Patent ID: 8153377
Filing Date: 2012-04-10
Classification: C12Q

Abstract:
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 or is suspected to comprise less than 10% of nucleic acids with the rare mutation; (b) in an amplification reaction amplifying the nucleic acid sample with primers flanking the sequence carrying the rare mutation; (c) removing excess dNTPs after the amplification reaction; (d) performing a primer extension reaction using 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 wildtype from the mutant nucleic acid molecule, and one dNTP and two ddNTPs, wherein the dNTP corresponds to a nucleoside adjacent to the detection primer in the nucleic acids with the rare mutation; and (e) detecting the consumption of dNTP, wherein the consumption of dNTP indicates the presence of the nucleic acid with a rare mutation, wherein the consumption of dNTP is detected using pyrosequencing.