Compositions and methods for cDNA synthesis

• US 20110151520A1
• Filed: 12/08/2009
• Published: 06/23/2011
• Est. Priority Date: 09/03/2002
• Status: Active Grant

• Current Assignee: Qiagen Beverly, Inc. d/b/a Enzymatics Inc. (Qiagen NV)
• Original Assignee: Quanta Biosciences Incorporated
• Inventors: Schuster, David, Rashtchian, Ayoub
• Granted Patent: US 8,420,324 B2
• Time in Patent Office: Days
• US Class Current: 435/91.5
• CPC Class Codes: C12N 15/1096 cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 1/6846 Common amplification features
• CPC Class Codes: C12Q 2521/107 RNA dependent DNA polymerase,(i.e. reverse transcriptase)
• CPC Class Codes: C12Q 2525/179 incorporating arbitrary or random nucleotide sequences
• CPC Class Codes: C12Q 2527/143 Concentration of primer or probe

Abstract

Methods for making cDNA molecules, for amplification of RNA by PCR and for preparation of cDNA libraries are provided. Kits for making cDNA molecules also are provided.

Compositions are also provided comprising mixtures of reagents, including reverse transcriptases, buffers, cofactors and other components, suitable for immediate use in conversion of RNA into cDNA and RT PCR without dilution or addition of further components. These compositions are useful, alone or in the form of kits, for cDNA synthesis or nucleic acid amplification (e.g., by the Polymerase Chain Reaction) or for any procedure utilizing reverse transcriptases in a variety of research, medical, diagnostic, forensic and agricultural applications.

Claims

1-15. -15. (canceled)

16. A reagent mixture comprising a ready to use reagent mixture that demonstrates prolonged stability when stored at −

20°

C., wherein said mixture comprises;

(a) glycerol in a concentration between about 10% and about 40%,

and

(b) a viral reverse transcriptase in a concentration sufficient for use in a reverse transcription reaction without adding additional reverse transcriptase, wherein said reverse transcriptase is selected from the group consisting of AMV RT, RSV RT, MMLV RT, HIV RT, EIAV RT, RAV2 RT, THERMOSCRIPT RT, ASLV and Rnase H⁻

mutants thereof, SUPERSCRIPT II RT, and SUPERSCRIPT I RT,

in a buffer suitable for use in a reverse transcription reaction, wherein said buffer further comprises;

a metal ion necessary for reverse transcriptase activity in a concentration sufficient for reverse transcriptase activity upon addition to a template solution.

17. The mixture according to claim 16, wherein said buffer further comprises at least one component selected from the group consisting of:

a monovalent cation selected from the group consisting of Li, Na, K and NH₄;

a reducing agent, nucleoside triphosphates, a primer and a detergent.

18. The mixture according to claim 17, wherein said buffer comprises at least one primer suitable for priming reverse transcription of a template by said reverse transcriptase.

19. The mixture according to claim 18, wherein said buffer comprises an RNAse inhibitor protein.

20. The mixture according to claim 16, wherein said buffer comprises a potassium salt, a magnesium salt, nucleoside triphosphates, DTT, at least one primer suitable for priming reverse transcription of a template by said reverse transcriptase, at least one non-ionic detergent, and an RNAse inhibitor protein.

21. The mixture according to claim 16, further comprising at least one thermostable DNA polymerase.

22. The mixture according to claim 21, further comprising at least one primer.

23. The mixture according to claim 16, wherein said metal ion necessary for reverse transcriptase activity is magnesium ion

24. The mixture according to claim 17, wherein said buffer comprises a monovalent cation selected from the group consisting of Li, Na, K and NH₄.

25. The mixture according to claim 17, wherein said buffer comprises a reducing agent.

26. The mixture according to claim 17, wherein said buffer comprises nucleoside triphosphates.

27. The mixture according to claim 17, wherein said buffer comprises a non-ionic detergent.

28. The mixture according to claim 21, further comprising a double-strand DNA-specific fluorescent dye.

29. The mixture according to claim 21, further comprising a fluorescent dual-labeled hybridization probe.

30. A method for reverse transcription of one or more nucleic acid molecules comprising contacting a solution comprising one or more nucleic acid templates with a reagent mixture, wherein said reagent mixture comprises a ready to use reagent mixture, wherein said ready to use reagent mixture comprises:

