Method and compositions for efficient and specific rolling circle amplification

US 20030165948A1
Filed: 12/19/2002
Published: 09/04/2003
Est. Priority Date: 03/09/2001
Status: Abandoned Application

First Claim

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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 the ligation operation is carried out in the presence of a set of open circle probes, wherein the set of open circle probes comprises a plurality of different 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, wherein the amplification operation is carried out in the presence of a plurality of detection rolling circle replication primers, a secondary DNA strand displacement primer, and a common rolling circle replication primer, wherein each detection rolling circle replication primer is associated with a peptide nucleic acid quencher or a peptide nucleic acid fluor, wherein each detection rolling circle replication primer corresponds to a different open circle probe in the set of open circle probes, wherein the secondary DNA strand displacement primer corresponds to all of the open circle probes in the set of open circle probes, wherein the common rolling circle replication primer corresponds to all of the open circle probes in the set of open circle probes, wherein two or more of the open circle probes in the set of open circle probes constitute a matched open circle probe set.

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Abstract

Disclosed are compositions and methods for nucleic acid amplification reactions that reduce, prevent, or eliminate artifacts; increase efficiency; increase specificity; and/or increase consistency. The disclosed method can combine, for example, the use of open circle probes that can form intramolecular stem structures; the use of matched open circle probe sets in the same amplification reaction; the use of detection primers and detection during the amplification reaction; the use of a plurality of detection rolling circle replication primer, a secondary DNA strand displacement primer and a common rolling circle replication primer in the same amplification reaction; and/or the use of peptide nucleic acid quenchers associated with detection rolling circle replication primers. Such combinations can produce, in the same amplification reaction, the benefits of each of the combined components.

