Sequencing and Genotyping Using Reversibly Terminating Nucleotides

• US 20080293071A1
• Filed: 12/20/2006
• Published: 11/27/2008
• Est. Priority Date: 12/21/2005
• Status: Active Grant

• Current Assignee: Roche Molecular Systems (Roche Holding AG)
• Original Assignee: Roche Molecular Systems (Roche Holding AG)
• Inventors: Gupta, Amar, Gelfand, David H.
• Granted Patent: US 9,206,476 B2
• Time in Patent Office: Days
• US Class Current: 435/6.12
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2521/301 Endonuclease
• CPC Class Codes: C12Q 2521/319 Exonuclease
• CPC Class Codes: C12Q 2521/525 Phosphatase
• CPC Class Codes: C12Q 2525/186 incorporating a non-extendable or blocking moiety

Abstract

The invention provides a method of determining the nucleotide sequence of a target nucleic acid using a reversibly terminating nucleotide that is modified at the 2′ position.

Claims

1. A method of sequencing at least a portion of a template nucleic acid, the method comprising:

(a) incubating at least one template nucleic acid with at least one polymerase, at least one 2′

-modified reversible terminator nucleotide, and at least one primer nucleic acid that is complementary to at least a subsequence of the template nucleic acid, whereby the polymerase extends the primer nucleic acid to produce at least one primer extension product that incorporates the 2′

-modified reversible terminator nucleotide at a 3′

-terminal end of the primer extension product;

(b) removing a blocking group from a 2′

position of the 2′

-modified reversible terminator nucleotide at the 3′

-terminal end of the primer extension product; and

,

(c) identifying the 2′

-modified reversible terminator nucleotide in the primer extension product prior to and/or during (b), whereby at least a portion of a base sequence of the template nucleic acid is determinable from the identified 2′

-modified reversible terminator nucleotide, thereby sequencing at least the portion of the template nucleic acid.

2. The method of claim 1, wherein the blocking group at the 2′

position is a phosphate.

3. The method of claim 2, wherein (b) comprises incubating the primer extension product with an activity that removes the phosphate at the 2′

position.

4. The method of claim 3, wherein the activity that removes the 2′

position phosphate is a phosphatase.

5. The method of claim 3, wherein the activity that removes the 2′

position phosphate is an exonuclease III.

6. The method of claim 3, wherein the activity that removes the 2′

position phosphate is an endonuclease IV.

7. The method of claim 3, wherein the activity that removes the 2′

position phosphate is polynucleotide kinase.

8. The method of claim 3, wherein the activity that removes the 2′

position phosphate is a phosphodiesterase or a combination of a phosphodiesterase and a phosphatase.

9. The method of claim 1, wherein the 2′

modified reversible terminator nucleotide is selected from the group consisting of;

10. The method of claim 1, wherein the 2′

-modified reversible terminator nucleotide is labeled with at least one labeling moiety.

11. The method of claim 10, wherein the labeling moiety comprises a fluorescent dye, a luminescent molecule, or a radioisotope.

12. The method of claim 11, wherein the labeling moiety is a fluorescent dye.

13. The method of claim 10, where in the labeling moiety is attached to the 2′

-modified reversible terminator nucleotide at the base via a cleavable linker, and the method further comprises a step of cleaving the linker.

14. The method of claim 10, wherein the labeling moiety is attached to a sugar residue of the 2′

-modified reversible terminator nucleotide.

15. The method of claim 10, where in the labeling moiety is attached to a phosphate present at the 2′

position of the 2′

-modified reversible terminator nucleotide.

16. The method of claim 10, wherein the 2′

-modified reversible terminator nucleotide is linked to two labeling moieties that comprise a donor and an acceptor.

17. The method of claim 16, wherein the donor and the acceptor are a reporter and a quencher pair.

18. The method of claim 16, wherein the two labeling moieties are capable of undergoing fluorescence resonance energy transfer.

19. The method of claim 17, wherein the quencher moiety is linked to the base of the 2′

-modified reversible terminator nucleotide and the reporter moiety is linked to a phosphate, with the proviso that the phosphate is not the alpha phosphate of the 2′

-modified reversible terminator nucleotide.

20. The method of claim 19, wherein the phosphate is a gamma phosphate.

21. The method of claim 19, wherein the phosphate is a phosphate present at the 2′

position of the 2′

-reversible terminator nucleotide.

22. The method of claim 16, wherein one labeling moiety is linked to a phosphate present at the 2′

position of the 2′

-modified reversible terminator nucleotide and the second labeling moiety is linked to a second phosphate.

23. The method of claim 22, wherein the second phosphate is a gamma phosphate.

24. The method of claim 1, wherein the reaction mixture comprises four different 2′

-modified reversible terminator nucleotides, each having a different base and labeled with a different labeling moiety.

25. The method of claim 1, wherein the detecting step comprises detecting pyrophosphate generated upon incorporation of the 2′

-modified reversible terminator nucleotide.

