Real-time PCR in micro-channels
The present invention relates to methods for amplifying nucleic acids in micro-channels. More specifically, the present invention relates to methods for performing a real-time polymerase chain reaction (PCR) in a continuous-flow microfluidic system and to methods for monitoring real-time PCR in such systems.
1. A method of performing real-time PCR comprising the steps of:
a) continuously moving a bolus of test solution containing real-time PCR reagents in a channel;
b) moving a carrier fluid in said channel, sequentially alternating with a test bolus;
c) cycling the temperature in a defined section of the channel in order to achieve PCR, wherein said defined section of the channel has a uniform temperature, which temperature cycles in time; and
d) measuring the intensity of the fluorescent signal at a plurality of locations along said defined section of said channel.
2. The method of claim 1 , which further comprises the step of measuring the average flow speed in the channel.
3. The method of claim 2 , wherein the average flow speed is measured by comparing sequential images of the fluorescent signal from the channel.
4. The method of claim 2 , wherein the average flow speed is measured by comparing sequential images of a marker in the channel.
5. The method of claim 4 , wherein the marker is in the test solution.
6. The method of claim 4 , wherein the marker is in the carrier fluid.
7. The method of claim 2 , which further comprises the step of adjusting the flow rate to control the timing of each PCR cycle.
8. The method of claim 7 , wherein the flow rate is adjusted by adjusting the pressure at an inlet or outlet of the channel.
9. The method of claim 1 , wherein the intensity of the fluorescent signal is measured at least once during each PCR cycle.
10. The method of claim 2 , wherein the intensity of the fluorescent signal is measured at least once during each PCR cycle.
11. The method of claim 7 , wherein the intensity of the fluorescent signal is measured at least once during each PCR cycle.
12. The method of claim 7 , wherein the average flow speed is measured by comparing sequential images of the fluorescent signal from the channel.
13. The method of claim 7 , wherein the average flow speed is measured by comparing sequential images of a marker in the channel.
14. The method of claim 13 , wherein the marker is in the test solution.
15. The method of claim 13 , wherein the marker is in the carrier fluid.
16. The method of claim 1 , wherein the step of cycling the temperature further includes the steps of:
i) heating said defined section of the channel to a first temperature;
ii) cooling said defined section of the channel to a second temperature; and
iii) heating said defined section of the channel to a third temperature.
17. The method of claim 16 , wherein the first temperature is in the range of about 85° C. to about 100° C., the second temperature is in the range of about 20° C. to about 70° C. and the third temperature is in the range of about 55° C. to about 80° C.
18. The method of claim 1 , wherein said defined section of the channel has a length suitable for completing 10 to 50 cycles of PCR.
19. A method for monitoring the progress of a polymerase chain reaction in a channel, comprising the steps of:
a) moving a bolus of test solution containing real-time PCR reagents in a channel;
b) moving a carrier fluid in said channel, sequentially alternating with a test bolus;
c) cycling the temperature in a defined section of the channel in order to achieve PCR, wherein said defined section of the channel has a uniform temperature, which temperature cycles in time; and
d) capturing an image of a reaction-dependent fluorescence signal along a section of the channel;
e) measuring the average flow speed in the channel; and
f) relating position of the test bolus to the number of temperature cycles experienced by a test bolus from the average flow speed.
20. The method as in claim 19 , wherein the average flow speed is measured by comparing sequential images of the reaction-dependent fluorescent signal from the channel.
21. The method as in claim 19 , wherein the average flow speed is measured by comparing sequential images of a reaction-independent flow marker from the channel.
22. The method as in claim 21 , wherein the reaction-independent flow marker is pre-mixed in the test bolus.
23. The method as in claim 21 , wherein the reaction-independent flow marker is introduced to the channel as a bolus alternating with the test bolus.
24. The method as in claim 21 , wherein the reaction-independent flow marker is pre-mixed in the carrier fluid.
25. The method as in claim 21 , wherein scattered light from the reaction-independent flow marker is resolvable from the reaction-dependent fluorescence by wavelength spectrum.
26. The method as in claim 21 , wherein scattered light from the reaction-independent flow marker is resolvable from the reaction-dependent fluorescence on the basis of fluorescence lifetime.
27. The method as in claim 21 , wherein the reaction-independent flow marker is further used to determine the flow dispersion of the test bolus.
28. The method as in claim 19 , wherein the image of reaction-dependent fluorescence is captured at least once per PCR cycle.
29. The method as in claim 19 , wherein the image of reaction-dependent fluorescence is captured sequentially by scanning a length of the channel on a time scale shorter than the duration of one PCR cycle.
30. The method as in claim 19 , wherein the image of reaction-dependent fluorescence is captured by acquiring signal from multiple points along the channel simultaneously.
31. The method as in claim 19 , wherein the flow rate measurements are part of a feedback loop for regulating the flow rate.
32. The method as in claim 19 , wherein the flow speed is measured through detecting a sample bolus entrance into and exit from a defined section of the channel.