Patent ID: 7459271

Claim:
A method for determining the presence or absence of a homozygous or heterozygous mutation in a gene, said method comprising: amplifying a target nucleotide sequence within the gene using a first and a second primer to produce an amplified product, wherein said first primer comprises one or more chemically modified nucleotides, bases or phosphodiester bonds such that a nucleotide strand which extends from said first primer is substantially resistant to exonuclease activity, and wherein said second primer comprises a nucleotide sequence which introduces in the strand extended by said first primer a tag sequence which is complementary to a capture oligonucleotide immobilized to a solid support and wherein one or both of said first or second primers introduces a restriction site in the target sequence when there is no mutation; digesting the amplified product with an exonuclease to digest the strand not amplified by the primer comprising the exonuclease-resistant nucleotides, bases or phosphodiester linkages to generate a single-stranded nucleic acid molecule; hybridizing to said single-stranded nucleic acid molecule a probe that contains complementarity to a region encompassing the restriction site that is introduced when no mutation is present to generate a partial double-stranded molecule wherein the probe comprises two reporter molecules capable of facilitating the provision of identifiable signals which can be distinguished from each other; contacting the partially double-stranded molecules with a restriction endonuclease which will digest the double-stranded molecules which do not contain the mutation and subjecting digested and/or non-digested molecules to conditions to permit annealing of the tag sequence to the capture oligonucleotide immobilized to the solid support; measuring the relative intensity of the signal by the reporter molecules; wherein an equal intensity of different signals represents a homozygous presence of the mutation; wherein one signal represents an absence of the mutation; and wherein a mixture of signals, in which the intensity of one signal is less than the intensity of the other signal thereby represents a heterozygous presence of the mutation.