Nanopore Sequencing
Our development of solid state nanopores and our studies of DNA translocation through these nanopores suggest how a nanopore could be the core of an instrument capable of inexpensive de novo sequencing. We are investigating and developing the basic science and technology required to build a nanopore based instrument that should be able to sequence a mammalian genome for <$1,000 and that meets the following requirements:
a.
High-speed sequential identification of the DNA's nucleotides directly on the basis of their distinct physical or electrical properties;
b.
Very long, indefinite length reads. Analysis and assembly is a bottle-neck in de novo sequencing and limits re-sequencing when copy number polymorphism or variable indels are to be identified in heterozygous genomes;
c.
The requisite sequence coverage (7.7-fold coverage, 6.5-fold coverage in Q20 bases) using genomic DNA from <106 cells with no amplification and minimal preparative steps. Otherwise, amplification or other preparatory steps become limiting.
The long term goal
We propose to investigate and develop the science and technology required to build a nanopore based instrument that meets the above requirements. Among the unique capabilities of this instrument, four already well demonstrated features stand out:
1) A nanoscale device that translocates polymer molecules in sequential monomer order through a very small volume of space
1-4, a small pore in an electrically biased membrane.
2) A single molecule detector that is also a very high throughput device. A nanopore can probe thousands of different molecules or thousands of identical molecules in a few minutes
5-7.
3) A detector that directly converts characteristic features of the translocating polymer into an electrical signal. Transduction and recognition occur in real time, on a molecule-by-molecule basis4.
4) A device that can probe very long lengths of DNA8. While practical considerations may limit the length of DNA that can be analyzed as it translocates through a nanopore, we are not aware of any theoretical limits.
Figure 1. A biased nanopore in an insulating membrane that separates two ionic solution-filled compartments translocates DNA molecules in sequencial nucleotide order between probes that serve as emitter and collector of a tunneling “microscope.” In response to a voltage bias (labeled “ - ” and “+”) across the membrane, ssDNA molecules (yellow) in the “-” compartment are driven, one at a time, into and through the nanopore. Elevated temperatures and denaturants maintain the DNA in an unstructured, single-stranded form.
The underlying principle of nanopore sequencing is that a single-stranded DNA or RNA molecule can be electrophoretically driven through a nano-scale pore in such a way that the molecule traverses the pore in strict linear sequence, as illustrated in Figure 1. Because a translocating molecule partially obstructs or blocks the nanopore, it alters the pore's electrical properties1. Until now, the translocation of nucleic acid polymers has been detected and converted into an electrical signal by sensing how the translocating polymer obstructs ionic current through the nanopore. Although this detection mode is extraordinarily sensitive and able to sense small differences in the base composition of the translocating molecule, theory and experiment show that measurement of ionic conductivity alone is unlikely to achieve the resolution required for rapid sequential detection of each nucleotide in a DNA molecule. Attaining such resolution requires that we incorporate a different, highly localizing detection mode into the nanopore. This can now be done with solid state nanopores which we have shown can detect single DNA molecules8,9. We propose to articulate these solid state nanopores with probes that will serve as atomic scale electrodes whose termini abut on opposite sides of the nanopore. A voltage bias between these probes will induce an electronic current stimulated by quantum mechanical tunneling to flow from one side of the nanopore to the other. The current, which will be sensed by an external circuit, will be modulated by the individual nucleotide bases as ssDNA molecules translocate through the nanopore. Articulating the nanopore, investigating the performance of these articulations, as well as other closely linked improvements that are critical for nanopore detection, define our major aims. These aims are:
1. Improve nanopore surfaces to reduce non-specific adsorption, pore clogging, and electrical noise;
2. Fabricate and test a nanopore detector articulated with integrated probes for molecular identification;
3. Investigate and optimize the electronic properties of probe-DNA interactions to control DNA translocation, orientation and nucleotide contrast;
4. Develop new enzymatic methods to better control and limit the rate of DNA translocation through articulated nanopores;
