Claims
- 1) A method for detecting a target nucleic acid sequence, the method comprising:
a) providing one or more target probes comprising a linear single-stranded DNA molecule, the target probes comprising at least two target-complementary sequences that are not joined to each other, wherein the 5′-end of a first target-complementary sequence is complementary to the 5′-end of the target nucleic acid sequence, and wherein the 3′-end of a second target-complementary sequence is complementary to the 3′-end of the target nucleic acid sequence, and wherein the target probe that comprises the first target-complementary sequence also comprises a sense promoter sequence that is joined to the 3′-end of the first target-complementary sequence; b) contacting the target probes with the target nucleic acid sequence and incubating under hybridization conditions, such that the target-complementary sequences anneal adjacently to the target nucleic acid sequence to form a target probe-target complex; c) contacting the target probe-target complex with a ligase under ligation conditions to form a ligation product; d) contacting the ligation product with an anti-sense promoter oligo and incubating under hybridization conditions, such that the anti-sense promoter oligo anneals to the sense promoter sequence to form a transcription substrate; e) contacting the transcription substrate with an RNA polymerase under transcription conditions to form a transcription product; f) optionally, repeating steps (a) through (f); and g) detecting the transcription product.
- 2) The method of claim 1, wherein the target nucleic acid sequence comprises a single-stranded DNA molecule obtained by reverse transcription of RNA.
- 3) The method of claim 1, wherein the target nucleic acid sequence comprises a DNA target nucleic acid in a sample.
- 4) The method of claim 1, wherein the one or more target probes comprise a bipartite target probe.
- 5) The method of claim 1, wherein the target probe comprising the second target-complementary sequence also comprises a signal sequence 5′-of the target-complementary sequence.
- 6) The method of claim 5, wherein the signal sequence comprises a substrate for Q-beta replicase.
- 7) The method of claim 5, wherein the signal sequence comprises a sequence that encodes a detectable protein.
- 8) The method of claim 7, wherein the detectable protein is green fluorescent protein.
- 9) The method of claim 5, wherein the signal sequence comprises a sequence that is detectable by a probe.
- 10) The method of claim 5, wherein the signal sequence comprises a sequence that is detectable by a molecular beacon.
- 11) The method of claim 1, wherein the ligase is selected from the group consisting of Ampligase® Thernostable DNA Ligase, Tfl DNA Ligase, Tsc DNA Ligase, Pfu DNA ligase, T4 DNA ligase and Tth DNA ligase.
- 12) The method of claim 1, wherein the anti-sense promoter oligo is attached to a solid support.
- 13) The method of claim 1, wherein the RNA polymerase is a T7-type RNA polymerase.
- 14) The method of claim 1, wherein the RNA polymerase is selected from the group consisting of T7 RNA polymerase, T3 RNA polymerase, SP6 RNA polymerase, Tth RNA polymerase, E. coli RNA polymerase, and SP6 or T7 R&DNA™ Polymerase.
- 15) The method of claim 1 comprising an additional step following step (c), the additional step comprising releasing the ligation product from the target nucleic acid sequence.
- 16) A method for detecting a target nucleic acid sequence, the method comprising:
a) providing a promoter target probe, wherein the 5′-end of the promoter target probe comprises a first target-complementary sequence that is complementary to the 5′-end of the target nucleic acid sequence, and wherein a sense promoter sequence is joined to the 3′-end of the first target-complementary sequence; b) providing a signal target probe comprising a second target complementary sequence, wherein the 3′-end of the second target-complementary sequence is complementary to the 3′-end of the target nucleic acid sequence; c) optionally, providing at least one additional target probe comprising a target-complementary sequence; d) contacting the target probes with the target nucleic acid sequence and incubating under hybridization conditions, such that the target-complementary sequences anneal adjacently to the target nucleic acid sequence to form a target probe-target complex; e) contacting the target probe-target complex with a ligase under ligation conditions to form a ligation product; f) contacting the ligation product with an anti-sense promoter oligo and incubating under hybridization conditions, such that the anti-sense promoter oligo anneals to the sense promoter sequence to form a transcription substrate; g) contacting the transcription substrate with an RNA polymerase under transcription conditions to form a transcription product; h) optionally, repeating steps (a) through (h); and i) detecting the transcription product.
