Characterization of individual polymer molecules based on monomer-interface interactions

• US 6,673,615 B2
• Filed: 02/20/2002
• Issued: 01/06/2004
• Est. Priority Date: 03/17/1995
• Status: Expired due to Term

• Current Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Original Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Inventors: Branton, Daniel, Meller, Amit, Denison, Timothy J., Golovchenko, Jene, Sauer, Alexis, Brandin, Eric
• Primary Examiner(s): Patterson, Jr., Charles L.
• Application Number:
  US10/079,178
• Publication Number:
  US 20030044816A1
• Time in Patent Office: 685 Days
• Field of Search: 436/5, 436/151, 435/6
• US Class Current: 436/2
• 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 method of detecting alterations or mutations in a polynucleic acid sequence, comprising:

providing a surface containing a channel of a dimension sufficient to allow sequential monomer-by-monomer passage of a single-stranded polynucleic acid, but not of a double-stranded polynucleic acid;

providing a source of a test polynucleic acid to be analyzed at one side of the surface;

inducing passage of the test polynucleic acid through the channel and monitoring the passage of the test polynucleic acid through the channel; and

wherein the method is performed at a temperature that allows a probe to hybridize to the test polynucleic acid if the probe is perfectly complementary to a portion of the polynucleic acid, but increases the probability of no hybridization occurring if the probe varies from being perfectly complementary to the test polynucleic acid.

2. The method of claim 1, wherein the source of the test polynucleic acid comprises an electrically conductive medium.

3. The method of claim 2, wherein inducing the passage of the test polynucleic acid comprises applying a voltage across the interface.

4. The method of claim 3, wherein ionic flow within the channel is monitored.

5. The method of claim 4, wherein duration of an ionic flow blockage is measured.

6. The method of claim 4, wherein magnitude of an ionic flow blockage is measured.

7. The method of claim 1, further comprising:

monitoring passage of the test polynucleic acid through the channel at a first temperature at which the test polynucleotide is unhybridized; and

monitoring passage of the test polynucleic acid through the channel at a second temperature at which the test polynucleic acid is hybridized.

8. A method for evaluating a polymer having improved sensitivity, comprising:

providing a surface containing a channel of a dimension sufficient to allow sequential monomer-by-monomer passage of a single-stranded polynucleic acid, but not of a double-stranded polynucleic acid;

providing a polynucleic acid source on one side of the surface, said polynucleic acid source being a hybridized polynucleic acid for which information related to a single strand thereof is desired;

applying a voltage across the channel to induce passage of a single-stranded polynucleic acid through the channel by subjecting the hybridized polynucleic acid to conditions in which the hybridized polynucleic acid undergoes base pair separation (melts) prior to its passage through the channel, such that the passage of the single-stranded polynucleic acid is characterized by higher duration time in the channel or higher ionic current flow through the channel, as compared to conditions suitable for inducing passage of a single-stranded polynucleic acid.

9. A method for evaluating a polymer molecule, the polymer molecule including linearly connected monomer residues, the method comprising:

providing a candidate polymer molecule in a liquid;

providing an interface in contact with the liquid, the interface capable of interacting with the candidate polymer molecule;

causing the candidate polymer molecule to traverse a region of the interface whereby individual monomer residues of the polymer molecule sequentially interact with the interface and data suitable to determine a monomer-dependent characteristic of the polymer molecule are obtained.

10. A method for evaluating a polymer molecule, the polymer molecule including linearly connected monomer residues, the method comprising:

providing a candidate polymer molecule in a liquid; and

causing the candidate polymer molecule to traverse an ion permeable passage in an otherwise impermeable barrier in the liquid, whereby individual monomer resides of the polymer molecule sequentially pass across the ion permeable passage and data suitable to determine a monomer-dependant characteristic of the polymer molecule is obtained.