Patent ID: 7090975

Claim:
A method for determining the presence or absence of a nucleic acid hybrid in a sample comprising the following steps: (a) providing a reaction mixture comprising (i) a sample that may contain a nucleic acid hybrid that comprises a 3′-terminus, (ii) pyrophosphate, (iii) an enzyme that catalyzes the release of a nucleotide from a nucleic acid hybrid, by pyrophosphorolysis of the 3′-terminus of a strand of the nucleic acid hybrid in the presence of pyrophosphate, and (iv) a suitable nucleotide that can be incorporated in the place of said released nucleotide; (b) maintaining said reaction mixture for a time period and under conditions that permit (i) pyrophosphorolysis of the 3′-terminus of a strand of a nucleic acid hybrid to produce a released nucleotide and a modified 3′-terminus as well as (ii) the incorporation of said suitable nucleotide into the modified 3′-terminus of the nucleic acid hybrid to produce an incorporated modified 3′-terminus, thereby forming a treated sample; and (c) assaying the treated sample to determine whether incorporation of said suitable nucleotide into the hybrid occurred and thereby determining the presence or absence of a nucleic acid hybrid in said sample.