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1. (WO2008061129) PROCÉDÉS ET COMPOSITIONS SE RAPPORTANT À UN GRADIENT PCR THERMIQUE À ÉCOULEMENT CONTINU
Note: Texte fondé sur des processus automatiques de reconnaissance optique de caractères. Seule la version PDF a une valeur juridique

CLAIMS
We claim:

1. A device for replicating nucleic acid, said device comprising: a microchannel extending from an inlet port to an outlet port; and a heater or heaters for producing a spatial temperature gradient.

2. The device of claim 1, wherein the microchannel forms a serpentine pattern over the temperature gradient.

3. The device of claim 1, wherein the temperature gradient allows for gradual heating of the sample.

4. The device of claim 1, wherein the temperature gradient is a steady-state gradient.

5. The device of claim 1, wherein the heater or heaters is disposed at the far edges of the device, away from the microchannel.

6. The device of claim 1, wherein the heater is disposed along a centerline of the microchannel.

7. The device of claim 1, wherein the device also comprises a cooling component.

8. The device of claim 7, wherein the cooling component cools by air.

9. The device of claim 7, wherein the cooling component cools by using cooling fins.

10. The device of claim 7, wherein the cooling component is a thermoelectric cooler.

11. The device of claim 1, wherein the spatial temperature gradient is between approximately 1°C and 500C or more per millimeter.

12. The device of claim 7, further comprising a pump for pumping fluid through the microchannel.

13. The device of claim 1, wherein the microchannel has a heating portion and a cooling portion.

14. The device of claim 13, wherein each portion of the microchannel has a width, wherein the width of the heating portion is wider than the width of the cooling portion.

15. The device of claim 13, wherein each portion of the microchannel has a width, wherein the width of the heating portion is narrower than the width of the cooling portion.

16. The device of claim 1, further having a light source for emitting light to thereby cause fluorescence.

17. The device of claim 16, further comprising a sensor for measuring the spatial distribution of fluorescence.

18. The device of claim 1, wherein the microchannel is formed in a thin film sandwiched between two plates.

19. The device of claim 1, wherein the microchannel has a channel depth of between 10 and 200 μm.

20. The device of claim 11, wherein the cooling portion of the microchannel has a width of approximately 30-500 μm.

21. The device of claim 1, wherein the heating portion of the microchannel has a width of approximately 5-5000 μm.

22. The device of claim 1, wherein the microchannel performs between 20 and 50 amplification cycles.

23. The device of claim 1, wherein the microchannel has a length of approximately 1 to 100 centimeters.

24. The device of claim 1, wherein the microchannel is formed using a Xurography process.

25. The device of claim 1, wherein the microchannel is formed using a wet etching process.

26. The device of claim 1, wherein the heater or heaters is maintained at a steady temperature.

27. A device for replicating a nucleic acid, said device comprising: a microchannel; two plates; and a heater; wherein the microchannel is sandwiched between the two plates and the heater is operable to form a spatial temperature gradient across the microchannel.

28. The device of claim 27, wherein the microchannel is comprised of a thin film.

29. The device of claim 28, wherein the microchannel is created using at least one of Xurography and glass etching.

30. The device of claim 28, wherein the microchannel has a serpentine pattern.

31. The device of claim 28, further comprising at least one fastener for holding the two plates together.

32. A device for replicating nucleic acid, said device comprising: a channel having a plurality of sections forming a continuous pattern; and a heater disposed along a centerline of the continuous pattern; wherein each section of the channel comprises a first portion and a second portion, the first portion of the channel being narrower than the second portion of the channel.

33. The device of claim 32, wherein the wide and narrow portions are determined by the heating and cooling sections of the device.

34. The device of claim 33, wherein the dimensions of the device determine the velocity of the fluid therein, thereby controlling the temperature ramp rates.

35. The device of claim 34, wherein the continuous pattern is a serpentine pattern.

36. The device of claim 34, further comprising a pump for pumping fluid through the channel.

37. A method of amplifying a nucleic acid, the method comprising the steps of:
a) forming a steady state temperature gradient on a device comprising microchannels; and d) exposing a nucleic acid to the temperature gradient in a manner conducive for amplification;
thereby amplifying a nucleic acid.

38. The method of claim 37, wherein the microchannel is in a serpentine pattern.

39. The method of claim 37, wherein the nucleic acid is amplified multiple times.

40. A method for monitoring nucleic acid replication using a microchip, said method comprising the steps of:
a) forming a temperature gradient across a device; and
b) exposing a nucleic acid to the temperature gradient in a manner conducive for amplification; and
c) detecting nucleic acid amplification using fluorescent monitoring;
thereby monitoring nucleic acid amplification using a microchip.

41. The method of claim 40, wherein the nucleic acid is detected by exposing the nucleic acid to a dye, then detecting interaction of the dye and the nucleic acid.

42. The method of claim 41, wherein the dye is fluorescent.

43. The method of claim 41, wherein the dye is intercalating.

44. The method of claim 40, wherein each cycle of nucleic acid replication can be detected.

45. The method of claim 40, wherein amplification can be detected with a single photograph.

46. The method of claim 44 further comprising the step of measuring the amount of fluorescence produced by the dye after each extension.

47. . The method of claim 46, wherein amplification is detected by monitoring the channel-wise growth in fluorescence and wherein the melting behavior of the amplicon is detected during each denaturing process.

48. The method of claim 47, further comprising the step of providing the information in real time.

49. The method of claim 40 wherein the nucleic acid is pumped into the
microchannel.

50. The method of claim 49 wherein the nucleic acid is pumped into the
microchannel using continuous flow.

51. The method of claim 40, wherein detecting nucleic acid further comprises determining information related to the denaturing or melting of the double stranded nucleic acid.

52. The method of claim 41, wherein the dye is at least of SYBR Green, LC Green, and LC Green Plus.

53. The method of claim 47, wherein a melting curve analysis is conducted on the nucleic acid.

54. The method of claim 40, wherein more than one nucleic acid sample is amplified at a time.

55. The method of claim 54, wherein the nucleic acid samples differ in sequence.

56. The method of claim 55, wherein an analysis of the spatial fluorescence and temperature distribution can distinguish between the multiple samples of nucleic acids.

57. A method of forming a chip with microchannels for use in continuous-flow PCR, said method comprising the steps of:
a) creating a digital image of the microchannels using a digital computing machine;
b) sending the digital image of the microchannels to a plotting device such that the plotting device forms the microchannels on a thin film; and
c) sandwiching the thin film between two plates;
thereby forming a chip with microchannels for use in continuous-flow PCR.

58. The method of claim 57, wherein the digital image of the microchannels includes a serpentine pattern.

58. The method of claim 57, further comprising the step of pre-drilling holes in the plates.

59. The method of claim 57, further comprising the step of applying pressure to the two plates.

60. The method of claim 57, further comprising the step of pre-coating the thin film with an adhesive.

61. The method of claim 57, further comprising the step curing the two plates and the thin film at an elevated temperature.

62. The method of claim 56, further comprising the step forming fluid interconnects in at least one of the two plates.

63. The method of claim 56, further comprising the step forming a spatial temperature gradient.

64. The method of claim 56, further comprising the step pumping a liquid having DNA therein through the microchannels.

65. The method of claim 64, further comprising the pumping the liquid at a constant volume flow rate.

66. The method of claim 65, further comprising varying the width of the
microchannels to control temperature ramping rates.