- 17) The method of claim 16, wherein the target nucleic acid sequence comprises a single-stranded DNA molecule obtained by reverse transcription of RNA.
- 18) The method of claim 16, wherein the target nucleic acid sequence comprises a DNA target nucleic acid in a sample.
- 19) The method of claim 16, wherein the signal target probe comprises a signal sequence 5′-of the target-complementary sequence.
- 20) The method of claim 19, wherein the signal sequence comprises a substrate for Q-beta replicase.
- 21) The method of claim 19, wherein the signal sequence comprises a sequence that encodes a detectable protein.
- 22) The method of claim 21, wherein the detectable protein is green fluorescent protein.
- 23) The method of claim 19, wherein the signal sequence comprises a sequence that is detectable by a probe.
- 24) The method of claim 19, wherein the signal sequence comprises a sequence that is detectable by a molecular beacon.
- 25) The method of claim 16, wherein the ligase is selected from the group consisting of Ampligase® Thermostable DNA Ligase, Tfl DNA Ligase, Tsc DNA Ligase, Pfu DNA ligase, T4 DNA ligase and Tth DNA ligase.
- 26) The method of claim 16, wherein the anti-sense promoter oligo is attached to a solid support.
- 27) The method of claim 16, wherein the RNA polymerase is a T7-type RNA polymerase.
- 28) The method of claim 16, wherein the RNA polymerase is selected from the group consisting of T7 RNA polymerase, T3 RNA polymerase, SP6 RNA polymerase, Tth RNA polymerase, E. coli RNA polymerase, and SP6 or T7 R&DNA™ Polymerase.
- 29) A method for detecting a target nucleic acid sequence, the method comprising:
a) providing one or more target probes comprising a linear single-stranded DNA molecule, the target probes comprising at least two target-complementary sequences that are not joined to each other, wherein the 5′-end of a first target-complementary sequence is complementary to the 5′-end of the target nucleic acid sequence, and wherein the 3′-end of a second target-complementary sequence is complementary to the 3′-end of the target nucleic acid sequence, and wherein the target probe that comprises the first target-complementary sequence also comprises a sense promoter sequence that is joined to the 3′-end of the first target-complementary sequence; b) contacting the target probes with the target nucleic acid sequence and incubating under hybridization conditions, such that the target probes anneal to the target nucleic acid sequence to form a target probe-target complex; c) contacting the target probe-target complex with a DNA polymerase under DNA polymerization conditions to form one or more DNA polymerase extension products that are adjacent to the 5′-end of a target-probe, such that a complex is formed; d) contacting the complex with a ligase under ligation conditions to form a transcription substrate; e) contacting the transcription substrate with an RNA polymerase; f) optionally, repeating steps (a) through (f); and g) detecting the transcription product.