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237 Claims

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 the ligation operation is carried out in the presence of a set of open circle probes, wherein the set of open circle probes comprises a plurality of different 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, wherein the amplification operation is carried out in the presence of a plurality of detection rolling circle replication primers, a secondary DNA strand displacement primer, and a common rolling circle replication primer, wherein each detection rolling circle replication primer is associated with a peptide nucleic acid quencher or a peptide nucleic acid fluor, wherein each detection rolling circle replication primer corresponds to a different open circle probe in the set of open circle probes, wherein the secondary DNA strand displacement primer corresponds to all of the open circle probes in the set of open circle probes, wherein the common rolling circle replication primer corresponds to all of the open circle probes in the set of open circle probes, wherein two or more of the open circle probes in the set of open circle probes constitute a matched open circle probe set.
- View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88)
- - 2. The method of claim 1 wherein each detection rolling circle replication primer comprises a complementary portion, wherein each open circle probe comprises a detection primer complement portion, wherein the complementary portion of the detection rolling circle replication primer is complementary to the detection primer complement portion of the open circle probe to which the detection rolling circle replication primer corresponds, wherein the complementary portion of the detection rolling circle replication primer is not substantially complementary to an open circle probe to which the detection rolling circle replication primer does not correspond.
  - 3. The method of claim 2 wherein the detection rolling circle replication primer is a peptide nucleic acid quenched primer, wherein the detection rolling circle replication primer further comprises a fluorescent moiety and a quencher complement portion, wherein each detection rolling circle replication primer is associated with a peptide nucleic acid quencher, wherein the peptide nucleic acid quencher is associated with the detection rolling circle replication primer via the quencher complement portion, wherein the peptide nucleic acid quencher comprises a quenching moiety, wherein association of the peptide nucleic acid quencher with the detection rolling circle replication primer quenches fluorescence from the fluorescent moiety, wherein the amplification operation results in disassociation of the peptide nucleic acid quenchers from the detection rolling circle replication primers, thereby allowing the fluorescent moiety of the detection rolling circle replication primers to fluoresce.
  - 4. The method of claim 3 wherein the quencher complement portion of each detection rolling circle replication primer is the same, wherein the peptide nucleic acid quencher associated with each detection rolling circle replication primer is the same.
  - 5. The method of claim 3 wherein the quencher complement portion of at least one of the detection rolling circle replication primers is different from the quencher complement portion of at least one of the other detection rolling circle replication primers.
  - 6. The method of claim 3 wherein the quencher complement portion of each detection rolling circle replication primer corresponding to an open circle probe in the set of open circle probes is the same, wherein the peptide nucleic acid quencher associated with each detection rolling circle replication primer is the same.
  - 7. The method of claim 3 wherein the quencher complement portion of at least one of the detection rolling circle replication primers corresponding to an open circle probe in the set of open circle probes is different from the quencher complement portion of at least one of the other detection rolling circle replication primers corresponding to an open circle probe in the set of open circle probes.
  - 8. The method of claim 3 wherein each detection rolling circle replication primer comprises a different fluorescent moiety.
  - 9. The method of claim 3 wherein the amplification operation results in disassociation of the peptide nucleic acid quenchers from the detection rolling circle replication primers, thereby allowing the fluorescent moiety of the detection rolling circle replication primers to fluoresce.
  - 10. The method of claim 2 wherein the detection rolling circle replication primer is a peptide nucleic acid quenched primer, wherein the detection rolling circle replication primer further comprises a quenching moiety and a quencher complement portion, wherein each detection rolling circle replication primer is associated with a peptide nucleic acid fluor, wherein the peptide nucleic acid fluor is associated with the detection rolling circle replication primer via the quencher complement portion, wherein the peptide nucleic acid fluor comprises a fluorescent moiety, wherein association of the peptide nucleic acid fluor with the detection rolling circle replication primer quenches fluorescence from the fluorescent moiety, wherein the amplification operation results in disassociation of the peptide nucleic acid fluors from the detection rolling circle replication primers, thereby allowing the fluorescent moiety of the peptide nucleic acid fluors to fluoresce.
  - 11. The method of claim 10 wherein the quencher complement portion of each detection rolling circle replication primer is the same, wherein the peptide nucleic acid fluor associated with each detection rolling circle replication primer is the same.
  - 12. The method of claim 10 wherein the quencher complement portion of at least one of the detection rolling circle replication primers is different from the quencher complement portion of at least one of the other detection rolling circle replication primers.
  - 13. The method of claim 10 wherein the quencher complement portion of each detection rolling circle replication primer corresponding to an open circle probe in the set of open circle probes is the same, wherein the peptide nucleic acid fluor associated with each detection rolling circle replication primer is the same.
  - 14. The method of claim 10 wherein the quencher complement portion of at least one of the detection rolling circle replication primers corresponding to an open circle probe in the set of open circle probes is different from the quencher complement portion of at least one of the other detection rolling circle replication primers corresponding to an open circle probe in the set of open circle probes.
  - 15. The method of claim 14 wherein each peptide nucleic acid fluor comprises a different fluorescent moiety.
  - 16. The method of claim 10 wherein the amplification operation results in disassociation of the peptide nucleic acid flours from the detection rolling circle replication primers, thereby allowing the fluorescent moiety of the peptide nucleic acid fluors to fluoresce.
  - 17. The method of claim 2 wherein the detection rolling circle replication primer is a hairpin quenched primer.
  - 18. The method of claim 1 wherein the ligation operation is carried out in the presence of one or more additional sets of open circle probes, wherein each set of open circle probes comprises a plurality of different open circle probes.
  - 19. The method of claim 18 wherein each detection rolling circle replication primer corresponds to a different open circle probe in all of the sets of open circle probes, wherein the secondary DNA strand displacement primer corresponds to all of the open circle probes in all of the sets of open circle probes, wherein the common rolling circle replication primer corresponds to all of the open circle probes in all of the sets of open circle probes.
  - 20. The method of claim 19 each detection rolling circle replication primer comprises a complementary portion, a fluorescent moiety, and a quencher complement portion.
  - 21. The method of claim 20 wherein each detection rolling circle replication primer corresponding to an open circle probe in the same set of open circle probes comprises a different fluorescent moiety.
  - 22. The method of claim 21 wherein at least one of the detection rolling circle replication primers corresponding to an open circle probe in one of the sets of open circle probes comprises the same fluorescent moiety as at least one of the detection rolling circle replication primers in a different one of the sets of open circle probes.
  - 23. The method of claim 20 wherein at least one of the detection rolling circle replication primers corresponding to an open circle probe in one of the sets of open circle probes comprises the same fluorescent moiety as a different detection rolling circle replication primer in the same set of open circle probes.
  - 24. The method of claim 18 wherein each detection rolling circle replication primer corresponds to a different open circle probe in all of the sets of open circle probes, wherein the common rolling circle replication primer corresponds to all of the open circle probes in all of the sets of open circle probes, wherein the amplification operation is carried out in the presence of a plurality of secondary DNA strand displacement primers, wherein each secondary DNA strand displacement primer corresponds to open circle probes in a different set of open circle probes, wherein a single secondary DNA strand displacement primer corresponds to all of the open circle probes in a given set of open circle probes.
  - 25. The method of claim 18 wherein each detection rolling circle replication primer corresponds to a different open circle probe in all of the sets of open circle probes, wherein the secondary DNA strand displacement primer corresponds to all of the open circle probes in all of the sets of open circle probes, wherein the amplification operation is carried out in the presence of a plurality of common rolling circle replication primers, wherein each common rolling circle replication primer corresponds to open circle probes in a different set of open circle probes, wherein a single common rolling circle replication primer corresponds to all of the open circle probes in a given set of open circle probes.
  - 26. The method of claim 18 wherein each detection rolling circle replication primer corresponds to a different open circle probe in all of the sets of open circle probes, wherein the amplification operation is carried out in the presence of a plurality of secondary DNA strand displacement primers, wherein each secondary DNA strand displacement primer corresponds to open circle probes in a different set of open circle probes, wherein a single secondary DNA strand displacement primer corresponds to all of the open circle probes in a given set of open circle probes, wherein the amplification operation is carried out in the presence of a plurality of common rolling circle replication primers, wherein each common rolling circle replication primer corresponds to open circle probes in a different set of open circle probes, wherein a single common rolling circle replication primer corresponds to all of the open circle probes in a given set of open circle probes.
  - 27. The method of claim 18 wherein all of the open circle probes in all of the sets of open circle probes are different.
  - 28. The method of claim 18 wherein each detection rolling circle replication primer corresponds to a different open circle probe in a given set of open circle probes.
  - 29. The method of claim 28 wherein at least one of the detection rolling circle replication primers corresponds to an open circle probe in each of at least two of the sets of open circle probes.
  - 30. The method of claim 18 wherein at least one of the detection rolling circle replication primers corresponds to an open circle probe in each of at least two of the sets of open circle probes.
  - 31. The method of claim 1 wherein the peptide nucleic acid quencher comprises peptide nucleic acid and a quenching moiety.
  - 32. The method of claim 31 wherein each detection rolling circle replication primer comprises a complementary portion, a fluorescent moiety, and a quencher complement portion, wherein the peptide nucleic acid quencher is associated with the detection rolling circle replication primers via the quencher complement portion.
  - 33. The method of claim 31 wherein each detection rolling circle replication primer comprises a complementary portion, a fluorescent moiety, and a quencher complement portion, wherein the amplification operation results in disassociation of the peptide nucleic acid quencher from the detection rolling circle replication primers, thereby allowing the fluorescent moiety of the detection rolling circle replication primers to fluoresce.
  - 34. The method of claim 1 wherein the peptide nucleic acid fluor comprises peptide nucleic acid and a fluorescent moiety.
  - 35. The method of claim 34 wherein each detection rolling circle replication primer comprises a complementary portion, a quenching moiety, and a quencher complement portion, wherein the peptide nucleic acid fluor is associated with the detection rolling circle replication primers via the quencher complement portion.
  - 36. The method of claim 1 wherein the open circle probes in the matched open circle probe set are targeted to different forms of the same target sequence.
  - 37. The method of claim 36 wherein the different forms of the same target sequence comprise a wild type form of the target sequence and a mutant form of the target sequence.
  - 38. The method of claim 37 wherein the different forms of the same target sequence further comprise a second mutant form of the target sequence.
  - 39. The method of claim 37 wherein the different forms of the same target sequence further comprise a plurality of different mutant forms of the target sequence.
  - 40. The method of claim 36 wherein the different forms of the same target sequence comprise a plurality of different mutant forms of the target sequence.
  - 41. The method of claim 36 wherein the different forms of the same target sequence comprise a normal form of the target sequence and a mutant form of the target sequence.
  - 42. The method of claim 41 wherein the different forms of the same target sequence further comprise a second mutant form of the target sequence.
  - 43. The method of claim 41 wherein the different forms of the same target sequence further comprise a plurality of different mutant forms of the target sequence.
  - 44. The method of claim 36 wherein the set of open circle probes comprises a plurality of matched open circle probe sets.
  - 45. The method of claim 44 wherein the open circle probes in each of the matched open circle probe sets are targeted to different forms of the same target sequence, wherein open circle probes in different matched open circle probe sets are targeted to different target sequences.
  - 46. The method of claim 45 wherein the different forms of the same target sequence comprise a wild type form of the target sequence and a mutant form of the target sequence.
  - 47. The method of claim 46 wherein the different forms of the same target sequence further comprise a second mutant form of the target sequence.
  - 48. The method of claim 46 wherein the different forms of the same target sequence further comprise a plurality of different mutant forms of the target sequence.
  - 49. The method of claim 45 wherein the different forms of the same target sequence comprise a plurality of different mutant forms of the target sequence.
  - 50. The method of claim 45 wherein the different forms of the same target sequence comprise a normal form of the target sequence and a mutant form of the target sequence.
  - 51. The method of claim 50 wherein the different forms of the same target sequence further comprise a second mutant form of the target sequence.
  - 52. The method of claim 50 wherein the different forms of the same target sequence further comprise a plurality of different mutant forms of the target sequence.
  - 53. The method of claim 45 wherein the different target sequences are in the same gene.
  - 54. The method of claim 45 wherein the different target sequences are associated with the same disease or condition.
  - 55. The method of claim 36 wherein the matched open circle probe set consists of two open circle probes, wherein one of the open circle probes in the matched open circle probe set is targeted to a wild type form of the target sequence, wherein the other open circle probe in the matched open circle probe set is targeted to a mutant form of the target sequence.
  - 56. The method of claim 36 wherein each detection rolling circle replication primer comprises a different fluorescent moiety.
  - 57. The method of claim 36 wherein each detection rolling circle replication primer corresponding to an open circle probes in the matched open circle probe set comprises a different fluorescent moiety.
  - 58. The method of claim 1 further comprising, following the ligation operation, heating the circularized open circle probes.
  - 59. The method of claim 58 wherein the circularized open circle probes are heated to about 95°
    - C. for about 10 minutes.
  - 60. The method of claim 1 wherein the open circle probes are each specific for a target sequence, wherein each target sequence comprises a 5′
    - region and a 3′
      
