Oscillating amplification reaction for nucleic acids

• US 9,428,781 B2
• Filed: 04/20/2012
• Issued: 08/30/2016
• 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 KANE
• Application Number:
  US14/113,200
• Publication Number:
  US 20140045221A1
• Time in Patent Office: 1,593 Days
• Field of Search: None
• US Class Current: 1/1
• 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 template of nucleic acid target sequence contained in a sample comprising:

contacting the sample with an amplification reaction mixture containing a primer complementary to the template of nucleic acid target 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 template of nucleic acid target 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 template of nucleic acid target sequence is RNA.

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

11. The method of claim 1 wherein the length of the target nucleic acid may be less than 1000 bp.

12. The method of claim 1 wherein the length of the target nucleic acid may be less than 250 bp.

13. The method of claim 1 wherein the length of the target nucleic acid may be less than 150 bp.

14. The method of claim 1 wherein the length of the target nucleic acid may be less than about 100 bp.

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

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

24. The method of claim 9 wherein the amplification reaction mixture comprises a reverse transcriptase and a DNA polymerase.

25. The method of claim 24 wherein the reverse transcriptase is a thermostable reverse transcriptase.

26. The method of claim 24 wherein the reverse transcriptase is selected from AMV-RT, Superscript II reverse transcriptase, Superscript III reverse transcriptase, or MMLV-RT.

30. The method of claim 1 wherein the destabilizing agent is dimethylsulfoxide (DMSO) or formamide.

33. The method of claim 1 wherein the amplification reaction mixture comprises an amplification reaction mixture buffer comprising:

single strand or double strand nucleic acid destabilizer;

monovalent cation;

divalent cation;

dNTPs; and

DNA Polymerase buffered at a pH to support activity.

34. The amplification reaction mixture of claim 33 further comprising a single stranded binding protein.

35. The method of claim 33 wherein the destabilizing agent is dimethylsulfoxide (DMSO) or formamide.

36. The method of claim 33 wherein the divalent cation is a salt selected from the group consisting of magnesium, manganese, copper, zinc or any combination thereof.

37. The method of claim 33 wherein the monovalent cation is a salt selected from the group consisting of sodium, potassium, lithium, rubidium, cesium, ammonium or any combination thereof.

38. The method of claim 33 wherein the DNA polymerase is a thermostable DNA polymerase.

39. The method of claim 33 wherein the DNA polymerase is selected from the group consisting of TAQ DNA polymerase, VentR DNA polymerase, and DeepVentR DNA polymerase.

40. The method of claim 33 wherein the DNA polymerase comprises a strand displacing activity.

41. The method of claim 33 wherein the DNA polymerase does not have 3′

→

5′

exonuclease activity.

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

43. The method of claim 42 wherein the pair of primers have a length and a GC content so that the melting temperature is ≧

65°

C.

44. The method of claim 42 wherein the pair of primers have a length and a GC content so that the melting temperature is ≧

70°

.

45. The method of claim 42 wherein the pair of primers have a length of between 35-70 base pairs.

46. The method of claim 42 wherein the melting temperature of each primer of the primer pair is between 70-80°

C.

47. The method of claim 42 wherein the pair of primers include a forward primer and a reverse primer each having a length of between 40-47 base pairs.

48. The method of claim 33 wherein a concentration of the nucleic acid destabilizer is 8-15 volume percent.

15. A method for amplifying a template of nucleic acid target sequence contained in a sample comprising:

contacting the sample with an amplification reaction mixture comprising;

a primer or a primer pair having a length of between 35-70 base pairs and complementary to the template of the nucleic acid target sequence and wherein the melting temperature of each primer of the primer pair is between 70-80°

C.;

DMSO;

monovalent cation;

divalent cation;

dNTPs; and

DNA Polymerase;

oscillating a temperature of the reaction 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; and

amplifying the template of nucleic acid target sequence.

16. The method of claim 15 wherein the divalent cation is a salt selected from the group consisting of magnesium, manganese, copper, zinc or any combination thereof.

17. The method of claim 15 wherein the monovalent cation is a salt selected from the group consisting of sodium, potassium, lithium, rubidium, cesium, ammonium or any combination thereof.

20. The method of claim 15 wherein the DNA polymerase is a thermostable DNA polymerase.

21. The method of claim 15 wherein the DNA polymerase is selected from the group consisting of TAQ DNA polymerase, VentR DNA polymerase, and DeepVentR DNA polymerase.

22. The method of claim 15 wherein the DNA polymerase comprises a strand displacing activity.

23. The method of claim 15 wherein the DNA polymerase does not have 3′

→

5′

exonuclease activity.

27. The method of claim 15 wherein the amplification reaction mixture comprises a single stranded binding protein.

28. The method of claim 27 wherein the single stranded binding protein is a thermal stable single stranded binding protein.

29. The method of claim 27 wherein the single stranded binding protein is a non-thermal stable single stranded binding protein.

31. The method of claim 15 wherein the sample is not alcohol free.

32. The method of claim 15 wherein the sample is not salt free.

49. The method of claim 15 wherein a concentration of the DMSO is 8-15 volume percent.