Rolling circle replication reporter systems
First Claim
1. A method of amplifying nucleic acid sequences, the method comprising,(a) mixing a rolling circle replication primer with one or more amplification target circles, to produce a primer-ATC mixture, and incubating the primer-ATC mixture under conditions that promote hybridization between the amplification target circles and the rolling circle replication primer in the primer-ATC mixture, wherein the amplification target circles each comprise a single-stranded, circular DNA molecule comprising a primer complement portion, and wherein the primer complement portion is complementary to the rolling circle replication primer, and wherein at least one of the amplification target circles is tethered to a specific binding molecule so that the amplification target circle can rotate freely, and (b) mixing DNA polymerase with the primer-ATC mixture, to produce a polymerase-ATC mixture, and incubating the polymerase-ATC mixture under conditions that promote replication of the amplification target circles, wherein replication of the amplification target circles results in the formation of tandem sequence DNA.
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Abstract
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.
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Citations
26 Claims
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1. A method of amplifying nucleic acid sequences, the method comprising,
(a) mixing a rolling circle replication primer with one or more amplification target circles, to produce a primer-ATC mixture, and incubating the primer-ATC mixture under conditions that promote hybridization between the amplification target circles and the rolling circle replication primer in the primer-ATC mixture, wherein the amplification target circles each comprise a single-stranded, circular DNA molecule comprising a primer complement portion, and wherein the primer complement portion is complementary to the rolling circle replication primer, and wherein at least one of the amplification target circles is tethered to a specific binding molecule so that the amplification target circle can rotate freely, and (b) mixing DNA polymerase with the primer-ATC mixture, to produce a polymerase-ATC mixture, and incubating the polymerase-ATC mixture under conditions that promote replication of the amplification target circles, wherein replication of the amplification target circles results in the formation of tandem sequence DNA.
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2. The method of claim 1 further comprising, following step (b), (c) detecting the presence of tandem sequence DNA.
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3. The method of claim 1 further comprising, following step (b), (c) measuring the amount of tandem sequence DNA formed.
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4. The method of claim 1 further comprising, simultaneous with, or following, step (b),
(c) mixing a secondary DNA strand displacement primer with the polymerase-ATC mixture, and incubating the polymerase-ATC mixture under conditions that promote hybridization between the tandem sequence DNA and the secondary DNA strand displacement primer, and replication of the tandem sequence DNA in the polymerase-ATC mixture, wherein replication of the tandem sequence DNA results in the formation of secondary tandem sequence DNA.
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5. The method of claim 4 further comprising, simultaneous with step (b), mixing a tertiary DNA strand displacement primer with the polymerase-ATC mixture.
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6. A method of selectively amplifying nucleic acid sequences related to one or more target sequences, the method comprising,
(a) mixing one or more different open circle probes with a target sample, to produce an OCP-target sample mixture, and incubating the OCP-target sample mixture under conditions that promote hybridization between the open circle probes and the target sequences in the OCP-target sample mixture, (b) mixing ligase with the OCP-target sample mixture, to produce a ligation mixture, and incubating the ligation mixture under conditions that promote ligation of the open circle probes to form amplification target circles, (c) mixing a rolling circle replication primer with the ligation mixture, to produce a primer-ATC mixture, and incubating the primer-ATC mixture under conditions that promote hybridization between the amplification target circles and the rolling circle replication primer in the primer-ATC mixture, and (d) mixing DNA polymerase with the primer-ATC mixture, to produce a polymerase-ATC mixture, and incubating the polymerase-ATC mixture under conditions that promote replication of the amplification target circles, wherein replication of the amplification target circles results in the formation of tandem sequence DNA, and simultaneous with, or following, step (d), (e) mixing a secondary DNA strand displacement primer with the polymerase-ATC mixture, and incubating the polymerase-ATC mixture under conditions that promote hybridization between the tandem sequence DNA and the secondary DNA strand displacement primer, and replication of the tandem sequence DNA in the polymerase-ATC mixture, wherein replication of the tandem sequence DNA results in the formation of secondary tandem sequence DNA.
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7. The method of claim 6 further comprising, simultaneous with step (c), mixing a tertiary DNA strand displacement primer with the polymerase-ATC mixture, wherein the secondary tandem sequence DNA is replicated, wherein replication of the secondary tandem sequence DNA results in the formation of tertiary tandem sequence DNA.
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8. The method of claim 6 further comprising detecting the presence of secondary tandem sequence DNA, or measuring the amount of secondary tandem sequence DNA formed.
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9. The method of claim 8 wherein the secondary tandem sequence DNA is detected at the site where the target sequence is located, and wherein detection of the secondary tandem sequence identifies the location of the target sequence in the target sample.
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10. The method of claim 9 wherein detection is mediated by detection probes or by a detection label incorporated in the secondary tandem sequence DNA.
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11. The method of claim 10 wherein the detection label is a ligand.
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12. The method of claim 11 wherein the ligand is biotin.
