Characterization of individual polymer molecules based on monomer-interface interactions

• US 6,362,002 B1
• Filed: 12/09/1999
• Issued: 03/26/2002
• 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: Sauer, Alexis, Branton, Daniel, Meller, Amit, Denison, Timothy J., Golovchenko, Jene, Brandin, Eric
• Primary Examiner(s): Patterson, Jr., Charles L.
• Application Number:
  US09/457,959
• Time in Patent Office: 838 Days
• Field of Search: 436/2, 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 for detecting hybridization of a polynucleic acid, 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 through the channel;

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

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

monitoring the passage characteristics of the test polynucleic acid through the channel to differentiate between a hybridized and unhybridized polynucleic acid.

2. The method of claim 1, wherein monitoring the passage characteristics of the test polynucleic acid includes comparing the passage characteristics of the test polynucleic acid to that of a control single-stranded polynucleic acid.

3. The method of claim 1, wherein the test polynucleic acid is provided in an electrically conductive medium.

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

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

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

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

8. The method of claim 4, wherein the applied voltage is variable.

9. The method of claim 8, wherein the applied voltage is varied between a first voltage that is below a threshold voltage for passage of a hybridized polynucleic acid and a second voltage that is above the threshold voltage for passage of a hybridized polynucleic acid.

10. The method of claim 1, wherein inducing passage of a hybridized polynucleic acid through the channel requires greater force or time than that required to pass an unhybridized polynucleic acid through the channel.

11. The method of claim 1, wherein the hybridized polynucleic acid comprises an intramolecular hybridization.

12. The method of claim 1, wherein the hybridized polynucleic acid comprises an intermolecular hybridization.

13. The method of claim 1, further comprising varying the temperature of the source of the test polynucleic acid.

14. The method of claim 1, wherein the passage of the hybridized polynucleic acid through the channel is slower for the passage of an unhybridized polynucleic acid.

15. A method for separating base pairs of a hybridized polynucleic acid, 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 hybridized polynucleic acid at the surface;

inducing passage of the polynucleic acid through the channel, whereby the hybridized polynucleic acid undergoes base pair separation (melts) prior to its passage.

16. The method of claim 15, wherein the double-stranded polynucleic acid is provided in an electrically conductive medium.

17. The method of claim 15, wherein the hybridized polynucleic acid comprises an intramolecular hybridization.

18. The method of claim 15, wherein the hybridized polynucleic acid comprises an intermolecular hybridization.

19. The method of claim 15, wherein passage of the polynucleic acid is induced by applying a voltage across the channel.

20. The method of claim 19, wherein the applied voltage is variable.

21. The method of claim 20, wherein the applied voltage is a pulsatile voltage.

22. The method of claim 20, wherein the applied voltage is varied between a first voltage that is below the voltage needed to melt the hybridized polynucleic acid and a second voltage that is above the voltage needed to melt the hybridized polynucleic acid.

23. The method of claim 15, wherein inducing passage of the hybridized polynucleic acid through the channel requires greater force or time than that required to pass an unhybridized polynucleic acid through the channel.

24. The method of claim 15, wherein passage of the polynucleic acid is induced by providing a polymerase or exonuclease at the channel, wherein the polymerase or exonuclease draws the nucleic acid through the channel.

25. The method of claim 21, wherein the applied voltage is varied between a first voltage that is below the voltage needed to melt the hybridized polynucleic acid and a second voltage at/or above the voltage needed to melt the hybridized polynucleic acid.