Nucleic acid reactions and related methods and compositions
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.
1. A method of detecting the presence or absence of a target nucleic acid analyte in a sample, the method comprising:
a) performing a polymerase chain reaction (PCR) amplification reaction on the sample;
b) measuring a signal generated during the amplification reaction and generating a kinetic signature from the measured signal; and
c) comparing the generated kinetic signature to a reference signature and determining whether the generated kinetic signature corresponds with the reference signature, thereby detecting the presence or absence of the target nucleic acid analyte in the sample,
wherein the reference signature corresponds with a kinetic signature generated by amplification of the target nucleic acid analyte under a set of constrained amplification parameters, the set of constrained parameters comprising one or more of (A) a range of forward and/or reverse primer concentrations; (B) chromophore concentration; and (C) chromophore type and wherein the amplification reaction is carried out within one or more of the constrained amplification parameters.
2. The method of claim 1 , wherein the kinetic signature comprises a signal selected from an electromagnetic signal, a wavelength, and a fluorescence emission signal.
3. The method of claim 1 , wherein the set of constrained parameters further comprises any combination of: (i) a range of polymerase concentrations; (ii) polymerase type; (iii) a range of nucleotide concentrations; (iv) a number of thermocycles; (v) rate of thermocycling; (vi) one or more thermocycle stage temperatures; (vii) one or more thermocycle stage time lengths; (viii) a range of target-specific molecular probes; (ix) primers associated with one or more target-specific probes; and (x) a concentration of one or more enzyme cofactors.