Single molecule amplification and detection of DNA

Abstract

Methods and systems for performing single molecule amplification for detection, quantification and statistical analysis of rare nucleic acids are provided.

1 - 117 . (Cancelled)  
     
     
         118 . A system for detecting low copy nucleic acids of interest in a sample, the system comprising: 
 a dilution module that dilutes the sample into multiple aliquots;    a microfluidic device comprising an amplification channel or chamber configured to thermocycle one or more of the multiple aliquots;    a detector integral with or proximal to the microfluidic device, which detector is configured to detect one or more amplified copies of the nucleic acid of interest in or on the microfluidic device; and,    system instructions that direct the dilution module to aliquot the sample into a plurality of aliquots, including a plurality of zero copy aliquots comprising no copies of the nucleic acids of interest and one or more single copy aliquot comprising a single copy of the nucleic acid of interest.    
     
     
         119 . The system of  claim 118 , further comprising system software that correlates a shape, length, width, volume or area occupied by amplified copies of the nucleic acid of interest, as detected by the detector, to the number of copies of the nucleic acid of interest present in one of the aliquots, or to the number of copies of the nucleic acid of interest present in the sample, or both.  
     
     
         120 . The system of  claim 118 , wherein the microfluidic device is configured to permit flow of the multiple aliquots though the amplification chamber while performing one or more amplification reaction on at least one of the multiple aliquots in the amplification channel or chamber.  
     
     
         121 . The system of  claim 118 , wherein the microfluidic device is configured to provide stopped flow conditions while performing one or more amplification reaction on at least one of the multiple aliquots in the amplification channel or chamber.  
     
     
         122 - 125 . (Cancelled)  
     
     
         126 . The system of  claim 118  wherein the dilution module is integral with the microfluidic device.  
     
     
         127 . The system of  claim 118 , wherein the microfluidic device comprises one or more electrodes positioned to flow electrical current into the microchamber or channel, whereby the flow of current into the microchamber or channel heats fluid in the microchamber or channel.  
     
     
         128 . The system of  claim 118 , wherein the microfluidic device comprises or is coupled to one or more heating element positioned within or proximal to the microchamber or channel, which heating element heats fluid in the microchamber or channel.  
     
     
         129 . The system of  claim 128 , wherein the heating element comprises a resistive heating element.  
     
     
         130 . The system of  claim 128 , wherein the heating element comprises a Peltier or Joule-Thompson device.  
     
     
         131 . The system of  claim 118 , wherein the detector is configured to detect one or more electromagnetic energy signal in or on the microfluidic device.  
     
     
         132 . The system of  claim 131 , wherein the detector detects fluorescence, luminescence or fluorescence polarization.  
     
     
         133 . The system of  claim 118 , wherein the system comprises statistical or probabilistic system software that performs one or more statistical or probabilistic analysis of signals received from one or more of the aliquots subjected to thermocycling.  
     
     
         134 . The system of  claim 133 , wherein the statistical or probabilistic analysis comprises one or more of: Poisson analysis, Monte Carlo analysis, application of a genetic algorithm, neural network training, Markov modeling, hidden Markov modeling, multidimensional scaling, PLS analysis, or PCA analysis.  
     
     
         135 . The system of  claim 133 , wherein the statistical or probabilistic analysis comprises quantitatively determining a concentration, proportion, or number of the nucleic acids of interest in the sample.  
     
     
         136 . The system of  claim 118 , further comprising a sample storage module that stores the sample until it is diluted by the dilution module.  
     
     
         137 . The system of  claim 136 , further comprising a sample retrieval module that retrieves the sample from the sample storage module and delivers it to the dilution module.