Systems and methods for monitoring the amplification and dissociation behavior of dna molecules

Abstract

The present invention relates to systems and methods for monitoring the amplification of DNA molecules and the dissociation behavior of the DNA molecules. The present invention in one embodiment provides a system that includes a microfluidic channel comprising a PCR processing zone and an HRTm analysis zone; and an image sensor having a first image sensor region having a first field of view and a second image sensor region having a second field of view, wherein the second field of view is different than the first field of view, wherein at least a portion of the PCR processing zone is within the first field of view; and at least a portion of the HRTm analysis zone is within the second field of view.

1 . A system, comprising:
 a micro fluidic channel wherein PCR processing and HRTm analysis occur; and   an imaging system, comprising an image sensor having a field of view, wherein   at least a portion of the micro fluidic channel where PCR processing and at least a portion of the HRTm analysis occurs is within the field of view.   
     
     
         2 . The system of  claim 1 , further comprising:
 a ft thermal generating apparatus configured to provide heat to and/or absorb heat from the PCR processing   wherein the thermal generating apparatus is configured to provide heat to and/or absorb heat from the HRTm analysis process.   
     
     
         3 . The system of  claim 2 , wherein the thermal generating apparatus is configured to cycle the temperature for PCR processing when a bolus of solution containing real-time PCR reagents is within the-microfluidic channel in order to achieve PCR. 
     
     
         4 . The system of  claim 3 , wherein the thermal generating apparatus is configured to perform PCR processing, such that the thermal generating apparatus heats the bolus to a first temperature, cools the bolus to a second temperature, and heats the bolus to a third temperature. 
     
     
         5 . The system of  claim 4 , wherein, the thermal generating apparatus is configured to provide to the bolus a substantially steadily increasing amount of heat at a thermal ramp rate of between about 0.1 to 1 degree Celsius (C) per second. 
     
     
         6 . The system of  claim 1 , further comprising a lens disposed between the channel and the image sensor, wherein the lens is configured to focus onto light coming from the bolus undergoing the PCR processing from the HRTm analysis. 
     
     
         7 . The system of  claim 1 , further comprising an excitation source for producing electromagnetic radiation directed at the channel. 
     
     
         8 . The system of  claim 1 , wherein the microfluidic channel is operable to receive at least one of a plurality of spaced apart boluses of solution containing real-time PCR reagents. 
     
     
         9 . The system of  claim 8 , further comprising an image sensor controller configured such that, for each of said bolus that undergoes HTRm processing, the image sensor controller captures at least about 10 images per second for at least about 1 minute while the bolus undergoes the HRTm analysis. 
     
     
         10 . The system of  claim 9 , wherein the image sensor controller is further configured so that, for each said bolus that undergoes PCR processing, the controller captures at least about 1 image every 90 seconds for at least about 10 minutes while the bolus is undergoing the PCR processing. 
     
     
         11 . The system of  claim 9 , wherein the image sensor controller is further configured so that, for each said bolus that undergoes PCR processing, the controller captures not more than 1 image every 1 second for at least about 10 minutes while the bolus undergoes the PCR processing. 
     
     
         12 . A system, comprising:
 a channel for receiving a bolus of solution containing real-time PCR reagents, wherein DNA amplification and a DNA melting are performed in the channel; and   an image sensor disposed in relation to the channel such that both the DNA melting and the DNA amplification are within the field of view of the sensor.   
     
     
         13 . The system of  claim 12 , further comprising:
 a thermal generating apparatus configured to provide heat to and/or absorb heat from the DNA amplification process; and   the thermal generating apparatus is configured to provide heat to and/or absorb heat from the DNA melting process.   
     
     
         14 . The system of  claim 12 , wherein the thermal generating apparatus is configured such that when the bolus undergoes DNA amplification, the thermal generating apparatus cycles the temperature for DNA amplification in order to achieve PCR. 
     
     
         15 . The system of  claim 14 , wherein the thermal generating apparatus is configured such that when the bolus undergoes the DNA amplification, the thermal generating apparatus heats the channel to a first temperature, cook the channel to a second temperature, and heats the channel to a third temperature. 
     
     
         16 . The system of  claim 15 , wherein the thermal generating apparatus is configured such that, when the DNA melting analysis is performed, the thermal generating apparatus provides to the-bolus a substantially steadily increasing amount of heat at a thermal ramp rate of between about 0.1 to 1 degree Celsius (C) per second. 
     
     
         17 . The system of  claim 12 , further comprising a lens disposed between the channel and the image sensor, wherein the lens is configured to focus onto the image sensor light coming from the DNA amplification process. 
     
     
         18 . The system of  claim 12 , further comprising an excitation source for producing electromagnetic radiation directed at the channel. 
     
     
         19 . The system of  claim 12 , wherein the channel is operable to receive a plurality of spaced apart boluses of solution containing real-time PCR reagents. 
     
