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) moving test solution containing real-time PCR reagents into a channel;
b) uniformly cycling the temperature in a defined section of the channel in order to achieve PCR; and
c) simultaneously measuring the intensity of the fluorescent signal emitted at a plurality of locations along said defined section of said channel at a specific temperature and time during a PCR temperature cycle, wherein each of the simultaneously measured intensities of the fluorescent signal emitted at each location along the channel corresponds to the same temperature and to the same point in time within a PCR cycle.
2. The method of claim 1 , wherein the test solution continuously flows in the channel.
3. The method of claim 1 , wherein the intensity of the fluorescent signal is measured at least once during each PCR cycle.
4. The method of claim 2 , wherein the intensity of the fluorescent signal is measured at least once during each PCR cycle.
5. 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.
6. The method of claim 5 , 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.
7. A method for monitoring the progress of a polymerase chain reaction in a channel, comprising the steps of:
a) moving test solution containing real-time PCR reagents into a channel;
b) uniformly cycling the temperature in a defined section of the channel in order to achieve PCR; and
c) simultaneously capturing an image of a reaction-dependent fluorescence signal emitted along a defined section of the channel at a specific temperature and time during a PCR temperature cycle, wherein each of simultaneously measured intensities of the fluorescent signal emitted at each location along the channel corresponds to the same temperature and to the same point in time within a PCR cycle.
8. The method of claim 7 , wherein the test solution continuously flows in the channel.
9. The method as in claim 7 , wherein scattered light from a reaction independent flow marker is resolvable from the reaction-dependent fluorescence by wavelength spectrum.
10. The method as in claim 7 , wherein the image of reaction-dependent fluorescence is captured at least once per PCR cycle.
11. The method as in claim 7 , 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.
12. The method as in claim 7 , wherein the image of reaction-dependent fluorescence is captured by acquiring signal from multiple points along the channel simultaneously.