Compositions and methods for labeling of nucleic acid molecules

• US 6,589,737 B1
• Filed: 05/19/2000
• Issued: 07/08/2003
• Est. Priority Date: 05/21/1999
• Status: Expired due to Term

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Invitrogen Corp. (Thermo Fisher Scientific Incorporated)
• Inventors: Shih, Po-Jen, Gruber, Christian E.
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US09/574,111
• Time in Patent Office: 1,145 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 536/25.3
• US Class Current: 435/6.1
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 1/68 involving nucleic acids
• CPC Class Codes: C12Q 1/6813 Hybridisation assays
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 1/6862 Ligase chain reaction [LCR]
• CPC Class Codes: C12Q 2521/107 RNA dependent DNA polymerase,(i.e. reverse transcriptase)
• CPC Class Codes: C12Q 2527/149 Concentration of an enzyme
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: G01N 27/44726 using specific dyes, markers or binding molecules

Abstract

The present invention is generally related to compositions, kits and methods for labeling nucleic acid molecules using reverse transcriptases, preferably multi-subunit reverse transcriptases such as ASLV reverse transcriptases. Specifically, the invention relates to methods, kits and compositions for fluorescently labeling nucleic acid molecules during nucleic acid synthesis. The labeled nucleic acid molecules produced in accordance with the invention are particularly suited as labeled probes for nucleic acid detection and diagnostics.

Claims

1. A composition for use in labeling one or more nucleic acid molecules, said composition comprising

(a) one or more enzymes having reverse transcriptase activity, wherein said enzymes are multi-subunit enzymes;

(b) one or more labeled nucleotides, with the proviso that said labeled nucleotides are not labeled dideoxynucleotides; and

(c) one or more nucleotides without labels.

2. The composition of claim 1, wherein said enzymes are heterodimers.

3. The composition of claim 1, wherein said enzymes are selected from the group consisting of ASLV reverse transcriptases.

4. The composition of claim 1, wherein said enzymes are reduced or substantially reduced in RNase H activity.

5. The composition of claim 1, wherein said enzymes are selected from the group consisting of RSV reverse transcriptase, AMV reverse transcriptase, RAV reverse transcriptase, MAV reverse transcriptase and HIV reverse transcriptase, and fragments or mutants thereof having DNA polymerase activity.

6. The composition of claim 1, wherein said one or more labeled nucleotides are one or more fluorescently labeled nucleotides.

7. The composition of claim 6, wherein said nucleotides are labeled with a fluorescent molecule or marker selected from the group consisting of rhodamine, fluorescein, Cy3 and Cy5.

8. The composition of claim 1, wherein said composition further comprises one or more DNA polymerases, and mutants and fragments thereof having DNA polymerase activity.

9. The composition of claim 1, wherein said composition further comprises a reverse transcription buffer and/or a nucleic acid template.

58. The composition of claim 1, wherein said one or more labeled nucleotides are selected from the group consisting of a ribonucleoside triphosphate, a deoxyribonucleoside triphosphate, dATP, dCTP, dITP, dUTP, dGTP, dTTP, [α

S]dATP, 7-deaza-dGTP, 7-deaza-dATP, [R6G]dUTP, [TAMRA]dUTP, [R110]dCTP, [R6G]dCTP, [TAMRA]dCTP, FluoroLink DeoxyNucleotides, FluoroLink Cy3-dCTP, FluoroLink Cy5-dCTP, FluoroLink FluorX-dCTP, FluoroLink Cy3-dUTP, FluoroLink Cy5-dUTP, Fluorescein-15-dATP, Fluorescein-12-dUTP, tetramethyl-rhodamine-6-dUTP, IR₇₇₀-9-dATP, Fluorescein-12-UTP, Fluorescein-15-2′

-dATP, ChromaTide Labeled Nucleotides, BODIPY-FL-14-UTP, BODIPY-FL-4-UTP, BODIPY-TMR-14-UTP, BODIPY-TMR-14-dUTP, BODIPY-TR-14-UTP, BODIPY-TR-14-dUTP, Cascade Blue-7-UTP, Cascade Blue-7-dUTP, fluorescein-12-UTP, fluorescein-12-dUTP, Oregon Green 488-5-dUTP, Rhodamine Green-5-UTP, Rhodamine Green-5-dUTP, tetramethylrhodamine-6-UTP, tetramethylrhodamine-6-dUTP, Texas Red-5-UTP, Texas Red-5-dUTP, and Texas Red-12-dUTP.

