Systems and methods for real-time PCR

• US 8,058,054 B2
• Filed: 06/29/2007
• Issued: 11/15/2011
• Est. Priority Date: 06/30/2006
• Status: Active Grant

• Current Assignee: Canon USA, Inc. (Canon Inc.)
• Original Assignee: Canon USA, Inc. (Canon Inc.)
• Inventors: Warfield, Dwayne W., Owen, Gregory H., Hasson, Kenton C., Dale, Gregory A., Zeng, Shulin
• Primary Examiner(s): WOOLWINE, SAMUEL C
• Application Number:
  US11/770,869
• Publication Number:
  US 20080176230A1
• Time in Patent Office: 1,600 Days
• Field of Search: None
• US Class Current: 435/283.1
• CPC Class Codes: B01L 2200/10 Integrating sample preparation and analysis in single entity, e.g. lab-on-a-chip concept
• CPC Class Codes: B01L 2200/143 Quality control, feedback systems
• CPC Class Codes: B01L 2300/0627 Sensor or part of a sensor is integrated
• CPC Class Codes: B01L 2300/0654 Lenses; Optical fibres
• CPC Class Codes: B01L 2300/0829 Multi-well plates; Microtitration plates
• CPC Class Codes: B01L 2300/18 Means for temperature control
• CPC Class Codes: B01L 2300/1811 using electromagnetic induction heating
• CPC Class Codes: B01L 2300/1822 using Peltier elements
• CPC Class Codes: B01L 2300/1827 using resistive heater
• CPC Class Codes: B01L 2300/1877 using chemical reactions
• CPC Class Codes: B01L 2400/0487 fluid pressure, pneumatics
• CPC Class Codes: B01L 3/5027 by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip B01L3/5023 takes precedence; micromixers B01F33/30; microreactors for synthesis B01J19/0093; microcapillary devices in general B81B1/00
• CPC Class Codes: B01L 3/502715 characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces
• CPC Class Codes: B01L 3/50273 characterised by the means or forces applied to move the fluids micropumps F04B19/006, of the membrane type F04B43/043
• CPC Class Codes: B01L 7/52 with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples amplification or hybridisation processes per se C12Q1/68; controlling sequential reactions for synthesis B01J19/0046
• CPC Class Codes: B01L 7/525 with physical movement of samples between temperature zones
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2561/113 Real time assay
• CPC Class Codes: C12Q 2565/629 being a microfluidic device
• CPC Class Codes: G01N 21/71 thermally excited
• CPC Class Codes: G01N 35/08 : using a stream of discrete samples flowing along a tube system, e.g. flow injection analysis
• CPC Class Codes: G06T 2207/10004 : Still image; Photographic image
• CPC Class Codes: G06T 2207/30072 : Microarray; Biochip, DNA array; Well plate
• CPC Class Codes: G06T 7/0012 : Biomedical image inspection
• CPC Class Codes: G06T 7/11 : Region-based segmentation
• CPC Class Codes: Y10T 436/115831 : Condition or time responsive
• CPC Class Codes: Y10T 436/117497 : with a continuously flowing sample or carrier stream

Abstract

In one aspect, the present invention provides a systems and methods for the real-time amplification and analysis of a sample of DNA.

Claims

1. A system for performing real-time PCR and melting of DNA, comprising:

a microfluidic device comprising;

a microfluidic channel having a PCR zone and a DNA melting zone, a first reagent well in fluid communication with the channel and a second reagent well in fluid communication with the channel;

a sipper connected to the microfluidic device, the sipper being in fluid communication with the channel;

a pump for forcing a sample to flow through the channel;

a well plate comprising a buffer well for storing

a buffer solution and a sample well for storing a sample solution containing a DNA sample;

a buffer solution storage container for storing a buffer solution, the buffer solution storage container being in fluid communication with the buffer well of the well plate;

a positioning system operable to position the well plate;

a temperature control system for cycling the temperature of a sample while the sample is in the PCR zone of the channel and for providing heat for melting the DNA contained in the sample while the sample is in the DNA melting zone;

an imaging system for detecting emissions from the PCR zone and from the DNA melting zone, the imaging system comprising;

a first excitation source and a detector configured and arranged to detect emissions from the PCR zone and a second excitation source and a detector configured and arranged to detect emissions from the DNA melting zone;

wherein the first excitation source for the PCR zone and the second excitation source for the DNA melting zone are different types; and

a main controller in communication with (a) the temperature control system, (b) the positioning system, (c) the imaging system, and (d) a display device.