     
         20 . The system of  claim 19 , further comprising an image sensor controller configured such that, for each said bolus where DNA melting is performed, the image sensor controller captures at least about 10 images per second for at least about 1 minute. 
     
     
         21 . The system of  claim 20 , wherein the image sensor controller is further configured so that, for each said bolus that undergoes PCR processing, the controller captures at least about 1 image every 90 seconds for at least about 10 minutes while the bolus is undergoing the PCR processing. 
     
     
         22 . The system of  claim 20 , wherein the image sensor controller is further configured so that, for each said bolus that undergoes PCR processing, the controller captures not more than 1 image every 1 second for at least about 10 minutes while the bolus undergoes the PCR processing. 
     
     
         23 . In a system comprising (a) an image sensor and (b) a microfluidic channel for performing a PCR processing and a HRTm analysis, a method comprising:
 (a) using the channel to achieve PCR;   (b) using the channel to perform an HRTm process;   (c) while performing steps (a) and (b), using the image sensor to obtain images of the HRTm analysis; and   PCR processing.   
     
     
         24 . The method of  claim 23 , wherein the step of PCR processing to achieve PCR comprises:
 moving a bolus of test solution containing real-time PCR reagents through the channel; and   while the bolus is in the channel, cycling the temperature of the bolus.   
     
     
         25 . The method of  claim 24 , wherein the step of cycling the temperature of the bolus comprises heating the bolus to a first temperature, cooling the bolus to a second temperature, and heating the bolus to a third temperature. 
     
     
         26 . The method of  claim 24 , wherein the step of performing the HRTm analysis comprises:
 moving a bolus of test solution containing real-time PCR reagents through the channel; and   steadily increasing the temperature of the bolus.   
     
     
         27 . The method of  claim 26 , wherein:
 the step of cycling the temperature of the bolus in order to achieve PCR comprises using a thermal generating apparatus to cycle the temperature, and   the step of steadily increasing the temperature of the bolus comprises using the thermal generating apparatus to steadily increase the temperature.   
     
     
         28 . The method of  claim 23 , further comprising using a lens to focus onto the image sensor light coming from a bolus undergoing PCR processing and to focus onto the image sensor light coming from a bolus undergoing HRTm analysis, wherein the light coming from the bolus undergoing PCR processing and the light coming from a bolus undergoing HRTm analysis is focused on the image sensor at separate times. 
     
     
         29 . The method of  claim 23 , further comprising using an excitation source to supply electromagnetic radiation to the PCR processing zone of the channel. 
     
     
         30 . (canceled) 
     
     
         31 . The method of  claim 30 , further comprising: using the image sensor to capture at least about 10 images of the bolus per second for at least about 1 minute while the bolus is undergoing the HRTm analysis zone. 
     
     
         32 . The method of  claim 31 , further comprising: using the image sensor to capture at least about 1 image of the bolus every 90 seconds for at least about 10 minutes while the bolus is undergoing  4  the PCR processing. 
     
     
         33 . A method, comprising:
 (a) performing a PCR process in a microfluidic channel;   (b) performing an HRTm process in said microfluidic channel;   (c) while performing step (a), focusing radiation from the channel onto an image sensor.   
     
     
         34 . The method of  claim 33 , wherein the step of performing the PCR process comprises:
 moving a bolus of test solution containing real-time PCR reagents through the channel; and   cycling the temperature of the bolus in order to achieve PCR.   
     
     
         35 . The method of  claim 34 , wherein the step of cycling the temperature of the bolus comprises heating the bolus to a first temperature, cooling the bolus to a second temperature, and heating the bolus to a third temperature. 
     
     
         36 . The method of  claim 34 , wherein the step of performing an HRTm process comprisesL
 moving a bolus of test solution containing real-time PCR reagents through the channel; and   steadily increasing the temperature of the bolus following completion of the PCR process.   
     
     
         37 . The method of  claim 36 , wherein:
 the step of cycling the temperature of the bolus in order to achieve PCR comprises using a thermal generating apparatus to cycle the temperature, and   the step of steadily increasing the temperature of the bolus comprises using the thermal generating apparatus.   
     
     
         38 . The method of  claim 33 , further comprising using a single lens to perform the focusing steps. 
     
     
         39 . The method of  claim 33 , further comprising using an excitation source to supply electromagnetic radiation to the PCR processing zone of the channel. 
     
     
         40 . The method of  claim 33 , further comprising causing a bolus of solution containing real-time PCR reagents to move through the channel. 
     
     
         41 . The method of  claim 40 , further comprising: using the image sensor to capture at least about 10 images of the bolus per second for at least about 1 minute. 
     
     
         42 . The method of  claim 31 , further comprising: using the image sensor to capture at least about 1 image of the bolus every 90 seconds for at least about 10 minutes or at least about 20 PCR cycles. 
     
     
         43 . A system, comprising:
 a micro fluidic channel comprising a PCR processing zone and an HRTm analysis zone; and   an imaging system, wherein   the imaging system obtains data from at least a portion of the microfluidic channel wherein PCR processing and HRTm occur.