    Berg HC (1993) Random walks in biology, Princeton University Press, Princeton, N.J.Google Scholar
  48. 48.
    Heng JB, Aksimentiev A, Ho C, Marks P, Grinkova YV, Sligar S, Schulten K, and Timp G, (2006) The Electromechanics of DNA in a Synthetic Nanopore. Biophys. J. 90, 1098–1106.CrossRefGoogle Scholar
  49. 49.
    Heng JB, Aksimentiev A, Ho C, Marks P, Grinkova YV, Sligar S, Schulten K, and Timp G (2005) Stretching DNA using the Field in a Synthetic Nanopore. Nano Let. 5(10), 1883–1888.CrossRefGoogle Scholar
  50. 50.
    Nakane J, Akeson M, and Marziali A (2002) Evaluation of nanopores as candidates for electronic analyte detection. Electrophoresis 23(16), 2592–2601.CrossRefGoogle Scholar
  51. 51.
    Durack G (2003) Cell Sorting Techniques and Technologies in Emerging Tools for Single Cell Analysis: Advances in optical measurement technologies. Wiley - Liss.Google Scholar
  52. 52.
    Scott R, Sethu P, Harnett CK (2008) 3D hydrodynamic focusing in a microfluidic. Rev. Sci. Instru. 79, 046104.CrossRefGoogle Scholar
  53. 53.
    Marcus JS, Anderson WF, and Quake SR (2006) Microfluidic single-cell MRNA isolation and analysis. Anal. Chem. 78(9), 3084–3089.CrossRefGoogle Scholar
  54. 54.
    Melin J and Quake SR (2007) Microfluidic Large-Scale Integration: The Evolution of Design Rules for Biological Automation. Annu. Rev. Biophys. Biomol. Struct. 36, 213–231.CrossRefGoogle Scholar
  55. 55.
    Luan B and Aksimentiev A (2008) Strain softening in stretched DNA. Physical Review Letters 101, 11.CrossRefGoogle Scholar
  56. 56.
    Li JL, Gershow M, Stein D, et al (2003) DNA molecules and configurations in a solid-state nanopore microscope. Nature Materials 2(9), 611–615.CrossRefGoogle Scholar
  57. 57.
    Zhao Q, Comer J, Dimitrov V, et al (2008) Stretching and unzipping nucleic acid hairpins using a synthetic nanopore. Nucleic Acids Research 36(5), 1532–1541.CrossRefGoogle Scholar
  58. 58.
    Comer J, Dimitrov V, Zhao Q, et al (2009) Microscopic Mechanics of Hairpin DNA Translocation through Synthetic Nanopores. Biophysical Journal 96(2), 593–608.CrossRefGoogle Scholar
  59. 59.
    Mirsaidov UM, Timp W, Zou X, et al (2009) Nanoelectromechanics of Methylated DNA in a Synthetic Nanopore. Biophysical Journal 96(4), L32–L34.CrossRefGoogle Scholar
  60. 60.
    Heinemann U and Hahn M (1992) CCAGGC-m5C-TGG, “Helical fine structure, hydration, and comparison with CCAGGCCTGG. Journal of Biological Chemistry 267, 7332–7341.Google Scholar
  61. 61.
    Derreumaux S, Chaoui M, Tevanian G, and Fermandjian S (2001) Impact of CpG methylation on structure, dynamics and solvation of cAMP DNA responsive element. Nucleic Acids Research 29, 2314–2326.CrossRefGoogle Scholar
  62. 62.
    Golovchenko JA, private communication.Google Scholar
  63. 63.
    Dawson JR and Harpst JA (1971) Light Scattering and Hydrodynamic Properties of Linear and Circular Bacteriophage Lambda DNA. Biopolymers 10, 2499–2508.CrossRefGoogle Scholar
  64. 64.
    Moffitt JR, Chemla YR, Izhaky D and Bustamante C (2006) Differential detection of dual traps improves the spatial resolution of optical tweezers. Proc. Natl. Acad. Sci, U.S.A. 103(24), 9006–9011.Google Scholar
  65. 65.
    Shim J, Timp W, Comer J, Wang D, Mirsaidov U, Aksimentiev A, and Timp G, unpublished.Google Scholar
  66. 66.
    Smeets RMM, Keyser U, Dekker N, et al (2008) Noise in solid-state nanopores. Proceedings of the National Academy of Sciences 105(2), 417.CrossRefGoogle Scholar
  67. 67.
    Smeets RMM, Dekker NH, and Dekker C (2009) Low-frequency noise in solid-state nanopores. Nanotechnology 20, 095501.Google Scholar
  68. 68.
    Coulter W (1953) Means for Counting Particles Suspended in a Fluid, USPTO.Google Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Gregory Timp
    • 1
  • Utkur Mirsaidov
  • Winston Timp
  • Jiwook Shim
  • Deqiang Wang
  • Valentin Dimitrov
  • Jan Scrimgeour
  • Chunchen Lin
  • Jeffrey Comer
  • Anthony H. Ho
  • Xueqing Zou
  • Aleksei Aksimentiev
  • Klaus Schulten
  1. 1.Stinson-Remick HallUniversity of Notre DameNotre DameUSA

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