2. The system of claim 1, wherein the positioning system comprises a positioner.

3. The system of claim 2, wherein the positioning system further comprises a stepping drive operable to drive the positioner.

4. The system of claim 3, wherein the positioning system further comprises a controller for controlling the stepping drive.

5. The system of claim 1, wherein the imaging system comprises:

a first light emitting diode directed to the PCR zone of the channel, a second light emitting diode directed to the PCR zone of the channel, and a laser directed to the melting zone of the channel.

6. The system of claim 5, wherein the first light emitting diode and the second light emitting diode are different colors, and wherein the first and second light emitting diodes are operated independently by the main controller.

7. The system of claim 1, wherein the second detector comprises a electron multiplying charge coupled device (EMCCD).

8. The system of claim 1, wherein the imaging system further comprises a detector controller interfaced with the first detector and the second detector.

9. The system of claim 8, wherein the detector controller is communicatively connected to the main controller.

10. The system of claim 9, wherein the detector controller is connected to the main controller through an Ethernet hub.

11. The system of claim 1, wherein the temperature control system comprises a first heater configured to heat a sample within the PCR zone and a second heater configured to heat a sample within the DNA melting zone.

12. The system of claim 11, wherein the first heater comprises a first thermoelectric cooler (TEC) and the second heater comprises a second thermoelectric cooler (TEC).

13. The system of claim 12, wherein the temperature control system further comprises a first temperature controller coupled to the first TEC and a second temperature controller coupled to the second TEC.

14. The system of claim 13, wherein the first temperature controller is coupled to the main controller and the second temperature controller is coupled to the main controller.

15. The system of claim 14, wherein the first temperature controller consists of a power amplifier.

16. The system of claim 11, wherein the temperature control system further comprises a source of electromagnetic radiation, which source is arranged to illuminate the PCR zone.

17. The system of claim 11, wherein the first or second heater is configured to produce infrared radiation.

18. The system of claim 1, wherein the main controller monitors the sample during PCR amplification.

19. The system of claim 1, wherein the main controller monitors the sample during DNA melting.

20. The system of claim 1, wherein the main controller adjusts one or more of the imaging system, the temperature control system, the positioning system or the pump.

21. The system of claim 1, wherein the main controller triggers one or more additional PCR cycles in response to the data received from the imaging system.

22. The system of claim 1, wherein the main controller triggers one or more additional DNA melts in response to the data received from the imaging system.

23. The system of claim 1, wherein the main controller is configured to operate the first excitation source and the second excitation source independently, and wherein the main controller is configured to operate the second excitation source in response to analysis of the emissions detected by the PCR zone detector.

24. A system for performing real-time PCR and melting of DNA, comprising:

a microfluidic device comprising;

a microfluidic channel having a PCR zone and a DNA melting zone, a first reagent well in fluid communication with the channel and a second reagent well in fluid communication with the channel;

a sipper connected to the microfluidic device, the sipper being in fluid communication with the channel;

a pump for forcing a sample to flow through the channel;

a well plate comprising a buffer well for storing a buffer solution and a sample well for storing a sample solution containing a DNA sample;

a buffer solution storage container for storing a buffer solution, the buffer solution storage container being in fluid communication with the buffer well of the well plate;

a positioning system operable to position the well plate;

a temperature control system for cycling the temperature of a sample while the sample is in the PCR zone of the channel and for providing heat for melting the DNA contained in the sample while the sample is in the DNA melting zone;

an imaging system for detecting emissions from the PCR zone and from the DNA melting zone, the imaging system comprising;

a first excitation source comprising a laser emitting diode and a detector configured and arranged to detect emissions from the PCR zone and a second excitation source comprising a laser and a detector configured and arranged to detect emissions from the DNA melting zone;

a main controller in communication with (a) the temperature control system, (b) the positioner, (c) the imaging system, and (d) a display device.