Nucleic acid amplification methods

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 method for detecting a target nucleic acid sequence in a biological sample during amplification comprising the steps of:
 adding a thermostable polymerase, a double-strand-specific DNA binding dye and primers configured for amplification of the target nucleic acid sequence to the biological sample;   amplifying the target nucleic acid sequence by polymerase chain reaction in the presence of the dye, the polymerase chain reaction comprising thermally cycling the biological sample between at least a denaturation temperature and an elongation temperature through a plurality of amplification cycles;   illuminating the biological sample comprising the amplified target nucleic acid sequence with light at a wavelength absorbed by the dye; and   detecting a fluorescent emission from the dye in the range of 520-580 nm, wherein the fluorescent emission is related to the quantity of the amplified target nucleic acid sequence in the sample.   
     
     
         2 . The method of  claim 1  wherein the fluorescent emission is monitored in the range of 520-550 nm. 
     
     
         3 . The method of  claim 1  wherein the illuminating is via light at a wavelength in the range of 450-490 nm. 
     
     
         4 . The method of  claim 1  wherein the illuminating is via visible light. 
     
     
         5 . The method of  claim 1  wherein the amplifying step includes using a rapid temperature cycling profile wherein 30 amplification cycles are completed in 10 to 30 minutes. 
     
     
         6 . The method of  claim 1  wherein the fluorescent emission is detected using a 520-580 nm band pass filter. 
     
     
         7 . The method of  claim 1  wherein the double-strand-specific DNA binding dye is SYBR® Green I. 
     
     
         8 . 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 emission in the range of 520-550 nm from a double-strand-specific DNA binding dye, and   a processor for recording and processing emissions from the dye.   
     
     
         9 . The system of  claim 8  wherein the sample container comprises a nucleic acid sample and a double-strand-specific DNA binding dye. 
     
     
         10 . The system of  claim 8  wherein the excitation source is configured to optically excite the sample using visible light. 
     
     
         11 . The system of  claim 8  wherein the heat exchange component comprises a forced air heater. 
     
     
         12 . A method of controlling temperature cycling parameters of a polymerase chain reaction comprising repeated cycles of annealing, extension, and denaturation phases of a polymerase chain reaction mixture comprising a nucleic acid binding fluorescent entity, wherein the parameters comprise duration of the annealing phase, duration of the denaturation phase, and number of cycles, comprising
 (a) illuminating the reaction mixture with a selected wavelength of light for eliciting fluorescence from the fluorescent entity and continuously monitoring fluorescence during the repeated annealing, extension, and denaturation phases;   (b) determining at least
 (i) duration for fluorescence to stop increasing during the extension phase, or 
 (ii) duration for fluorescence to decrease to a baseline level during the denaturation phase, or 
 (iii) a number of cycles for fluorescence to reach a preselected level during the extension phase; and 
   (c) adjusting the length of the extension phase according to the length of time for fluorescence to stop increasing during the extension phase, the length of the denaturation phase according to the length of time for fluorescence to decrease to the baseline level during the denaturation phase, or the number of cycles according to the number of cycles for fluorescence to reach the preselected level during the extension phase.   
     
     
         13 . A method of controlling the number of temperature cycles of a polymerase chain reaction comprising repeated cycles of a denaturation phase and an extension phase of a polymerase chain reaction mixture comprising a nucleic-acid-binding fluorescent entity, the method comprising
 (a) illuminating the reaction mixture with a selected wavelength of light for eliciting fluorescence from the fluorescent entity and monitoring fluorescence during the extension phase of each of the repeated temperature cycles;   (b) determining a number of cycles for the fluorescence to reach a preselected level during the extension phase; and   (c) adjusting the number of cycles according to the number of cycles for the fluorescence to reach the preselected level during the extension phase.   
     
     
         14 . The method of  claim 13  wherein the adjusting step comprises terminating the polymerase chain reaction when the fluorescence indicates that the polymerase chain reaction has reached a plateau phase. 
     
     
         15 . The method of  claim 14  wherein the preselected level is a level wherein fluorescence acquired during the extension phase of a particular cycle is at least 1.2 times the lowest fluorescence obtained, and the increase of fluorescence of the particular cycle over the preceding cycle falls below a defined ratio. 
     
     
         16 . The method of  claim 15  wherein the defined ratio is from about 1.00 to about 1.02. 
     
     
         17 . The method of  claim 13  wherein the extension phase is a combined annealing/extension phase. 
     
     
         18 . The method of claim  31  wherein fluorescence is continually monitored throughout temperature cycling. 
     
     
         19 . The method of  claim 13  wherein the fluorescent entity comprises a double strand specific nucleic acid binding dye. 
     
     
         20 . The method of  claim 13  wherein the fluorescent entity comprises a pair of oligonucleotide probes, one of said probes being labeled with an acceptor fluorophore and the other probe labeled with a donor fluorophore of a fluorescence energy transfer pair.