SYSTEM AND METHOD FOR NUCLEIC ACIDS SEQUENCING BY PHASED SYNTHESIS

• US 20090312188A1
• Filed: 06/10/2009
• Published: 12/17/2009
• Est. Priority Date: 06/16/2008
• Status: Active Grant

• Current Assignee: LDIP, LLC
• Original Assignee: iNDx Lifecare, Inc.
• Inventors: Herron, James, Duer, Reuven
• Granted Patent: US 8,747,751 B2
• Time in Patent Office: Days
• US Class Current: 506/6
• CPC Class Codes: B01J 2219/00529 DNA chips
• CPC Class Codes: B01J 2219/00596 Solid-phase processes
• CPC Class Codes: B01J 2219/00659 Two-dimensional arrays
• CPC Class Codes: B01J 2219/00704 integrated with the reactor apparatus
• CPC Class Codes: B01J 2219/00711 Light-directed synthesis
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: B01L 2300/0654 Lenses; Optical fibres
• CPC Class Codes: B01L 2300/0819 Microarrays; Biochips
• CPC Class Codes: B01L 3/502715 characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces
• CPC Class Codes: B01L 7/52 with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples amplification or hybridisation processes per se C12Q1/68; controlling sequential reactions for synthesis B01J19/0046
• CPC Class Codes: B82Y 30/00 Nanotechnology for materials or surface science, e.g. nanocomposites
• CPC Class Codes: C12N 15/1068 Template (nucleic acid) mediated chemical library synthesis, e.g. chemical and enzymatical DNA-templated organic molecule synthesis, libraries prepared by non ribosomal polypeptide synthesis [NRPS], DNA/RNA-polymerase mediated polypeptide synthesis
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2523/319 Photocleavage, photolysis, photoactivation
• CPC Class Codes: C12Q 2535/107 Maxam and Gilbert method, i.e. sequential release and detection of nucleotides
• CPC Class Codes: C12Q 2565/607 being a sensor, e.g. electrode
• CPC Class Codes: G01N 2021/6441 with two or more labels
• CPC Class Codes: G01N 21/6452 Individual samples arranged in a regular 2D-array, e.g. multiwell plates
• CPC Class Codes: G01N 21/648 using evanescent coupling or surface plasmon coupling for the excitation of fluorescence
• CPC Class Codes: G02B 27/56 : Optics using evanescent waves, i.e. inhomogeneous waves
• CPC Class Codes: G02B 6/24 : Coupling light guides

Abstract

A system and methods of sequencing a nucleic acid by detecting the identity of a fluorescent nucleotide analogue incorporated at the 3′ end of a growing nucleic acid strand are provided. One method includes the steps of (a) immobilizing a plurality of complexes comprising a template nucleic acid, a primer configured to hybridize to the template and a polymerase, at a plurality of optical sensing sites of a substrate, wherein the substrate is part of a waveguide-based optical scanning system; (b) extending the primer by a single nucleotide with the polymerase and one or more fluorescent nucleotide analogues using a polymerase extension reaction, wherein each type of fluorescent nucleotide analogue comprises a unique fluorescent tag optionally configured to inhibit further primer extension and/or a blocking agent at the 3′ end and wherein incorporation of the fluorescent nucleotide analogue reversibly terminates the polymerase extension reaction; (c) detecting the unique tag of the fluorescent nucleotide analogue by optically scanning the substrate using the optical scanning system to identify the fluorescent nucleotide analogue incorporated by the polymerase reaction; (d) recording the results of the optical scanning of the substrate; (e) reversing the termination of the polymerase extension reaction by providing a photo-cleaving pulse of light to one or more of the optical sensing sites of the substrate to cleave the fluorescent tag or the blocking agent; and (f) repeating steps (b) through (e).

