Patent ID: 8440405

Claim:
A method for detecting the presence or absence or amount of a target nucleic acid or at least one variant nucleotide in one or more target nucleic acids contained in a sample, said method comprising: (a) treating the sample with a first oligonucleotide primer for a first region of a target nucleic acid sequence under hybridisation conditions, wherein the nucleotide sequence of said first primer is such that it is substantially complementary to said first region of the target nucleic acid such as to anneal thereto thereby creating a mixture of duplexes comprising the first oligonucleotide primer annealed to the target nucleic, and wherein said first region is a diagnostic portion comprising a suspected variant nucleotide, and wherein a nucleotide at, or within 1, 2, or 3 nucleotides of, the 3′ terminus is complementary to the suspected variant nucleotide such that when the first primer anneals to the target region containing the suspected variant nucleotide, the annealed first primer is extendable, and wherein when the first primer anneals to the target region containing the normal nucleotide, the annealed first primer is non-extendable; (b) maintaining the mixture of step (a) under extension conditions, which comprise appropriate nucleoside triphosphates and a nucleic acid polymerase to extend the annealed first primer, if extendable, to synthesize an extension product of the first primer; and (c) maintaining the mixture of step (b) under degradation conditions such that if an extension product of the first primer is present, this is degraded under the degradation conditions, thereby generating detectable signals which are indicative of the presence and\or amount of the target nucleic acid or the variant nucleotide in the target nucleic acid, and if no extension product of the first primer is generated, no degradation of the extension product of the first primer occurs under the degradation conditions, thereby no detectable signals are generated, which is indicative of the absence of the target nucleic acid or the variant nucleotide in the target nucleic acid, wherein said degradation conditions comprise an exonuclease activity, and wherein (i) at least one of the said nucleoside triphosphates is labelled, and\or (ii) the first primer is labelled, in each case such that degrading the extension product of the first primer generates detectable signals from said label.