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13. A method of selectively amplifying nucleic acid sequences related to a target sequence, the method comprising,
(a) mixing a first open circle probe with a target sample, to produce an OCP-target sample mixture, and incubating the OCP-target sample mixture under conditions that promote hybridization between the first open circle probe and a target sequence in the OCP-target sample mixture, wherein the target sequence comprises a 5′ - region and a 3′
region, andwherein the first open circle probe comprises a single-stranded, linear DNA molecule comprising, from 5′
end to 3′
end, a 5′
phosphate group, a right target probe portion, a spacer portion, a left target probe portion, and a 3′
hydroxyl group, wherein the left target probe portion is complementary to the 3′
region of the primary target sequence and the right target probe portion is complementary to the 5′
region of the primary target sequence,(b) mixing ligase with the OCP-target sample mixture, to produce a first ligation mixture, and incubating the first ligation mixture under conditions that promote ligation of the first open circle probe resulting in the formation of a first amplification target circle, (c) mixing a second open circle probe with the first ligation mixture, to produce an OCP-ATC mixture, and incubating the OCP-ATC mixture under conditions that promote hybridization between the second open circle probe and the first ATC, (d) mixing ligase with the OCP-ATC mixture, to produce a second ligation mixture, and incubating the second ligation mixture under conditions that promote ligation of the second open circle probe to form a second amplification target circle, (e) mixing a rolling circle replication primer with the second ligation mixture, to produce a primer-ATC mixture, and incubating the primer-ATC mixture under conditions that promote hybridization between the second amplification target circle and the rolling circle replication primer in the primer-ATC mixture, and (f) mixing DNA polymerase with the primer-ATC mixture, to produce a polymerase-ATC mixture, and incubating the polymerase-ATC mixture under conditions that promote replication of the second amplification target circle, wherein replication of the second amplification target circle results in the formation of tandem sequence DNA.
- region and a 3′
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14. A method of amplifying nucleic acid sequences, the method comprising,
(a) mixing a rolling circle replication primer with one or more amplification target circles, to produce a primer-ATC mixture, and incubating the primer-ATC mixture under conditions that promote hybridization between the amplification target circles and the rolling circle replication primer in the primer-ATC mixture, wherein the amplification target circles each comprise a single-stranded, circular DNA molecule comprising a primer complement portion, wherein the primer complement portion is complementary to the rolling circle replication primer, (b) mixing DNA polymerase with the primer-ATC mixture, to produce a polymerase-ATC mixture, and incubating the polymerase-ATC mixture under conditions that promote replication of the amplification target circles, wherein replication of the amplification target circles results in the formation of tandem sequence DNA, and simultaneous with, or following, step (b), (c) mixing a secondary DNA strand displacement primer with the polymerase-ATC mixture, and incubating the polymerase-ATC mixture under conditions that promote hybridization between the tandem sequence DNA and the secondary DNA strand displacement primer, and replication of the tandem sequence DNA in the polymerase-ATC mixture, wherein replication of the tandem sequence DNA results in the formation of secondary tandem sequence DNA.
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15. The method of claim 14 further comprising, simultaneous with step (c), mixing a tertiary DNA strand displacement primer with the polymerase-ATC mixture, wherein the secondary tandem sequence DNA is replicated, wherein replication of the secondary tandem sequence DNA results in the formation of tertiary tandem sequence DNA.
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16. The method of claim 14 further comprising detecting the presence of secondary tandem sequence DNA, or measuring the amount of secondary tandem sequence DNA formed.
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17. The method of claim 16 wherein the secondary tandem sequence DNA is detected at the sites where the amplification target circles are located, and wherein detection of the secondary tandem sequence identifies the locations of the amplification target circles in the target sample.
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18. The method of claim 17 wherein detection is mediated by detection probes or by a detection label incorporated in the secondary tandem sequence DNA.
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19. The method of claim 18 wherein the detection label is a ligand.
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20. The method of claim 19 wherein the ligand is biotin.
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21. A method of amplifying nucleic acid sequences, the method comprising,
(a) mixing a rolling circle replication primer with one or more amplification target circles, to produce a primer-ATC mixture, and incubating the primer-ATC mixture under conditions that promote hybridization between the amplification target circles and the rolling circle replication primer in the primer-ATC mixture, wherein the amplification target circles each comprise a single-stranded, circular DNA molecule comprising a primer complement portion, wherein the primer complement portion is complementary to the rolling circle replication primer, (b) mixing DNA polymerase with the primer-ATC mixture, to produce a polymerase-ATC mixture, and incubating the polymerase-ATC mixture under conditions that promote replication of the amplification target circles, wherein replication of the amplification target circles results in the formation of tandem sequence DNA; wherein at least one of the amplification target circles is tethered to a specific binding molecule via a tether loop so that the amplification target circle can rotate freely.
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22. The method of claim 21 further comprising detecting the presence of tandem sequence DNA, or measuring the amount of tandem sequence DNA formed.
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23. A method of amplifying nucleic acid sequences, the method comprising,
(a) mixing a rolling circle replication primer with one or more amplification target circles, to produce a primer-ATC mixture, and incubating the primer-ATC mixture under conditions that promote hybridization between the amplification target circles and the rolling circle replication primer in the primer-ATC mixture, wherein the amplification target circles each comprise a single-stranded, circular DNA molecule comprising a primer complement portion, wherein the primer complement portion is complementary to the rolling circle replication primer, (b) mixing DNA polymerase with the primer-ATC mixture, to produce a polymerase-ATC mixture, and incubating the polymerase-ATC mixture under conditions that promote replication of the amplification target circles, wherein replication of the amplification target circles results in the formation of tandem sequence DNA, wherein the amplification target circles are topologically locked, wherein the tandem sequence DNA is detected at the sites where the amplification target circles are located, and wherein detection of the tandem sequence identifies the locations of the amplification target circles.
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24. The method of claim 23 wherein detection is mediated by detection probes or by a detection label incorporated in the tandem sequence DNA.
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25. The method of claim 24 wherein the detection label is a ligand.
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26. The method of claim 25 wherein the ligand is biotin.
Specification