Claims

1. A scanning sensor system for sequencing a nucleic acid comprising:

a substrate comprising a plurality of substantially parallel excitation waveguides, and a plurality of substantially parallel collection waveguides, the excitation waveguides and collection waveguides crossing to form a two-dimensional array of intersection regions where an excitation waveguide and a collection waveguide cross and provide optical communication with the intersection region at each crossing;

a plurality of optical sensing sites arranged in optical communication with the intersection regions;

one or more switchable light sources coupled to and in optical communication with the excitation waveguides of the substrate, and comprising a scanning light source input and a photo-cleaving light source input;

a light dispersive module coupled to and in optical communication with the collection waveguides of the substrate, and comprising an array of elements;

a detector coupled to and in optical communication with the light dispersive module.

2. The system of claim 1, wherein a scanning light source is coupled to a first switchable light source coupled to and in optical communication with the excitation waveguides at a first side of the substrate and a photo-cleaving light source coupled to a second switchable light source coupled to and in optical communication with the excitation waveguides at a second side of the substrate.

5. The system of any preceding claim, wherein the dispersive module is configured to disperse light from one or more of the collection waveguides to a plurality of elements in the detector.

6. The system of claim 5, wherein the dispersive module is configured to disperse light from a given collection waveguide to four or more elements in the detector.

7. The system of claim 6, wherein the light dispersed from the dispersive module comprises a plurality of light wavelengths.

8. The system of claim 7, wherein the plurality of wavelengths comprises four or more light wavelengths.

9. The system of claim 1, 2, 3 or 4, wherein the photo-cleaving light source emits light having a wavelength ranging between 400 nm and 2000 nm.

10. The system of claim 9, wherein the photo-cleaving light source input is coupled to an ultra-violet light source.

11. The system of claim 10, wherein the ultra-violet light source emits light having a wavelength ranging between 100 nm and 400 nm.

3. A scanning sensor system for sequencing a nucleic acid comprising:

a substrate comprising a plurality of substantially parallel excitation waveguides, and a plurality of substantially parallel collection waveguides, the excitation waveguides and collection waveguides crossing to form a two-dimensional array of intersection regions where an excitation waveguide and a collection waveguide cross and provide optical communication with the intersection region at each crossing;

a plurality of optical sensing sites arranged in optical communication with the intersection regions;

a switchable light source in optical communication with the excitation waveguides, and comprising a scanning light source input

a photo-cleaving light source and light delivery system arranged external to the substrate;

a light dispersive module coupled to and in optical communication with the collection waveguides of the substrate, and comprising an array of elements;

a detector coupled to and in optical communication with the light dispersive module.

4. The system of claim 3 wherein the light delivery system comprises a photo-cleaving light source input.

12. A method of sequencing a nucleic acid by detecting the identity of a fluorescent nucleotide analogue incorporated at the 3′

end of a growing nucleic acid strand comprising;

(a) immobilizing a plurality of complexes comprising a template nucleic acid, a primer configured to hybridize to the template and a polymerase, at a plurality of optical sensing sites of a substrate, wherein the substrate is part of a waveguide-based optical scanning system;

(b) extending the primer by a single nucleotide with the polymerase and one or more fluorescent nucleotide analogues using a polymerase extension reaction, wherein each type of fluorescent nucleotide analogue comprises a unique fluorescent tag optionally configured to inhibit further primer extension and/or a blocking agent at the 3′

end and wherein incorporation of the fluorescent nucleotide analogue reversibly terminates the polymerase extension reaction;

(c) detecting the unique tag of the fluorescent nucleotide analogue by optically scanning the substrate using the optical scanning system to identify the fluorescent nucleotide analogue incorporated by the polymerase reaction;

(d) recording the results of the optical scanning of the substrate;

(e) reversing the termination of the polymerase extension reaction by providing a photo-cleaving pulse of light to one or more of the optical sensing sites of the substrate to cleave the fluorescent tag or the blocking agent; and

(f) repeating steps (b) through (e).

13. The method of claim 12 wherein the primer is immobilized at the plurality of optical sensing sites prior to formation and immobilization of the plurality of complexes.

14. The method of claim 13, wherein the primers are covalently immobilized at the optical sensing sites.

15. The method of claim 13, wherein the primers are immobilized using a photo-cleavable linker at the optical sensing sites.

16. The method of claim 12 wherein the polymerase is immobilized at the plurality of optical sensing sites prior to formation and immobilization of the plurality of complexes.

