Method for improved reverse transcription at high temperatures

• US 6,271,004 B1
• Filed: 06/26/2000
• Issued: 08/07/2001
• Est. Priority Date: 06/25/1999
• Status: Expired due to Fees

• Current Assignee: Azign Bioscience A/S
• Original Assignee: Display Systems Biotech A/S
• Inventors: Warthoe, Peter
• Primary Examiner(s): Horlick, Kenneth R.
• Assistant Examiner(s): Spiegler, Alexander H.
• Application Number:
  US09/603,185
• Time in Patent Office: 407 Days
• Field of Search: 435/91.51, 435/91.1, 435/188, 435/91.2, 435/6, 536/25.3, 536/24.33
• US Class Current: 435/91.51
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2521/107 RNA dependent DNA polymerase,(i.e. reverse transcriptase)
• CPC Class Codes: C12Q 2527/125 Specific component of sample, medium or buffer
• CPC Class Codes: C12Q 2563/119 the label being proteinic

Abstract

The invention relates to a method for enzyme stabilization. A method for improved reverse transcription at high temperatures is provided, wherein a thermostable heat shock protein (HSPs) stabilizes a reverse transcriptase, as well as reduces the RNase H activity of said reverse transcriptase. The present invention thus relates to a stabilizing agent, that prevents thermal denaturing and enhances thermostability of a reverse transcriptase. The invention further relates to a method of producing a polypeptide complex consisting of a Chaperonin and a Moloney murine leukemia virus (MMVL) reverse transcriptase, characterized by having enhanced thermostability as well as reduced RNase H activity, compared to a (MMVL) reverse transcriptase alone. The invention further relates to a kit for the preparation of cDNA from mRNA, comprising either both stabilizing agent and reverse transcriptase or the polypeptide complex of the invention.

Claims

1. A method for reverse transcribing RNA into DNA comprising contacting the RNA with a reverse transcriptase, and a chaperone protein, at a temperature above 42°

C., whereby the reverse transcriptase has enhanced thermostability and substantially reduced RNase H activity.

13. A method according to any one of claim 1-10 wherein the DNA is cDNA.

14. A method according to any one of claims 1-10 wherein the RNA is mRNA.

15. A method according to any one of claims 1-10 wherein the DNA is full length cDNA.

16. A method according to any one of claims 1-10 wherein the RNA is mRNA and the DNA is cDNA.

17. A method according to any one of claims 1-10 additionally comprising isolating the DNA.

18. A method according to any one of claims 1-10 wherein the RNA is mRNA, and the DNA is cDNA and the method additionally comprises isolating the cDNA.

19. A method according to any one of claims 1-10 wherein the chaperone protein is CpkB.

20. A method according to any one of claims 1-10 wherein the chaperone protein is a Beta subunit of a chaperonin.

21. A method according to any one of claims 1-10 wherein the chaperone protein is from Hyperthermophilic Archaeon Pyrococcus sp.

22. A method according to any of claims 1-10, wherein the reverse transcriptase is selected from the group consisting of reverse transcriptases from AMV (Avian Myeloblastosis Virus), M-MuLV (murine M-MuLV pol gene), HIV-1 (HIV virus), and degenerated or truncated or mutated versions thereof.

23. A method according to claim 22, wherein the reverse transcriptase is derived from Moloney murine leukemia virus.

24. A method according to any one of claims 1-10 wherein the RNase H activity is reduced to substantially no RNase H activity.

25. A method according to any one of claims 1-10 comprising separately adding the reverse transcriptase and the chaperone protein to the RNA.

26. A method according to any one of claims 1-10 comprising adding a mixture of the reverse transcriptase and the chaperone protein to the RNA, or simultaneously adding the reverse transcriptase and the chaperone protein to the RNA.

2. In a method for reverse transcribing RNA into DNA comprising contacting the RNA with a reverse transcriptase, wherein the improvement comprises performing said contacting in the presence of a chaperone protein, whereby the reverse transcriptase has enhanced thermostability and substantially reduced RNase H activity.

3. A method for enhancing thermal stability and substantially reducing RNase H activity of a reverse transcriptase comprising employing said reverse transcriptase, in a reverse transcription reaction of RNA to DNA, in the presence of a chaperone protein.

