Characterization of individual polymer molecules based on monomer-interface interactions

• US 7,189,503 B2
• Filed: 12/18/2003
• Issued: 03/13/2007
• Est. Priority Date: 03/17/1995
• Status: Expired due to Fees

• Current Assignee: President and Fellows of Harvard College (Harvard Corporation), Regents of the University of California (University of California)
• Original Assignee: President and Fellows of Harvard College (Harvard Corporation), Regents of the University of California (University of California)
• Inventors: Deamer, David W., Branton, Daniel, Church, George, Akeson, Mark
• Primary Examiner(s): Patterson, Jr.; Charles L.
• Application Number:
  US10/739,585
• Publication Number:
  US 20050053961A1
• Time in Patent Office: 1,181 Days
• Field of Search: None
• US Class Current: 435/2
• CPC Class Codes: B82Y 15/00 Nanotechnology for interacting, sensing or actuating, e.g. quantum dots as markers in protein assays or molecular motors
• CPC Class Codes: B82Y 30/00 Nanotechnology for materials or surface science, e.g. nanocomposites
• CPC Class Codes: C07K 14/705 Receptors; Cell surface antigens; Cell surface determinants tumour specific antigens C07K14/4748
• CPC Class Codes: C12N 9/1241 Nucleotidyltransferases (2.7.7)
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2523/307 Denaturation or renaturation by electric current/voltage
• 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 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
• CPC Class Codes: G01N 33/68 involving proteins, peptides or amino acids involving lipoproteins G01N33/92
• CPC Class Codes: Y10S 977/795 Composed of biological material
• CPC Class Codes: Y10S 977/88 with arrangement, process, or apparatus for testing
• CPC Class Codes: Y10S 977/953 Detector using nanostructure
• CPC Class Codes: Y10S 977/957 Of chemical property or presence
• CPC Class Codes: Y10S 977/958 Of biomolecule property

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. A polymer analysis system comprising:

(a) a structure comprising an ion-permeable passage connecting a first pool and a second pool, wherein a polymer to be analyzed is placed in the first pool;

(b) a molecular motor disposed adjacent to the ion-permeable passage, wherein the molecular motor is a processive nucleic acid translocation molecule, wherein said processive nucleic acid translocation molecule is not a bacteriophage receptor capable of transporting bacteriophage DNA; and

(c) a detection system operative to detect a property of a target polymer moving relative to the ion-permeable passage.

2. The polymer analysis system of claim 1, wherein a protein comprises said ion-permeable passage.

3. The polymer analysis system of claim 2, wherein the molecular motor is fused to the protein.

4. The polymer analysis system of claim 1, wherein the structure comprises a lipid bilayer.

5. The polymer analysis system of claim 1, wherein the translocation molecule comprises a DNA polymerase, a RNA polymerase, an exonuclease, or a ribosome.

6. The polymer analysis system of claim 5, wherein the DNA polymerase is an E. coli DNA polymerase I or a T7 DNA polymerase.

7. The polymer analysis system of claim 5, wherein the RNA polymerase is T7 RNA polymerase.

8. The polymer analysis system of claim 5, wherein the exonuclease is exonuclease Lambda.

9. The polymer analysis system of claim 1, wherein the molecular motor is disposed on the side of the structure cis to the first pool.

10. The polymer analysis system of claim 1, wherein the molecular motor is disposed on the side of the structure trans to the first pool.

11. The polymer analysis system of claim 1, wherein the ion permeable passage has a diameter of about 1.0 to 4.0 nanometers.

12. The polymer analysis system of claim 1, wherein the ion-permeable passage has a diameter of about 0.5 to 2 nanometers.

13. The polymer analysis system of claim 1, wherein the detection system is operative to detect an electrical property of the target polymer.