10. A method for reverse transcription of one or more nucleic acid molecules comprising

(a) mixing one or more nucleic acid templates with (i) one or more enzymes having reverse transcriptase activity, wherein said enzymes are multi-subunit enzymes, (ii) one or more labeled nucleotides, with the proviso that said labeled nucleotides are not labeled dideoxynucleotides, and (iii) one or more nucleotides without labels;

and

(b) incubating said mixture under conditions sufficient to make one or more nucleic acid molecules complementary to all or a portion of said one or more templates, wherein at least one of said first nucleic acid molecules is detectably labeled.

11. The method of claim 10, wherein said nucleic acid template is a messenger RNA (mRNA) molecule or a population of mRNA molecules.

12. The method of claim 10, said method further comprising incubating said one or more first nucleic acid molecules under conditions sufficient to make one or more second nucleic acid molecules complementary to all or a portion of said one or more first nucleic acid molecules.

13. The method of claim 10, wherein said enzymes are selected from the group consisting of ASLV reverse transcriptases.

14. The method of claim 10, wherein said enzymes are selected from the group consisting of RSV RT, AMV RT, ThermoScript™

and ThermoScript™

II.

15. The method of claim 10, wherein said detectable label is a fluorescent label.

16. The method of claim 15, wherein said fluorescent label is selected from the group consisting of fluorescein, rhodamine, Cy3 and Cy5.

17. The method of claim 10, wherein said detectably labeled first nucleic acid molecules comprise one or more fluorescently labeled nucleotides.

18. The method of claim 10, wherein said detectable labels are the same or different.

19. The method of claim 18, wherein said detectable labels are fluorescent labels.

20. A labeled nucleic acid molecule prepared according to the method of claim 10.

59. The method of claim 10, wherein said one or more labeled nucleotides are selected from the group consisting of a ribonucleoside triphosphate, a deoxyribonucleoside triphosphate, dATP, dCTP, dITP, dUTP, dGTP, dTTP, [α

S]dATP, 7-deaza-dGTP, 7-deaza-dATP, [R6G]dUTP, [TAMRA]dUTP, [R110]dCTP, [R6G]dCTP, [TAMRA]dCTP, FluoroLink DeoxyNucleotides, FluoroLink Cy3-dCTP, FluoroLink Cy5-dCTP, FluoroLink FluorX-dCTP, FluoroLink Cy3-dUTP, FluoroLink Cy5-dUTP, Fluorescein-15-dATP, Fluorescein-12-dUTP, tetramethyl-rhodamine-6-dUTP, IR₇₇₀-9-dATP, Fluorescein-12-UTP, Fluorescein-15-2′

-dATP, ChromaTide Labeled Nucleotides, BODIPY-FL-14-UTP, BODIPY-FL-4-UTP, BODIPY-TMR-14-UTP, BODIPY-TMR-14-dUTP, BODIPY-TR-14-UTP, BODIPY-TR-14-dUTP, Cascade Blue-7-UTP, Cascade Blue-7-dUTP, fluorescein-12-UTP, fluorescein-12-dUTP, Oregon Green 488-5-dUTP, Rhodamine Green-5-UTP, Rhodamine Green-5-dUTP, tetramethylrhodamine-6-UTP, tetramethylrhodamine-6-dUTP, Texas Red-5-UTP, Texas Red-5-dUTP, and Texas Red-12-dUTP.

21. A kit for use in labeling one or more nucleic acid molecules, said kit comprising

(a) one or more enzymes having reverse transcriptase activity, wherein said enzymes are multi-subunit enzymes;

(b) one or more labeled nucleotides, with the proviso that said labeled nucleotides are not labeled dideoxynucleotides; and

(c) one or more nucleotides without labels.

