Rolling circle replication reporter systems
Disclosed are compositions and a method for of amplifying nucleic acid sequences useful for detecting the presence of molecules of interest. The method is useful for detecting specific nucleic acids in a sample with high specificity and sensitivity. The method also has an inherently low level of background signal. A preferred form of the method consists of a DNA ligation operation, an amplification operation, and a detection operation. The DNA ligation operation circularizes a specially designed nucleic acid probe molecule. This operation is dependent on hybridization of the probe to a target sequence and forms circular probe molecules in proportion to the amount of target sequence present in a sample. The amplification operation is rolling circle replication of the circularized probe. A single round of amplification using rolling circle replication results in a large amplification of the circularized probe sequences. Following rolling circle replication, the amplified probe sequences are detected and quantified using any of the conventional detection systems for nucleic acids such as detection of fluorescent labels, enzyme-linked detection systems, antibody-mediated label detection, and detection of radioactive labels. Because, the amplified product is directly proportional to the amount of target sequence present in a sample, quantitative measurements reliably represent the amount of a target sequence in a sample. Major advantages of this method are that the ligation step can be manipulated to obtain allelic discrimination, the DNA replication step is isothermal, and signals are strictly quantitative because the amplification reaction is linear and is catalyzed by a highly processive enzyme. In multiplex assays, the primer oligonucleotide used for the DNA polymerase reaction can be the same for all probes. Also described are modes of the method in which additional amplification is obtained using a cascade of strand displacement reactions.
1. A method comprising
a DNA ligation operation and an amplification operation,
wherein the DNA ligation operation comprises circularization of an open circle probe, wherein circularization of the open circle probe is dependent on hybridization of the open circle probe to a target sequence,
wherein the amplification operation comprises a) rolling circle replication of the circularized open circle probe to produce tandem sequence DNA and b) secondary DNA strand displacement,
wherein secondary DNA strand displacement is accomplished by hybridizing secondary DNA strand displacement primers to the tandem sequence DNA produced by the rolling circle replication of the circularized open circle probe to produce secondary tandem sequence DNA, and
wherein the tandem sequence DNA as well as the secondary tandem sequence DNA is detected.
2. The method of claim 1 wherein the tandem sequence DNA is detected using molecular beacon detection probes.
3. The method of claim 1 wherein the tandem sequence DNA is detected using detection probes labeled with fluorescent moieties where the fluorescent moieties fluoresce only when the detection probe is hybridized, wherein the detection probes hybridize to the tandem sequence DNA.
4. The method of claim 1 wherein the target sequence is coupled to a specific binding molecule, wherein the specific binding molecule can interact with a target molecule.
5. The method of claim 4 wherein the target molecule is a protein.
6. The method of claim 4 wherein the target molecule is a nucleic acid molecule.
7. A method comprising
a) tethering an oligonucleotide portion of a reporter binding agent to a specific binding molecule portion of the reporter binding agent, wherein the oligonucleotide portion of the reporter binding agent is an amplification target circle; and
b) performing an amplification operation,
wherein the specific binding molecule portion of the reporter binding agent binds to a target molecule,
wherein the oligonucleotide portion of the reporter binding agent is not the target molecule, and
wherein the amplification operation comprises amplification of the amplification target circle via rolling circle replication to produce tandem sequence DNA.
8. The method of claim 7 wherein the tandem sequence DNA is detected using molecular beacon detection probes.
9. The method of claim 7 wherein the tandem sequence DNA is detected using detection probes labeled with fluorescent moieties where the fluorescent moieties fluoresce only when the detection probe is hybridized, wherein the detection probes hybridize to the tandem sequence DNA.