26. The method of claim 1, wherein the 2′

-modified reversible terminator nucleotide comprises a 2′

-monophosphate-3′

-hydroxyl nucleotide.

27. The method of claim 1, comprising repeating (a)-(c) one or more times.

28. A kit for sequencing by synthesis, comprising:

at least one 2′

-modified reversible terminator nucleotide, and

a reagent for removing the modification at the 2′

position of the 2′

-modified reversible terminator nucleotide.

29. The kit of claim 28, wherein the 2′

modified reversible terminator nucleotide as a phosphate as the 2′

modification, and the reagent is selected from the group consisting of a phosphatase, an exonuclease III, an endonuclease IV, and a polynucleotide kinase.

30. The kit of claim 28, further comprising a polymerase.

31. The kit of claim 28, wherein the 2′

-modified reversible terminator nucleotide is attached to a label.

32. The kit of claim 28, further comprising at least one extendable nucleotide.

33. A method of genotyping, comprising:

a) incubating a template nucleic acid in a reaction mixture comprising a primer, a polymerase and a 2′

-modified reversible terminator nucleotide, said modified reversible terminator nucleotide comprising one of the four naturally occurring bases or analogs thereof and further comprising a modification at the 2′

position that terminates synthesis under conditions in which the primer anneals to the template nucleic acid and is extended by the polymerase present in the reaction to form an extended product;

b) determining if a signal indicative of incorporation of the 2′

-modified reversible terminator nucleotide into the extended product is present;

c) if said signal is not present, repeating steps a) and b) with a different 2′

-modified reversible terminator nucleotide, said different 2′

-modified reversible terminator nucleotide comprising a different one of the four naturally occurring bases or analogs thereof,

d) incubating the extended product with an activity that removes the modification at the 2′

position present on the reversible terminator nucleotide,

e) repeating steps a) to d) a desired number of times to determine the sequence of a portion of said template nucleic acid, and

f) comparing the sequence of the portion of the template nucleic acid to a known sequence of a polymorphic site.

34. A system for sequencing a nucleic acid, comprising:

a thermal cycler, comprising a reaction chamber wherein a template nucleic acid is incubated in a reaction mixture comprising a primer, a polymerase and a 2′

-modified reversible terminator nucleotide, said modified reversible terminator nucleotide comprising one of the four naturally occurring bases or analogs thereof and further comprising a modification at the 2′

position that terminates synthesis, the thermal cycler being effective to create conditions in which the primer anneals to the template nucleic acid and is extended by the polymerase present in the reaction to form an extended product;

said reaction chamber having an inlet port and an outlet port for adding and removing reagents; and

a detector, the detector being effective to detect a signal indicative of incorporation of the 2′

-modified reversible terminator nucleotide into the extended product.

35. A labeled 2′

-modified reversible terminator nucleotide selected from the group consisting of;

36. A method of sequencing at least a portion of a template nucleic acid, comprising:

a) incubating the template nucleic acid in a reaction mixture comprising a primer, a polymerase, and a 2′

-modified reversible terminator nucleotide, under conditions in which the primer anneals to the template nucleic acid and is extended by the polymerase present in the reaction to form an extended product;

b) determining the presence of a signal indicative of incorporation of the 2′

-modified reversible terminator nucleotide into the extended product;

c) incubating the extended product with an activity that removes the modification at the 2′

position present on the reversible terminator nucleotide;

d) repeating steps a) to c) a desired number of times to determine the sequence of at least portion of the template nucleic acid.

37. The method of claim 36, wherein step (a) is carried out using a plurality of different 2′

-modified reversible terminator nucleotides in a single reaction mixture, each different 2′

-modified reversible terminator nucleotide comprising a different base.

38. The method of claim 37, wherein each different 2′

-modified reversible terminator nucleotide is added sequentially until a signal indicative of incorporation of the 2′

-modified reversible terminator nucleotide into the extended product is detected.

39. The method of claim 37, wherein each different 2′

-modified reversible terminator nucleotide is added simultaneously and each different 2′

-modified reversible terminator nucleotide is labeled with a different labeling moiety.

40. A method of sequencing at least a portion of a template nucleic acid, comprising:

a) incubating the template nucleic acid in a reaction mixture comprising a primer, a polymerase, and a 2′

-modified reversible terminator nucleotide, the modified reversible terminator nucleotide comprising one of the four naturally occurring bases or analogs thereof and further comprising a modification at the 2′

position that terminates synthesis, under conditions in which the primer anneals to the template nucleic acid and is extended by the polymerase present in the reaction to form an extended product;

b) determining if a signal indicative of incorporation of the 2′

-modified reversible terminator nucleotide into the extended product is present;

c) if the signal is not present, repeating steps a) and b) with a different 2′

-modified reversible terminator nucleotide, said different 2′

-modified reversible terminator nucleotide comprising a different one of the four naturally occurring bases or analogs thereof,

d) incubating the extended product with an activity that removes the modification at the 2′

position present on the reversible terminator nucleotide, and

e) repeating steps a) to d) a desired number of times to determine the sequence of at least a portion of the template nucleic acid.