22. The kit of claim 21, said kit further comprising one or more components selected from the group consisting of one or more DNA polymerases, a suitable buffer and one or more primers.

23. The kit of claim 22, wherein at least one or said nucleotides are fluorescent nucleotides.

24. The kit of claim 21, wherein said enzymes are heterodimers.

25. The kit of claim 21, wherein said enzymes are selected from the group of ASLV reverse transcriptases.

26. The kit of claim 21, wherein said enzymes are reduced or substantially reduced in RNase H activity.

27. The kit of claim 21, wherein said enzymes are selected from the group consisting of RSV reverse transcriptase, AMV reverse transcriptase, RAV reverse transcriptase, MAV reverse transcriptase and HIV reverse transcriptase, and fragments or mutants thereof having DNA polymerase activity.

28. The kit of claim 23, wherein said nucleotides are labeled with a fluorescent molecule or marker selected from the group consisting of rhodamine, fluorescein, Cy3 and Cy5.

60. The kit of claim 21, wherein said one or more labeled nucleotides are selected from the group consisting of a ribonucleoside triphosphate, a deoxyribonucleoside triphosphate, dATP, dCTP, dITP, dUTP, dGTP, dTTP, [α

S]dATP, 7-deaza-dGTP, 7-deaza-dATP, [R6G]dUTP, [TAMRA]dUTP, [R110]dCTP, [R6G]dCTP, [TAMRA]dCTP, FluoroLink DeoxyNucleotides, FluoroLink Cy3-dCTP, FluoroLink Cy5-dCTP, FluoroLink FluorX-dCTP, FluoroLink Cy3-dUTP, FluoroLink Cy5-dUTP, Fluorescein-15-dATP, Fluorescein-12-dUTP, tetramethyl-rhodamine-6-dUTP, IR₇₇₀-9-dATP, Fluorescein-12-UTP, Fluorescein-15-2′

-dATP, ChromaTide Labeled Nucleotides, BODIPY-FL-14-UTP, BODIPY-FL-4-UTP, BODIPY-TMR-14-UTP, BODIPY-TMR-14-dUTP, BODIPY-TR-14-UTP, BODIPY-TR-14-dUTP, Cascade Blue-7-UTP, Cascade Blue-7-dUTP, fluorescein-12-UTP, fluorescein-12-dUTP, Oregon Green 488-5-dUTP, Rhodamine Green-5-UTP, Rhodamine Green-5-dUTP, tetramethylrhodamine-6-UTP, tetramethylrhodamine-6-dUTP, Texas Red-5-UTP, Texas Red-5-dUTP, and Texas Red-12-dUTP.

29. A composition for use in labeling one or more nucleic acid molecules, said composition comprising

(a) one or more enzymes having reverse transcriptase activity, wherein said enzymes are multi-subunit enzymes;

(b) one or more labeled nucleotides, with the proviso that said labeled nucleotides are not labeled dideoxynucleotides;

(c) one or more nucleotides without labels; and

(d) one or more RNA templates.

30. The composition of claim 29, wherein said one or more RNA templates is a messenger RNA (mRNA) molecule or a population of mRNA molecules.

31. The composition of claim 29, wherein said enzymes are heterodimers.

32. The composition of claim 29, wherein said enzymes are selected from the group consisting of ASLV reverse transcriptases.

33. The composition of claim 29, wherein said enzymes are reduced or substantially reduced in RNase H activity.

34. The composition of claim 29, wherein said enzymes are selected from the group consisting of RSV reverse transcriptase, AMV reverse transcriptase, RAV reverse transcriptase, MAV reverse transcriptase and HIV reverse transcriptase, and fragments or mutants thereof having DNA polymerase activity.

35. The composition of claim 29, wherein said one or more labeled nucleotides are one or more fluorescently labeled nucleotides.

36. The composition of claim 35, wherein said nucleotides are labeled with a fluorescent molecule or marker selected from the group consisting of rhodamine, fluorescein, Cy3 and Cy5.

37. The composition of claim 29, wherein said composition further comprises one or more DNA polymerases, and mutants and fragments thereof having DNA polymerase activity.

