Systems and methods for real-time PCR

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.