Oscillating amplification reaction for nucleic acids

• US 10,316,358 B2
• Filed: 08/25/2016
• Issued: 06/11/2019
• Est. Priority Date: 04/20/2011
• Status: Active Grant

• Current Assignee: Mesa Biotech, Inc (Thermo Fisher Scientific Incorporated)
• Original Assignee: Mesa Biotech, Inc (Thermo Fisher Scientific Incorporated)
• Inventors: Cai, Hong, Cobb, Nathan J.
• Primary Examiner(s): Mummert, Stephanie K
• Application Number:
  US15/247,728
• Publication Number:
  US 20160362725A1
• Time in Patent Office: 1,020 Days
• Field of Search: None
• CPC Class Codes: B01L 3/502723 characterised by venting arrangements
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2522/101 Single or double stranded nucleic acid binding proteins
• CPC Class Codes: C12Q 2525/204 specific length of the oligonucleotides
• CPC Class Codes: C12Q 2527/107 Temperature of melting, i.e. Tm
• CPC Class Codes: C12Q 2527/125 Specific component of sample, medium or buffer
• CPC Class Codes: C12Q 2527/127 the enzyme inhibitor or activator used

Abstract

One embodiment of the present invention provides for a method for amplifying a template of nucleic acid target sequence contained in a sample. The method includes contacting the sample with an amplification reaction mixture containing a primer complementary to the template of nucleic acid target sequence. A temperature of the reaction is oscillated between an upper temperature and a lower temperature wherein the change in temperature is no greater than about 20° C. during a plurality of temperature cycles. The template of nucleic acid target sequence is amplified.

Claims

1. A method for amplifying a target nucleic acid sequence contained in a sample, the method comprising:

contacting the sample with an amplification reaction mixture comprising a primer complementary to the target nucleic acid sequence;

oscillating a temperature of the reaction between an upper temperature and a lower temperature wherein a change in temperature is no greater than 20°

C. during a plurality of temperature cycles; and

amplifying the target nucleic acid sequence.

2. The method of claim 1 wherein the change in temperature is no greater than about 15°

C. during a plurality of temperature cycles.

3. The method of claim 1 wherein the change in temperature is no greater than about 10°

C. during a plurality of temperature cycles.

4. The method of claim 1 wherein the change in temperature is no greater than about 5°

C. during a plurality of temperature cycles.

5. The method of claim 1 wherein upon reaching the upper temperature or the lower temperature, the temperature is maintained for a set period of time within a temperature fluctuation.

6. The method of claim 1 wherein upon reaching an upper or lower temperature within the temperature range, the temperature is varied to the other temperature.

7. The method of claim 1 wherein the lower temperature is no less than about 50°

C.

8. The method of claim 1 wherein the upper temperature is no greater than about 85°

C.

9. The method of claim 1 wherein the target nucleic acid sequence comprises single stranded DNA or RNA.

10. The method of claim 1 wherein the target nucleic acid sequence comprises double stranded DNA or RNA.

11. The method of claim 1 wherein the target nucleic acid sequence is RNA.

12. The method of claim 1 wherein the target nucleic acid sequence is DNA.

13. The method of claim 1 wherein the length of the target nucleic acid sequence is less than about 1000 bp.

14. The method of claim 1 wherein the length of the target nucleic acid sequence is less than about 250 bp.

15. The method of claim 1 wherein the length of the target nucleic acid sequence is less than about 150 bp.

16. The method of claim 1 wherein the length of the target nucleic acid sequence is be less than about 100 bp.

17. The method of claim 1 wherein the amplification reaction mixture comprises a pair of primers which bind to opposite strands of the target nucleic acid sequence.

18. The method of claim 17 wherein the each primer of the primer pair has a length and a GC content so that its melting temperature is 65°

C.

19. The method of claim 18 wherein the each primer of the primer pair has a length and a GC content so that the melting temperature is ≥

70°

.

20. The method of claim 19 wherein the melting temperature of each primer of the primer pair is between 70-80°

C.

21. The method of claim 17 wherein the each primer of the primer pair has a length of 35-70 base pairs.

22. The method of claim 17 wherein the pair of primers comprise a forward primer and a reverse primer each having a length of 40-47 base pairs.

23. The method of claim 1 wherein the amplification reaction mixture comprises a nucleic acid destabilizing agent.

24. The method of claim 23 wherein the destabilizing agent is selected from the group consisting of dimethylsulfoxide (DMSO), formamide, Betaine, and combinations thereof.

25. The method of claim 23 wherein a concentration of the nucleic acid destabilizer is from 8-15 volume percent.

26. The method of claim 23 wherein the destabilizing agent is a single strand or double strand nucleic acid destabilizing agent.

27. The method of claim 1 wherein the amplification reaction mixture comprises a DNA polymerase.

28. The method of claim 27 wherein the DNA polymerase is buffered at a pH to support activity.

29. The method of claim 27 wherein the DNA polymerase is a thermostable DNA polymerase.

30. The method of claim 27 wherein the DNA polymerase is selected from the group consisting of TAQ DNA polymerase, VentR DNA polymerase, and DeepVentR DNA polymerase.

31. The method of claim 27 wherein the DNA polymerase comprises a strand displacing activity.

32. The method of claim 27 wherein the DNA polymerase does not have 3′

→

5′

exonuclease activity.

33. The method of claim 1 wherein the amplification reaction mixture comprises a reverse transcriptase.

34. The method of claim 33 wherein the reverse transcriptase is a thermostable reverse transcriptase.

35. The method of claim 33 wherein the reverse transcriptase is selected from AMV-RT, Superscript II reverse transcriptase, Superscript III reverse transcriptase, or MMLV-RT.

36. The method of claim 1 wherein the amplification reaction mixture comprises a single stranded binding protein.

37. The method of claim 36 wherein the single stranded binding protein is a thermal stable single stranded binding protein.

38. The method of claim 36 wherein the single stranded binding protein is a non-thermal stable single stranded binding protein.

39. The method of claim 1 wherein the sample is not alcohol free.

40. The method of claim 1 wherein the sample is not salt free.

41. The method of claim 1 wherein the upper temperature is a denaturation temperature and a lower temperature is an annealing temperature.

42. The method of claim 41 wherein the upper denaturation temperature is sufficiently high to completely denature the target nucleic acid sequence.

43. The method of claim 1 further comprising mixing a solution comprising a divalent cation together with other components to form the amplification reaction mixture.

44. The method of claim 43 wherein the solution is a buffer.

45. The method of claim 43 wherein the divalent cation is a salt comprising an element selected from the group consisting of magnesium, manganese, copper, zinc and any combination thereof.

46. The method of claim 43 wherein the solution comprises a monovalent cation.

47. The method of claim 46 wherein the monovalent cation is a salt comprising an element selected from the group consisting of sodium, potassium, lithium, rubidium, cesium, ammonium and any combination thereof.