(a) glycerol in a concentration between about 10% and about 40%,

and

(b) a viral reverse transcriptase in a concentration sufficient for use in a reverse transcription reaction without adding additional reverse transcriptase, wherein said reverse transcriptase is selected from the group consisting of AMV RT, RSV RT, MMLV RT, HIV RT, EIAV RT, RAV2 RT, THERMOSCRIPT RT, ASLV and Rnase H⁻

mutants thereof, SUPERSCRIPT II RT, and SUPERSCRIPT I RT,

in

(c) a buffer suitable for use in a reverse transcription reaction, wherein said buffer further comprises at least one component selected from the group consisting of;

a metal ion necessary for reverse transcriptase activity in a concentration sufficient for reverse transcriptase activity and

nucleoside triphosphates;

and incubating the resulting solution at a temperature suitable for cDNA synthesis.

31. The method according to claim 30, wherein said buffer comprises a magnesium salt metal in a concentration sufficient for reverse transcriptase activity.

32. The method according to claim 30, wherein said buffer comprises nucleoside triphosphates.

33. The method according to claim 31, wherein said buffer comprises nucleoside triphosphates.

34. The mixture according to claim 30, wherein said buffer further comprises at least one component selected from the group consisting of:

a monovalent cation selected from the group consisting of Li, Na, K and NH₄;

a reducing agent, a primer, and-a detergent.

35. The method according to claim 34, wherein said buffer comprises at least one primer suitable for priming reverse transcription of a template by said reverse transcriptase.

36. The method according to claim 30, wherein said buffer comprises an RNAse inhibitor protein.

37. The method according to claim 30, wherein said buffer comprises a potassium salt, a magnesium salt, nucleoside triphosphates, DTT, at least one primer suitable for priming reverse transcription of a template by said reverse transcriptase, at least one detergent, and an RNAse inhibitor protein.

38. The method according to claim 30, wherein said buffer further comprises at least one thermostable DNA polymerase.

39. The method according to claim 38, wherein said buffer further comprises at least one primer.

40. The method according to claim 30, wherein said buffer further comprises a double-strand DNA-specific fluorescent dye.

41. The method according to claim 30, wherein said buffer further comprises a fluorescent dual-labeled hybridization probe.

42. The method according to claim 30, wherein said ready to use reagent mixture was stored at −

20°

C. prior to contacting said template solution comprising one or more nucleic acid templates with said reagent mixture.

43. A method for preparing a ready to use reverse transcriptase reagent mixture suitable for reverse transcription of one or more nucleic acid molecules, comprising preparing a mixture containing

(a) glycerol in a concentration between about 10% and about 40%,

(b) a viral reverse transcriptase in a concentration sufficient for use in a reverse transcription reaction without adding additional reverse transcriptase, wherein said reverse transcriptase is selected from the group consisting of AMV RT, RSV RT, MMLV RT, HIV RT, EIAV RT, RAV2 RT, THERMOSCRIPT RT, ASLV and Rnase H⁻

mutants thereof, SUPERSCRIPT II RT, and SUPERSCRIPT I RT, and

(c) a metal ion necessary for reverse transcriptase activity in a concentration sufficient for reverse transcriptase activity upon addition to a template solution;

in a buffer suitable for use in a reverse transcription reaction,

and storing said mixture at about −

20°

C.

44. The method according to claim 43, wherein said buffer further comprises nucleoside triphosphates.

45. The method according to claim 43 wherein said buffer further comprises a monovalent cation selected from the group consisting of Li, Na, K and NH₄

46. The method according to claim 43, wherein said buffer further comprises a reducing agent.

47. The method according to claim 43, wherein said buffer further comprises a detergent.

48. The method according to claim 43, wherein said buffer further comprises at least one primer suitable for priming reverse transcription of a template by said reverse transcriptase.

49. The method according to claim 43, wherein said buffer further comprises an RNAse inhibitor protein.

50. The method according to claim 43, wherein said buffer further comprises a potassium salt, nucleoside triphosphates, DTT, at least one primer suitable for priming reverse transcription of a template by said reverse transcriptase, and at least one detergent.

51. The method according to claim 43, wherein said buffer further comprises at least one thermostable DNA polymerase.

52. The method according to claim 51, wherein said buffer further comprises a double-strand DNA-specific fluorescent dye.

53. The method according to claim 51, wherein said buffer further comprises a fluorescent dual labeled hybridization probe.

54. The method according to claim 52, wherein said buffer further comprises at least one primer.

55. The method according to claim 52, wherein said buffer further comprises at least one primer.