METHODS AND APPARATUS FOR CHARACTERIZING POLYNUCLEOTIDES

• US 20120094278A1
• Filed: 05/18/2011
• Published: 04/19/2012
• Est. Priority Date: 03/23/2004
• Status: Active Grant

• Current Assignee: Agilent Technologies, Inc., President and Fellows of Harvard College (Harvard Corporation), Regents of the University of California (University of California)
• Original Assignee: Agilent Technologies, Inc.
• Inventors: Akeson, Mark, Branton, Daniel, Deamer, David W., Sampson, Jeffrey R.
• Granted Patent: US 8,673,556 B2
• Time in Patent Office: Days
• US Class Current: 435/6.1
• CPC Class Codes: B01L 2300/0896 Nanoscaled
• CPC Class Codes: B01L 2400/0475 specific mechanical means and fluid pressure
• CPC Class Codes: B01L 3/502707 characterised by the manufacture of the container or its components by shaping or joining plastic parts B29C59/00, B29C65/00; by laminating B32B37/00; manufacture of microstructural devices in general B81C
• CPC Class Codes: B01L 3/502761 specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules investigating characteristics of particles G01N15/00
• CPC Class Codes: B82Y 30/00 Nanotechnology for materials or surface science, e.g. nanocomposites
• CPC Class Codes: B82Y 5/00 Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2521/543 Immobilised enzyme(s)
• CPC Class Codes: C12Q 2565/631 being a biochannel or pore
• CPC Class Codes: G01N 33/48721 Investigating individual macromolecules, e.g. by translocation through nanopores Coulter counters in general G01N15/12; fabrication methods for nanoscale apertures B81B1/00; sequencing of nucleic acids C12Q1/68

Abstract

Systems and methods for analysis of polymers, e.g., polynucleotides, are provided. The systems are capable of analyzing a polymer at a specified rate. One such analysis system includes a structure having a nanopore aperture and a molecular motor, e.g., a polymerase, adjacent the nanopore aperture.

Claims

1. (canceled)

2. An analysis system comprising:

a structure having a nanopore aperture that separates two independent adjacent pools of solution;

a helicase that is capable of moving a polynucleotide with respect to the nanopore aperture at a rate of less than 250 Hz and that is located in at least one of the pools; and

a detector capable of measuring transport properties of the polynucleotide as it moves with respect to the nanopore.

3. The system of claim 2, wherein the nanopore aperture is solid-state.

4. The system of claim 2, wherein the nanopore aperture comprises a biological nanopore.

5. The system of claim 2, wherein the transport properties are the amplitude or duration of individual conductance or electron tunneling current changes across the nanopore aperture.

6. The system of claim 2, wherein the helicase is selected from the group consisting of E-coli bacteriophage T7 gp4 and T4 gp41 gene proteins, and the E. coli proteins DnaB, RuvB, and rho.

7. The system of claim 2, wherein the detector comprises electrodes at or near the nanopore aperture.

8. The system of claim 2, wherein the detector comprises electrodes placed within the adjacent pools.

9. The system of claim 2, wherein the rate of the helicase is less than or equal to 200 Hz.

10. The system of claim 2, wherein the rate of the helicase is less than or equal to 150 Hz.

11. The system of claim 2, wherein the helicase is capable of moving the polynucleotide through the nanopore.

12. A kit comprising:

(i) a device comprising a structure having a nanopore aperture that separates two independent adjacent pools of solution; and

(ii) a helicase that is capable of moving a polynucleotide with respect to the nanopore aperture a rate of less than 250 Hz.

13. The kit of claim 12, wherein the nanopore aperture is solid-state.

14. The kit of claim 12, wherein the nanopore aperture comprises a biological nanopore.

15. The kit of claim 12, wherein the helicase is selected from the group consisting of E-coli bacteriophage T7 gp4 and T4 gp41 gene proteins, and the E. coli proteins DnaB, RuvB, and rho.

16. The kit of claim 12, wherein the nanopore device further comprises electrodes at or near the nanopore aperture.

17. The kit of claim 12, wherein the nanopore device further comprises electrodes placed within the adjacent pools.

18. The kit of claim 12, further comprising a biological nanopore.

19. The kit of claim 12, wherein the rate of the helicase is less than or equal to 200 Hz.

20. The kit of claim 12, wherein the rate of the helicase is less than or equal to 150 Hz.

21. The kit of claim 12, wherein the helicase is capable of moving the polynucleotide through the nanopore.

22. A method for analyzing a polynucleotide, said method comprising:

(i) providing a structure having a nanopore aperture that separates two independent adjacent pools of solution, wherein a helicase is located in at least one of the pools;

(ii) placing the polynucleotide in one of the pools and allowing the helicase to move the polynucleotide with respect to the nanopore aperture at a rate of less than 250 Hz; and

(iii) measuring transport properties of the polynucleotide as it moves with respect to the nanopore, thereby analyzing the polynucleotide.

23. The method of claim 22, wherein the nanopore aperture is solid-state.

24. The method of claim 22, wherein the nanopore aperture comprises a biological nanopore.

25. The method of claim 22, wherein the transport properties are the amplitude or duration of individual conductance or electron tunneling current changes across the nanopore aperture.

26. The method of claim 22, wherein the helicase is selected from the group consisting of E-coli bacteriophage T7 gp4 and T4 gp41 gene proteins, and the E. coli proteins DnaB, RuvB, and rho.

27. The method of claim 22, wherein the device further comprises electrodes that are at or near the nanopore aperture and that measure the transport properties of the polynucleotide.

28. The method of claim 22, wherein the device further comprises electrodes that placed within the adjacent pools and that measure the transport properties of the polynucleotide.

29. The method of claim 22, wherein the rate of the helicase is less than or equal to 200 Hz.

30. The method of claim 22, wherein the rate of the helicase is less than or equal to 150 Hz.

31. The method of claim 22, further comprising determining the sequence of the polynucleotide from the measured transport properties.

32. The method of claim 22, wherein, in step (ii), the helicase moves the polynucleotide through the nanopore.