4. A method for reverse transcribing RNA into DNA comprising contacting the RNA with a reverse transcriptase, and a chaperone protein at a temperature above 42°

C., whereby the reverse transcriptase has substantially reduced RNase H activity.

5. In a method for reverse transcribing RNA into DNA comprising contacting the RNA with a reverse transcriptase, wherein the improvement comprises performing said contacting in the presence of a chaperone protein, whereby the reverse transcriptase has substantially reduced RNase H activity.

6. A method for substantially reducing RNase H activity of a reverse transcriptase comprising employing said reverse transcriptase, in a reverse transcription reaction of RNA to DNA, in the presence of a chaperone protein.

7. A method for preparing DNA from RNA comprising contacting the RNA with a reverse transcriptase, wherein the contacting is in the presence of a chaperone protein, whereby the reverse transcriptase has substantially reduced RNase H activity and enhanced thermostability.

8. A method for preparing DNA from RNA comprising contacting the RNA with a reverse transcriptase, wherein the contacting is in the presence of a chaperone protein, whereby the reverse transcriptase has substantially reduced RNase H activity.

9. A method for reverse transcribing RNA to DNA comprising contacting the RNA with a reverse transcriptase, and a chaperone protein, whereby the reverse transcriptase has enhanced thermostability and substantially reduced RNase H activity.

10. A method for reverse transcribing RNA into DNA comprising contacting the RNA with a reverse transcriptase, and a chaperone protein, whereby the reverse transcriptase has substantially reduced RNase H activity.

11. A method of any one of claims 2-3 or 5-10 wherein the method is performed at a temperature above 42°

C.

12. A method of claim 11 wherein he method is performed at a temperature above 65°

C.

27. A method for preparing a DNA molecule, said method comprising

a) mixing an mRNA template with one or more cDNA primers to form a first mixture,

b) adding to the first mixture a chaperone protein and a reverse transcriptase, separately, or simultaneously, or adding to the first mixture a chaperone protein-reverse transcriptase;

combination or mixture, to thereby form a second mixture and substantially reduce any RNase H activity of the reverse transcriptase,

c) incubating said second mixture under conditions sufficient to transcribe a first DNA molecule complementary to said mRNA template.

28. A method according to claim 27, wherein said one or more cDNA primer does not include a poly or oligo dT tail in the 5′

end.

29. A method according to claim 28, wherein said one or more cDNA primer has the following structure 5′

-N_xTTA-3′

or 5′

-N_xCTA-3′

or 5′

-N_xTCA-3, wherein N is A, G, T, or C, and x is an integer 1≦

x≦

20.

30. A method according to any one of claims 27-29, wherein said first DNA molecule is a full length cDNA.

31. A method according to any one of claims 27-29, further comprising incubating said first DNA molecule under conditions sufficient to transcribe a second DNA molecule complementary to said first DNA molecule.

32. A method according to claim 31, wherein said first DNA molecule is a full length cDNA.

33. A method according to claim 31, wherein said first and second DNA molecules form a double stranded DNA molecule.

34. A method according to claim 33, wherein said first DNA molecule is a full length cDNA.

35. A method according to claim 33, wherein said double stranded DNA molecule is a full-length cDNA.

36. A composition comprising CpkB and a reverse transcriptase.

37. A kit for the preparation of cDNA comprising a first container containing CpkB and a second container containing a reverse transcriptase;

or a container containing CpkB and a reverse transcriptase mixture.

38. A kit according to claim 37, further comprising one or more additional containers selected from the group consisting of:

(a) a container containing one or more nucleoside triphosphates,

(b) a container containing an oligo (dT) primer, and

(c) a container containing a buffer suitable for use in transcribing a cDNA.

39. A composition for reverse transcription of a target ribonucleic acid (RNA) comprising a single lyophilizate comprising:

a) an effective amount of a reverse transcriptase;

b) CpkB;

c) deoxyribonucleotide triphosphates and ribonucleotide triphosphates, wherein when said lyophilizate is reconstituted by addition of an aqueous solvent, and the resulting solution will amplify a single-stranded RNA molecule having a target nucleotide sequence region when contacted with one or more suitable oligonucleotide primers under appropriate nucleic acid amplification conditions.