38. The composition of claim 29, wherein said composition further comprises a reverse transcription buffer.

61. The composition of claim 29, wherein said one or more labeled nucleotides are selected from the group consisting of a ribonucleoside triphosphate, a deoxyribonucleoside triphosphate, dATP, dCTP, dITP, dUTP, dGTP, dTTP, [α

S]dATP, 7-deaza-dGTP, 7-deaza-dATP, [R6G]dUTP, [TAMRA]dUTP, [R110]dCTP, [R6G]dCTP, [TAMRA]dCTP, FluoroLink DeoxyNucleotides, FluoroLink Cy3-dCTP, FluoroLink Cy5-dCTP, FluoroLink FluorX-dCTP, FluoroLink Cy3-dUTP, FluoroLink Cy5-dUTP, Fluorescein-15-dATP, Fluorescein-12-dUTP, tetramethyl-rhodamine-6-dUTP, IR₇₇₀-9-dATP, Fluorescein-12-UTP, Fluorescein-15-2′

-dATP, ChromaTide Labeled Nucleotides, BODIPY-FL-14-UTP, BODIPY-FL-4-UTP, BODIPY-TMR-14-UTP, BODIPY-TMR-14-dUTP, BODIPY-TR-14-UTP, BODIPY-TR-14-dUTP, Cascade Blue-7-UTP, Cascade Blue-7-dUTP, fluorescein-12-UTP, fluorescein-12-dUTP, Oregon Green 488-5-dUTP, Rhodamine Green-5-UTP, Rhodamine Green-5-dUTP, tetramethylrhodamine-6-UTP, tetramethylrhodamine-6-dUTP, Texas Red-5-UTP, Texas Red-5-dUTP, and Texas Red-12-dUTP.

39. A method for reverse transcription of one or more nucleic acid molecules comprising

(a) mixing one or more RNA templates with (i) one or more enzymes having reverse transcriptase activity, wherein said enzymes are multi-subunit enzymes, (ii) one or more labeled nucleotides, with the proviso that said labeled nucleotides are not labeled dideoxynucleotides, and (iii) one or more nucleotides without labels;

and

(b) incubating said mixture under conditions sufficient to make one or more nucleic acid molecules complementary to all or a portion of said one or more templates, wherein at least one of said first nucleic acid molecules is detectably labeled.

40. The method of claim 39, wherein said one or more RNA templates is a messenger RNA (mRNA) molecule or a population of mRNA molecules.

41. The method of claim 39, said method further comprising incubating said one or more first nucleic acid molecules under conditions sufficient to make one or more second nucleic acid molecules complementary to all or a portion of said one or more first nucleic acid molecules.

42. The method of claim 39, wherein said enzymes are selected from the group consisting of ASLV reverse transcriptases.

43. The method of claim 39, wherein said enzymes are selected from the group consisting of RSV RT, AMV RT, ThermoScript™

and ThermoScript™

II.

44. The method of claim 39, wherein said detectable label is a fluorescent label.

45. The method of claim 44, wherein said fluorescent label is selected from the group consisting of fluorescein, rhodamine, Cy3 and Cy5.

46. The method of claim 39, wherein said detectably labeled first nucleic acid molecules comprise one or more fluorescently labeled nucleotides.

47. The method of claim 39, wherein said detectable labels are the same or different.

48. The method of claim 47, wherein said detectable labels are fluorescent labels.

49. A labeled nucleic acid molecule prepared according to the method of claim 39.

62. The method of claim 39, wherein said one or more labeled nucleotides are selected from the group consisting of a ribonucleoside triphosphate, a deoxyribonucleoside triphosphate, dATP, dCTP, dITP, dUTP, dGTP, dTTP, [α

S]dATP, 7-deaza-dGTP, 7-deaza-dATP, [R6G]dUTP, [TAMRA]dUTP, [R110]dCTP, [R6G]dCTP, [TAMRA]dCTP, FluoroLink DeoxyNucleotides, FluoroLink Cy3-dCTP, FluoroLink Cy5-dCTP, FluoroLink FluorX-dCTP, FluoroLink Cy3-dUTP, FluoroLink Cy5-dUTP, Fluorescein-15-dATP, Fluorescein-12-dUTP, tetramethyl-rhodamine-6-dUTP, IR₇₇₀-9-dATP, Fluorescein-12-UTP, Fluorescein-15-2′

