Patent ID: 7906287

Claim:
A method of detecting a nucleotide variation within a nucleic acid of interest comprising: (a) a step comprising generating a set of extension products from a first nucleic acid and causing modified nucleotides to be present in the extension products, wherein the modified nucleotides comprise a single type of base, (b) a step comprising hybridizing the extension products to a second nucleic acid, (c) a step comprising contacting the hybridizing nucleic acid with one or more agents that collectively remove the modified nucleotides, (d) a step comprising contacting the hybridizing nucleic acid with a first enzyme that extends the extension products in the presence of three types of nuclease-resistant nucleotides and not in the presence of nucleotides that comprise the same type of base as the modified nucleotides, wherein each of the three types of nuclease-resistant nucleotides comprises a different type of base, (e) a step comprising contacting the hybridizing nucleic acid with a second enzyme that removes nucleotides from the 3′ end of the extension products, wherein the second enzyme does not remove the nuclease-resistant nucleotides, (f) a step comprising contacting the hybridizing nucleic acid with a third enzyme that extends the extension products in the presence of nucleotides, (g) a step comprising distinguishing those extension products comprising a nuclease-resistant nucleotide from those extension products not comprising a nuclease-resistant nucleotide, wherein the first nucleic acid is a reference nucleic acid and the second nucleic acid is the nucleic acid of interest or wherein the first nucleic acid is the nucleic acid of interest and the second nucleic acid is a reference nucleic acid, thereby detecting nucleotide variation in the nucleic acid of interest.