Microwave Assisted PCR Amplification of DNA

Abstract

A method of microwave assisted nucleic acid amplification by PCR is disclosed. The method includes denaturing, annealing, and extending a nucleic acid sample, with at least the denaturing and extension steps being carried out under the influence of microwave radiation, while preventing the temperature of the sample from varying more than 40° C. from start to finish, and while maintaining the temperature of the sample from start to finish at no more than 60° C.

Description

Claims

1. A method of carrying out PCR amplification of DNA comprising: maintaining a composition of DNA and primer and replicating enzyme at a temperature less than 60° C.;directing a first pulse of microwave radiation to the DNA sufficient to denature the DNA while maintaining the DNA at a temperature less than 60° C.;annealing the primer onto the separate DNA strands while maintaining the DNA at a temperature less than 60° C.;directing a second pulse of microwave radiation to the annealed DNA strands sufficient to replicate and extend the primary sequence while maintaining the DNA at a temperature less than 60° C.; andcarrying out the denaturing, annealing and extension steps concurrently on a plurality of compositions of DNA and primer and replicating enzyme.

2. A method according to claim 1 comprising amplifying the DNA with a replicating enzyme that denatures at temperatures above about 60° C.

3. A method according to claim 1 comprising amplifying the DNA with a replicating enzyme that denatures at temperatures above about 45° C.

4. A method according to claim 1 comprising maintaining the composition at a temperature below about 45° C. during the denaturing, annealing and extension steps.

5. (canceled)

6. A method according to claim 5 comprising carrying out the concurrent denaturing, annealing and extension steps on a plurality of identical samples.

7. A method according to claim 5 comprising carrying out the concurrent denaturing, annealing and extension steps on at least two different samples.

8. A method according to claim 1 comprising annealing the primer at a temperature lower than the temperature of the denaturing step.

9. A method according to claim 1 comprising annealing the primer at a temperature lower than the temperature of the extension step.

10. A method according to claim 1 comprising proactively cooling the composition of DNA and primer and replicating enzyme to maintain the desired temperatures during the denaturation, annealing and extension steps.

11. A method according to claim 1 wherein the step of directing said first pulse or said second pulse comprises applying a pulse of between about 50 and 250 watts for no more than one second.

12. A method according to claim 1 comprising maintaining the desired temperature by proactively cooling the sample with an inert gas.

13. A method according to claim 1 comprising maintaining the desired temperature by proactively cooling the sample with a liquid

14. A method according to claim 1 further comprising directing a pulse of microwave radiation to the composition during the annealing step.

15. A method of microwave assisted nucleic acid amplification by PCR comprising: at least two cycles of denaturing, annealing, and extending a nucleic acid sample, with at least the denaturing and extension steps being carried out under the influence of microwave radiation;while preventing the temperature of the sample from varying more than 40° C. from start to finish;and while maintaining the temperature of the sample from start to finish at no more than 60° C.

16. A method according to claim 15 comprising preventing the temperature of the sample from varying more than 20° C. from start to finish.

17. A method according to claim 15 comprising preventing the temperature of the sample from varying more than 10° C. from start to finish.

18. A method according to claim 15 comprising maintaining the temperature of the sample from start to finish at no more than 50° C.

19. A method according to claim 15 comprising maintaining the temperature of the sample from start to finish at no more than 45° C.

20. (canceled)

21. A method according to claim 15 comprising at least 30 cycles of denaturing, annealing, and extending.

22. A method according to claim 15 comprising denaturing, annealing, and extending a DNA sample.

23. A method according to claim 22 comprising denaturing, annealing, and extending a single DNA sample.

24. A method according to claim 22 comprising denaturing, annealing, and extending at least two DNA samples.

25. A method according to claim 24 comprising denaturing, annealing and extending different samples of DNA.

26. A method according to claim 22 comprising denaturing, annealing, and extending at least 96 DNA samples.

27. A method according to claim 26 comprising denaturing, annealing and extending different samples of DNA.

28. A method according to claim 15 where in the step of carrying out the cycle under the influence of microwave radiation comprises applying pulse radiation to the DNA sample.

29. A method according to claim 28 comprising applying a first pulse of microwave radiation for the denaturing step and applying a second pulse of microwave radiation for the extension step.

30. A method of microwave assisted DNA amplification by PCR comprising: at least 20 PCR cycles, with each cycle including,denaturing, annealing, and extending a DNA sample, with at least the denaturing and extension steps being carried out under the influence of microwave radiation;while preventing the temperature of the sample from varying more than 20° C. from start to finish; andwhile maintaining the temperature of the sample from start to finish at no more than 50° C.

31. A method according to claim 30 comprising carrying out the 20 cycles in less than one hour.

32. A method according to claim 30 comprising carrying out the 20 cycles in less than 30 minutes.

33. A method according to claim 30 comprising an efficiency of 0.6 to 0.9.

34. A method of carrying out PCR DNA amplification cycles comprising: directing a first pulse of microwave radiation to a composition of DNA and primer and replicating enzyme that is sufficient to denature the DNA in the presence of a replicating enzyme that denatures at temperatures above 60° C.;annealing the primer onto the separate DNA strands;directing a second pulse of microwave radiation to the annealed DNA strands sufficient to replicate and extend the primary sequence; andrepeating the denaturing, annealing and extension cycle at least twice without refreshing the replicating enzyme.

35. A method according to claim 34 comprising maintaining the composition of DNA and primer and replicating enzyme at a temperature less than about 60° C. throughout each PCR cycle.

36. A method according to claim 34 comprising maintaining the composition of DNA and primer and replicating enzyme at a temperature less than about 45° C. throughout each PCR cycle.

37. A method according to claim 34 comprising directing the first pulse of microwave radiation to a composition that includes a replicating enzyme that denatures above about 45° C.

38. A method according to claim 34 comprising repeating the denaturing, annealing and extension cycle at least 20 times without refreshing the replicating enzyme.

39. A method according to claim 34 comprising repeating the denaturing, annealing and extension cycle at least 30 times without refreshing the replicating enzyme.