17. The method of claim 16, wherein the polymerases are covalently immobilized at the optical sensing sites prior to immobilizing the plurality of complexes.

18. The method of claim 12, wherein the primers are immobilized using a photo-cleavable linker at the optical sensing sites and the polymerases are covalently immobilized at the optical sensing sites prior to formation and immobilization of the plurality of complexes.

19. The method of claim 18, wherein prior to step (b) immobilized primer and template duplexes are formed, and a photo-cleaving pulse of light is provided to cleave the photo-cleavable linker and release the duplexes, wherein the released duplexes subsequently bind to the immobilized polymerases and form the immobilized plurality of complexes.

20. The method of claim 12, wherein step (b) is performed before step (c) without a washing step between steps (b) and (c).

21. The method of claim 12, wherein step (f) further comprises performing step (e) before repeating step (b) without a washing step between steps (e) and (b).

22. The method of claim 12, wherein the nucleic acid is DNA.

23. The method of claim 12, wherein the fluorescent nucleotide analogs comprise four different dNTPs, wherein each dNTP is labeled with a different fluorescent tag.

24. The method of claim 23, wherein the fluorescent tags are attached to the dNTPs through a photo-cleavable chemical bond.

25. A method of sequencing a single nucleic acid molecule by detecting the identity of a fluorescent nucleotide analogue after the nucleotide analogue is incorporated into a growing nucleic acid strand comprising:

(a) immobilizing a complex comprising a template nucleic acid, a primer configured to hybridize to the template and a polymerase, at an optical sensing sites of a substrate, wherein the substrate is part of a waveguide-based optical scanning system;

(b) extending the primer by a single nucleotide with the polymerase and one or more fluorescent nucleotide analogues using a polymerase extension reaction, wherein each fluorescent nucleotide analogue comprises a fluorescent tag optionally configured to inhibit further primer extension and/or a blocking agent at the 3′

end of the nucleotide analog and wherein incorporation of the fluorescent nucleotide analogue terminates the polymerase extension reaction;

(c) detecting the unique label attached to the fluorescent nucleotide analogue by optically scanning the substrate using the optical scanning system to identify the fluorescent nucleotide analogue incorporated by the polymerase reaction;

(d) recording the results of the optical scanning of the substrate;

(e) providing a photo-cleaving pulse of light to one or more of the optical sensing sites of the substrate to cleave the fluorescent tag and/or the blocking agent; and

(f) repeating steps (b) through (e).

26. The method of claim 25 wherein the primer is immobilized at the plurality of optical sensing sites prior to formation and immobilization of the complex.

27. The method of claim 26, wherein the primer is covalently immobilized at the optical sensing sites.

28. The method of claim 26, wherein the primer is immobilized using a photo-cleavable linker at the optical sensing sites.

29. The method of claim 25 wherein the polymerase is immobilized at the plurality of optical sensing sites prior to formation and immobilization of the complex.

30. The method of claim 27, wherein the polymerase is covalently immobilized at the optical sensing sites prior to immobilizing the complex.

31. The method of claim 25, wherein the primer is immobilized using a photo-cleavable linker at the optical sensing sites and polymerase is covalently immobilized at the optical sensing sites prior to formation and immobilization of the complex.

32. The method of claim 31, wherein prior to step (b) immobilized primer and template duplexes are formed, and a photo-cleaving pulse of light is provided to cleave the photo-cleavable linker and release the duplexes, wherein the released duplexes subsequently bind to the immobilized polymerase and form the immobilized complex.

33. The method of claim 25, wherein step (b) is performed before step (c) without a washing step between steps (b) and (c).

34. The method of claim 25, wherein step (f) further comprises performing step (e) before repeating step (b) without a washing step between steps (e) and (b).

35. The method of claim 25, wherein the nucleic acid is DNA.

36. The method of claim 25, wherein the fluorescent nucleotide analogs comprise four different dNTPs, wherein each is labeled with a different fluorescent tag.

37. The method of claim 36, wherein the fluorescent tags are attached to the dNTPs through a photo-cleavable chemical bond.