- 30) A method for detecting a target nucleic acid sequence, the method comprising:
a) providing a target sequence amplification probe (TSA probe) comprising a linear single-stranded DNA molecule comprising a 5′-end portion and a 3′-end portion that are not joined, wherein the 5′-end portion is complementary to the 5′-end of the target sequence, and wherein the 3′-end portion is complementary to the 3′-end of the target sequence; b) providing a primer that is complementary to the TSA probe; c) providing one or more target probes comprising a second linear single-stranded DNA molecule, the target probes comprising at least two target-complementary sequences that are not joined to each other, wherein the 5′-end of a first target-complementary sequence is complementary to the 5′-end of the target nucleic acid sequence, and wherein the 3′-end of a second target-complementary sequence is complementary to the 3′-end of the target nucleic acid sequence, and wherein the target probe that comprises the first target-complementary sequence also comprises a sense promoter sequence that is joined to the 3′-end of the first target-complementary sequence; d) contacting the TSA probe with the target nucleic acid sequence and incubating under hybridization conditions, such that the end portions anneal adjacently to the target nucleic acid sequence to form a complex; e) contacting the complex with a ligase under ligation conditions, such that a target sequence amplification circle (TSA circle) is formed; f) contacting the TSA circle with the primer and incubating under hybridization conditions to form a TSA circle-primer complex; g) contacting the TSA circle-primer complex with a strand-displacing DNA polymerase under strand-displacing polymerization conditions, such that a rolling circle replication product comprising multiple copies of the target nucleic acid sequence is formed; h) contacting the target probes with the rolling circle replication product and incubating under hybridization conditions, such that the target-complementary sequences anneal adjacently to the rolling circle replication product to form a target probe-rolling circle replication product complex; i) contacting the target probe-rolling circle replication product complex with the ligase under ligation conditions to form a ligation product; j) optionally, releasing the ligation product from the rolling circle replication product complex, k) contacting the ligation product with an anti-sense promoter oligo and incubating under hybridization conditions, such that the anti-sense promoter oligo anneals to the sense promoter sequence to form a transcription substrate; l) contacting the transcription substrate with an RNA polymerase under transcription conditions to form a transcription product; m) optionally, repeating steps (a) through (m); and n) detecting the transcription product.
- 31) The method of claim 30, wherein the target nucleic acid sequence comprises a single-stranded DNA molecule obtained by reverse transcription of RNA.
- 32) The method of claim 30, wherein the target nucleic acid sequence comprises a DNA target nucleic acid in a sample.
- 33) The method of claim 30, wherein the target probe comprising the second target-complementary sequence also comprises a signal sequence 5′-of the target-complementary sequence.
- 34) The method of claim 33, wherein the signal sequence comprises a substrate for Q-beta replicase.
- 35) The method of claim 33, wherein the signal sequence comprises a sequence that encodes a detectable protein.
- 36) The method of claim 35, wherein the detectable protein is green fluorescent protein.
- 37) The method of claim 33, wherein the signal sequence comprises a sequence that is detectable by a probe.
- 38) The method of claim 33, wherein the signal sequence comprises a sequence that is detectable by a molecular beacon.
- 39) The method of claim 30, wherein the ligase is selected from the group consisting of Ampligase® Thermostable DNA Ligase, Tfl DNA Ligase, Tsc DNA Ligase, Pfu DNA ligase, T4 DNA ligase and Tth DNA ligase.
- 40) The method of claim 30, wherein the strand-displacing DNA polymerase is selected from the group consisting of RepliPHI™ phi29 DNA polymerase, phi29 DNA polymerase, rBst DNA polymerase large fragment, IsoTherm™ DNA polymerase, BcaBEST™ DNA polymerase, SequiTherm™ DNA polymerase, phage M2 DNA polymerase, phage phi PRD1 DNA polymerase, VENT® DNA polymerase, Klenow fragment of DNA polymerase I, T5 DNA polymerase, PRD1 DNA polymerase, and T7 DNA polymerase in the presence of a T7 helicase/primase complex.
- 41) The method of claim 30, wherein the anti-sense promoter oligo is attached to a solid support.
- 42) The method of claim 30, wherein the RNA polymerase is a T7-type RNA polymerase.
- 43) The method of claim 30, wherein the RNA polymerase is selected from the group consisting of T7 RNA polymerase, T3 RNA polymerase, SP6 RNA polymerase, Tth RNA polymerase, E. coli RNA polymerase, and SP6 or T7 R&DNA™ Polymerase.
- 44) The method of claim 30, comprising an additional step following step (e), the additional step comprising releasing the TSA circle from the target nucleic acid sequence.
