System for fluorescence monitoring

Abstract

The present invention is directed to devices for performing PCR and monitoring the reaction of a sample comprising a nucleic acid and a fluorescent dye. Illustrative devices comprise a heat exchange component for heating and cooling the sample, a control device for repeatedly operating the heat exchange component to subject the sample to thermal cycling, an excitation source for optically exciting the sample to cause the sample to fluoresce, a photodetector for detecting temperature-dependent fluorescence levels from the sample, and a processor configured to record and process emissions from the fluorescent dye.

1 . A system for performing PCR and monitoring the reaction during temperature cycling comprising:
 a sample container for holding a PCR sample,   a heat exchange component for heating and cooling the sample,   a control device for repeatedly operating the heat exchange component to subject the PCR sample to thermal cycling,   an excitation source for optically exciting the sample to cause the sample to fluoresce,   a photodetector configured for detecting fluorescent emissions from SYBR® Green I, and   a processor for recording and processing emissions from SYBR Green I.   
     
     
         2 . The system of  claim 1  wherein the photodetector includes a filter for the detection of SYBR® Green I. 
     
     
         3 . The system of  claim 1  wherein the filter is a 520-580 nm band pass filter. 
     
     
         4 . The system of  claim 1  wherein the sample container has a small volume and a large surface area. 
     
     
         5 . The system of  claim 1  wherein the photodetector is configured to monitor fluorescence from SYBR® Green I at least once per amplification cycle. 
     
     
         6 . The system of  claim 1  wherein the photodetector is configured to monitor fluorescence from SYBR® Green I continuously throughout each amplification cycle. 
     
     
         7 . The system of  claim 1  wherein the photodetector is configured to measure fluorescence from SYBR® Green I every 200 msec. 
     
     
         8 . The system of  claim 1  wherein the photodetector is configured to produce a melting curve during or subsequent to amplification. 
     
     
         9 . The system of  claim 1  wherein the sample container comprises SYBR® Green I during thermal cycling. 
     
     
         10 . The system of  claim 1  wherein the processor is configured to generate a melting curve during or subsequent to amplification. 
     
     
         11 . The system of  claim 10  wherein the melting curve is a derivative plot. 
     
     
         12 . The system of  claim 10  wherein the processor is further configured to generate an amplification curve. 
     
     
         13 . The system of  claim 1  wherein the device further comprises a chamber in thermal communication with the heat exchange component, and the chamber is further configured for receiving the sample container. 
     
     
         14 . The system of  claim 13  wherein the heat exchange component further comprises a heater and a fan. 
     
     
         15 . The system of  claim 1  wherein the heat exchange component comprises a heat block device. 
     
     
         16 . The system of  claim 1  wherein the photodetector is configured to detect temperature-dependent fluorescence levels from the sample during amplification. 
     
     
         17 . The system of  claim 1  wherein the control device operates the heat exchange device to heat the sample at a rate of >0.1° C./second.