Open circle probes with intramolecular stem structures
First Claim
1. A method of amplifying nucleic acid sequences, the method comprising a DNA ligation operation and an amplification operation, wherein the DNA ligation operation comprises circularization of one or more open circle probes, wherein each open circle probe comprises two ends, wherein at least one of the ends of at least one of the open circle probes can form an intramolecular stem structure, wherein circularization of the open circle probes that can form an intramolecular stem structure is dependent on hybridization of the open circle probe to a target sequence, wherein the amplification operation comprises rolling circle replication of the circularized open circle probes.
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Accused Products
Abstract
Disclosed are compositions and methods for reducing or eliminating generation of unwanted, undesirable, or non-specific amplification products in nucleic acid amplification reactions, such as rolling circle amplification. One form of composition is an open circle probe that can form an intramolecular stem structure, such as a hairpin structure, at one or both ends. The stem structure allows the open circle probe to be circularized when hybridized to a legitimate target sequence but results in inactivation of uncircularized open circle probes. This inactivation, which preferably involves stabilization of the stem structure, extension of the end of the open circle probe, or both, reduces or eliminates the ability of the open circle probe to prime nucleic acid synthesis or to serve as a template for rolling circle amplification. The disclosed method is useful for detection, quantitation, and/or location of any desired analyte, such as proteins and peptides.
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Citations
163 Claims
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1. A method of amplifying nucleic acid sequences, the method comprising
a DNA ligation operation and an amplification operation, wherein the DNA ligation operation comprises circularization of one or more open circle probes, wherein each open circle probe comprises two ends, wherein at least one of the ends of at least one of the open circle probes can form an intramolecular stem structure, wherein circularization of the open circle probes that can form an intramolecular stem structure is dependent on hybridization of the open circle probe to a target sequence, wherein the amplification operation comprises rolling circle replication of the circularized open circle probes.
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69. 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; -
wherein at least one of the open circle probes comprises two ends, wherein at least one of the ends of the open circle probe can form an intramolecular stem structure. - View Dependent Claims (70, 71, 72, 73, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105)
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74. The method of 73
wherein the intramolecular stem structure prevents the open circle probe that can form an intramolecular stem structure from priming nucleic acid replication.
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75. The method of 73
wherein the intramolecular stem structure prevents the open circle probe that can form an intramolecular stem structure from serving as a template for rolling circle replication.
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76. The method of 69
wherein the intramolecular stem structure prevents the open circle probe that can form an intramolecular stem structure from priming nucleic acid replication.
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77. The method of 69
wherein the intramolecular stem structure prevents the open circle probe that can form an intramolecular stem structure from serving as a template for rolling circle replication.
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106. A method of amplifying nucleic acid sequences, the method comprising
an amplification operation, wherein the amplification operation comprises rolling circle replication of one or more amplification target circles, wherein rolling circle replication is primed by one or more rolling circle replication primers, wherein each rolling circle replication primer comprises two ends, wherein at least one of the ends of at least one of the rolling circle replication primers can form an intramolecular stem structure, wherein priming by the rolling circle replication primers that can form an intramolecular stem structure is dependent on hybridization of the rolling circle replication primers to the amplification target circles.
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115. A method of amplifying nucleic acid sequences, the method comprising
an amplification operation, wherein the amplification operation comprises rolling circle replication of one or more amplification target circles, wherein the amplification operation produces tandem sequence DNA, wherein the amplification operation further comprises secondary DNA strand displacement.
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125. An open circle probe
wherein the open circle probe comprises two ends, wherein at least one of the ends of the open circle probe can form an intramolecular stem structure.
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133. The open circle probe of 132
wherein the intramolecular stem structure prevents the open circle probe from priming nucleic acid replication.
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134. The open circle probe of 132
wherein the intramolecular stem structure prevents the open circle probe from serving as a template for rolling circle replication.
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135. The open circle probe of 125
wherein the intramolecular stem structure prevents the open circle probe from priming nucleic acid replication.
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136. The open circle probe of 125
wherein the intramolecular stem structure prevents the open circle probe from serving as a template for rolling circle replication.
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145. A kit for selectively detecting one or more target sequences or selectively amplifying nucleic acid sequences related to one or more target sequences, the kit comprising,
one or more open circle probes each comprising two ends, wherein at least one of the ends of one of the open circle probe can form an intramolecular stem structure, wherein portions of each open circle probe are complementary to the one or more target sequences, and one or more rolling circle replication primers, wherein all or a portion of each rolling circle replication primer is complementary to a portion of one or more of the open circle probes.
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161. A kit for selectively detecting one or more target sequences or selectively amplifying nucleic acid sequences related to one or more target sequences, the kit comprising,
a plurality of open circle probes each comprising two ends, wherein at least one of the ends of one of the open circle probe can form an intramolecular stem structure, wherein portions of each open circle probe are complementary to the one or more target sequences, wherein the portions of the open circle probes that are complementary to the target sequence are complementary to a different target sequence for each of a plurality of the open circle probes, one or more rolling circle replication primers, wherein all or a portion of each rolling circle replication primer is complementary to a portion of one or more of the open circle probes, and a plurality of detection probes, wherein a portion of each of a plurality of the detection probes has sequence matching or complementary to a portion of a different one of the open circle probes.
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162. A kit for selectively detecting one or more target sequences or selectively amplifying nucleic acid sequences related to one or more target sequences, the kit comprising,
a plurality of open circle probes each comprising two ends, wherein at least one of the ends of one of the open circle probe can form an intramolecular stem structure, wherein portions of each open circle probe are complementary to the one or more target sequences, wherein the portions of the open circle probes that are complementary to the target sequences are complementary to a different target sequence for each of a plurality of the open circle probes, one or more rolling circle replication primers, wherein all or a portion of each rolling circle replication primer is complementary to a portion of one or more of the open circle probes, and a plurality of reporter binding agents each comprising a specific binding molecule and an oligonucleotide portion, wherein the oligonucleotide portion comprises one of the target sequences.
Specification