Methods for detection of a target nucleic acid by forming a cleavage structure using a reverse transcriptase
View Patent ↗The invention relates to and methods for generating a signal indicative of the presence of a target nucleic acid in a sample. The compositions and methods include a reverse transcriptase, a nuclease, an upstream primer and downstream probe.
1. A method for detecting a target nucleic acid sequence in a sample comprising, mixing an upstream primer, a downstream probe, wherein the downstream probe comprises a 5′ region and a 3′ region, wherein the 3′ region is at least partially complementary to the target nucleic acid and the 5′ region forms a flap, a reverse transcriptase, a cleavage agent and a target nucleic acid under conditions which are permissive for the steps of
(i) annealing of the upstream primer and downstream probe,
(ii) extending the upstream primer wherein the reverse transcriptase synthesizes a primer extension product and forms a cleavage structure with the downstream probe, and
(iii) cleaving the cleavage structure with the cleavage agent to generate a detectable signal
(iv) detecting and/or measuring said detectable signal, wherein detecting and/or measuring of said detectable signal is indicative of the presence of said target nucleic acid.
2. The method of claim 1 , wherein the cleavage agent is a FEN nuclease.
3. The method of claim 1 , wherein the reverse transcriptase is thermostable.
4. The method of claim 1 wherein the reverse transcriptase is selected from the group consisting of: Moloney Murine Leukemia Virus (M-MLV) RT, Human Immunodeficiency Virus (HIV) RT, Avian Sarcoma-Leukosis Virus (ASLV) RT, Rous Sarcoma Virus (RSV) RT, Avian Myeloblastosis Virus (AMV) RT, Avian Erythroblastosis Virus (AEV), Helper Virus MCAV RT, Avian Myelocytomatosis Virus, MC29 Helper Virus MCAV RT, Avian Reticuloendotheliosis Virus (REV-T), Helper Virus REV-A RT, Avian Sarcoma Virus, UR2 Helper Virus UR2AV RT, Avian Sarcoma Virus, Y73 Helper Virus YAV RT, Rous Associated Virus (RAV) RT, and Myeloblastosis Associated Virus (MAV) RT.
5. The method of claim 2 , wherein the FEN nuclease is thermostable.
6. The method of claim 2 , wherein the FEN nuclease is a flap-specific nuclease.
7. A method of detecting a target nucleic acid, comprising:
(a) providing:
an upstream primer that is at least partially complementary to a first region of a target nucleic acid, and
a downstream oligonucleotide comprising a 5′ region and a 3′ region, wherein the 3′ region is at least partially complementary to a second region of the target nucleic acid;
(b) mixing the target nucleic acid and the upstream primer and downstream oligonucleotide under conditions which permit formation of a duplex between the target nucleic acid and the upstream primer and the 3′ region of the downstream oligonucleotide;
(c) subjecting the duplex to a reverse transcriptase polymerization activity under conditions which permit extension of the upstream primer by polymerization of a DNA strand complementary to a length of the extension region sufficient to form a cleavage structure;
(d) providing a cleavage agent under conditions such that cleavage of the cleavage structure occurs at a site located within the downstream oligonucleotide in a manner dependent upon the formation of the cleavage structure, thereby permitting cleavage of the downstream oligonucleotide;
(e) detecting the cleavage of the downstream oligonucleotide, wherein detecting said cleavage of said downstream oligonucleotide is indicative of the presence of said target nucleic acid.
8. The method of claim 7 , wherein the primer and the downstream oligonucleotide hybridize to non-overlapping regions of the target nucleic acid.
9. The method of claim 7 , wherein the cleavage agent is a FEN nuclease.
10. The method of claim 7 , wherein the reverse transcriptase polymerization activity comprises strand displacement activity.
11. The method of claim 7 , wherein the method is carried out isothermally.
12. The method of claim 7 , wherein the reverse transcriptase is selected from the group consisting of: Moloney Murine Leukemia Virus (M-MLV) RT, Human Immunodeficiency Virus (HIV) RT, Avian Sarcoma-Leukosis Virus (ASLV) RT, Rous Sarcoma Virus (RSV) RT, Avian Myeloblastosis Virus (AMV) RT, Avian Erythroblastosis Virus (AEV), Helper Virus MCAV RT, Avian Myelocytomatosis Virus, MC29 Helper Virus MCAV RT, Avian Reticuloendotheliosis Virus (REV-T), Helper Virus REV-A RT, Avian Sarcoma Virus, UR2 Helper Virus UR2AV RT, Avian Sarcoma Virus, Y73 Helper Virus YAV RT, Rous Associated Virus (RAV) RT, and Myeloblastosis Associated Virus (MAV) RT.
13. The method of claim 7 , wherein the reverse transcriptase polymerization activity polymerizes nucleotides complementary to a length of extension region sufficient to form a cleavage structure, the polymerized complementary nucleic acid is adjacent at its 3′ end to the downstream oligonucleotide in the duplex.
14. The method of claim 7 , wherein the reverse transcriptase activity polymerizes nucleotides complementary to a length of extension region sufficient to form a cleavage structure, the length of the extension region is a length such that the extension region is fully duplex nucleic acid.
15. The method of claim 7 , wherein the 5′ region of the downstream probe forms a 5′ flap.