Open circle probes with intramolecular stem structures
First Claim
1. A method of amplifying nucleic acid sequences, the method comprisinga 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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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.
51 Citations
105 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)
wherein if the open circle probe that can form an intramolecular stem structure is not ligated to form an amplification target circle the end of the open circle probe is extended during replication of the amplification target circles using the open circle probe as a template. -
71. The method of claim 70
wherein extension of the end of the open circle probe prevents the extended open circle probes from serving as a template for rolling circle replication. -
72. The method of claim 70
wherein extension of the end of the open circle probe stabilizes the intramolecular stem structure, wherein the stabilized intramolecular stem structure prevents the extended open circle probes from priming nucleic acid replication. -
73. The method of claim 69
wherein the intramolecular stem structure can form under the conditions used for replication of the amplification target circles. -
78. The method of claim 69
wherein the intramolecular stem structure forms a hairpin structure. -
79. The method of claim 69
wherein the intramolecular stem structure forms a stem and loop structure. -
80. The method of claim 79
wherein the two ends of the open circle probe that can form an intramolecular stem structure together form the intramolecular stem structure. -
81. The method of claim 69
wherein one of the ends of the open circle probe that can form an intramolecular stem structure is a 3′ - end,
wherein the 3′
end of the open circle probe can form an intramolecular stem structure.
- end,
-
82. The method of claim 81
wherein the other end of the open circle probe is a 5′ - end,
wherein the 5′
end of the open circle probe can form an intramolecular stem structure.
- end,
-
83. The method of claim 80
wherein one of the ends of the open circle probe is a 5′ - end,
wherein the 5′
end of the open circle probe can form an intramolecular stem structure.
- end,
-
84. The method of claim 69
wherein the target sequence to which the open circle probe that can form an intramolecular stem structure can hybridize comprises a 5′ - region and a 3′
region,wherein the open circle probe comprises a single-stranded, linear DNA molecule, wherein the single-stranded, linear DNA molecule comprises, 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 target sequence,wherein the right target probe portion is complementary to the 5′
region of the target sequence.
- region and a 3′
-
85. The method of claim 84
wherein the intramolecular stem structure forms a stem and loop structure. -
86. The method of claim 85
wherein a portion of one of the target probe portions is in the loop of the stem and loop structure, wherein the portion of the target probe portion in the loop can hybridize to the target sequence, wherein hybridization of the target probe portion in the loop to the target sequence disrupts the intramolecular stem structure. -
87. The method of claim 86
wherein disruption of the intramolecular stem structure allows the end of the open circle probe that can form an intramolecular stem structure to hybridize to the target sequence. -
88. The method of claim 87
wherein a hybrid between the target sequence and the target probe portion at the end of the open circle probe that can form an intramolecular stem structure is more stable than the intramolecular stem structure. -
89. The method of claim 84
wherein a hybrid between the target sequence and the target probe portion at the end of the open circle probe that can form an intramolecular stem structure is more stable than the intramolecular stem structure. -
90. The method of claim 84
wherein the spacer portion comprises a primer complement portion. -
91. The method of claim 84
wherein the target sequence further comprises a central region located between the 5′ - region and the 3′
region,wherein neither the left target probe portion of the open circle probe nor the right target probe portion of the open circle probe is complementary to the central region of the target sequence.
- region and the 3′
-
92. The method of claim 91
wherein step (a) further comprises mixing one or more gap oligonucleotides with the target sample, wherein each gap oligonucleotide comprises a single-stranded, linear DNA molecule comprising a 5′ - phosphate group and a 3′
hydroxyl group, wherein each gap oligonucleotide is complementary all or a portion of the central region of the target sequence.
- phosphate group and a 3′
-
93. The method of claim 91
wherein a complement to the central region of the target sequence is synthesized during step (b). -
94. The method of claim 69
wherein at least one of the target sequences is coupled to a specific binding molecule. -
95. The method of claim 94
wherein the method further comprises bringing into contact the specific binding molecule and a target molecule, wherein the specific binding molecule binds to a target molecule. -
96. The method of claim 95
wherein the specific binding molecule and target molecule are brought into contact prior to step (a). -
97. The method of claim 95
wherein the specific binding molecule and target molecule are brought into contact following step (b). -
98. The method of claim 95
wherein the specific binding molecule and target molecule are brought into contact following step (d). -
99. The method of claim 95
wherein the target molecule is a peptide, protein, carbohydrate, lipid, nucleic acid, or metabolite. -
100. The method of claim 95
wherein the target molecule is present in, or derived from, tissue, bodily fluid, or cells. -
101. The method of claim 95
wherein the target molecule is present in, or derived from, tissue. -
102. The method of claim 94
wherein the specific binding molecule is an antibody. -
103. The method of claim 94
wherein the rolling circle replication primer is coupled to a specific binding molecule, wherein the specific binding molecule is bound to a target molecule. -
104. The method of claim 69
wherein the method further comprises detecting the tandem sequence DNA. -
105. The method of claim 69
wherein the tandem sequence DNA is replicated to form secondary tandem sequence DNA, wherein the method further comprises detecting the tandem sequence DNA, the secondary tandem sequence DNA, or both.
-
-
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.
-
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.
-
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.
-
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.
Specification