Patent ID: 9376714
Filing Date: 2016-06-28
CPC Classification: C12Q

Claim Text:
1. A method of detecting the presence of one or more nucleic acids with a rare mutation in a sample, wherein said rare mutation includes any change from a wildtype sequence including polymorphisms that are present in less than 10% of the nucleic acid molecules in the sample, comprising the steps of; (a) providing a nucleic acid sample comprising a mixture of nucleic acids wherein the mixture of nucleic acids comprises at least one nucleic acid with a rare mutation that is present in less than 10% of nucleic acids in the sample; (b) in an amplification reaction amplifying the nucleic acid sample with primers flanking the sequence carrying the rare mutation, wherein the primers allele-specifically amplify the nucleic acid with the rare mutation; (c) removing excess dNTPs after the amplification reaction; (d) performing a primer extension reaction using (i) one or more detection primers which are designed so that the 3′ end of the detection primer is immediately adjacent to a nucleic acid which differentiates the rare mutation carrying nucleic acid from a wildtype nucleic acid molecule, and (ii) only one dNTP or only one ddNTP selected according to the rare mutation, wherein the dNTP or the ddNTP corresponds to a nucleoside adjacent to the detection primer in the nucleic acids with the rare mutation; and (e) detecting the presence of the primer extension product(s) after the primer extension reaction and/or detecting the consumption of the dNTP or the ddNTP, wherein the presence of a primer extension product in the reaction or the consumption of the dNTP or the ddNTP indicates the presence of the nucleic acid with a rare mutation.