Systems and methods for real-time pcr

Abstract

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

1 . (canceled) 
     
     
         2 . 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 where PCR will be performed;   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 into the channel;   cycling the temperature of the sample according to configuration information included in the script to amplify the DNA sample;   illuminating the sample with a first excitation source and using a first image detector to obtain more than one image of the sample as the sample undergoes PCR;   processing the images;   determining whether another PCR cycle is needed, and if so, causing additional temperature cycling to occur; melting the amplified DNA, illuminating the sample with a second excitation source, and using a second image detector to obtain more than one image of the sample.   
     
     
         3 . The method of  claim 2 , further comprising configuring an imaging system prior to obtaining said images of the sample, wherein the imaging system is used to obtain said images. 
     
     
         4 . The method of  claim 3 , wherein the step of configuring the imaging system comprises:
 (a) obtaining an image of the channel where PCR occurs using a detector;   (b) displaying the image 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).   
     
     
         5 . The method of  claim 1 , wherein the step of cycling the temperature of the sample, comprises:
 causing the temperature of the channel where PCR occurs to reach a denature stage temperature; once the temperature of the channel reaches the denature stage temperature, maintaining 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 channel to reach an annealing stage temperature; once the temperature reaches the annealing stage temperature, maintaining the annealing stage temperature for a second predetermined period of time; immediately after the second predetermined period of time elapses,   causing the temperature to reach an extension stage temperature; and once the temperature reaches the extension stage temperature, maintaining the extension stage temperature for a third predetermined period of time.   
     
     
         6 . The method of  claim 1 , wherein the step of obtaining images of the sample as the sample undergoes PCR 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. 
   
     
     
         7 . The method of  claim 6 , wherein the first excitation source is a blue or red light emitting diode. 
     
     
         8 . The method of  claim 6 , wherein the following steps are performed either simultaneously with steps (b)-(d) of  claim 6  or following step (d) of  claim 6 :
 (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. 
 
     
     
         9 . The method of  claim 1 , further comprising the steps of:
 obtaining an image of the sample as it undergoes melting;   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 sample as it undergoes melting, and, for each image,   recording only the pixels that are within the determined pixel window.   
     
     
         10 . The method of  claim 9 , further comprising using information contained in the images of the sample undergoing melting to create a graph that plots measured fluorescence intensity versus temperature. 
     
     
         11 . The method of  claim 9 , 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 sample undergoing melting, and, for each image,   recording only the pixels that are within the determined pixel window.