TEMPERATURE-SELECTABLE FRET CASSETTE SIGNALING
A multiplexed nucleic acid amplification and detection system useful for detecting the presence of multiple specific nucleic acid sequences or single nucleotide polymorphisms (i.e., “SNPs”) in a temperature-dependent fashion using only a single fluorescence detection channel of a nucleic acid analyzer. The technique can be carried out using standard PCR instrumentation equipped for fluorescence detection or monitoring.
1 . A method of analyzing a sample comprising target nucleic acids, the method comprising the steps of:
(a) contacting, in a reaction mixture, any of a first target nucleic acid of the sample with a first primary probe oligonucleotide comprising a sequence complementary thereto, and a FEN-1 endonuclease under conditions such that if the first primary probe oligonucleotide is hybridized to the first target nucleic acid, the first primary probe is cleaved by the FEN-1 endonuclease to generate a first primary cleaved flap,
wherein the first primary cleaved flap hybridizes to a cleaved flap-hybridizing sequence of a first FRET cassette oligonucleotide contained in the reaction mixture to form an invasive cleavage structure that is cleaved by the FEN-1 endonuclease at a cleavage site between a first fluorophore moiety and a first quencher moiety of the first FRET cassette oligonucleotide to release a first cassette cleaved flap comprising the first fluorophore moiety,
wherein a first masking oligonucleotide comprising a second quencher moiety hybridizes to the first cassette cleaved flap to form a duplex at a first temperature that is below a first Tm of the first masking oligonucleotide and the first cassette cleaved flap,
wherein fluorescence emission from the first fluorophore moiety of the duplex is quenched by the second quencher moiety, and
wherein at a second temperature that is above the first Tm, the first masking oligonucleotide and the first cassette cleaved flap do not form a stable duplex;
(b) detecting any fluorescence emitted from the first fluorophore moiety at the second temperature; and
(c) determining either that
the sample comprises the first target nucleic acid if fluorescence emitted from the first fluorophore moiety is detected in step (b), or
the sample does not comprise the first target nucleic acid if fluorescence emitted from the first fluorophore moiety is not detected in step (b).
2 . The method of claim 1 ,
wherein step (a) further comprises contacting, in the reaction mixture, any of a second target nucleic acid of the sample with a second primary probe oligonucleotide comprising a sequence complementary thereto, and the FEN-1 endonuclease under conditions such that if the second primary probe oligonucleotide hybridizes to the second target nucleic acid, the second primary probe is cleaved by the FEN-1 endonuclease to generate a second primary cleaved flap that is different from the first primary cleaved flap,
wherein the second primary cleaved flap hybridizes to the cleaved flap-hybridizing sequence of a second FRET cassette oligonucleotide contained in the reaction mixture to form an invasive cleavage structure that is cleaved by the FEN-1 endonuclease at a cleavage site between a second fluorophore moiety and a third quencher moiety of the second FRET cassette oligonucleotide to release a second cassette cleaved flap comprising the second fluorophore moiety,
wherein at a third temperature that is below a second Tm, a second masking oligonucleotide comprising a fourth quencher moiety hybridizes to the second cassette cleaved flap to form a duplex,
wherein fluorescence emission from the second fluorophore moiety of the duplex is quenched by the fourth quencher moiety,
wherein at a fourth temperature that is above the second Tm, the second masking oligonucleotide and the second cassette cleaved flap do not form a stable duplex, and
wherein the first Tm and the second Tm differ from each other by at least 5° C.;
wherein step (b) further comprises detecting any fluorescence emitted from the second fluorophore moiety at the fourth temperature; and
wherein step (c) further comprises determining either that
the sample comprises the second target nucleic acid if fluorescence emitted from the second fluorophore moiety of a 5′ flap cleavage product of the second FRET cassette oligonucleotide is detected in step (b), or
the sample does not comprise the first target nucleic acid if fluorescence emitted from the second fluorophore moiety of the 5′ flap cleavage product of the second FRET cassette oligonucleotide is not detected in step (b).
3 . The method of claim 1 ,
wherein step (a) further comprises contacting, in the reaction mixture, any of a second target nucleic acid of the sample with a second primary probe oligonucleotide comprising a sequence complementary thereto, and the FEN-1 endonuclease under conditions such that if the second primary probe oligonucleotide hybridizes to the second target nucleic acid, the second primary probe is cleaved by the FEN-1 endonuclease to generate a second primary cleaved flap,
wherein the second primary cleaved flap hybridizes to the cleaved flap-hybridizing sequence of a second FRET cassette oligonucleotide contained in the reaction mixture to form an invasive cleavage structure that is cleaved by the FEN-1 endonuclease at a cleavage site between a second fluorophore moiety and a third quencher moiety of the second FRET cassette oligonucleotide to release a cleavage product comprising the second fluorophore moiety,
wherein the cleavage product does not hybridize to any masking oligonucleotide in the reaction mixture to result in fluorescence quenching;
wherein step (b) further comprises detecting any fluorescence emitted from the second fluorophore moiety of the cleavage product; and
wherein step (c) further comprises determining either that
the sample comprises the second target nucleic acid if fluorescence emitted from the second fluorophore moiety is detected in step (b), or
the sample does not comprise the first target nucleic acid if fluorescence emitted from the second fluorophore moiety is not detected in step (b).
4 . The method of claim 2 , wherein step (b) comprises detecting with a single channel of a fluorescence monitoring device any fluorescence emitted from the first and second fluorophore moieties.
5 . The method of claim 4 , wherein step (b) is performed while a nucleic acid amplification reaction is occurring in the reaction mixture, and wherein products of the nucleic acid amplification reaction comprise the first target nucleic acid and the second target nucleic acid.
6 . The method of claim 5 , wherein the nucleic acid amplification reaction comprises steps for thermocycling, and wherein the reaction mixture further comprises a thermostable DNA polymerase.
7 . The method of claim 1 , wherein step (b) is performed as the temperature of the reaction mixture decreases to permit annealing of masking oligonucleotides and complementary cassette cleaved flaps.
8 . The method of 4 , wherein the first and second fluorophore moieties are the same as each other.
9 . The method of claim 4 , wherein the step of (b) detecting any fluorescence comprises measuring any fluorescence.
10 . The method of claim 9 , further comprising the step of either detecting or measuring fluorescence at the first temperature.
11 . The method of claim 2 , wherein the first fluorophore moiety and the second fluorophore moiety are both detectable in the same fluorescence detection channel of an energy sensor device.
12 . The method of claim 11 , wherein the second fluorophore moiety is the same as the first fluorophore moiety.
13 . The method of claim 11 , wherein the second fluorophore moiety is not the same as the first fluorophore moiety.
14 . The method of claim 1 , wherein fluorescence from the second fluorophore is detected and/or measured at the first temperature.
15 . The method of claim 2 , wherein the third quencher moiety is the same as the first quencher moiety and/or the second quencher moiety.
16 . The method of claim 1 , wherein the reaction mixture comprises primer oligonucleotides that amplify target nucleic acids, wherein at least one primer oligonucleotide acts as an invasive oligonucleotide in the presence of a primary probe oligonucleotide and target nucleic acid and/or target amplicon to form an invasive cleavage structure that is cleaved by the FEN-1 endonuclease.
17 . The method of claim 1 , wherein step (c) comprises determining with a computer programmed with software.