CHARACTERIZATION OF INDIVIDUAL POLYMER MOLECULES BASED ON MONOMER-INTERFACE INTERACTIONS

• US 20120160687A1
• Filed: 07/20/2011
• Published: 06/28/2012
• Est. Priority Date: 03/17/1995
• Status: Abandoned Application

• Current Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Original Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Inventors: Akeson, Mark, Branton, Daniel, Church, George, Deamer, David W.
• Application Number:
  US13/186,966
• Publication Number:
  US 20120160687A1
• Time in Patent Office: Days
• US Class Current: 204/540
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2565/607 being a sensor, e.g. electrode
• CPC Class Codes: C12Q 2565/631 being a biochannel or pore
• CPC Class Codes: G01N 27/447 using electrophoresis
• 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

The invention relates to a method for detecting a double-stranded region in a nucleic acid by (1) providing two separate, adjacent pools of a medium and a interface between the two pools, the interface having a channel so dimensioned as to allow sequential monomer-by-monomer passage of a single-stranded nucleic acid, but not of a double-stranded nucleic acid, from one pool to the other pool; (2) placing a nucleic acid polymer in one of the two pools; and (3) taking measurements as each of the nucleotide monomers of the single-stranded nucleic acid polymer passes through the channel so as to differentiate between nucleotide monomers that are hybridized to another nucleotide monomer before entering the channel and nucleotide monomers that are not hybridized to another nucleotide monomer before entering the channel.

Claims

1-24. -24. (canceled)

25. A method for characterising a nucleic acid polymer, the method comprising:

a. providing two separate pools of solution and an impermeable barrier between the two pools, the barrier containing an ion-permeable protein pore;

b. placing the nucleic acid polymer in one of the two pools;

c. measuring current flow across the ion-permeable protein pore in the presence of a voltage differential between the two pools; and

d. determining sequence-dependent characteristics of the nucleic acid polymer.

26. The method of claim 25, wherein the passage is a bacterial porin.

27. The method of claim 25, wherein the passage is a mutant bacterial porin.

28. The method of claim 25, wherein the solution is an aqueous solution.

29. The method of claim 25, wherein the nucleic acid polymer moves through the protein pore.

30. The method of claim 29, wherein the movement of the nucleic acid polymer is controlled by a molecular motor.

31. The method of claim 30, wherein the molecular motor is provided in the same pool as the nucleic acid polymer to be characterized.

32. The method of claim 25, wherein step (c) is carried out with at least a portion of the nucleic acid polymer in the pore.

33. The method of claim 25, wherein the impermeable barrier is an amphiphilic layer.