Apparatus and method for sequencing a nucleic acid

• US 7,335,762 B2
• Filed: 08/15/2002
• Issued: 02/26/2008
• 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: McDade, Keith, Colangelo, Christopher M., Dewell, Scott B., Bader, Joel S., Weiner, Michael P., Rothberg, Jonathan M., Berka, Jan, Simpson, John W.
• Primary Examiner(s): Kim; Young J.
• Application Number:
  US10/222,592
• Publication Number:
  US 20030100102A1
• Time in Patent Office: 2,021 Days
• Field of Search: 422/80, 422/82.05, 435/6, 435/91.1, 435/91.2, 536/24.3, 536/24.33
• US Class Current: 536/24.3
• 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 cuvette for simultaneously monitoring and recording an individual light emission in an array of reaction chambers comprising:

(a) a detection chamber, comprising an opening which comprises an array comprised from a cavitated fiber optic wafer formed from a fused bundle of a plurality of individual optical fibers, wherein 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 400,000 reaction chambers, wherein said reaction chambers 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 reaction chamber ranges from between one half the diameter of an individual optical fiber and three times the diameter of an individual optical fiber;

said detection chamber further comprising at least one surface or opening that allows transmission of said light emission, said reaction chamber adapted to receive at least one analyte;

(b) a receptacle within said detection chamber to align said array with an optical detector; and

(c) affluent and effluent outlets connected to said detection chamber to allow said array to be in fluid communication with one or more reagent reservoirs.

2. The cuvette of claim 1 further comprising a plurality of optical fibers that forms an optical fiber bundle;

wherein said bundle is capable of transmitting light emitted from said reaction chamber to said optical detector; and

wherein each optical fiber is aligned to transmitted the light emission of only one said reaction chamber.

3. The cuvette of claim 2 wherein the optical fiber bundle contains at least one optical fiber per reaction chamber.

4. The cuvette of claim 2 wherein the optical fiber bundle contains at least 4 optical fibers per reaction chamber.

5. The cuvette of claim 1 wherein said detection chamber is adapted to receive a fluid optical coupler to transmit said light emission to said optical detector.

6. The cuvette of claim 5 wherein said fluid optical coupler is microscope oil.

7. The cuvette of claim 5 wherein said fluid optical coupler is an aqueous solution.

8. The cuvette of claim 1 wherein said array is in optical communication with said optical detector through an optical lens.

9. The cuvette of claim 8 wherein said optical lens is a compound lens containing more than one optical element.

10. The cuvette of claim 1 further comprising an optical detection system adapted to detect light emission from each said reaction chamber.

11. The cuvette of claim 10 wherein said optical detection system is a CCD camera.

12. The cuvette of claim 11 wherein said CCD camera comprises at least one individually addressable pixel per said reaction chamber.

13. The cuvette of claim 11 wherein said CCD camera comprises at least 4 individually addressable pixel per said reaction chamber.

14. The cuvette of claim 1 further comprising thermo coupling means to connect said array to a thermal source.

15. The cuvette of claim 14 wherein said thermal source is selected from the group consisting of a heating source, a cooling source, and a combination thereof.

16. The cuvette of claim 1 further comprising a reagent delivery means between said affluent outlet and said one or more reagent reservoir.

17. The cuvette of claim 16 wherein said reagent delivery means is capable of delivering an activated nucleotide 5′

triphosphate precursor of one known nitrogenous base to each said reaction chamber.

18. The cuvette of claim 17 wherein said activated nucleotide 5′

triphosphate is selected from the group consisting of ATP, GTP, TTP, CTP and UTP.

19. The cuvette of claim 16 wherein said reagent delivery means is connected to one or more reagent reservoirs to sequentially add two or more activated nucleotide 5′

triphosphate precursor of one known nitrogenous base to each said reaction chamber.

20. The cuvette of claim 1 further containing reversible coupling means at said affluent and effluent outlets to reversibly connect said cuvette to said one or more reagent reservoirs.

21. The cuvette of claim 1 further comprising a data collection system in communication with the optical detection system.

22. The cuvette of claim 1, wherein the optical detector is a CCD camera.

23. The cuvette of claim 1, wherein the analyte is nucleic acid.

24. The cuvette of claim 1, wherein the analyte is immobilized on one or more mobile solid supports that are disposed in the reaction chamber.

25. A cuvette for simultaneously monitoring and recording an individual light emission in an array of reaction chambers comprising:

(a) a detection chamber, comprising an opening which comprises an array comprised from a cavitated fiber optic wafer formed from a fused bundle of a plurality of individual optical fibers, wherein 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 400,000 reaction chambers, wherein said reaction chambers 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 reaction chamber ranges from between one half the diameter of an individual optical fiber and three times the diameter of an individual optical fiber;

said detection chamber further comprising at least one surface or opening that allows transmission of said light emission, said reaction chamber adapted to receive at least one analyte;

(b) a receptacle within said detection chamber to align said array with an optical detector;

(c) affluent and effluent outlets connected to said detection chamber to allow said array to be in fluid communication with one or more reagent reservoirs; and

(d) wherein 50% to 100% of the reaction chambers have a mobile solid support disposed therein, said mobile solid support having at least one pyrophosphate sequencing reagent immobilized thereon.

26. The cuvette of claim 25, wherein the diameter of each individual optical fiber in the cavitated wafer is between 6-50 μ

m.

27. The cuvette of claim 25, wherein a plurality of the reaction chambers have beads disposed therein, said beads having nucleic acids immobilized thereon.