Patent ID: 9005932
Filing Date: 2015-04-14
Classification: C12Q

Abstract:
1. A method of preferentially amplifying and detecting a first allele of a locus having at least first and second alleles, the method comprising: a) providing a reaction mix comprising b) maintaining the reaction mix at a temperature between the probe:first allele Tm and the probe:second allele Tm, such that the probe preferentially hybridises to the second allele; c) carrying out a thermal cycling amplification on the reaction mix, the amplification including a melt phase, an annealing phase, and an extension phase, in which the temperatures of the extension and annealing phases are between the probe:first allele Tm and the probe:second allele Tm, such that the probe is hybridised to the second allele during these phases; to thereby amplify the first allele; and d) detecting hybridisation of the probe to the sample at a temperature at or below the probe:first allele Tm; detecting hybridisation of the probe to the sample at a higher temperature at or below the probe:second allele Tm; and comparing the two; to thereby detect the amplified first allele; wherein the probe is labelled with a fluorescent label which generates a differential signal depending on whether the probe has hybridised to a target strand; wherein one of the primers is provided in a rate-limiting amount, and the amplification reaction is asymmetric PCR; and wherein one or both of the primers overlap with the probe binding site such that the probe competes with the primer for binding, to thereby prevent binding of the primer and hence strand extension.