10. The method of claim 7 wherein the target molecule is a protein.
11. The method of claim 7 wherein the target molecule is a nucleic acid molecule.
12. The method of claim 7 wherein the specific binding molecule is an antibody.
13. A method comprising
a DNA ligation operation and an amplification operation,
wherein the DNA ligation operation comprises circularization of an open circle probe, wherein circularization of the open circle probe is dependent on hybridization of the open circle probe to a target sequence,
wherein the amplification operation comprises rolling circle amplification of the circularized open circle probe to produce tandem sequence DNA, wherein the tandem sequence DNA is detected, wherein rolling circle amplification is primed by a rolling circle replication primer, a secondary DNA strand displacement primer, and a tertiary DNA strand displacement primer,
wherein the rolling circle replication primer and tertiary DNA strand displacement primer are complementary to the circularized open circle probe, wherein the secondary DNA strand displacement primer is complementary to sequences in the tandem sequence DNA, wherein the secondary DNA strand displacement primer is not complementary to the complement of either the rolling circle replication primer or the tertiary DNA strand displacement primer.
14. The method of claim 13 wherein the tandem sequence DNA is detected using molecular beacon detection probes.
15. The method of claim 13 wherein the tandem sequence DNA is detected using detection probes labeled with fluorescent moieties where the fluorescent moieties fluoresce only when the detection probe is hybridized, wherein the detection probes hybridize to the tandem sequence DNA.
16. The method of claim 13 wherein the target sequence is coupled to a specific binding molecule, wherein the specific binding molecule can interact with a target molecule.
17. The method of claim 16 wherein the target molecule is a protein.
18. The method of claim 16 wherein the target molecule is a nucleic acid molecule.
19. A method comprising
an amplification operation,
wherein a reporter binding agent and a target molecule are brought into contact, wherein the reporter binding agent comprises a specific binding molecule and an oligonucleotide, wherein the specific binding molecule interacts with the target molecule, wherein an amplification target circle and the reporter binding agent are brought into contact,
wherein the amplification operation comprises rolling circle amplification of the amplification target circle to produce tandem sequence DNA, wherein the tandem sequence DNA is detected, wherein rolling circle amplification is primed by the oligonucleotide of the reporter binding agent, a secondary DNA strand displacement primer, and a tertiary DNA strand displacement primer.
20. The method of claim 19 wherein the tandem sequence DNA is detected using molecular beacon detection probes.
21. The method of claim 19 wherein the tandem sequence DNA is detected using detection probes labeled with fluorescent moieties where the fluorescent moieties fluoresce only when the detection probe is hybridized, wherein the detection probes hybridize to the tandem sequence DNA.
22. The method of claim 19 wherein the target molecule is a protein.
23. The method of claim 19 wherein the target molecule is a nucleic acid molecule.
24. A method comprising
an amplification operation,
wherein a reporter binding agent and a target molecule are brought into contact, wherein the reporter binding agent comprises a specific binding molecule and an oligonucleotide, wherein the oligonucleotide comprises an amplification target circle, wherein the specific binding molecule interacts with the target molecule,
wherein the amplification operation comprises rolling circle amplification of the amplification target circle to produce tandem sequence DNA, wherein the tandem sequence DNA is detected, wherein rolling circle amplification is primed by a rolling circle replication primer, a secondary DNA strand displacement primer, and a tertiary DNA strand displacement primer,
wherein the rolling circle replication primer and tertiary DNA strand displacement primer, are complementary to the amplification target circle, wherein the rolling circle replication primer and tertiary DNA strand displacement primer are not complementary to the same sequence on the amplification target circle, wherein the secondary DNA strand displacement primer is complementary to sequences in the tandem sequence DNA, wherein the secondary DNA strand displacement primer is not complementary to the complement of either the rolling circle replication primer or the tertiary DNA strand displacement primer.
25. The method of claim 24 wherein the tandem sequence DNA is detected using molecular beacon detection probes.
26. The method of claim 24 wherein the tandem sequence DNA is detected using detection probes labeled with fluorescent moieties where the fluorescent moieties fluoresce only when the detection probe is hybridized, wherein the detection probes hybridize to the tandem sequence DNA.
27. The method of claim 24 wherein the target molecule is a protein.
28. The method of claim 24 wherein the target molecule is a nucleic acid molecule.