System and methods for monitoring the amplification and dissociation behavior of DNA molecules

• US 20080003593A1
• Filed: 11/30/2006
• Published: 01/03/2008
• Est. Priority Date: 06/30/2006
• Status: Active Grant

• Current Assignee: Canon USA, Inc. (Canon Inc.)
• Original Assignee: Canon US Life Sciences Inc (Canon Inc.)
• Inventors: Hasson, Kenton C., Dale, Gregory A.
• Granted Patent: US 9,573,132 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• 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

The present invention relates to systems and methods for monitoring the amplification of DNA molecules and the dissociation behavior of the DNA molecules. A method according to one embodiment of the invention may include the steps of: forcing a sample of a solution containing real-time PCR reagents to move though a channel; and while the sample is moving through an analysis region of the channel, performing the steps of: (a) cycling the temperature of the sample until the occurrence of a predetermined event; (b) after performing step (a), causing the sample'"'"'s temperature to gradually increase from a first temperature to a second temperature; and (c) while the step of gradually increasing the sample'"'"'s temperature is performed, using an image sensor to monitor emissions from the sample.

Claims

1. A method, comprising:

(a) introducing into a microchannel at least one bolus containing nucleic acid;

(b) forcing the bolus to move through the microchannel;

(c) while the bolus is moving through the microchannel, amplifying nucleic acid contained in the bolus and determining whether predetermined event has occurred;

(d) if the predetermined event has not occurred, then repeating step (c), otherwise, while the bolus is still moving through the microchannel, causing dsDNA within the bolus to transition to ssDNA and using an image sensor to capture images of the bolus.

2. The method of claim 1, wherein the step of determining whether the predetermined event has occurred comprises using the image sensor to capture an image of the bolus.

3. The method of claim 2, further comprising the step of processing the captured image data to determine the intensity of light emitted from the bolus.

4. The method of claim 3, further comprising the step of (a) comparing a value representing the determined intensity to a threshold value or (b) determining the rate of change in the intensity of the emissions from the bolus.

5. The method of claim 4, further comprising the step of initiating a process for causing the dsDNA within the bolus to transition to ssDNA if the result of the comparison indicates that the value representing the determined intensity is greater than the threshold value.

6. The method of claim 5, wherein the step of initiating the process comprises configuring a heating system to gradually increase the temperature of the bolus.

7. The method of claim 6, wherein the temperature is increased at or about a constant rate.

8. The method of claim 7, wherein the constant rate is between about 0.1 and 2 degree Celsius per second.

9. The method of claim 2, wherein the step of using the image sensor to capture an image of the bolus comprises determining a region of interest and then reading out only those pixels of the image sensor that are within the region of interest.

10. The method of claim 1, wherein the step of determining whether the predetermined event has occurred comprises determining whether (a) the bolus has been exposed to at least a predetermined number of PCR temperature cycles, (b) at least a predetermined amount of time has elapsed since a particular point in time, and/or (c) the bolus has entered a predefined region of the microchannel.

11. An optical analysis system, comprising:

a substrate comprising a microfluidic channel;

a thermal generating unit operable to provide heat to and absorb heat from at least a portion of the microfluidic channel;

an image sensor disposed in relation to the substrate such that said portion of the channel is within the field of view of the image sensor; and

an image processing system coupled to the image sensor and configured to;

(i) receive image data from the image sensor and (ii) use image data from the image sensor to determine whether (a) the intensity of emissions from a nucleic acid sample traveling through said portion of the channel equals or exceeds a predetermined emission intensity threshold or (b) the rate of change in the intensity of emissions from the nucleic acid sample traveling through said portion of the channel equals or is less than a predetermined threshold; and

a temperature controller configured to control the thermal generating unit and configured to cause the thermal generating unit to gradually ramp the temperature of the portion of the channel from a first temperature to a second temperature in response to the image processing system determining that (a) the intensity of emissions from a nucleic acid sample traveling through said portion of the channel equals or exceeds a predetermined emission intensity threshold or (b) the rate of change in the intensity of emissions from the nucleic acid sample equals or is less than the predetermined threshold.

12. The system of claim 11, wherein the temperature controller is further configured to cause the thermal generating unit to cycle the temperature of the portion of the channel in response to a predetermined input.

13. The system of claim 12, further comprising an image sensor controller, wherein the image sensor controller is operable to cause the image sensor to capture images of at least a segment of said portion of the channel that is within the field of view of the image sensor while the thermal generating unit cycles the temperature of the portion of the channel.

