Chromophore-based characterization and detection methods

A kit for detecting at least one genetic variation in a polynucleotide analyte in a sample, comprising a first primer and a second primer, wherein a fluorophore is attached to the first primer, a quencher is attached to the second primer, the first primer and the second primer are specific for the polynucleotide analyte, at least one of the first primer and the second primer hybridizes to a region of the polynucleotide analyte encoding the at least one genetic variation, the fluorophore is different from the quencher, and the first primer and the second primer is used to detect the least one genetic variation according to the steps of:

• a) measuring a first signal generated by the fluorophore and quencher;
• b) performing at least one polymerase chain reaction (PCR) reaction with the first primer and the second primer using the polynucleotide analyte as a template, thereby amplifying the template to generate a PCR amplification product comprising the at least one genetic variation;
• c) measuring a second signal generated by the fluorophore and quencher from the PCR amplification product;
• d) comparing the first and second signals to determine a change between the first and second signals;
• and
• e) determining the presence or absence of the at least one genetic variation based i) on the change in signal as determined in step e);
• and ii) by comparing said change to the change in signal observed upon PCR amplification for a corresponding polynucleotide analyte lacking the at least one genetic variation in the presence of the first primer and the second primer.