Instrument for monitoring polymerase chain reaction of DNA

Abstract

An optical instrument monitors PCR replication of DNA in a reaction apparatus having a temperature cycled block with vials of reaction ingredients including dye that fluoresces in presence of double-stranded DNA. A beam splitter passes an excitation beam to the vials to fluoresce the dye. An emission beam from the dye is passed by the beam splitter to a CCD detector from which a processor computes DNA concentration. A reference strip with a plurality of reference emitters emit reference beams of different intensity, from which the processor selects an optimum emitter for compensating for drift. Exposure time is automatically adjusted for keeping within optimum dynamic ranges of the CCD and processor. A module of the beam splitter and associated optical filters is associated with selected dye, and is replaceable for different dyes.

1 . A method of monitoring replication of DNA in a reaction apparatus, comprising: 
 generating an excitation beam with a light source;    directing the excitation beam through a beam splitter and simultaneously into a plurality of vials;    directing emission beams from two or more of the plurality of vials through the beam splitter and onto a detector;    generating primary data signals representative of the emission beams, each primary data signal corresponding to a respective concentration of DNA in a respective one of the plurality of vials;    emitting reference light from a reference emitter in response to the excitation beam;    focusing at least a portion of the reference light as a reference beam onto the detector;    generating a reference signal based on the reference beam received by the detector;    processing the reference signal to compute reference data; and    normalizing the primary data signals with the reference data for a chosen point in the replication of DNA, to correct for instrument drift during the monitoring.    
     
     
         2 . The method of  claim 1 , wherein the reference emitter comprises a plurality of fluorescent reference emitters each emitting a reference beam of different intensity in response to the excitation beam.  
     
     
         3 . The method of  claim 2 , further comprising focusing the reference beams of different intensities onto the detector.  
     
     
         4 . The method of  claim 2 , wherein the reference data is computed from a ratio of a first reference data signal corresponding to the reference emitter having the highest signal intensity that is less than a predetermined maximum, and from a second reference data signal corresponding to the reference emitter having the next highest signal intensity.  
     
     
         5 . The method of  claim 1 , wherein the generated reference signal is converted to the reference data by an analog/digital device.  
     
     
         6 . The method of  claim 1 , further comprising: 
 generating a source beam comprising a secondary excitation frequency; and    directing the source beam at a standard concentration of a passive dye that emits light substantially without influence from DNA to cause the passive dye to emit light at a secondary emission frequency and to emit a secondary emission beam focused onto the detector.    
     
     
         7 . The method of  claim 6 , further comprising generating secondary data signals based on the secondary emission beam.  
     
     
         8 . The method of  claim 7 , further comprising: 
 processing the secondary data signals to compute secondary data; and    normalizing the primary data, whereby the computed concentration of DNA is normalized to the standard concentration of the passive dye.    
     
     
         9 . The method of  claim 6 , wherein the standard concentration of passive dye is included in at least one vial of the plurality of vials.  
     
     
         10 . The method of  claim 6 , wherein the standard concentration of passive dye is included in each vial of the plurality of vials.  
     
     
         11 . The method of  claim 6 , wherein the standard concentration of passive dye comprises a nucleic acid sequence labeled with Rhodamine and fluorescein dye derivatives.  
     
     
         12 . The method of  claim 6 , wherein the standard concentration of passive dye comprises Rox dye.  
     
     
         13 . The method of  claim 1 , wherein the processing comprises processing with a computer processor.  
     
     
         14 . The method of  claim 1 , wherein the plurality of vials comprises a plurality of wells.  
     
     
         15 . The method of  claim 1 , wherein the plurality of vials comprises 96 wells in a plastic unitary tray.  
     
     
         16 . The method of  claim 1 , further comprising replicating DNA in cycles, wherein the normalizing comprises normalizing the primary data in real time as a ratio to the reference data from an initial cycle of the DNA replication.  
     
     
         17 . The method of  claim 1 , further comprising replicating DNA in cycles, wherein the normalizing comprises normalizing the primary data in real time as a ratio to the reference data from a previous cycle of the DNA replication.  
     
     
         18 . The method of  claim 1 , wherein the replication of DNA comprises a polymerase chain reaction.  
     
     
         19 . A method of monitoring replication of DNA in a reaction apparatus, comprising: 
 generating an excitation beam with a light source;    directing the excitation beam into a plurality of vials;    directing emission beams from two or more of the plurality of vials onto a detector;    generating primary data signals representative of the emission beams, each primary data signal corresponding to a respective concentration of DNA in a respective one of the plurality of vials;    generating dark signal data; and    correcting the primary data signals with the dark signal data.    
     
     
         20 . The method of  claim 19 , wherein the correcting comprises subtracting the dark signal data from the primary data signals to yield dark-corrected data.  
     
     
         21 . The method of  claim 19 , wherein the detector comprises pixels, and the generating primary data signals comprises collecting multiple normal exposures for each pixel.  
     
     
         22 . The method of  claim 19 , further comprising converting both the primary data signals and the dark signal data from analog signals to digital signals using an analog/digital device.  
     
     
         23 . The method of  claim 19 , further comprising: 
 generating a source beam comprising a secondary excitation frequency; and    directing the source beam at a standard concentration of a passive dye that emits light substantially without influence from DNA, to cause the passive dye to emit light at a secondary emission frequency and to emit a secondary emission beam focused onto the detector.    
     
     
         24 . The method of  claim 23 , further comprising generating secondary data signals based on the secondary emission beam.  
     
     
         25 . The method of  claim 24 , further comprising: 
 processing the secondary data signals to compute secondary data; and    normalizing the primary data, whereby the computed concentration of DNA is normalized to the standard concentration of the passive dye.    
     
     
         26 . The method of  claim 19 , further comprising replicating DNA in cycles, wherein the normalizing comprises normalizing the primary data in real time from an initial cycle of the DNA replication.