Methods for nucleic acid sequence determination

• US 20050239085A1
• Filed: 04/23/2004
• Published: 10/27/2005
• Est. Priority Date: 04/23/2004
• Status: Abandoned Application

• Current Assignee: Helicos Biosciences Corporation
• Original Assignee: Helicos Biosciences Corporation
• Inventors: Ickes, Rebecca Adele, Buzby, Philip Richard, DiMeo, James Joseph
• Application Number:
  US10/831,214
• Publication Number:
  US 20050239085A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 2521/101 DNA polymerase
• CPC Class Codes: C12Q 2527/125 Specific component of sample, medium or buffer

Abstract

Methods of the invention comprise methods for nucleic acid sequence determination. Generally, the invention relates to sequencing a target nucleic acid by exposing the target nucleic acid to a primer and a polymerase. Such methods may involve determining the sequence of a target nucleic acid by using a thermophilic polymerase, such as a variant of said 9° N DNA polymerase.

Claims

1. A method for nucleic acid sequence determination, the method comprising the steps of:

(a) exposing a target nucleic acid to a primer that is complementary to at least a portion of the target, a thermophilic polymerase, and at least one nucleotide for extension of said primer;

(b) conducting a primer extension at a temperature of about 20-70°

C.;

(c) detecting incorporation of said nucleotide in said primer; and

,

(d) repeating steps (a), (b) and (c), thereby to determine a sequence of said target.

2. The method of claim 1, wherein said polymerase is a 9°

N DNA polymerase.

3. The method of claim 1, wherein said polymerase is a variant of said 9°

N DNA polymerase.

4. The method of claim 3, wherein said polymerase is a 9°

N A485L (exo-) DNA polymerase.

5. The method of claim 1, wherein said variant is a thermostable polymerase with enhanced ability to incorporate a modified nucleotide.

6. The method of claim 5, wherein said variant is an Archaeon polymerase.

7. The method of claim 1, wherein the primer extension is conducted at a temperature of about 20-70°

C.

8. The method of claim 1, wherein the primer extension is conducted at a temperature of about 30-40°

C.

9. The method of claim 1, wherein the primer extension is conducted at a temperature of about 37°

C.

10. The method of claim 5, wherein said modified nucleotide is a nucleotide analog.

11. The method of claim 5, wherein said nucleotide analog is selected from the group consisting of a deoxynucleotide, a ribonucleotide, and analog thereof.

12. The method of claim 5, wherein said nucleotide analog comprises a cleavable linker.

13. The method of claim 12, wherein the cleavage of said linker is done using photolysis or chemical hydrolysis.

14. The method of claim 5, wherein said nucleotide analog lacks a 3′

hydroxyl group.

15. The method of claim 14, wherein the nucleotide analog is a 2′

,3′

-dideoxynucleotide, acyclonucleotide, or analog thereof.

16. The method of claim 1, wherein said polymerase has a decreased 3′

to 5′

proofreading exonuclease activity.

17. The method of claim 1, wherein said nucleotide comprises a detectable label.

18. The method of claim 17, wherein said label is a fluorescent label.

19. The method of claim 18, wherein the detectable label is selected from the group consisting of cyanine, rhodamine, fluorescein, coumarin, BODIPY, alexa, or conjugated multi-dyes.

20. The method of claim 12, further comprising the step of removing or neutralizing said label subsequent to said detecting step.

21. The method of claim 1, wherein said detecting step comprises optically detecting incorporation of said nucleotide.

22. The method of claim 1, wherein said target is attached to a substrate.

23. The method of claim 1, further comprising the step of washing an unincorporated nucleotide.

24. The method of claim 22, wherein a plurality of said target nucleic acids are spaced apart such that each target is optically resolvable.

25. The method of claim 21, wherein said detecting step comprises detecting a fluorescent label attached to said nucleotide.

26. The method of claim 25, wherein said label represents a single nucleic acid molecule.

27. The method of claim 1, further comprising the step of compiling a sequence of a complement of said target based upon sequential incorporation of said nucleotides into said primer.

28. The method of claim 27, further comprising the step of compiling a sequence of said target based upon said complement sequence.

29. The method of claim 24, wherein each member of said plurality is covalently attached to a surface comprising glass or fused silica.

30. The method of claim 29, wherein each member of said plurality is covalently attached to a surface that has reduced background fluorescence with respect to polished glass or fused silica.

31. The method of claim 30, wherein said surface is polytetrafluoroethylene or a derivative of polytetrafluoroethylene.

32. The method of claim 31, wherein said derivative is silanized.

33. The method of claim 19, wherein said label is selected from a cyanine 5 dye and a cyanine 3 dye.

34. The method of claim 17, wherein said nucleotide comprises a first fluorescent label and said polymerase comprises a second fluorescent label.

35. The method of claim 34, wherein said detecting step comprises detecting coincident fluorescence emission of said first fluorescent label and said second fluorescent label.

36. The method of claim 35, wherein the coincident fluorescence emission spectrum is between about 400 nm to about 900 nm.

37. The method of claim 36, wherein said coincident detection represents the presence of a single labeled molecule.

38. The method of claim 5, wherein said nucleotide is a non-chain terminating nucleotide.

39. The method of claim 38, wherein said non-chain terminating nucleotide is a deoxynucleotide selected from the group consisting of dATP, dTTP, dUTP, dCTP, and dGTP.

40. The method of claim 38, wherein said non-chain terminating nucleotide is a ribonucleotide selected from the group consisting of ATP, UTP, CTP, and GTP.