Method of sequencing a nucleic acid

• US 7,244,559 B2
• Filed: 03/21/2001
• Issued: 07/17/2007
• Est. Priority Date: 09/16/1999
• Status: Expired due to Fees

• Current Assignee: 454 Life Sciences Corporation (Roche Holding AG)
• Original Assignee: 454 Life Sciences Corporation (Roche Holding AG)
• Inventors: Rothberg, Jonathan M., Berka, Jan, McDade, Keith, Simpson, John W., Colangelo, Christopher M., Dewell, Scott B., Bader, Joel S.
• Primary Examiner(s): Kim; Young J.
• Application Number:
  US09/814,338
• Publication Number:
  US 20020012930A1
• Time in Patent Office: 2,309 Days
• Field of Search: 359/30, 359/110, 359/341.1, 435/6, 435/91.1, 435/91.2, 435/8, 435/283.1, 435/287.1, 435/288.4, 435/288.7, 435/287.2, 536/23.1, 536/24.3, 536/24.33, 438/16, 438/60, 385/115, 385/116
• US Class Current: 435/5
• CPC Class Codes: B01L 2300/0636 Integrated biosensor, microarrays
• CPC Class Codes: B01L 2300/0654 Lenses; Optical fibres
• CPC Class Codes: B01L 2300/0816 Cards, e.g. flat sample carriers usually with flow in two horizontal directions
• CPC Class Codes: B01L 2300/0819 Microarrays; Biochips
• CPC Class Codes: B01L 2300/0822 Slides
• CPC Class Codes: B01L 2300/0877 Flow chambers
• CPC Class Codes: B01L 2300/0893 having a very large number of wells, microfabricated wells
• 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: B01L 3/502715 characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces
• CPC Class Codes: B01L 9/527 for microfluidic devices, e.g. used for lab-on-a-chip
• 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: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• 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 2525/307 Circular oligonucleotides
• CPC Class Codes: C12Q 2531/125 Rolling circle
• CPC Class Codes: C12Q 2533/101 Primer extension
• CPC Class Codes: C12Q 2535/101 : Sanger sequencing method, i.e. oligonucleotide sequencing using primer elongation and dideoxynucleotides as chain terminators
• CPC Class Codes: C12Q 2565/107 : Alteration in the property of hybridised versus free label oligonucleotides
• CPC Class Codes: C12Q 2565/301 : Pyrophosphate (PPi)
• CPC Class Codes: C12Q 2565/519 : characterised by the capture moiety being a single stranded oligonucleotide
• CPC Class Codes: G01N 21/0303 : Optical path conditioning in cuvettes, e.g. windows; adapted optical elements or systems; path modifying or adjustment G01N21/031 - G01N21/15 take precedence
• CPC Class Codes: G01N 21/6452 : Individual samples arranged in a regular 2D-array, e.g. multiwell plates
• CPC Class Codes: G01N 21/7703 : using reagent-clad optical fibres or optical waveguides using measurement of total internal reflection or attenuated total reflection G01N21/552; optical fibres or waveguides per se G02B

Abstract

Disclosed herein are methods and apparatuses for sequencing a nucleic acid. These methods permit a very large number of independent sequencing reactions to be arrayed in parallel, permitting simultaneous sequencing of a very large number (>10,000) of different oligonucleotides.

Claims

1. A substrate for analyzing a nucleic acid, the substrate comprising:

a cavitated fiber optic wafer fonried from a fused bundle of a plurality of individual optical fibers, each individual optical fiber having a diameter between 3 and 100 μ

m, the wafer comprising a top surface and a bottom surface, the top surface comprising at least 10,000 wells, wherein said wells are etched into the top surface of the cavitated fiber optic wafer and wherein the thickness of the wafer between the top surface and the bottom surface is between 0.5 mm and 5.0 mm in thickness;

wherein the depth of each well ranges from between one half the diameter of an individual optical fiber and three times the diameter of an individual optical fiber; and

wherein a plurality of wells on the top surface of the cavitated wafer have a nucleic acid disposed therein; and

a plurality of beads disposed within wells on the top surface of the cavitated wafer, said beads having a pyrophosphate sequencing reagent attached thereto.

