Invasion assay
View Patent ↗The invention relates to improved methods of detecting and characterizing polynucleotide sequences and variations in polynucleotide sequences. The invention further relates to a device that can be used to detect and characterize polynucleotide sequences and variations in polynucleotide sequences.
1. A method of detecting a target polynucleotide which comprises the steps of:
(a) contacting a target polynucleotide having a first portion and a second portion immediately contiguous to one another with:
(i) an invader oligonucleotide, at least a part of which is capable of specifically hybridizing to the first portion of the target polynucleotide;
(ii) a probe oligonucleotide comprising a first region that is capable of specifically hybridizing to the second portion of the target polynucleotide and a flap region located adjacent to the first region; and
(iii) a reagent that is capable of cleaving the flap region of the probe oligonucleotide when the probe oligonucleotide is hybridized to the second portion of the target polynucleotide and the invader oligonucleotide is hybridized to the first portion of the polynucleotide;
under conditions such that the cleaved flap region of the probe oligonucleotide and the reagent can come into contact with a reporter precursor to which the flap region of the probe oligonucleotide is capable of hybridizing to form a complex that can be cleaved by the reagent to provide a reporter capable of being detected;
(b) obtaining a data set (t, S(t)), wherein t is time and S(t) is signal as a function of time, by detecting the reporter at a plurality of times; and
(c) transforming (t, S(t)) to provide a transformed data set (t*, S*(t*)) wherein t* and S*(t*) have minima of zero and maxima of unity, and determining whether the transformed data set exhibits non-linear behavior;
wherein the target polynucleotide is detected if the transformed data set exhibits non-linear behavior.
2. The method of claim 1 wherein the invader oligonucleotide comprises a first region that is capable of specifically hybridizing to the first portion of the target polynucleotide, and a flap region located adjacent to the first region.
3. The method of claim 2 wherein the flap region of the invader oligonucleotide is capable of specifically hybridizing to the target polynucleotide.
4. The method of claim 2 wherein flap region of the invader oligonucleotide is not capable of specifically hybridizing to the target polynucleotide.
5. The method of claim 2 wherein flap region of the invader oligonucleotide comprises a first section that is not capable of specifically hybridizing to the target polynucleotide, and a second section that is capable of specifically hybridizing to the target polynucleotide.
6. The method of claim 1 wherein the second portion of the target polynucleotide is located immediately 3′ to the first portion of the target polynucleotide.
7. The method of one of claims 2 - 5 wherein the flap region of the invader oligonucleotide is located immediately 3′ to the first region of the invader oligonucleotide, and the flap region of the probe is located immediately 5′ to the first region of the probe.
8. The method of claim 1 wherein the signal is fluorescence or phosphorescence.
9. The method of claim 1 wherein the determination of whether the signal exhibits a specific behavior as a function of time is performed in real time.
10. The method of claim 1 wherein the non-linear behavior is quadratic.
11. A method of detecting a target polynucleotide which comprises the steps of:
(a) contacting a target polynucleotide having a first portion and a second portion immediately contiguous to one another with:
(i) an invader oligonucleotide, at least a part of which is capable of specifically hybridizing to the first portion of the target polynucleotide;
(ii) a probe oligonucleotide comprising a first region that is capable of specifically hybridizing to the second portion of the target polynucleotide and a flap region located adjacent to the first region; and
(iii) a reagent that is capable of cleaving the flap region of the probe oligonucleotide when the probe oligonucleotide is hybridized to the second portion of the target polynucleotide and the invader oligonucleotide is hybridized to the first portion of the polynucleotide;
under conditions such that the cleaved flap region of the probe oligonucleotide and the reagent can come into contact with a reporter precursor to which the flap region of the probe oligonucleotide is capable of hybridizing to form a complex that can be cleaved by the reagent to provide a reporter capable of being detected;
(b) obtaining a data set (t, S(t)), wherein t is time and S(t) is signal as a function of time, by detecting the reporter at a plurality of times; and
(c) fitting (t, S(t)) to a quadratic function, transforming the quadratic function to yield a transformed function having independent and dependent variables having minima of zero and maxima of unity, and determining whether the transformed function exhibits non-linear behavior;
wherein the target polynucleotide is detected if the transformed function exhibits non-linear behavior.
12. The method of claim 11 wherein the non-linear behavior is quadratic.