Nucleic acid sequencing by signal stretching and data integration

• US 20030232346A1
• Filed: 06/17/2002
• Published: 12/18/2003
• Est. Priority Date: 06/17/2002
• Status: Active Grant

• Current Assignee: Intel Corporation
• Original Assignee: Intel Corporation
• Inventors: Su, Xing
• Granted Patent: US 6,952,651 B2
• Time in Patent Office: Days
• US Class Current: 435/6.12
• CPC Class Codes: B82Y 5/00 Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2565/631 being a biochannel or pore
• CPC Class Codes: Y10S 977/853 Biological sample

Abstract

The methods and apparatus 100 disclosed herein concern DNA sequencing. In some embodiments of the invention, the methods comprise measuring the distance between labeled nucleotides 220, such as nucleotides labeled with bulky groups. The methods may further comprise placing identical template DNA 200 into four reaction chambers 110, 120, 130, 140, each containing a different labeled nucleotide precursor, synthesizing complementary strands 230, 240, 250 and detecting labeled nucleotides 220. The distances between labeled nucleotides 220 may be used to construct 450 distance maps 310, 320, 330, 340 for each type of labeled nucleotide 220. The distance maps 310, 320, 330, 340 may be aligned 520 to obtain a nucleic acid sequence 210. Overlapping data analysis and frequency analysis may be used to construct 450 the distance maps 310, 320, 330, 340 and non-overlapping data analysis may be used to align 520 the distance maps into a sequence 210.

Claims

1. ) A method comprising:

(a) separating a template nucleic acid into four samples, each sample to contain a different type of labeled nucleotide;

(b) synthesizing labeled nucleic acid strands complementary in sequence to the template nucleic acids;

(c) measuring the distances between the labeled nucleotides;

(d) constructing a distance map for the labeled nucleic acid strands in each sample; and

(e) assembling the distance maps into a nucleic acid sequence of the complementary strands.

2. ) The method of claim 1, further comprising analyzing the measured distances by overlapping data analysis and frequency analysis to construct the distance maps.

3. ) The method of claim 1, wherein the distance maps are assembled into a sequence by non-overlapping data analysis.

4. ) The method of claim 1, further comprising (i) attaching the labeled nucleic acid strands to a surface;

and (ii) orienting the labeled nucleic acid strands parallel to each other.

5. ) The method of claim 1, wherein the nucleotides are labeled with bulky groups.

6. ) The method of claim 5, wherein the bulky groups are attached to the nucleotides before or after synthesis of the complementary nucleic acid strands.

7. ) The method of claim 6, wherein the bulky groups are selected from the group consisting of organic groups, quantum dots, antibodies and metallic groups.

8. ) The method of claim 7, wherein the metallic groups are nanoparticles.

9. ) The method of claim 8, wherein the nanoparticles are gold or silver, said nanoparticles between about 0.5 and 5.0 nanometer (nm) in size.

10. ) The method of claim 5, wherein the distance between bulky groups is measured by scanning probe microscopy.

11. ) The method of claim 10, wherein the distance between bulky groups is measured by atomic force microscopy or scanning tunneling microscopy.

12. ) The method of claim 1, wherein one end of the labeled nucleic acid strands is attached to biotin and the biotin binds to a surface, said surface comprising streptavidin and/or avidin.

13. ) The method of claim 1, wherein each sample comprises about 100,000 copies of the template nucleic acid.

14. ) The method of claim 1, further comprising hybridizing oligonucleotide primers to said template nucleic acids, the 5′

end of said primers attached to biotin.

15. ) The method of claim 1, wherein the labeled nucleic acid strands are synthesized by a polymerase.

16. ) A method comprising:

(a) separating a template nucleic acid into four chambers, each chamber to contain a different type of labeled nucleotide;

(b) synthesizing labeled nucleic acid strands complementary in sequence to the template nucleic acids;

(c) moving the labeled nucleic acid strands through nanopores;

(d) measuring the distances between the labeled nucleotides;

(e) constructing a distance map for the labeled nucleic acid strands in each chamber; and

(f) assembling the distance maps into a sequence for the complementary nucleic acid strands.

17. ) The method of claim 16, further comprising analyzing the measured distances by overlapping data analysis and frequency analysis to construct the distance maps.

18. ) The method of claim 16, wherein the distance maps are assembled into a sequence by non-overlapping data analysis.

19. ) The method of claim 16, wherein the nanopores are between 1 nm and 50 nm in diameter.

20. ) The method of claim 19, wherein the nanopores are between 3 nm and 10 nm in diameter.

21. ) The method of claim 19, wherein the nanopore diameter is selected to allow a single labeled nucleic acid strand to pass through a single nanopore at one time.

22. ) The method of claim 21, wherein the labeled nucleic acid strand is hybridized to a template nucleic acid.

23. ) The method of claim 16, wherein at least one end of each nucleic acid strand is attached to a distinguishable label.

24. ) The method of claim 16, wherein each nanopore is operably coupled to a detector.

25. ) The method of claim 24, wherein the detector is selected from the group consisting of an ion-channel-lipid bilayer sensor, a photodetector, an electrical detector and a mass detector.

26. ) An apparatus comprising:

(a) at least four reaction chambers, each chamber to contain a different labeled nucleotide;

(b) multiple nanopores in each reaction chamber; and

(c) a detector operably coupled to each nanopore.

27. ) The apparatus of claim 26, further comprising:

(i) an information processing system; and

(ii) a database.

28. ) The apparatus of claim 26, wherein said detectors are capable of detecting metal nanoparticles attached to labeled nucleic acid strands.

29. ) The apparatus of claim 26, wherein the reaction chambers and nanopores are part of an integrated chip.

30. ) The apparatus of claim 26, wherein the detector is selected from the group consisting of an ion-channel-lipid bilayer sensor, a photodetector, an electrical detector and a mass detector.