Method for preventing false positives in methods employing ddNTP's

A method of identifying a target nucleotide sequence in a test polynucleotide comprising:

• (a) contacting said test polynucleotide with a ddNTP and an unlabelled labelling probe, which probe comprises from 5 to 200 nucleotides and hybridizes to said target nucleotide sequence, when present, immediately 5′ to a base which is complementary to said ddNTP in said target nucleotide sequence, in the presence of a polymerase, wherein said ddNTP carries a first label and when said target sequence is present in said test polynucleotide said unlabelled labelling probe hybridizes to said test polynucleotide and is extended in the 3′ direction by said polymerase to attach said labelled ddNTP to said unlabelled labelling probe to form a labelled labelling probe;
• (b) separating the labelling probe which is a first polynucleotide, which may be labelled or unlabelled, from the test polynucleotide;
• (c) hybridizing said labelling probe to a reporter probe, which is a second polynucleotide which comprises from 5 to 200 nucleotides and, carries a second label to form a double stranded third polynucleotide, wherein the first or second label is a fluorophore and the other label is a molecule which affects the fluorescence of said fluorophore when in proximity to said fluorophore, wherein said first and second polynucleotides are complementary to one another over 80-100% of the total length of their sequences and wherein when said labelling probe is unlabelled, at the end closest to the second label said double stranded third polynucleotide is (i) blunt ended;
• (ii) has a protruding 5′ end or (iii) has a protruding 3′ end, and at the end distal to the second label said double stranded third polynucleotide is (i) blunt ended;
• (ii) has a protruding 5′ end or (iii) has a protruding 3′ end, wherein said protruding 3′ or 5′ end consists of a single stranded region and when said single stranded region provides a 5′ protruding sequence it does not contain a base complementary to said ddNTP immediately adjacent to the double stranded region;
• (d) avoiding the generation of a false melting temperature for the double stranded third polynucleotide and/or avoiding the generation of false FRET effects by inactivating or degrading said polymerase in the period during the extension in step a) or after step a) and before or during generation of said double stranded third polynucleotide in step c), wherein when said inactivation or degradation is performed during the extension in step a) the polymerase is not immediately inactivated;
• and
• (e) determining if said labelling probe carries a first label by assessing the signal associated with the double stranded third polynucleotide, wherein said double stranded third polynucleotide is subject to melting curve analysis in which melting temperature and/or FRET effects are assessed and in which a change in the first or second label's signal during dissociation which occurs during said melting curve analysis is indicative of the presence of said target sequence in said test polynucleotide.