      region, wherein each 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.
  - 61. The method of claim 60 wherein at least one of the target sequences 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 specific for the target sequence nor the right target probe portion of the open circle probe specific for the target sequence is complementary to the central region of the target sequence.
  - 62. The method of claim 61 wherein the ligation operation comprises mixing the open circle probes and one or more gap oligonucleotides with one or more target samples, and incubating under conditions that promote hybridization between the open circle probes and the gap oligonucleotides and the target sequences, and ligation of the open circle probes and gap oligonucleotides to form the circularized open circle probes, 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.
  - 63. The method of claim 61 wherein a complement to the central region of the target sequence is synthesized during the ligation operation.
  - 64. The method of claim 60 wherein a plurality of the open circle probes are each specific for a different target sequence.
  - 65. The method of claim 64 wherein a plurality of different target sequences are detected.
  - 66. The method of claim 64 wherein the amplification operation produces amplified nucleic acid, wherein the method further comprises detecting the amplified nucleic acid with one or more detection probes.
  - 67. The method of claim 66 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, wherein a plurality of different amplified nucleic acids are detected using the plurality of detection probes.
  - 68. The method of claim 60 wherein the spacer portion comprises a detection primer complement portion.
  - 69. The method of claim 60 wherein the spacer portion comprises a common primer complement portion.
  - 70. The method of claim 60 wherein the intramolecular stem structure of at least one of the open circle probes forms a stem and loop structure.
  - 71. The method of claim 60 wherein a portion of one of the target probe portions of at least one of the open circle probes 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.
  - 72. The method of claim 60 wherein a hybrid between the target sequence and the target probe portion at the end of the open circle probes that can form an intramolecular stem structure is more stable than the intramolecular stem structure.
  - 73. The method of claim 1 wherein if one or more of the open circle probes that can form an intramolecular stem structure are not circularized, the end of at least one of the uncircularized open circle probes that forms the intramolecular stem structure is extended during the amplification operation using the open circle probe as a template.
  - 74. The method of claim 1 wherein the intramolecular stem structure can form under the conditions used for the amplification operation.
  - 75. The method of claim 1 wherein the intramolecular stem structure prevents the open circle probes from priming nucleic acid replication.
  - 76. The method of claim 1 wherein the intramolecular stem structure prevents the open circle probes from serving as a template for rolling circle replication.
  - 77. The method of claim 1 wherein the intramolecular stem structure forms a hairpin structure.
  - 78. The method of claim 1 wherein the intramolecular stem structure forms a stem and loop structure.
  - 79. The method of claim 1 wherein one of the ends of the open circle probes is a 3′
    - end, wherein the 3′
      
