| 主权项 |
1. A method of detecting the absence of a wild type allele of a locus in the JAK2 gene having at least first mutant and wild type alleles, the method comprising:
a) providing a reaction mix comprising
i) a sample including nucleic acid representing at least a portion of the JAK2 gene;ii) an oligonucleotide probe which hybridises to the mutant allele with a lower melting temperature (Tm) than that with which it hybridises to the wild type 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:mutant allele; b) maintaining the reaction mix at a temperature between the probe:mutant allele Tm and the probe:wild type allele Tm, such that the probe preferentially hybridises to the wild type 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:mutant allele Tm and the probe:wild type allele Tm, such that the probe is hybridised to the wild type allele during these phases; to thereby amplify the mutant allele; and d) detecting hybridisation of the probe to the sample at a temperature at or below the probe:mutant allele Tm; detecting hybridisation of the probe to the sample at a higher temperature at or below the probe:wild type allele Tm; and comparing the two; to thereby detect the presence or absence of the wild type allele. |