2. The substrate of claim 1, wherein the nucleic acid immobilized on the wells or on said beads.

3. The substrate of claim 1, wherein the diameter of each individual optical fiber in the cavitated wafer is between 6-50 μ

m.

4. The substrate of claim 3, wherein the fiber optic surface includes two or more nucleic acids separated by approximately 10 μ

m to approximately 200 μ

m.

5. The substrate of claim 3, wherein the fiber optic surface includes two or more nucleic acids separated by approximately 10 μ

m to approximately 150 μ

m.

6. The substrate of claim 3, wherein the fiber optic surface includes two or more nucleic acids separated by approximately 150 μ

m.

7. The substrate of claim 1 wherein the wafer further comprises 10³ or more groups of nucleic acid sequences in said wells.

8. The substrate of claim 7, wherein said substrate comprises 10⁴ or more different groups of nucleic acid sequences in discrete known regions.

9. The substrate of claim 7, wherein said substrate comprises 10⁵ or more different groups of nucleic acid sequences in discrete known regions.

10. The substrate of claim 7, wherein the nucleic acid sequences are attached to the wells or beads by a linker.

11. The substrate of claim 7, wherein the nucleic acid sequences are covalently attached to the wells or beads.

16. The substrate of claim 1 wherein the substrate has a polished fiber optic surface opposite to the cavitated fiber optic surface.

17. The substrate of claim 16 wherein the polished surface allows for optical coupling to a second optical fiber.

18. The substrate of claim 1 wherein the cavitated fiber optic wafer is coated.

19. The substrate of claim 18 wherein the coating is selected from the group consisting of plastic, gold layers, organosilane reagents, photoreactive linkers, hydrophilic polymer gels and pluronic polymers.

20. The substrate of claim 1 wherein said sequencing reagent is luciferase.

21. The substrate of claim 1 wherein said sequencing reagent is sulfurylase.

12. An apparatus for processing a plurality of nucleic acids, the apparatus comprising:

a flow chamber having disposed therein a cavitated fiber optic wafer;

a cavitated fiber optic wafer formed from a fused bundle of a plurality of individual optical fibers, each individual optical fiber having a diameter between 3 and 100 μ

m, the wafer comprising a top surface and a bottom surface, the top surface comprising at least 10,000 wells, wherein said wells are etched into the top surface of the cavitated fiber optic wafer and wherein the thickness of the wafer between the top surface and the bottom surface is between 0.5 mm and 5.0 mm in thickness;

wherein the depth of each well ranges from between one half the diameter of an individual optical fiber and three times the diameter of an individual optical fiber; and

wherein a plurality of wells on the on the top surface of the cavitated wafer have a nucleic acid disposed therein;

a plurality of beads disposed within wells on the top surface of the cavitated wafer, said beads having a pyrophosphate sequencing reagent attached thereto;

fluid means for delivering pyrophosphate sequencing reagents, including sequential delivery of nucleotide triphosphates, from one or more reservoirs to the flow chamber so the nucleic acids disposed on beads in the wells on the top surface of the fiber optic wafer are exposed to the reagents; and

detection means for detecting optical signals from each well, wherein said detection means is in communication with the wells, each optical signal being indicative of reaction of the pyrophosphate sequencing reagents with the nucleic acid in a well.

13. The apparatus of claim 12, wherein the diameter of each individual optical fiber in the cavitated wafer is between 6-50 μ

m.

14. The apparatus of claim 13, wherein said detection means is a CCD camera.

15. The apparatus of claim 12, wherein the nucleic acid is DNA.

22. The apparatus of claim 12 wherein the cavitated fiber optic wafer is coated.

23. The apparatus of claim 22 wherein the coating is selected from the group consisting of plastic, gold layers, organosilane reagents, photoreactive linkers, hydrophilic polymer gels and pluronic polymers.

24. The apparatus of claim 12 wherein said sequencing reagent is luciferase.

25. The apparatus of claim 12 wherein said pyrophosphate sequencing reagent is sulfurylase.