Tagged extendable primers and extension products

• US 6,200,748 B1
• Filed: 06/07/1995
• Issued: 03/13/2001
• Est. Priority Date: 01/16/1984
• Status: Expired

• Current Assignee: California Institute of Technology
• Original Assignee: California Institute of Technology
• Inventors: Hood, Leroy E., Hunkapiller, Tim J., Smith, Lloyd M., Connell, Charles R., Hunkapiller, Michael W.
• Primary Examiner(s): Houtteman, Scott W.
• Application Number:
  US08/484,340
• Time in Patent Office: 2,106 Days
• Field of Search: 435/6, 435/91.1, 435/91.51, 435/91.2, 536/24.33, 536/26.6
• US Class Current: 435/6.12
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2535/101 Sanger sequencing method, i.e. oligonucleotide sequencing using primer elongation and dideoxynucleotides as chain terminators
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: G01N 27/44721 by optical means
• CPC Class Codes: G01N 27/44726 using specific dyes, markers or binding molecules

Abstract

This invention provides a duplex comprising an oligonucleotide primer and a template, wherein the primer is coupled chemically to a chromophore or fluorophore so as to allow chain extension by a polymerase. In one embodiment, the primer is extended by a polymerase to generate the complement of the template. In a further embodiment, the extended primer is separated from the template for use in a number of methods, including sequencing reactions. Methods of generating these compositions of matter are further provided.

Claims

1. A duplex comprising an oligonucleotide primer and a template, wherein the primer is covalently coupled to a chromophore or fluorophore so as to allow chain extension by a polymerase.

2. A duplex comprising an extended oligonucleotide primer and a template, produced by providing a duplex according to claim 1 and extending the oligonucleotide primer with a polymerase.

3. A single-stranded labeled polynucleotide produced by separating the extended oligonucleotide primer from the duplex of claim 2.

4. A set of duplexes comprising two or more of the duplexes of claim 1.

5. A set of duplexes comprising two or more of the duplexes of claim 2.

6. A set of polynucleotides comprising two or more single-stranded labeled polynucleotides of claim 3.

11. The duplex of claim 1, prepared by a method comprising hybridizing an oligonucleotide primer to a template, wherein the primer is covalently coupled to a chromophore or fluorophore so as to allow chain extension by a polymerase.

12. The duplex of claim 11, wherein the chromophore or fluorophore is covalently coupled to the primer through an amine linkage.

13. The duplex of claim 11, wherein the chromophore or fluorophore is covalently coupled to the primer at its 5′

end.

14. A single-stranded labeled polynucleotide produced by the method comprising the steps of extending the oligonucleotide primer of the duplex of claim 1 by a polymerase to produce a labeled polynucleotide and separating the labeled polynucleotide from the template.

15. The polynucleotide of claim 14, wherein the chromophore or fluorophore is covalently coupled to the oligonucleotide through an amine linkage.

16. The polynucleotide of claim 14, wherein the chromophore or fluorophore is covalently coupled to the oligonucleotide at its 5′

end.

17. A chain termination DNA sequencing method comprising extending the primer of the duplex of claim 1 by a polymerase to produce a labeled polynucleotide, and separating the labeled polynucleotide from the template.

18. A chain termination DNA sequencing method comprising extending the primers of the set of duplexes of claim 4 by a polymerase to produce a set of labeled polynucleotides.

19. The chain termination DNA sequencing method of claim 18, wherein the set of duplexes comprises four DNA sequencing reactions, wherein each labeled polynucleotide is distinguishable by spectral characteristics of the chromophore or fluorophore covalently coupled thereto.

20. The oligonucleotide primer of claim 1, wherein the primer is DNA.

21. The oligonucleotide primer of claim 1 wherein the chromophore or fluorophore is detectable by exposure to a high-intensity monochromatic light source.

22. The duplex of either of claims 1 or 2, wherein the chromophore or fluorophore is detectable by exposure to a laser.

23. The set of duplexes of either of claims 4 or 5, wherein the primers are DNA.

24. The set of duplexes of either of claims 4 or 5, wherein the chromophore or fluorophore is detectable by exposure to a high-intensity monochromatic light source.

25. The set of duplexes of either of claims 4 or 5, wherein the chromophore or fluorophore is detectable by exposure to a laser.

29. The polynucleotide of any of claims 14 to 16, wherein the primer is DNA.

30. The polynucleotide of any of claims 14 to 16, wherein the chromophore or fluorophore is detectable by exposure to a high-intensity monochromatic light source.

31. The polynucleotide of any of claims 14 to 16, wherein the chromophore or fluorophore is detectable by exposure to a laser.

