Method of sequencing a nucleic acid

• US 7,211,390 B2
• Filed: 02/26/2004
• Issued: 05/01/2007
• 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., Dewell, Scott B., Colangelo, Christopher M., Bader, Joel S.
• Primary Examiner(s): Kim; Young J.
• Application Number:
  US10/788,529
• Publication Number:
  US 20040248161A1
• Time in Patent Office: 1,160 Days
• Field of Search: None
• US Class Current: 435/6.1
• 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 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 30/00 Nanotechnology for materials or surface science, e.g. nanocomposites
• 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 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: G01N 21/6452 Individual samples arranged in a regular 2D-array, e.g. multiwell plates
• CPC Class Codes: G01N 2201/08 Optical fibres; light guides
• CPC Class Codes: G01N 2201/0846 : Fibre interface with sample, e.g. for spatial resolution

Abstract

Disclosed herein are methods and apparatuses for sequencing a nucleic acid. In one aspect, the method includes annealing a population of circular nucleic acid molecules to a plurality of anchor primers linked to a solid support, and amplifying those members of the population of circular nucleic acid molecules which anneal to the target nucleic acid, and then sequencing the amplified molecules by detecting the presence of a sequence byproduct such as pyrophosphate.

Claims

1. A method for sequencing a nucleic acid, the method comprising:

providing a substrate which comprises;

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 top surface of the cavitated wafer have a nucleic acid therein; and

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

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

detecting optical signals from each well using a detection means that 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, thereby sequencing the nucleic acid.

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

3. The method of claim 1, wherein the-nucleic acid is DNA.

4. The method of claim 1, wherein the-nucleic acid is genomic DNA or cDNA.

5. The method of claim 1, wherein the nucleic acid is 10–

1000 nucleotides in length.

6. The method of claim 1, wherein pyrophosphate is produced as a sequencing byproduct.

7. The method of claim 6, wherein the pyrophosphate is detected by contacting the sequencing byproduct with a sulfurylase under conditions that allow formation of ATP.

8. The method of claim 7, wherein the sulfurylase is a thermostable sulfurylase.

9. The method of claim 6, further comprising adding apyrase to degrade unreacted nucleotide triphosphates.

10. The method of claim 6, further comprising washing the top surface of the fiber optic wafer with a buffer between each delivery of the nucleotide triphosphates.

11. The method of claim 10, wherein the-buffer includes apyrase.

12. The method of claim 1, wherein the diameter of each individual optical fiber in the cavitated wafer is between 6–

50 μ

m.

13. The method of claim 1, wherein the nucleic acid is sequenced in the presence of a dATP analog.

14. The method of claim 13, wherein the dATP analog is a thio ATP.

15. The method of claim 1, wherein the fiber optic surface includes two or more nucleic acids separated by approximately 10 μ

m to approximately 200 μ

m.

16. The method of claim 15, wherein the fiber optic surface includes two or more nucleic acids separated by approximately 50 μ

m to approximately 150 μ

m.

17. The method of claim 15, wherein the fiber optic surface includes two or more nucleic acids separated by approximately 100 μ

m to approximately 150 μ

m.

18. The method of claim 15, wherein the fiber optic surface includes two or more nucleic acids separated by approximately 150 μ

m.

19. The method of claim 1, wherein said detection means is a CCD camera.

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

21. The method of claim 20, wherein the polished surface allows for optical coupling to a second optical fiber.

22. The method of claim 1, wherein the cavitated fiber optic wafer is coated.

23. The method 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 method of claim 1, wherein said pyrophosphate sequencing reagent is luciferase.

25. The method of claim 1, wherein said pyrophosphate sequencing reagent is sulfurylase.

26. The method of claim 1, wherein said substrate further comprises 10³ or more nucleic acids in said wells.

27. The method of claim 1, wherein said substrate comprises 10⁴ or more nucleic acids in said wells.

28. The method of claim 1, wherein said substrate comprises 10⁵ or more nucleic acids in said wells.

29. The method of claim 26, wherein the nucleic acids are attached to the wells or beads by a linker.

30. The method of claim 26, wherein the nucleic acids are covalently attached to the wells or beads.