5. Develop algorithms for signal feature detection and base identification from articulated nanopores;
6. Demonstrate single base sensitivity and resolution on single-stranded DNA translocating through a nanopore.
Having studied single molecule transport through nanopores1-14
and single atoms with electron current transport in tunneling microscopes15-24, it is clear to us that integrating the two approaches holds significant potential for rapid electronic sequencing. Transporting and unraveling DNA molecules with solid state nanopores is now well documented7,12
as is the use of various scanning probe microscopies in vacuum and liquid environments and on dsDNA molecules25-30. In contrast to the small, picoamp ionic current through a nanopore, typical electron currents in tunneling microscopes are nanoamps. Combining these larger electron currents (and the attendant greater signal to noise) available from electronic signals with the high throughput and spatial confinement of a nanopore opens the prospect of high bandwidth sequencing. For example, tunneling microscopes have been reported that scan surfaces with atomic resolution at speeds approaching 104 nm/sec32, which translates to ~ 3 x 104 bases per second if the microscope scan was spatially confined to travel along only the length of the DNA molecule. Furthermore, this speed of a tunneling microscope is limited by the capabilities of the piezoelectric scanning mechanism, and only a small area (100 nm x 100 nm) can be scanned. The important electronic limitation on scan speed is determined by the capabilities of the tunneling current preamplifier. A state of the art preamplifier, as developed at IBM32, is sensitive to nanoamp currents at bandwiths of ~ 106 Hz. This tells us that the rate at which nucleotide bases could be sensed by the currents of a tunneling microscope would be ~ 105 – 106/bases per second if the microscope could follow the molecule's length at that rate and over a sufficient range. But a tunneling microscope cannot do this, and earlier efforts to use tunneling microscopes for sequencing
25-30
were abandoned in favor of the then more promising gel-based methods. The problem of confining the scan to the length of the DNA molecule is solved by integrating tunneling probe electrodes into a nanopore device and by having ssDNA molecules electrophoretically drawn into and through the confining volume of a nanopore at rates commensurate with the potential sensitivity and bandwidth of the electronics. Because resolving the various bases will depend on each having its own characteristic tunneling signal, the observed contrast between the bases may require slower translocation speeds than 106 bases per second, e.g. 104 bases/sec, but as a starting point the numbers look very promising. These considerations motivate our primary focus on sequencing DNA molecules by translocating them through a tunneling gap fabricated within a solid state nanopore.
A nanopore based instrument can satisfy the requirements for a $1,000/mammalian genome assay because it will directly transduce the sequence of bases into an electrical signal, and because it is a single molecule approach that can achieve the requisite sequence coverage and over sampling with DNA from <106 target genomes. This corresponds to about 2 nanograms of human genomic material which can be directly obtained without amplification using standard sampling methods. If we are able to resolve each base as it passes through a nanopore at the rate of 104 bases/sec, an instrument with an array of 100 such nanopores could produce high-quality draft sequence of one mammalian genome in ~20 hours.
References:
1. Kasianowicz, J.J., Brandin, E., Branton, D. & Deamer, D.W. Characterization of individual polynucleotide molecules using a membrane channel. Proc. Natl. Acad. Sci. U.S.A. 93, 13770-13773 (1996).
2. Deamer, D.W. & Akeson, M. Nanopores and nucleic acids: prospects for ultrarapid sequencing. Trends in Biotechnology 18, 131-180 (2000).
3. Deamer, D. & Branton, D. Characterization of nucleic acids by nanopore analysis. Acc. Chem. Res. 35, 817-825 (2002).
4. Meller, A. & Branton, D. Single molecule measurements of DNA transport through a nanopore. Electrophoresis 23, 2583-2591 (2002).
5. Akeson, M., Branton, D., Kasianowicz, J.J., Brandin, E. & Deamer, D.W. Microsecond time-scale discrimination among polycytidylic acid, polyadenylic acid, and polyuridylic acid as homopolymers or as segments within single RNA molecules. Biophys. J. 77, 3227-3233 (1999).
6. Meller, A., Nivon, L., Brandin, E., Golovchenko, J. & Branton, D. Rapid nanopore discrimination between single oligonucleotide molecules. The Proceedings of the National Academy of Science 97, 1079-1084 (2000).
7. Vercoutere, W., Winters-Hilt, S., Olsen, H., Deamer, D., Haussler, D. & Akeson, M. Rapid discrimination among individual DNA hairpin molecules at single-nucleotide resolution using an ion channel. Nature Biotechnology 19 , 248-252 (2001).
8. Chen, P., Gu, J., Brandin, E., Kim, Y.-R., Wang, Q. & Branton, D. Probing single DNA molecule transport using fabricated nanopores. Nano Letters 4, 2293-2298 (2004).
9. Li, J., Gershow, M., Stein, D., Brandin, E. & Golovchenko, J. DNA molecules and configurations in a solid-state nanopore microscope. Nature Materials 2, 611-615 (2003).
10. Meller, A., Nivon, L. & Branton, D. Voltage-driven DNA translocations through a nanopore. Phys. Rev. Lett. 86, 3435-3438 (2001).
11. Li, J., Stein, D., McMullan, C., Branton, D., Aziz, M.J. & Golovchenko, J.A. Ion-beam sculpting at nanometre length scales. Nature 412, 166-169 (2001).