      end of at least one of the open circle probes can form an intramolecular stem structure.
  - 80. The method of claim 1 wherein rolling circle replication is primed by one or more detection rolling circle replication primers, wherein each detection rolling circle replication primer comprises two ends, wherein at least one of the ends of at least one of the detection rolling circle replication primers can form an intramolecular stem structure, wherein priming by the detection rolling circle replication primers that can form an intramolecular stem structure is dependent on hybridization of the detection rolling circle replication primers to the circularized open circle probes.
  - 81. The method of claim 1 wherein the amplification operation produces tandem sequence DNA, wherein the amplification operation further comprises secondary DNA strand displacement.
  - 82. The method of claim 1 wherein rolling circle replication is primed by one or more common rolling circle replication primers, wherein each common rolling circle replication primer comprises two ends, wherein at least one of the ends of at least one of the common rolling circle replication primers can form an intramolecular stem structure, wherein priming by the common rolling circle replication primers that can form an intramolecular stem structure is dependent on hybridization of the common rolling circle replication primers to the circularized open circle probes.
  - 83. The method of claim 1 wherein the amplification operation produces tandem sequence DNA, wherein the method further comprises detecting the tandem sequence DNA.
  - 84. The method of claim 83 wherein the tandem sequence DNA is detected via one or more fluorescent change probes.
  - 85. The method of claim 84 wherein the fluorescent change probes are hairpin quenched probes, cleavage quenched probes, cleavage activated probes, fluorescent activated probes, or a combination.
  - 86. The method of claim 83 wherein the tandem sequence DNA is detected via one or more fluorescent change primers.
  - 87. The method of claim 86 wherein the fluorescent change primers are stem quenched primers, hairpin quenched primers, or a combination.
  - 88. The method of claim 1 wherein the amplification operation produces tandem sequence DNA and secondary tandem sequence DNA, wherein the method further comprises detecting the tandem sequence DNA, the secondary tandem sequence DNA, or both.