14. The system of claim 13, wherein the image sensor controller is operable to cause the image sensor to capture images of at least a segment of said portion of the channel that is within the field of view of the image sensor while the thermal generating unit ramps the temperature of the portion of the channel.

15. The system of claim 14, wherein the image sensor controller is operable to cause the image sensor to capture at least 1 image every 90 seconds of at least a segment of said portion of the channel that is within the field of view of the image sensor while the thermal generating unit cycles the temperature of the portion of the channel.

16. The system of claim 15 wherein the image sensor controller is operable to cause the image sensor to capture at least 5 images per second of at least a segment of said portion of the channel that is within the field of view of the image sensor while the thermal generating unit ramps the temperature of the portion of the channel.

17. The system of claim 16, wherein the thermal generating unit is configured to ramp the temperature of the portion of the channel by steadily increasing the amount of heat provided to the portion of the channel at a thermal ramp rate of between about 0.1 to 2 degree Celsius (C) per second.

18. The system of claim 11, further comprising an excitation source for producing electromagnetic radiation directed at the portion of the channel.

19. The system of claim 18, further comprising a second excitation source for illuminating at least a segment of the portion of the channel in response to the image processing system determining that (a) the intensity of emissions from a nucleic acid sample traveling through said portion of the channel equals or exceeds a predetermined emission intensity threshold or (b) the rate of change in the intensity of emissions from the nucleic acid sample equals or is less than the predetermined threshold.

20. The system of claim 19, wherein the image sensor is a CMOS image sensor and the second excitation source is a laser.

21. In a system comprising a substrate comprising a channel, a method comprising:

forcing a sample of a solution containing real-time PCR reagents to move though the channel; and

while the sample is moving through an analysis region of the channel, performing the steps of;

(a) cycling the temperature of the sample until the occurrence of a predetermined event;

(b) after performing step (a), causing the sample'"'"'s temperature to gradually increase from a first temperature to a second temperature; and

(c) while the step of gradually increasing the sample'"'"'s temperature is performed, using an image sensor to monitor emissions from the sample.

22. The method of claim 21, further comprising the step of determining whether the predetermined event has occurred.

23. The method of claim 22, wherein the step of determining whether the predetermined event has occurred comprises determining whether nucleic acid in the sample has been sufficiently amplified.

24. The method of claim 22, wherein the step of determining whether the nucleic acid in the sample has been sufficiently amplified comprises determining the intensity of light emitted from the sample and comparing the determined intensity to an intensity threshold value.

25. The method of claim 24, wherein the step of determining the intensity of light emitted from the sample comprises using an image sensor to capture an image of the sample.

26. The method of claim 25, wherein the step of using the image sensor to capture an image of the sample comprises determining a region of interest and then reading out only those pixels of the image sensor that are within the region of interest.

27. The method of claim 21, wherein the step of cycling the temperature of the sample comprises performing the following steps one or more times:

(i) holding the temperature of the sample at or about a first temperature for at least a first period of time;

(ii) after step (i), using a heating source to heat the temperature of the sample from the first temperature to a second temperature;

(iii) after step (ii), holding the temperature of the sample at or about the second temperature for at least a second period of time;

(iv) after step (iii), using the heating source to heat the temperature of the sample from the second temperature to a third temperature;

(v) after step (iv), holding the temperature of the sample at or about the third temperature for at least a third period of time.

28. The method of claim 21, wherein the step of gradually increasing the temperature of the sample from the first temperature to the second temperature comprises increasing the temperature at or about a constant rate.

29. The method of claim 28, wherein the constant rate is between about 0.1 and 1 degrees Celsius per second.

30. The method of claim 29, wherein increasing the temperature at or about a constant rate comprises increasing the temperature at said rate for at least about 1 minute.

31. The method of claim 21, further comprising the step of keeping a record of the number of temperature cycles to which the sample is exposed.

32. The method of claim 31, wherein the step of determining whether the predetermined event has occurred comprises determining whether the sample has been exposed to at least a predetermined number of temperature cycles.

33. The method of claim 21, wherein the step of determining whether the predetermined event has occurred comprises determining whether at least a predetermined amount of time has elapsed since a particular point in time.

34. The method of claim 21, wherein the step of determining whether the predetermined event has occurred comprises determining whether the sample has entered a predefined region of the channel.