Sequencing and genotyping using reversibly terminating nucleotides

• US 9,206,476 B2
• Filed: 12/20/2006
• Issued: 12/08/2015
• 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: Gelfand, David H., Gupta, Amar
• Primary Examiner(s): MUMMERT, STEPHANIE KANE
• Application Number:
  US12/158,257
• Publication Number:
  US 20080293071A1
• Time in Patent Office: 3,275 Days
• Field of Search: None
• US Class Current: 1/1
• 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 in a reaction mixture at least one template nucleic acid with at least one DNA polymerase, a plurality of different 2′

-modified reversible terminator nucleotides, each different 2′

-modified reversible terminator nucleotide comprising a different base and further comprising a blocking group at a 2′

position of the sugar moiety, wherein the blocking group is a phosphate, 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 in a sequence specific manner the correct 2′

-modified reversible terminator nucleotide at a 3′

-terminal end of the primer extension product;

(b) removing the blocking group from the 2′

position of the 2′

-modified reversible terminator nucleotide at the 3′

-terminal end of the primer extension product with an enzyme that removes the phosphate;

(c) identifying the correct 2′

-modified reversible terminator nucleotide in the primer extension product prior to and/or during (b); and

(d) repeating (a)-(c) one or more times,

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 enzyme that removes the 2′

position phosphate is a phosphatase.

3. The method of claim 1, wherein the enzyme that removes the 2′

position phosphate is an exonuclease III.

4. The method of claim 1, wherein the enzyme that removes the 2′

position phosphate is an endonuclease IV.

5. The method of claim 1, wherein the enzyme that removes the 2′

position phosphate is polynucleotide kinase.

6. The method of claim 1, wherein the enzyme that removes the 2′

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

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

modified reversible terminator nucleotide is

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

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

9. The method of claim 8, wherein the labeling moiety comprises a fluorescent dye, a luminescent molecule, or a radioisotope.

10. The method of claim 9, wherein the labeling moiety is a fluorescent dye.

11. The method of claim 8, 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.

12. The method of claim 8, wherein the 2′

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

13. The method of claim 12, wherein the donor and the acceptor are a reporter and a quencher pair.

14. The method of claim 12, wherein the two labeling moieties are capable of undergoing fluorescence resonance energy transfer.

15. 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.

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

-modified reversible terminator nucleotide.

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

-modified reversible terminator nucleotide comprises a 2′

-monophosphate-3′

-hydroxyl nucleotide.

18. 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 plurality of different 2′

-modified reversible terminator nucleotides, each different 2′

-modified reversible terminator nucleotide comprising a different base, and further comprising a phosphate as a blocking group at the 2′

position of the sugar moiety, 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 sequence specific incorporation of the correct 2′

-modified reversible terminator nucleotide into the extended product;

c) incubating the extended product with an enzyme that removes the phosphate 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.

19. The method of claim 18, wherein each different 2′

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

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

20. The method of claim 18, 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.

21. 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 plurality of different 2′

-modified reversible terminator nucleotides, each different 2′

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

position of the sugar moiety 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 sequence specific incorporation of the correct 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 enzyme that removes the phosphate 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.