Compositions and methods for labeling of nucleic acid molecules

• US 7,037,661 B2
• Filed: 04/03/2003
• Issued: 05/02/2006
• Est. Priority Date: 05/21/1999
• Status: Expired due to Term

• Current Assignee: Invitrogen Corp. (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:
  US10/405,608
• Publication Number:
  US 20030190661A1
• Time in Patent Office: 1,125 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 535/25.3, 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 and are reduced or substantially reduced in RNase H activity; and

b) one or more deoxynucleotides labeled with a fluorescent label selected from the group consisting of 5-carboxyfluorescein (FAM), 2′

7′

-dimethoxy-4′

5-dichloro-6-carboxyfluorescein (JOE), 6-carboxyrhodamine (R6G), N,N,N′

,N′

-tetramethyl-6-carboxyrhodamine (TAMRA), 6-carboxy-X-rhodamine (ROX), 4-(4′

dimethylaminophenylazo) benzoic acid (DABCYL), Cascade Blue, Oregon Green, Cyanine, 5-(2′

-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS), Cy3, Cy5, ChromaTide, BODIPY-FL and BODIPY-TMR.

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 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 reverse transcriptase activity.

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

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

18. The composition of claim 1, wherein said enzymes have less than about 30% of the RNase H activity of the corresponding RNase H⁺ enzymes.

19. The composition of claim 1, wherein said enzymes have less than about 25% of the RNase H activity of the corresponding RNase H⁺ enzymes.

20. The composition of claim 1, wherein said enzymes have less than about 20% of the RNase H activity of the corresponding RNase H⁺ enzymes.

21. The composition of claim 1, wherein said enzymes have less than about 15% of the RNase H activity of the corresponding RNase H⁺ enzymes.

22. The composition of claim 1, wherein said enzymes have less than about 10% of the RNase H activity of the corresponding RNase H⁺ enzymes.

23. The composition of claim 1, wherein said enzymes have less than about 7.5% of the RNase H activity of the corresponding RNase H⁺ enzymes.

24. The composition of claim 1, wherein said enzymes have less than about 5% of the RNase H activity of the corresponding RNase H⁺ enzymes.

25. The composition of claim 1, wherein said enzymes have less than about 2% of the RNase H activity of the corresponding RNase H⁺ enzymes.

7. A method for reverse transcription of one or more nucleic acid molecules, said method 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 and are reduced or substantially reduced in RNase H activity; and

(ii) one or more deoxynucleotides labeled with a fluorescent label selected from the group consisting of 5-carboxyfluorescein (FAM), 2′

7′

-dimethoxy-4′

5-dichloro-6-carboxyfluorescein (JOE), 6-carboxyrhodamine (R6G), N,N,N′

,N′

-tetramethyl-6-carboxyrhodamine (TAMRA), 6-carboxy-X-rhodamine (ROX), 4-(4′

dimethylaminophenylazo) benzoic acid (DABCYL), Cascade Blue, Oregon Green, Cyanine, 5-(2′

-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS), Cy3, Cy5, ChromaTide, BODIPY-FL and BODIPY-TMR; and

(b) incubating said mixture under conditions sufficient to make one or more first 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 fluorescently labeled.

8. The method of claim 7, wherein said nucleic acid template is a messenger RNA (mRNA) molecule or a population of mRNA molecules.

9. The method of claim 7, 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.

10. The method of claim 7, wherein said enzymes are selected from the group consisting of ASLV reverse transcriptases.

11. The method of claim 7, wherein said enzymes are selected from the group consisting of RSV RT, AMY RT, ThermoScript™

and ThermoScript™

II.

12. The method of claim 7, wherein said detectably labeled first nucleic acid molecules comprise one or more fluorescently labeled nucleotides.

13. The method of claim 7, wherein said detectably labeled first nucleic acid molecules comprise more than one labeled nucleotides that are different from one another.

14. The method of claim 13, wherein said more than one labeled nucleotides are fluorescently labeled nucleotides.

15. A labeled nucleic acid molecule prepared according to the method of claim 7.

26. The method of claim 7, wherein said enzymes have less than about 30% of the RNase H activity of the corresponding RNase H⁺ enzymes.

27. The method of claim 7, wherein said enzymes have less than about 25% of the RNase H activity of the corresponding RNase H⁺ enzymes.

28. The method of claim 7, wherein said enzymes have less than about 20% of the RNase H activity of the corresponding RNase H⁺ enzymes.

29. The method of claim 7, wherein said enzymes have less than about 15% of the RNase H activity of the corresponding RNase H⁺ enzymes.

30. The method of claim 7, wherein said enzymes have less than about 10% of the RNase H activity of the corresponding RNase H⁺ enzymes.

31. The method of claim 7, wherein said enzymes have less than about 7.5% of the RNase H activity of the corresponding RNase H⁺ enzymes.

32. The method of claim 7, wherein said enzymes have less than about 5% of the RNase H activity of the corresponding RNase H⁺ enzymes.

33. The method of claim 7, wherein said enzymes have less than about 2% of the RNase H activity of the corresponding RNase H⁺ enzymes.

16. A kit for use in labeling one or more nucleic acid molecules, said kit comprising one or more enzymes having reverse transcriptase activity, and one or more deoxynucleotides labeled with a fluorescent label selected from the group consisting of 5-carboxyfluorescein (FAM), 2′

7′

-dimethoxy-4′

5-dichloro-6-carboxyfluorescein (JOE), 6-carboxyrhodamine (R6G), N,N,N′

,N′

-tetramethyl-6-carboxyrhodamine (TAMRA), 6-carboxy-X-rhodamine (ROX), 4-(4′

dimethylaminophenylazo) benzoic acid (DABCYL), Cascade Blue, Oregon Green, Cyanine, 5-(2′

-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS), Cy3, Cy5, ChromaTide, BODIPY-FL and BODIPY-TMR, wherein said enzymes are multi-subunit enzymes and are reduced or substantially reduced in RNase H activity.

17. The kit of claim 16, 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.

34. The kit of claim 16, wherein said enzymes have less than about 30% of the RNase H activity of the corresponding RNase H⁺ enzymes.

35. The kit of claim 16, wherein said enzymes have less than about 25% of the RNase H activity of the corresponding RNase H⁺ enzymes.

36. The kit of claim 16, wherein said enzymes have less than about 20% of the RNase H activity of the corresponding RNase H⁺ enzymes.

37. The kit of claim 16, wherein said enzymes have less than about 15% of the RNase H activity of the corresponding RNase H⁺ enzymes.

38. The kit of claim 16, wherein said enzymes have less than about 10% of the RNase H activity of the corresponding RNase H⁺ enzymes.

39. The kit of claim 16, wherein said enzymes have less than about 7.5% of the RNase H activity of the corresponding RNase H⁺ enzymes.

40. The kit of claim 16, wherein said enzymes have less than about 5% of the RNase H activity of the corresponding RNase H⁺ enzymes.

41. The kit of claim 16, wherein said enzymes have less than about 2% of the RNase H activity of the corresponding RNase H⁺ enzymes.