Methods for fast nucleic acid amplification
Devices are provided for performing polymerase chain reaction (PCR).
1. A device for performing PCR comprising;
a sample container for holding a PCR sample, wherein the sample container comprises
a target nucleic acid,
dNTPs,
a polymerase provided at a concentration of at least 0.5 μM, and
a pair of primers configured for amplifying the target nucleic acid, wherein the primers are each provided at a concentration of at least 2 μM;
means for heating the sample;
means for cooling the sample; and
control means for repeatedly subjecting the sample container to the means for heating the sample and the means for cooling the sample to thermal cycle the sample at a ramp rate of at least 200° C./s.
2. The device of claim 1 , further comprising means for optically exciting the sample in the monitoring position to cause the sample to fluoresce; and means for detecting the fluorescence of the excited sample during amplification.
3. The device of claim 2 , wherein the ramp rate is at least 300° C./s.
4. The device of claim 3 , wherein the ramp rate is at least 400° C./s.
5. The device of claim 1 , wherein the means for heating is a hot water bath and the means for cooling is a cool water bath.
6. The device of claim 1 , wherein the device is a microfluidics system.
7. A device for performing PCR comprising;
a sample container for holding a PCR sample, wherein the sample container comprises
a target nucleic acid,
dNTPs,
a polymerase provided at a concentration of at least 0.5 μM, and
a pair of primers configured for amplifying the target nucleic acid, wherein the primers are each provided at a concentration of at least 2 μM;
a heat transfer device for changing temperature of the sample in the sample container; and
a temperature controller for controlling the heat transfer device to repeatedly heat and cool the sample at a ramp rate of at least 200° C./s.
8. The device of claim 7 , wherein
the heat transfer device comprises a plurality of water baths, and
the temperature controller is a motor attached to an arm that moves the sample container between the water baths.
9. The device of claim 7 , wherein
the heat transfer device comprises a plurality of temperature zones,
the sample container defines a path in each of the temperature zones, and
the temperature controller controls flow of the sample to each of the temperature zones.
10. The device of claim 7 , wherein
the heat transfer device comprises heated air.
11. The device of claim 7 , wherein
the heat transfer device includes Joule heating.
12. The device of claim 7 , wherein the heat transfer device is a heat block.
13. The device of claim 7 , wherein the sample container further comprises a dye, and the device further comprises an optics block configured to detect emissions from the dye.
14. The device of claim 13 , wherein the optics block detects emissions from the dye in real-time.
15. The device of claim 7 , wherein the temperature controller is triggered by sample temperature.
16. The device of claim 7 , wherein the temperature controller is programmed to hold the sample at a temperature for a specific duration of time.
17. The device of claim 16 , wherein the specific duration of time is less than one second.
18. The device of claim 7 , wherein the device is controlled by a CPU.
19. The device of claim 7 , wherein transfer between temperature zones is achieved in less than 100 msec.
20. A device for performing PCR comprising;
a sample container for holding a PCR sample, wherein the sample container comprises eukaryotic genomic DNA and further comprises a polymerase provided at a concentration of at least 0.5 μM;
a heat transfer device for changing temperature of the sample in the sample container; and
a temperature controller for controlling the heat transfer device to repeatedly heat and cool the sample at a ramp rate of at least 200° C./s.
21. The device of claim 20 , wherein the sample container further comprises a pair of primers configured for amplifying the target nucleic acid, wherein the primers are each provided at a concentration of at least 2 μM.
22. The device of claim 20 , wherein the sample container further comprises primers for amplification of a target nucleic acid sequence of the eukaryotic genomic DNA, wherein the polymerase to primer ratio is 0.03 to 0.4 polymerase: total primer concentration.