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 composition comprising a fluorescence resonance energy transfer (FRET) cassette reporter system, the composition comprising:
(i) a 5′ flap FRET cassette oligonucleotide comprising:
a 5′ flap portion comprising a first fluorophore moiety,
a stem-loop portion comprising a first quencher moiety, wherein fluorescence emission from the first fluorophore moiety of the 5′ flap FRET cassette is quenched by the first quencher moiety, and
a 3′ portion comprising a cleaved flap-hybridizing sequence,
wherein hybridization of a cassette-specific invasive oligonucleotide complementary to the cleaved flap-hybridizing sequence of the 5′ flap FRET cassette oligonucleotide forms an invasive cleavage structure cleavable by a flap structure-specific endonuclease-1 (FEN-1) endonuclease at a cleavage site between the first fluorophore moiety and the first quencher moiety,
wherein cleavage of the 5′ flap FRET cassette oligonucleotide at the cleavage site produces a cassette cleaved flap comprising the 5′ flap portion and the first fluorophore moiety; and
(ii) a masking oligonucleotide comprising a second quencher moiety,
wherein at least a portion of the masking oligonucleotide is specifically hybridizable to the 5′ flap portion of the FRET cassette oligonucleotide,
wherein hybridization of the masking oligonucleotide to the cassette cleaved flap forms a duplex having a first melting temperature exhibiting a first melting peak, and
wherein fluorescence emission from the first fluorophore moiety in the duplex is quenched by the second quencher moiety.
2. The composition of claim 1 , wherein the first quencher moiety and the second quencher moiety are the same as each other.
3. The composition of claim 1 , further comprising a FEN-1 endonuclease.
4. The composition of claim 3 , wherein the FEN-1 endonuclease is a thermostable FEN-1 endonuclease.
5. The composition of claim 4 , wherein the thermostable FEN-1 endonuclease is from an archaeal organism.
6. The composition of claim 1 , further comprising a first target-specific invasive oligonucleotide and a first target-specific primary probe oligonucleotide,
wherein each of the first target-specific invasive oligonucleotide and the first target-specific primary probe oligonucleotide comprise sequences configured to hybridize to a target nucleic acid to form an invasive cleavage structure cleavable by a FEN-1 endonuclease to produce a primary cleaved flap, and
wherein the primary cleaved flap is a cassette-specific invasive oligonucleotide configured to hybridize to the cleaved flap-hybridizing sequence of the 5′ flap FRET cassette oligonucleotide to form an invasive cleavage structure cleavable by the FEN-1 endonuclease.
7. The composition of claim 6 , further comprising the target nucleic acid.
8. The composition of claim 7 , further comprising deoxynucleoside triphosphates (dNTPs), a thermostable DNA polymerase, and primers having 3′ ends extendable by the thermostable DNA polymerase using the target nucleic acid as the template in a template-dependent nucleic acid amplification reaction.
9. The composition of claim 1 , further comprising:
(iii) a second FRET cassette oligonucleotide comprising
a 5′ portion comprising a second fluorophore moiety,
a stem-loop portion comprising a third quencher moiety, and
a 3′ portion comprising a second cleaved flap-hybridizing sequence,
wherein hybridization of a second cassette-specific invasive oligonucleotide to the second cleaved flap-hybridizing sequence of the second FRET cassette oligonucleotide forms an invasive cleavage structure cleavable by a FEN-1 endonuclease at a cleavage site between the second fluorophore moiety and the third quencher moiety, and
wherein cleavage of the second FRET cassette oligonucleotide at the cleavage site produces a second cassette cleavage product comprising the second fluorophore moiety.
10. The composition of claim 9 , wherein the second FRET cassette oligonucleotide is a second 5′ flap FRET cassette comprising a 5′ flap portion, wherein the cassette cleavage product is a second cassette cleaved flap comprising the second fluorophore, and wherein the composition further comprises:
(iv) a second masking oligonucleotide comprising a fourth quencher moiety,
wherein at least a portion of the second masking oligonucleotide is specifically hybridizable to the 5′ flap portion of the second FRET cassette oligonucleotide,
wherein hybridization of the second masking oligonucleotide to the second cassette cleaved flap forms a second duplex having a second melting temperature that is higher than the first melting temperature, and
wherein fluorescence emission from the second fluorophore moiety in the second duplex is quenched by the fourth quencher moiety.
11. The composition of claim 10 , wherein the third quencher moiety and the fourth quencher moiety are the same as each other.
12. The composition of claim 9 , wherein the second cassette cleavage product comprises no more than 5 nucleotides.
13. The composition of claim 9 , wherein the second cassette cleavage product comprises no more than 4 nucleotides.
14. The composition of claim 9 , wherein the second cassette cleavage product comprises no more than 3 nucleotides.
15. The composition of claim 9 , wherein the second cassette cleavage product comprises no more than 2 nucleotides.
16. The composition of claim 9 , wherein emission signals from the first fluorophore moiety and the second fluorophore moiety are detectable in the same fluorescence detection channel of a fluorescence-monitoring apparatus.
17. The composition of claim 9 , wherein the first fluorophore moiety and the second fluorophore moiety are the same as each other.
18. The composition of claim 9 , wherein the first fluorophore moiety and the second fluorophore moiety are not the same as each other.