Mutational analysis

A method for detecting somatic mutations in a sample from a subject, the sample comprising nucleic acid from a locus having at least first and second alleles, the first allele being a mutant allele and the second allele being a wild type allele, the method comprising

• a) providing a reaction mix comprising
• i) the sample;
• ii) an oligonucleotide probe which hybridises to the first allele with a lower melting temperature (Tm) than that with which it hybridises to the second allele;
• iii) a pair of oligonucleotide primers for nucleic acid amplification, the primers hybridising to the nucleic acid in the sample at first and second sites flanking the oligonucleotide probe binding site;
• wherein the Tm of the primer:sample is higher than the Tm of the probe:first allele;
• b) maintaining the reaction mix at a temperature between the probe:first allele Tm and the probe:secod 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;
• d) maintaining the temperature of the reaction mix at or below the Tm probe:mutant allele;
• e) maintaining the temperature of the reaction mix above the temperature in step d), but at or below the Tm probe :
• wild type allele;
• and
• f) detecting hybridised probe in steps d) and e), to thereby determine the presence or absence of the mutant allele in the sample;
• 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.