-dATP, ChromaTide Labeled Nucleotides, BODIPY-FL-14-UTP, BODIPY-FL-4-UTP, BODIPY-TMR-14-UTP, BODIPY-TMR-14-dUTP, BODIPY-TR-14-UTP, BODIPY-TR-14-dUTP, Cascade Blue-7-UTP, Cascade Blue-7-dUTP, fluorescein-12-UTP, fluorescein-12-dUTP, Oregon Green 488-5-dUTP, Rhodamine Green-5-UTP, Rhodamine Green-5-dUTP, tetramethylrhodamine-6-UTP, tetramethylrhodamine-6-dUTP, Texas Red-5-UTP, Texas Red-5-dUTP, and Texas Red-12-dUTP.

50. A kit for use in labeling one or more nucleic acid molecules, said kit comprising

(a) one or more enzymes having reverse transcriptase activity, wherein said enzymes are multi-subunit enzymes;

(b) one or more labeled nucleotides, with the proviso that said labeled nucleotides are not labeled dideoxynucleotides;

(c) one or more nucleotides without labels; and

(d) one or more RNA templates.

51. The kit of claim 50, said kit further comprising one or more components selected from the group consisting of one or more DNA polymerases, a suitable buffer and one or more primers.

52. The kit of claim 50, wherein said enzymes are heterodimers.

53. The kit of claim 50, wherein said enzymes are selected from the group of ASLV reverse transcriptases.

54. The kit of claim 50, wherein said enzymes are reduced or substantially reduced in RNase H activity.

55. The kit of claim 50, wherein said enzymes are selected from the group consisting of RSV reverse transcriptase, AMV reverse transcriptase, RAV reverse transcriptase, MAV reverse transcriptase and HIV reverse transcriptase, and fragments or mutants thereof having DNA polymerase activity.

56. The kit of claim 52, wherein said nucleotides are labeled with a fluorescent molecule or marker selected from the group consisting of rhodamine, fluorescein, Cy3 and Cy5.

57. The kit of claim 50, wherein said one or more RNA templates is a messenger RNA (mRNA) molecule or a population of mRNA molecules.

63. The kit of claim 50, wherein said one or more labeled nucleotides are selected from the group consisting of a ribonucleoside triphosphate, a deoxyribonucleoside triphosphate, dATP, dCTP, dITP, dUTP, dGTP, dTTP, [α

S]dATP, 7-deaza-dGTP, 7-deaza-dATP, [R6G]dUTP, [TAMRA]dUTP, [R110]dCTP, [R6G]dCTP, [TAMRA]dCTP, FluoroLink DeoxyNucleotides, FluoroLink Cy3-dCTP, FluoroLink Cy5-dCTP, FluoroLink FluorX-dCTP, FluoroLink Cy3-dUTP, FluoroLink Cy5-dUTP, Fluorescein-15-dATP, Fluorescein-12-dUTP, tetramethyl-rhodamine-6-dUTP, IR₇₇₀-9-dATP, Fluorescein-12-UTP, Fluorescein-15-2′

-dATP, ChromaTide Labeled Nucleotides, BODIPY-FL-14-UTP, BODIPY-FL-4-UTP, BODIPY-TMR-14-UTP, BODIPY-TMR-14-dUTP, BODIPY-TR-14-UTP, BODIPY-TR-14-dUTP, Cascade Blue-7-UTP, Cascade Blue-7-dUTP, fluorescein-12-UTP, fluorescein-12-dUTP, Oregon Green 488-5-dUTP, Rhodamine Green-5-UTP, Rhodamine Green-5-dUTP, tetramethylrhodamine-6-UTP, tetramethylrhodamine-6-dUTP, Texas Red-5-UTP, Texas Red-5-dUTP, and Texas Red-12-dUTP.