Use of reversible extension terminator in nucleic acid sequencing

• US 20060183145A1
• Filed: 01/27/2006
• Published: 08/17/2006
• Est. Priority Date: 01/31/2005
• Status: Active Grant

• Current Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Original Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Inventors: Turner, Stephen
• Granted Patent: US 7,476,504 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: B01L 2200/0668 Trapping microscopic beads
• CPC Class Codes: B01L 2300/0819 Microarrays; Biochips
• CPC Class Codes: B01L 2400/0454 radiation pressure, optical tweezers
• CPC Class Codes: B01L 3/5085 for multiple samples, e.g. microtitration plates
• CPC Class Codes: B82Y 30/00 Nanotechnology for materials or surface science, e.g. nanocomposites
• 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 2525/186 incorporating a non-extendable or blocking moiety
• CPC Class Codes: G02B 6/02 Optical fibres with cladding with or without a coating

Abstract

The present invention relates to the uses of reversible extension terminator in conjunction with optical confinements in nucleic acid sequencing. The apparatus and methods embodied in the present invention are particularly useful for high-throughput and low-cost nucleic acid sequencing.

Claims

1. A method for identifying a base in a template nucleic acid, comprising:

(a) providing an optical confinement;

(b) providing a reaction mixture within the optical confinement, the reaction mixture comprising the template nucleic acid, a polymerase enzyme, and at least a first nucleotide analog, wherein the first nucleotide analog comprises a removable blocking group that terminates chain extension of a nascent nucleic acid strand when the first nucleotide analog is incorporated into the nascent nucleic acid strand; and

(c) identifying the base in the template nucleic acid strand by detecting the incorporated nucleotide analog.

2. The method of claim 1, further comprising:

removing the removable blocking group; and

repeating the providing and identifying steps of (b) and (c) with at least a second nucleotide analog that comprises a removable blocking group that terminates chain extension of a nascent nucleic acid strand when the second nucleotide analog is incorporated into the nascent nucleic acid strand.

3. The method of claim 1, wherein the first nucleotide analog is present in the reaction mixture at a concentration greater than about 1 micromolar.

4. The method of claim 1, wherein the first nucleotide analog is present in the reaction mixture at a concentrations greater than about 50 micromolar.

5. The method of claim 1, wherein the first nucleotide analog is present in the reaction mixture at a concentration greater than about 100 micromolar.

6. The method of claim 1, wherein the first nucleotide analog comprises a fluorescent label.

7. The method of claim 1, wherein the removable blocking group comprises a photolabile blocking group.

8. The method of claim 1, wherein the removable blocking group comprises the fluorescent label.

9. The method of claim 1 wherein the removable blocking group is coupled to a 3′

position of the nucleotide analog.

10. The method of claim 2, wherein the first and second nucleotide analogs are concurrently present in the reaction mixture.

11. The method of claim 10, wherein the first and second nucleotide analogs each comprise a fluorescent label.

12. The method of claim 1, wherein the reaction mixture comprises at least four different nucleotide analogs.

13. The method of claim 12, wherein each of the four different nucleotide analogs comprises a different fluorescent label.

14. The method of claim 1, wherein the optical confinement provides an effective observation volume that is less than 100 zeptoliters.

15. The method of claim 1, wherein the optical confinement provides an observation volume that is less than 50 zeptoliters.

16. The method of claim 1, wherein the optical confinement provides an observation volume that is less than 10 zeptoliters.

17. The method of claim 1, wherein the optical confinement comprises a zero mode waveguide.

18. The method of claim 1, further comprising providing a plurality of optical confinements, providing a reaction mixture within each of the plurality of optical confinements, each reaction mixture comprising the template nucleic acid, a polymerase enzyme, and at least a first nucleotide analog, wherein the first nucleotide analog comprises a removable blocking group that terminates chain extension of a nascent nucleic acid strand when the first nucleotide analog is incorporated into the nascent nucleic acid strand.

19. The method of claim 18, wherein the identifying step comprises detecting incorporation of the at least first nucleotide analog into a nascent nucleic acid strand in one or more of the plurality of optical confinements.

20. The method of claim 18, wherein the identifying step comprises detecting incorporation in a subset of the plurality of optical confinements at a time.

21. The method of claim 18, wherein the identifying step comprises detecting incorporation in one optical confinement at a time.

22. The method of claim 18, wherein the identifying step comprises monitoring a plurality of optical confinements at a time.

30. The method of claim 1, wherein a single complex of the template nucleic acid and the polymerase enzyme is contained in said optical confinement.

23. A system, comprising:

an optical confinement;

a reaction mixture disposed within the optical confinement, the reaction mixture comprising a template nucleic acid, a polymerase enzyme, and at least a first nucleotide analog, wherein the first nucleotide analog comprises a removable blocking group that terminates chain extension of a nascent nucleic acid strand when the first nucleotide analog is incorporated into the nascent nucleic acid strand.

24. The system of claim 23, comprising a detector positioned proximal to the optical confinement to detect incorporation of the first nucleotide analog into the nascent nucleic acid strand.

25. The system of claim 24, wherein the first nucleotide analog comprises a fluorescent label, and the detector comprises a fluorescence detector.

26. The system of claim 23, wherein the optical confinement comprises a zero mode waveguide disposed upon or within a substrate.

27. The system of claim 23, further comprising an array of zero mode waveguides disposed upon or within a substrate.

32. The system of claim 23, wherein a single complex of the template nucleic acid and the polymerase enzyme is contained in said optical confinement.

28. A method for identifying a base in a template nucleic acid, comprising:

(a) providing a reaction mixture comprising the template nucleic acid, a polymerase enzyme, and at least a first nucleotide analog, wherein the first nucleotide analog comprises a removable blocking group that terminates chain extension of a nascent nucleic acid strand when the first nucleotide analog is incorporated into the nascent nucleic acid strand;

(b) identifying the base in the template nucleic acid strand by detecting the incorporated nucleotide analog;

(c) removing the removable blocking group from the incorporated nucleotide analog; and

(d) repeating at least the identifying and removing steps with at least a second nucleotide analog without depleting at least one reagent from the reaction mixture of (a).

29. The method of claim 28, wherein the reaction mixture is provided in an optical confinement.

31. The method of claim 29, wherein a single complex of the template nucleic acid and the polymerase enzyme is contained in said optical confinement.