Quantitating high titer samples by digital pcr

Abstract

The present invention provides systems, devices, methods, kits, and compositions for nucleic acid analysis using digital PCR. In particular, methods are provided to analyze high titer samples that cannot be divided into a sufficient number of partitions containing zero nucleic acid molecules per partition to allow for Poisson analysis (digital PCR analysis).

We claim: 
     
         1 . A system, comprising:
 a) a reaction mixture, comprising:
 i) at least one target nucleic acid; 
 ii) at least one internal standard nucleic acid at a concentration sufficient to produce a number of partitions containing zero copies of said internal standard nucleic acid per partition wherein λ IS ≈0.01-1, wherein λ IS  is the average number of internal standard copies per partition to allow for Poisson analysis, wherein said internal standard nucleic acid has at least one identical primer binding sequence as said target nucleic acid; 
 iii) at least one primer oligonucleotide specific for said target nucleic acid and for said internal standard nucleic acid; 
 iv) at least one amplification reagent; 
   b) a partitioning device;   c) an amplification device; and   d) a detection device.   
     
     
         2 . The system of  claim 1 , wherein said target nucleic acid is at a concentration that cannot be divided into a sufficient number of partitions containing zero nucleic molecules per partition to allow for Poisson analysis. 
     
     
         3 . The system of  claim 1 , wherein said partitioning device comprises one or more of a droplet microactuator, an emulsification device, a microfluidics platform, a continuous-flow microfluidics device, an array micro-chamber, a microspray device, one or more microplate wells, a reagent immobilization device, an electrical manipulation device, a thermal manipulation device, a mechanical manipulation device, an optical manipulation device, a chemical manipulation device and combinations thereof. 
     
     
         4 . The system of  claim 1 , wherein said at least one amplification reagent comprises one or more of a polymerase, an oligonucleotide, a buffer and a salt. 
     
     
         5 . The system of  claim 1 , wherein said at least one amplification reagent comprises one excess primer and one limiting primer. 
     
     
         6 . The system of  claim 1 , wherein said amplification device is a thermal cycler. 
     
     
         7 . The system of  claim 1 , further comprising at least one probe. 
     
     
         8 . The system of  claim 7 , wherein said at least one probe comprises a first labeled probe configured to bind to said target nucleic acid and a second labeled probe configured to bind to said internal standard nucleic acid, wherein said first labeled probe and said second labeled probe are differentially labeled. 
     
     
         9 . The system of  claim 8 , wherein said first labeled probe and said second labeled probe comprise different fluorescent labels. 
     
     
         10 . The system of  claim 7 , wherein one or more of said at least one target nucleic acid, said at least one internal standard nucleic acid, said at least one primer oligonucleotide and/or said at least one probe is immobilized to a surface. 
     
     
         11 . The system of  claim 10 , wherein said surface is a substrate, a plate, an array, a bead and/or a particle. 
     
     
         12 . The system of  claim 1 , wherein said detection device comprises a fluorescence detection device, a luminescence detection device or a radioactivity detection device. 
     
     
         13 . The system of  claim 1 , further comprising a sorting device. 
     
     
         14 . The system of  claim 13 , wherein said sorting device is a micro-manipulator, an electrophoresis device, a flow cytometry device, a fluorescence-activated cell sorting (FACS) device or an amplicon isolation device. 
     
     
         15 . The system of  claim 1 , further comprising a sample processing device comprising a cell lysis device and a least one cell lysis reagent, a restriction digestion device and at least one restriction digestion reagent, a nucleic acid purification device and at least one nucleic acid purification reagent, a nucleic acid precipitation device and at least one nucleic acid precipitation reagent, and/or a nucleic acid resuspension device and/or at least one nucleic acid resuspension reagent. 
     
     
         16 . The system of  claim 1 , wherein said amplification device is a digital polymerase chain reaction linear-after-the-exponential (dPCR LATE) amplification device. 
     
     
         17 . The system of  claim 1 , further comprising an amplicon quantification reagent. 
     
     
         18 . The system of  claim 1 , further comprising one or more of a channel, a network of channels, a valve, an internal pump, an external pump and/or a centrifugal force element. 
     
     
         19 . The system of  claim 1 , further comprising a dispensing device, a splitting device, a transporting device, a merging device, a mixing device and/or an agitating device. 
     
     
         20 . The system of  claim 1 , further comprising a calibration sample at one or more known concentrations.