12. Sauer-Budge, A.F., Nyamwanda, J.A., Lubensky, D.K. & Branton, D. Unzipping kinetics of double-stranded DNA in a nanopore. Phys. Rev. Lett. 90, 238101-238101 - 238101-238104 (2003).
13. Chen, P., Mitsui, T., Farmer, D.B., Golovchenko, J., Gordon, R.G. & Branton, D. Atomic layer deposition to fine-tune the surface properties and diameters of fabricated nanopores. Nano Letters 4, 133-1337 (2004).
14. Wang, H., Dunning, J.E., Huang, A.P.-H., Nyamwanda, J.A. & Branton, D. DNA heterogeneity and phosphorylation unveiled by single-molecule electrophoresis. Proc. Natl. Acad Sci. USA 101, 13472-13477 (2004).
15. Becker, R.S., Golovchenko, J.A. & Swartzentruber, B.S. Tunneling images of the 5x5 surface reconstruction on GeSi(III). Phys. Rev. B 32, 8455-8457 (1985).
16. Becker, R.S., Golovchenko, J.A., Hamann, D.R. & Swartzentruber, B.S. Real space observation of surface states of Si(III) 7x7 with the tunneling microscope. Phys. Rev. Lett. 55, 2032-2034 (1985).
17. Becker, R.S., Golovchenko, J.A., McRae, E.G. & Swartzentruber, B.S. Tunneling images of atomic steps on the Si(III) 7x7 surface. Phys. Rev. Lett. 55, 2028-2031 (1985).
18. Becker, R.S., Golovchenko, J.A. & Swartzentruber, B.S. Tunneling images of germanium reconstructions and phase boundaries. Phys. Rev. Lett. 54, 2678-2680 (1985).
19. Becker, R.S., Golovchenko, J.A., Higashi, G.S. & Swartzentruber, B.S. New reconstructions on silicon (III) surfaces. Phys. Rev. Lett. 57, 1020-1023 (1986).
20. Patel, J.R., Freeland, R.E., Golovchenko, J.A., Kortan, A.R., Chadi, D.J. & Quian, G.-X. Normal displacements on a reconstructed silicon (III) surface: An x-ray standing wave study. Phys. Rev. Lett. 57, 3077-3080 (1986).
21. Golovchenko, J. The tunneling microscope: A new look at the atomic world. Science 232, 48-53 (1986).
22. Becker, R.S., Golovchenko, J.A. & Swartzentruber, B.S. Atomic scale surface modification using a tunneling microscope. Nature 325, 419-421 (1987).
23. Chen, D., Golovchenko, J.A., Bedrossian, P. & Mortensen, K. Tunneling images of Gallium on a silicon surface: Reconstruction, superlattices and incommensuration. Phys. Rev. Lett. 61, 2867-2870 (1988).
24. Zegenhagen, J. et al. X-Ray standing wave and tunneling microscope location of gallium atoms on a silicon surface. Phys. Rev. B. 39, 1298-1301 (1989).
25. Drake, B., Sonnenfeld, R., Schneir, J. & Hansma, P.K. Scanning Tunneling Microscopy of Processes at Liquid Solid Interfaces. Surface Science 181, 92-97 (1987).
26. Driscoll, R.J., Youngquist, M.G. & Baldeschwieler, J.D. Atomic-scale imaging of DNA using scanning tunnelling microscopy. Nature 346, 294-296 (1990).
27. Allison, D.P., Thundat, T., Jacobson, K.B., Bottomley, L.A. & Warmack, R.J. Imaging Entire Genetically Functional DNA-Molecules with the Scanning Tunneling Microscope. Journal of Vacuum Science & Technology a-Vacuum Surfaces and Films 11, 816-819 (1993).
28. Mou, J.X., Czajkowsky, D.M., Zhang, U.U. & Shao, Z.F. High-resolution atomic-force microscopy of DNA: the pitch of the double helix. FEBS Lett. 371, 279- (1995).
29. Ikai, A. STM and AFM of biolorganic molecules and structures. Surface Science Reports 26, 263-332 (1996).
30. Dunlap, D.D. & Bustamante, C. Images of single-stranded nucleic acids by scanning tunnelling microscopy. Nature 342, 204-206 (1989).
31. Curtis, R., Mitsui, T. & Ganz, E. An ultrahigh vacuum high speed scanning tunneling microscope. Reviews of Scientific Instruments 68, 2790-2796 (1997).
32. Michel, B., Novotny, L. & Durig, U. Low-temperature compatible I-V converter. Ultramicroscopy 42-44, 1647-1652 (1992).