- 45) A method for detecting a target nucleic acid sequence, the method comprising:
a) providing a bipartite target probe comprising a linear single-stranded DNA molecule, the target probes comprising at least two target-complementary sequences that are not joined to each other, wherein the 5′-end of a first target-complementary sequence is complementary to the 5′-end of the target nucleic acid sequence, and wherein the 3′-end of a second target-complementary sequence is complementary to the 3′-end of the target nucleic acid sequence; b) optionally, providing at least one additional target probe comprising a target-complementary sequence; c) contacting the target probe with the target nucleic acid sequence and incubating under hybridization conditions, such that the target-complementary sequences anneal adjacently to the target nucleic acid sequence to form a target probe-target complex; d) contacting the target probe-target complex with a ligase under ligation conditions to form a transcription substrate; e) contacting the transcription substrate with an RNA polymerase under transcription conditions to form a transcription product; f) optionally, repeating steps (a) through (f); and g) detecting the transcription product.
- 46) A method for detecting a target nucleic acid sequence, the method comprising:
a) providing one or more target probes comprising a linear single-stranded DNA molecule, the target probes comprising at least two target-complementary sequences that are not joined to each other, wherein the 5′-end of a first target-complementary sequence is complementary to the 5′-end of the target nucleic acid sequence, and wherein the 3′-end of a second target-complementary sequence is complementary to the 3′-end of the target nucleic acid sequence, and wherein the target probe that comprises the first target-complementary sequence also comprises a pseudopromoter that is joined to the 3′-of the first target-complementary sequence; b) contacting the target probes with the target nucleic acid sequence and incubating under hybridization conditions, such that the target-complementary sequences anneal adjacently to the target nucleic acid sequence to form a target probe-target complex; c) contacting the target probe-target complex with a ligase under ligation conditions to form a transcription substrate; d) contacting the transcription substrate with an RNA polymerase; e) optionally, repeating steps (a) through (e); and f) detecting the transcription product.
- 47) A method for detecting an analyte in a sample, the method comprising:
a) providing a target nucleic acid sequence comprising a target sequence tag that is joined to an analyte-binding substance; b) contacting the analyte-binding substance with the analyte to form a specific binding pair; c) separating the specific binding pair from analyte-binding substance molecules that are not bound to the analyte; d) providing one or more target probes comprising a linear single-stranded DNA molecule, the target probes comprising at least two target-complementary sequences that are not joined to each other, wherein the 5′-end of a first target-complementary sequence is complementary to the 5′-end of the target nucleic acid sequence, and wherein the 3′-end of a second target-complementary sequence is complementary to the 3′-end of the target nucleic acid sequence, and wherein the target probe that comprises the first target-complementary sequence also comprises a promoter that is joined to the 3′-end of the first target-complementary sequence; e) contacting the target probes with the target nucleic acid sequence and incubating under hybridization conditions, such that the target-complementary sequences anneal adjacently to the target nucleic acid sequence to form a target probe-target complex; f) contacting the target probe-target complex with a ligase under ligation conditions to form a ligation product; g) contacting the ligation product with an anti-sense promoter oligo and incubating under hybridization conditions, such that the anti-sense promoter oligo anneals to the sense promoter sequence to form a transcription substrate; h) contacting the transcription substrate with an RNA polymerase under transcription conditions to form a transcription product; i) optionally, repeating steps (d) through (i); and j) detecting the transcription product.
- 48) The method of claim 47, wherein the analyte is selected from the group consisting of a biochemical molecule, a biopolymer, a protein, a glycoprotein, a lipoprotein, an enzyme, a hormone, a biochemical metabolite, a receptor, an antigen, an antibody, a nucleic acid, a DNA molecule, an RNA molecule, a polysaccharide and a lipid.
- 49) The method of claim 47, wherein the analyte-binding substance is selected from the group consisting of a nucleic acid, a polynucleotide, an oligonucleotide, a segment of a nucleic acid or polynucleotide, a DNA molecule, an RNA molecule, a molecule comprising both DNA and RNA mononucleotides, modified DNA mononucleotides, a molecule obtained by a method termed “SELEX”, a nucleic acid molecule having an affinity for protein molecules, a polynucleotide molecule having an affinity for protein molecules, an operator, a promoter, an origin of replication, a ribosomal nucleic acid sequence, a sequence recognized by steroid hormone-receptor complexes, a peptide nucleic acid (PNA), a nucleic acid and a PNA, a molecule prepared by using a combinatorial library of randomized peptide nucleic acids, an oligonucleotide or polynucleotide with a modified backbone that is not an amino acid, a lectin, a receptor for a hormone, a hormone, and an enzyme inhibitor.
