Method for preventing false positives in methods employing ddNTP's
View Patent ↗The present invention relates to a method which prevents undesirable binding of ddNTPs to double stranded polynucleotides when in the presence of a polymerase. Such methods may be used to prevent the appearance of false positives in methods employing ddNTPs, e.g. in sequence detection methods. The present invention also provides a method of avoiding a false Tm reading or false FRET effects (such as false positive quenching), for example in a melting curve analysis method. In particular a method is provided in which a target nucleotide sequence in a test polynucleotide is detected using a method in which a double stranded molecule is generated which may or may not comprise two labels depending on whether the target sequence is present in which the presence of the two labels is determined, preferably by performing a melting curve analysis.
1 . 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.
2 . The method of claim 1 wherein said first and/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 and/or said labelling probe and/or reporter probe is from 10-100 nucleotides in length.
3 . The method of claim 1 wherein said polymerase is inactivated or degraded with proteinase K or SDS.
4 . The method of claim 1 wherein said target nucleotide sequence is a single nucleotide polymorphism (SNP).
5 . The method of claim 1 wherein at least two target nucleotide sequences are detected in said method.
6 . The method of claim 1 wherein at least two ddNTPs which are labelled are used.
7 . The method of claim 1 wherein at least two reporter probes and/or labelling probes are used.
8 . The method of claim 7 wherein the at least two reporter probe(s) and the at least two labelling probe(s) are hybridized to one another generating at least two double stranded third polynucleotides with different melting temperatures.
9 . The method of claim 6 wherein multiple pairs of labels are provided, wherein each pair of labels comprises a first label on a ddNTP and a second label on a reporter probe, and wherein each pair of labels is different from every other pair of labels.
10 . The method of claim 1 wherein the reporter probe is labelled at one of the three nucleotides at its 5′ end.
11 . The method of claim 1 wherein said first or second label is a fluorophore and the other label is a quencher.
12 . The method of claim 1 wherein when said single stranded region provides a 5′ protruding sequence it does not contain a base complementary to said ddNTP anywhere in said single stranded region.
13 . The method of claim 1 wherein said first and second label is a fluorophore.
14 . The method of claim 5 wherein at least two SNPs are detected in said method.
15 . The method of claim 6 wherein the first labels on the at least two ddNTPs are different.
16 . The method of claim 7 wherein the second labels on the at least two reporter probes are different.
17 . The method of claim 9 wherein each pair of the multiple pairs of labels contains first and second labels which are different to any first or second label found in any other pair of labels.
18 . The method of claim 10 wherein the reporter probe is labelled at the 5′ terminus.