32. The duplex of any of claims 11 to 13, wherein the primer is DNA.

33. The duplex of any of claims 11 to 13, wherein the chromophore or fluorophore is detectable by exposure to a high-intensity monochromatic light source.

34. The duplex of any of claims 11 to 13, wherein the chromophore or fluorophore is detectable by exposure to a laser.

35. The duplex of either of claims 1 or 2, wherein the chromophore or fluorophore is covalently coupled to the primer through an amine linkage.

36. The set of duplexes of either of claims 4 or 5, wherein the chromophore or fluorophore is covalently coupled to the primer through an amine linkage.

38. The duplex of either of claims 1 or 2, wherein the chromophore or fluorophore is covalently coupled to the primer at its 5′

end.

39. The set of duplexes of either of claims 4 or 5, wherein the chromophore or fluorophore is covalently coupled to the primer at its 5′

end.

41. The polynucleotide of claim 3, wherein the chromophore or fluorophore is covalently coupled to the primer through an amine linkage.

42. The polynucleotide of claim 3, wherein the chromophore or fluorophore is covalently coupled to the primer at its 5′

end.

43. The polynucleotide of claim 3, wherein the chromophore or fluorophore is detectable by exposure to a laser.

44. The set of polynucleotides of claim 6, wherein the primers are DNA.

45. The set of polynucleotides of claim 6, wherein the chromophore or fluorophore is detectable by exposure to a high-intensity monochromatic light source.

46. The set of polynucleotides of claim 6, wherein the chromophore or fluorophore is detectable by exposure to a laser.

47. The set of polynucleotides of claim 6, wherein the chromophore or fluorophore is covalently coupled to the primer through an amine linkage.

48. The set of polynucleotides of claim 6, wherein the chromophore or fluorophore is covalently coupled to the primer at its 5′

end.

7. A set of reagents comprising oligonucleotide primers covalently coupled to one or more chromophores or fluorophores so as to allow chain extension by a polymerase, and a polymerase.

26. The set of reagents of claim 7, wherein the primers are DNA.

27. The set of reagents of claim 7, wherein the chromophore or fluorophore is detectable by exposure to a high-intensity monochromatic light source.

28. The set of reagents of claim 7, wherein the chromophore or fluorophore is detectable by exposure to a laser.

37. The set of reagents of claim 7, wherein the chromophore or fluorophore is covalently coupled to the primer through an amine linkage.

40. The set of reagents of claim 7, wherein the chromophore or fluorophore is covalently coupled to the primer at its 5′

end.

8. A single-stranded labeled polynucleotide comprising a first portion and a second portion,

wherein the first portion comprises an oligonucleotide primer covalently coupled to a chromophore or fluorophore;

and

wherein the second portion is produced by extension of the first portion along a complementary template.

9. The polynucleotide of claim 8, wherein the chromophore or fluorophore is covalently coupled to the first portion through an amine linkage.

10. The polynucleotide of claim 8, wherein the chromophore or fluorophore is covalently coupled to the first portion at its 5′

end.

49. A duplex comprising an oligonucleotide primer and a template, wherein the primer hybridizes to a specific region of the template and wherein the primer is covalently coupled to a chromophore or fluorophore so as to allow chain extension by a polymerase.

50. A plurality of identical oligonucleotide primers of defined length and base sequences wherein each primer is covalently coupled to a fluorophore or chromophore so as to allow chain extension by a polymerase.

51. The plurality of claim 50 wherein said primers have a free 3′

hydroxyl group.

52. The plurality of claim 51 wherein the chromophore or fluorophore is covalently coupled to the primer at its 5′

end.

53. The plurality of claim 50 wherein said primers are coupled to said fluorophore or chromophore by an amine linkage.

54. A composition comprising the plurality of claim 50.

55. The composition of claim 54 further comprising a buffer.

56. A set of reagents comprising the plurality of claim 50 and a polymerase.

57. A set of reagents comprising two or more pluralities of oligonucleotide primers of claim 50 wherein each plurality has a different emission spectra.

58. A plurality of single-stranded labeled polynucleotides produced by the method comprising the steps of hybridizing the plurality of oligonucleotide primers of claim 50 to a template thereby forming a plurality of duplexes;

extending the primers of said duplexes by a polymerase thereby forming labeled polynucleotides; and

separating said labeled polynucleotides from said duplexes.

59. A set of single stranded labeled polynucleotides comprising two or more pluralities of polynucleotides of claim 58, wherein each plurality has a different emission spectra.

60. The plurality of claim 50 wherein the chromophore or fluorophore is detectable by exposure to a high-intensity monochromatic light source.

61. The plurality of claim 50 wherein the chromophore or fluorophore is detectable by exposure to a laser.