US 2004/0171047 A1 · Target-dependent transcription

Abstract

The present invention comprises novel methods, compositions and kits comprising monopartite or bipartite target probes and an RNA polymerase to detect and quantify analytes comprising one or multiple target nucleic acid sequences, including target sequences that differ by as little as one nucleotide, or to detect and quantify non-nucleic acid analytes by detecting a target sequence tag that is joined to an analyte-binding substance. The method, called “target-dependent transcription,” consists of an annealing process, a DNA ligation process, a transcription process, and a detection process. The invention also comprises novel methods, compositions and kits for amplifying RNA, including strand-displacement reverse transcription and rolling circle reverse transcription.

Claims66

Dependent 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.

5 more elements

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.

7 more elements

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.

12 more elements

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.

8 more elements

Dependencies are inferred from the claim text.

Target-dependent transcription

Life of this patent

Target-dependent transcription

Life of this patent

Abstract

Claims66