Nucleic acid reactions and related methods and compositions

Abstract

The present invention relates to methods of nucleic acid analyte detection by PCR. In particular, methods and kits for the detection of a plurality of nucleic acid analytes and the generation of kinetic signatures are provided. Further provided are methods and kits of nested PCR and PCR using limiting primers.

What is claimed is: 
     
         1 . A method of detecting multiple distinct target nucleic acid analytes, the method comprising:
 obtaining, from a non-transitory computer readable storage medium, a reference signature corresponding to a reference signal generated when one of the multiple distinct target nucleic acid analytes is amplified with a primer set by a thermocycling reaction comprising a set of temperature cycles;   adding primer pairs to a bio-sample, wherein one primer pair comprises a limiting primer, and each primer pair defines a region of a target nucleic acid analyte to be amplified;
 performing an amplification reaction on the bio-sample; 
 detecting, with a detector of a PCR system, a signal generated during the amplification reaction; 
 determining a kinetic signature from the signal; 
 comparing the kinetic signature to the reference signature; and 
 determining if each of the plurality of target nucleic acid analytes is present based on a curve fit of the kinetic signature to the reference signature. 
   
     
     
         2 . The method of  claim 1 , wherein the signal is selected from an electromagnetic signal, a wavelength, and a fluorescence emission signal. 
     
     
         3 . The method of  claim 1 , wherein the kinetic signature is plotted as a curve. 
     
     
         4 . The method of  claim 3 , wherein the curve comprises at least one of an exponential region, a linear region, and a plateau region. 
     
     
         5 . The method of  claim 1 , wherein the kinetic signature comprises recorded values. 
     
     
         6 . The method of  claim 1 , wherein the values are recorded in a table. 
     
     
         7 . The method of  claim 1 , wherein the amplification reaction is a polymerase chain reaction (PCR). 
     
     
         8 . The method of  claim 1 , wherein the reference signature corresponds with a kinetic signature generated by amplification of the target nucleic acid analyte under one or more members of a set of constrained amplification parameters. 
     
     
         9 . The method of  claim 8 , wherein the set of constrained amplification parameters comprises any combination of: (i) a range of forward and/or reverse primer concentrations; (ii) a range of polymerase concentrations; (iii) polymerase type; (iv) a range of nucleotide concentrations; (v) chromophore concentration; and (vi) chromophore type; (vii) a number of thermocycles; (viii) rate of thermocycling; (ix) one or more thermocycle stage temperatures; and (x) one or more thermocycle stage time lengths; (xi) a range of target-specific molecular probes; (xii) primers associated with one or more target-specific probes; and (xiii) a concentration of one or more enzyme cofactors. 
     
     
         10 . The method of  claim 9 , wherein the amplification reaction is carried out within one or more of the constrained amplification parameters. 
     
     
         11 . The method of  claim 1 , wherein the nucleic acid analyte is from a source selected from the group consisting of an animal, a plant, a bacterium, a parasite, and a virus. 
     
     
         12 . The method of  claim 11 , wherein the animal is a human. 
     
     
         13 . The method of  claim 1 , wherein the nucleic acid analyte comprises bacterial DNA. 
     
     
         14 . The method of  claim 1 , wherein the nucleic acid analyte comprises viral DNA. 
     
     
         15 . The method of  claim 1 , wherein the nucleic acid analyte comprises genomic DNA. 
     
     
         16 . The method of  claim 15 , wherein the genomic DNA is an oncological biomarker. 
     
     
         17 . The method of  claim 15 , wherein the genomic DNA is an autoimmune disease biomarker. 
     
     
         18 . The method of  claim 3 , wherein the at least one of an exponential region, a linear region, and a plateau region occurs over at least 4 cycles. 
     
     
         19 . The method of  claim 3 , wherein the at least one of an exponential region, a linear region, and a plateau region occurs over at least 8 seconds.