Use of reversible extension terminator in nucleic acid sequencing

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 photolabile 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 photolabile blocking group; and

repeating the providing and identifying steps of (b) and (c) with at least a second nucleotide analog that comprises a photolabile 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 the fluorescent label.

8. The method of claim 1 wherein the removable blocking group is coupled to a 3′ position of the nucleotide analog.

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

10. The method of claim 9 , wherein the first and second nucleotide analogs each comprise a fluorescent label.

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

12. The method of claim 11 , wherein each of the four different nucleotide analogs comprises a different fluorescent label.

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

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

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

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

17. 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 photolabile 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.

18. The method of claim 17 , 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.

19. The method of claim 17 , wherein the identifying step comprises detecting incorporation in a subset of the plurality of optical confinements at a time.

20. The method of claim 17 , wherein the identifying step comprises detecting incorporation in one optical confinement at a time.

21. The method of claim 17 , wherein the identifying step comprises monitoring a plurality of optical confinements at a time.

22. 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 photolabile 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.

23. The system of claim 22 , comprising a detector positioned proximal to the optical confinement to detect incorporation of the first nucleotide analog into the nascent nucleic acid strand.

24. The system of claim 23 , wherein the first nucleotide analog comprises a fluorescent label, and the detector comprises a fluorescence detector.

25. The system of claim 22 , wherein the optical confinement comprises a zero mode waveguide disposed upon or within a substrate.

26. The system of claim 22 , further comprising an array of zero mode waveguides disposed upon or within a substrate.

27. 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).

28. The method of claim 27 , wherein the reaction mixture is provided in an optical confinement.

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

30. The method of claim 28 , wherein a single complex of the template nucleic acid and the polymerase enzyme is contained in said optical confinement.

31. The system of claim 22 , wherein a single complex of the template nucleic acid and the polymerase enzyme is contained in said optical confinement.

32. The method of claim 27 , wherein the first nucleotide analog is present in the reaction mixture at a concentration greater than about 1 micromolar.

33. The method of claim 27 , wherein the first nucleotide analog is present in the reaction mixture at a concentrations greater than about 50 micromolar.

34. The method of claim 27 , wherein the first nucleotide analog is present in the reaction mixture at a concentration greater than about 100 micromolar.

35. The method of claim 27 , wherein the first nucleotide analog comprises a fluorescent label.

36. The method of claim 27 , wherein the removable blocking group comprises the fluorescent label.

37. The method of claim 27 , wherein the removable blocking group is coupled to a 3′ position of the nucleotide analog.

38. The method of claim 27 , wherein the first and second nucleotide analogs are concurrently present in the reaction mixture.

39. The method of claim 27 , wherein the first and second nucleotide analogs each comprise a fluorescent label.

40. The method of claim 27 , wherein the reaction mixture comprises at least four different types of nucleotide analogs.

41. The method of claim 40 , wherein each of the four different nucleotide analogs comprises a different fluorescent label.

42. The method of claim 28 , wherein the optical confinement provides an effective observation volume that is less than 100 zeptoliters.

43. The method of claim 28 , wherein the optical confinement provides an observation volume that is less than 50 zeptoliters.

44. The method of claim 28 , wherein the optical confinement provides an observation volume that is less than 10 zeptoliters.

45. The method of claim 28 , wherein the optical confinement comprises a zero mode waveguide.

46. The method of claim 28 , 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.

47. The method of claim 46 , 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.

48. The method of claim 46 , wherein the identifying step comprises detecting incorporation in a subset of the plurality of optical confinements at a time.

49. The method of claim 46 , wherein the identifying step comprises detecting incorporation in one optical confinement at a time.

50. The method of claim 46 , wherein the identifying step comprises monitoring a plurality of optical confinements at a time.

51. The method of claim 2 or 27 , wherein the step of removing the removable blocking group is effected by illuminating the optical confinement with an incident light beam to cleave the blocking group.