Method and apparatus for nucleic acid sequencing and identification

• US 7,005,264 B2
• Filed: 05/20/2002
• Issued: 02/28/2006
• Est. Priority Date: 05/20/2002
• Status: Active Grant

• Current Assignee: Intel Corporation
• Original Assignee: Intel Corporation
• Inventors: Su, Xing, Berlin, Andrew A.
• Primary Examiner(s): Horlick, Kenneth R.
• Assistant Examiner(s): Kim, Young J.
• Application Number:
  US10/153,125
• Publication Number:
  US 20060019247A1
• Time in Patent Office: 1,380 Days
• Field of Search: 435/6, 435/91.1, 435/91.2
• US Class Current: 435/6.17
• CPC Class Codes: B01L 3/5027 by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip B01L3/5023 takes precedence; micromixers B01F33/30; microreactors for synthesis B01J19/0093; microcapillary devices in general B81B1/00
• CPC Class Codes: B82Y 30/00 Nanotechnology for materials or surface science, e.g. nanocomposites
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2527/113 Time
• CPC Class Codes: C12Q 2563/155 Particles of a defined size, e.g. nanoparticles
• 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

The methods and apparatus 100 disclosed herein are of use for sequencing and/or identifying nucleic acids 230, 310. Nucleic acids 230, 310 containing labeled nucleotides 235, 245, 315 may be synthesized and passed through nanopores 255, 310. Detectors 257, 345 operably coupled to the nanopores 255, 310 may detect the labeled nucleotides 235, 245, 315. By determining the time intervals at which labeled nucleotides 235, 245, 315 are detected, distance maps 140 for each type of labeled nucleotide 235, 245, 315 may be compiled. The distance maps 140 in turn may be used to sequence 150 and/or identify 160 the nucleic acid 230, 310. In different embodiments of the invention, luminescent nucleotides 235, 245 or nanoparticles 315 may be detected using photodetectors 257 or electrical detectors 310. Apparatus 100 and sub-devices 200, 300 of use for nucleic acid 230, 310 sequencing 150 and/or identification 160 are also disclosed herein.

Claims

1. A method comprising:

a) placing a template nucleic acid into four chambers, each chamber being associated with different nanopores and each chamber comprising a different labeled nucleotide;

b) synthesizing a labeled nucleic acid from the template nucleic acid;

c) contacting the labeled nucleic acid in each chamber with its associated nanopore, wherein the labeled nucleic acid passes through the associated nanopore;

d) detecting a labeled nucleotide;

e) compiling a nucleotide distance map for each type of labeled nucleotide; and

f) determining the sequence of the nucleic acid from the nucleotide distance maps.

2. The method of claim 1, wherein each nanopore is operably coupled to a detector.

3. The method of claim 1, wherein only one labeled nucleic acid passes through a nanopore at a time.

4. The method of claim 1, wherein a first chamber contains labeled deoxyadenosine-5′

-triphosphate, a second chamber contains labeled deoxyguanosine-5′

-triphosphate, a third chamber contains labeled deoxycytidine-5′

-triphosphate and a fourth chamber contains labeled deoxythymidine-5′

-triphosphate or labeled deoxyuridine-5′

-triphosphate.

5. The method of claim 1, wherein the labeled nucleotide comprises between about 1% and 50% of the total amount of the nucleotide in said each chamber.

6. The method of claim 5, wherein the labeled nucleotide comprises between about 10% and 20% of the total amount of the nucleotide in said each chamber.

7. The method of claim 1, wherein the length of time between passage of a first-labeled nucleotide and a second labeled nucleotide through a nanopore corresponds to the distance along the labeled nucleic acid between the first and second labeled nucleotides.

8. The method of claim 1, wherein the labels of said labeled nucleotides are selected from the group consisting of luminescent labels, fluorescent labels, phosphorescent labels, chemiluminescent labels, conductive labels, nuclear magnetic resonance labels, mass spectroscopy labels, electron spin resonance labels, electron paramagnetic resonance labels and Raman labels.

9. The method of claim 1, wherein at least one end of the labeled nucleic acid is attached to an identifiable label.

10. The method of claim 1, wherein said labeled nucleic acid comprises luminescent labels and said labeled nucleotides are detected with a photodetector.

11. The method of claim 1, wherein said labeled nucleic acid comprises nanoparticles and said labeled nucleotides are detected with an electrical detector.

12. The method of claim 1, further comprising analyzing a multiplicity of labeled nucleic acids from each chamber.

13. A method comprising:

a) placing a template nucleic acid into four sub-devices, each sub-device comprising an upper chamber and a lower chamber separated by a sensor layer having one or more nanopores, the upper and lower chambers of each sub-device in fluid communication through the nanopores, and each upper chamber containing a different labeled nucleotide;

b) synthesizing a labeled nucleic acid from the template nucleic acid in each upper chamber;

c) passing the labeled nucleic acid in each upper chamber through its associated nanopore;

d) detecting labeled nucleotides passing through the associated nanopore with a detector, the detector being operably coupled to the associated nanopore; and

e) compiling a nucleotide distance map for each type of labeled nucleotide.

14. The method of claim 13, further comprising:

f) determining the sequence of the template nucleic acid from the nucleotide distance maps.

15. The method of claim 13, further comprising:

f) identifying the template nucleic acid from the nucleotide distance maps.

16. The method of claim 13, wherein only one labeled nucleic acid passes through a nanopore at a time.

17. The method of claim 13, wherein a first upper chamber contains labeled deoxyadenosine-5′

-triphosphate, a second upper chamber contains labeled deoxyguanosine-5′

-triphosphate, a third upper chamber contains labeled deoxycytidine-5′

-triphosphate and a fourth upper chamber contains labeled deoxythymidine-5′

-triphosphate or labeled deoxyuridine-5′

-triphosphate.

18. The method of claim 13, wherein the labeled nucleotide comprises between about 1% and 50% of the total amount of the nucleotide in said each chamber.

19. The method of claim 13, wherein the length of time between passage of a first-labeled nucleotide and a second labeled nucleotide through a nanopore corresponds to the distance along the labeled nucleic acid between the first and second labeled nucleotides.

20. The method of claim 13, wherein the labels of said labeled nucleotides are selected from the group consisting of luminescent labels, fluorescent labels, phosphorescent labels, chemiluminescent labels, conductive labels, nuclear magnetic resonance labels, mass spectroscopy labels, electron spin resonance labels, electron paramagnetic resonance labels and Raman labels.

21. The method of claim 13, wherein at least one end of the labeled nucleic acid is attached to an identifiable label.

22. The method of claim 13, wherein the labeled nucleic acid comprises luminescent labels and the detector is a photodetector.

23. The method of claim 13, wherein the labeled nucleic acid comprises nanoparticles and the detector is an electrical detector.

24. The method of claim 13, wherein the detector is capable of separately detecting signals from all sub-devices.

25. The method of claim 13, wherein the detector is operably coupled to a computer.

26. The method of claim 13, wherein the sensor layers comprise one or more support layers, photon-sensing layers, and light opaque layers.

27. The method of claim 26, wherein the detector is a photodetector operably coupled to the photon-sensing layer.

28. The method of claim 13, wherein the sensor layers comprise at least two insulating layers and at least one conducting layer.

29. The method of claim 28, wherein the detector is an electrical detector operably coupled to the conducting layer.

30. The method of claim 13, wherein one or more of the sub-devices includes electrodes in the upper and lower chambers operably coupled to a voltage regulator configured to generate an electrical field that drives labeled nucleic acids from the upper to the lower chamber through the nanopores.