Mutation analysis

A method for detecting the presence of a variant nucleic acid sequence in a target nucleic acid sequence that is polymorphic in that it is present in multiple alleles within a given population, the method comprising

• a) providing a reaction mix comprising:
• an oligonucleotide probe that hybridizes to the target nucleic acid sequence at a region where individual alleles within the multiple alleles differ from one another, which region includes a first site where individual alleles within the multiple alleles differ by at least one non-silent mutation desired to be detected and also includes a second site where individual alleles within the multiple alleles differ by at least one phenotypically silent mutation or conservative mutation not desired to be detected, wherein the oligonucleotide probe has a first melting temperature (Tm) when hybridized to a first allele of the target sequence, which first allele includes the non-silent mutation at the first site, and a second, different Tm when hybridized to a second allele of the target sequence, which second allele does not include the non-silent mutation at the first site, and further wherein the probe comprises at least one universal base at the second site where variants are not desired to be detected, and wherein the site where variants are not desired to be detected is a residue where mutations are phenotypically silent mutations or is a residue where mutations give rise to conservative replacements which do not alter the properties of the expressed peptide, and wherein the probe does not include a quencher moiety;
• and
• a sample comprising nucleic acid whose nucleotide sequence comprises the target sequence;
• b) allowing the probe to hybridize to nucleic acids in the sample;
• and
• c) determining the Tm of the probe when hybridized to the nucleic acids in the sample;
• to thereby determine whether a variant nucleic acid sequence is present in the sample.