- 50) The method of claim 47, wherein the target probe comprising the second target-complementary sequence also comprises a signal sequence 5′-of the target-complementary sequence.
- 51) The method of claim 50, wherein the signal sequence comprises a substrate for Q-beta replicase.
- 52) The method of claim 50, wherein the signal sequence comprises a sequence that encodes a detectable protein.
- 53) The method of claim 52, wherein the detectable protein is green fluorescent protein.
- 54) The method of claim 50, wherein the signal sequence comprises a sequence that is detectable by a probe.
- 55) The method of claim 50, wherein the signal sequence comprises a sequence that is detectable by a molecular beacon.
- 56) The method of claim 47, wherein the ligase is selected from the group consisting of Ampligase® Thermostable DNA Ligase, Tfl DNA Ligase, Tsc DNA Ligase, Pfu DNA ligase, T4 DNA ligase and Tth DNA ligase.
- 57) The method of claim 47, wherein the anti-sense promoter oligo is attached to a solid support.
- 58) The method of claim 47, wherein the RNA polymerase is a T7-type RNA polymerase.
- 59) The method of claim 47, wherein the RNA polymerase is selected from the group consisting of T7 RNA polymerase, T3 RNA polymerase, SP6 RNA polymerase, Tth RNA polymerase, E. coli RNA polymerase, and SP6 or T7 R&DNA™ Polymerase.
- 60) The method of claim 47, comprising an additional step following step (c) the additional step comprising releasing the ligation product from the target nucleic acid sequence.
- 61) A method for selectively transcribing a target nucleic acid sequence, the method comprising a DNA ligation operation and a transcription operation, wherein the DNA ligation operation comprises ligation of one or more target probes comprising a sense promoter sequence that is joined to the 3′-end of a target complementary sequence to form a ligation product, wherein the ligation is dependent on hybridization of the target probes to the target nucleic acid sequence, and wherein the transcription operation comprises contacting the ligation product with an RNA polymerase.
- 62) A kit for detecting a target nucleic acid sequence, the kit comprising:
a) one or more target probes comprising a linear single-stranded DNA molecule, the target probes comprising at least two target-complementary sequences that are not joined to each other, wherein the 5′-end of a first target-complementary sequence is complementary to the 5′-end of the target nucleic acid sequence, and wherein the 3′-end of a second target-complementary sequence is complementary to the 3′-end of the target nucleic acid sequence, and wherein the target probe that comprises the first target-complementary sequence also comprises a promoter that is joined to the 3′-of the first target-complementary sequence; b) a ligase; and c) an RNA polymerase.
- 63) The kit of claim 62, further comprising a reverse transcriptase.
- 64) The kit of claim 62, further comprising a target sequence amplification probe (TSA probe) comprising a linear single-stranded DNA molecule comprising a 5′-end portion and a 3′-end portion that are not joined, wherein the 5′-end portion is complementary to the 5′-end of the target sequence, and wherein the 3′-end portion is complementary to the 3′-end of the target sequence.
- 65) The kit of claim 62, further comprising a DNA polymerase.
- 66) The kit of claim 62, further comprising a target sequence tag that is joined to an analyte-binding substance.
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application claims priority to U.S. Provisional Patent Application Serial No. 60/436,062 filed Dec. 23, 2002. The entire disclosure of all priority applications is specifically incorporated herein by reference.
Provisional Applications (1)
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Number |
Date |
Country |
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60436062 |
Dec 2002 |
US |
Continuation in Parts (1)
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Number |
Date |
Country |
Parent |
10153219 |
May 2002 |
US |
Child |
10744815 |
Dec 2003 |
US |