Methods and apparatus for characterizing polynucleotides

• US 7,947,454 B2
• Filed: 11/25/2009
• Issued: 05/24/2011
• 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., President and Fellows of Harvard College (Harvard Corporation), Regents of the University of California (University of California)
• Inventors: Akeson, Mark, Deamer, David W., Sampson, Jeffrey R., Branton, Daniel
• Primary Examiner(s): WHISENANT, ETHAN C
• Application Number:
  US12/626,014
• Publication Number:
  US 20100267026A1
• Time in Patent Office: 545 Days
• Field of Search: 435/6, 536/23.1, 536/24.3
• US Class Current: 435/6.12
• 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. A method for analyzing a target polynucleotide, comprising the steps of:

(a) introducing the target polynucleotide to a nanopore analysis system comprising a nanopore aperture;

(b) allowing the target polynucleotide to move with respect to the nanopore aperture to produce a signal at a rate of 350-2000 Hz; and

(c) monitoring the signal corresponding to the movement of the target polynucleotide with respect to the nanopore aperture, thereby analyzing the target polynucleotide.

2. The method of claim 1, wherein the nanopore analysis system further comprises a molecular motor, wherein the molecular motor moves the polynucleotide with respect to the nanopore aperture.

3. The method of claim 2, wherein the molecular motor comprises a DNA polymerase, a RNA polymerase, a ribosome, an exonuclease, or a helicase.

4. The method of claim 3, wherein the DNA polymerase is selected from E. coli DNA polymerase I, E. coli DNA polymerase I Large Fragment (Klenow fragment), phage T7 DNA polymerase, Phi-29 DNA polymerase, Thermus aquaticus (Taq) DNA polymerase, Thermus flavus (Tfl) DNA polymerase, Thermus Thermophilus (Tth) DNA polymerase, Thermococcus litoralis (Tli) DNA polymerase, Pyrococcus furiosus (Pfu) DNA polymerase, Vent™

DNA polymerase, Bacillus stearothermophilus (Bst) DNA polymerase, AMV reverse transcriptase, MMLV reverse transcriptase, and HIV-1 reverse transcriptase.

5. The method of claim 3, wherein the RNA polymerase is selected from T7 RNA polymerase, T3 RNA polymerase, SP6 RNA polymerase, and E. coli RNA polymerase.

6. The method of claim 3, wherein the exonuclease is selected from exonuclease Lambda, T7 Exonuclease, Exo III, RecJ₁ Exonuclease, Exo I, and Exo T.

7. The method of claim 3, wherein the helicase is selected from E. coli bacteriophage T7 gp4 and T4 gp41 gene proteins, E. coli protein DnaB, E. coli protein RuvB, and E. coli protein rho.

8. The method of claim 1, wherein step (c) comprises measuring a monomer-dependent characteristic of the target polynucleotide while the target polynucleotide moves with respect to the nanopore aperture.

9. The method of claim 8, wherein the monomer dependent property is the identity of a nucleotide or the number of nucleotides in the polynucleotide.

10. The method of claim 1, further comprising altering the rate of movement of the polynucleotide before, during, or after the monitoring in step (c).

11. A method for analyzing a target polynucleotide, comprising the steps of:

(a) introducing the target polynucleotide to a nanopore analysis system comprising a nanopore aperture and a molecular motor disposed adjacent the nanopore aperture, wherein the molecular motor comprises a DNA polymerase, a ribosome, an exonuclease, or a helicase;

(b) allowing the molecular motor to move the target polynucleotide with respect to the nanopore aperture to produce a signal at a rate of 50-2000 Hz; and

(c) monitoring the signal corresponding to the movement of the target polynucleotide with respect to the nanopore aperture, thereby analyzing the target polynucleotide.

15. The method of claim 11, wherein in step (b) signal is produced at a rate of 75-2000 Hz.

16. The method of claim 11, wherein in step (b) signal is produced at a rate of 50-1500 Hz.

12. A method for analyzing a target polynucleotide, comprising the steps of:

(a) introducing the target polynucleotide to a nanopore analysis system comprising a nanopore aperture and a molecular motor disposed adjacent the nanopore aperture;

(b) allowing the molecular motor to move the target polynucleotide with respect to the nanopore aperture to produce a signal; and

(c) monitoring the signal corresponding to the movement of the target polynucleotide with respect to the nanopore aperture, wherein before, during, or after the monitoring, the rate of movement of the polynucleotide is altered, thereby analyzing the target polynucleotide.

13. The method of claim 12, wherein the rate of movement of the polynucleotide is increased, decreased, initiated, or stopped.

14. The method of claim 12, wherein the rate of movement of the polynucleotide is altered, at least in part, by a change in voltage, pH, temperature, viscosity, or concentration of a chemical species.