Systems and methods for real-time PCR

• US 9,283,563 B2
• Filed: 10/28/2008
• Issued: 03/15/2016
• Est. Priority Date: 06/30/2006
• Status: Active Grant

• Current Assignee: Canon USA, Inc. (Canon Inc.)
• Original Assignee: Canon USA, Inc. (Canon Inc.)
• Inventors: Owen, Gregory H., Dale, Gregory A., Hasson, Kenton C., Warfield, Dwayne W., Zeng, Shulin
• Primary Examiner(s): WOOLWINE, SAMUEL C
• Application Number:
  US12/259,609
• Publication Number:
  US 20090053726A1
• Time in Patent Office: 2,695 Days
• Field of Search: None
• US Class Current: 1/1
• 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 real-time DNA amplification and analysis method, comprising:

creating a script, wherein the script contains configuration information;

preparing a microfluidic device having a microfluidic channel having a PCR zone and a DNA

melting zone;

reading the script to obtain the configuration information;

positioning a well plate relative to the device, the well plate having a buffer well containing a buffer solution and sample well containing a sample solution containing a DNA sample;

activating a pump, wherein the pump is configured to create a pressure differential that causes the sample solution to flow through the channel, wherein the sample flows through the PCR zone prior to flowing through the DNA melting zone;

while the sample is flowing through the channel;

cycling the temperature of the sample according to configuration information included in the script as the sample flows through the PCR zone to amplify the DNA sample;

illuminating the sample with a first excitation source as the sample flows through the PCR zone and using a first image detector to obtain more than one image of the sample;

processing the images;

determining whether another PCR cycle is needed, and if so, causing additional temperature cycling to occur; and

melting the amplified DNA, illuminating the sample with a second excitation source as the sample flows through the DNA melting zone and using a second image detector to obtain more than one image of the sample, and wherein the first and second excitation sources are of different types.

2. The method of claim 1, wherein the step of preparing the device comprises:

filling a first well located in the device with a first reagent;

filling a second well located in the device with a second reagent.

3. The method of claim 1, further comprising determining the speed at which the sample flows through the channel and adjusting the pump if the speed is too fast or too slow.

4. The method of claim 3, wherein the step of determining the speed at which the sample flows through the channel comprises processing images of the sample that were taken of the sample while the sample was flowing through the channel.

5. The method of claim 1, further comprising configuring an imaging system prior to obtaining said images of the sample, wherein the imaging system is used to obtain said images.

6. The method of claim 5, wherein the step of configuring the imaging system comprises:

(a) obtaining an image of the PCR zone using a detector;

(b) displaying the image of the PCR zone and displaying a reference image;

(c) determining whether the detector is focused adequately;

(d) if the detector is not adequately focused, then adjusting the focus of the detector and repeating steps (a) through (d).

7. The method of claim 1, wherein the step of positioning the well plate comprises:

determining a well of the well plate based on the script;

positioning the well plate so that a sipper connected to the device is placed in the determined well.

8. The method of claim 7, further comprising repositioning the well plate relative to the device after a predetermined amount of time has elapsed after positioning the well plate and/or activating the pump.

9. The method of claim 8, wherein the step of repositioning the well plate comprises positioning the well plate relative to the device so that the sipper is placed in the buffer well.

10. The method of claim 1, wherein the step of cycling the temperature of the sample, comprises:

causing the temperature of the PCR zone to reach a denature stage temperature;

once the temperature of the PCR zone reaches the denature stage temperature, maintaining the PCR zone at about the denature stage temperature for a first predetermined period of time;

immediately after the first predetermined period of time elapses, causing the temperature of the PCR zone to reach an annealing stage temperature;

once the temperature of the PCR zone reaches the annealing stage temperature, maintaining the PCR zone at about the annealing stage temperature for a second predetermined period of time;

immediately after the second predetermined period of time elapses, causing the temperature of the PCR zone to reach an extension stage temperature; and

once the temperature of the PCR zone reaches the extension stage temperature, maintaining the PCR zone at about the extension stage temperature for a third predetermined period of time.

11. The method of claim 1, wherein the step of obtaining images of the sample as the sample flows through the PCR zone, comprises:

determining whether an image trigger point is reached; and

performing the following steps if it is determined that the image trigger point is reached;

(a) waiting for a predetermined amount of time to elapse;

(b) once the predetermined amount of time has elapsed, turning on, or increasing the output of, the first excitation source;

(c) after step (b), acquiring an image of the sample; and

(d) turning off, or lowering the output of, the first excitation source.

12. The method of claim 11, wherein the first excitation source is a blue or red light emitting diode.

13. The method of claim 11, wherein the following steps are performed either simultaneously with steps (b)-(d) of claim 11 or following step (d) of claim 11:

(e) turning on, or increasing the output of, the second excitation source;

(f) after step (e), acquiring an image of the sample; and

(g) turning off, or lowering the output of, the second excitation source.

14. The method of claim 1, further comprising the steps of:

obtaining an image of the DNA melting zone;

displaying the image on a display screen;

enabling a user to use the image to specify a region of interest;

determining a pixel window that includes the region of interest; and

operating a detector to obtain more than one image per second of the DNA melting zone, and, for each image, recording only the pixels that are within the determined pixel window.

15. The method of claim 14, further comprising using information contained in the images of the DNA melting zone to create a graph that plots measured fluorescence intensity versus temperature.

16. The method of claim 14, further comprising the step of:

determining whether the melt is complete;

wherein, if the melt is not complete, the method additionally comprises performing the step of operating a detector to obtain more than one image per second of the DNA melting zone, and, for each image, recording only the pixels that are within the determined pixel window.