89. 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 the ligation operation is carried out in the presence of a set of open circle probes, wherein the set of open circle probes comprises a plurality of different open circle probes, wherein the amplification operation comprises rolling circle replication of the circularized open circle probes, wherein the amplification operation is carried out in the presence of a plurality of detection rolling circle replication primers, a secondary DNA strand displacement primer, and a common rolling circle replication primer, wherein each detection rolling circle replication primer corresponds to a different open circle probe in the set of open circle probes, wherein the secondary DNA strand displacement primer corresponds to all of the open circle probes in the set of open circle probes, wherein the common rolling circle replication primer corresponds to all of the open circle probes in the set of open circle probes.
- View Dependent Claims (90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118)
- - 90. The method of claim 89 wherein each detection rolling circle replication primer comprises a complementary portion, wherein each open circle probe comprises a detection primer complement portion, wherein the complementary portion of the detection rolling circle replication primer is complementary to the detection primer complement portion of the open circle probe to which the detection rolling circle replication primer corresponds, wherein the complementary portion of the detection rolling circle replication primer is not substantially complementary to an open circle probe to which the detection rolling circle replication primer does not correspond.
  - 91. The method of claim 90 wherein the detection rolling circle replication primer is a peptide nucleic acid quenched primer, wherein the detection rolling circle replication primer further comprises a fluorescent moiety and a quencher complement portion, wherein each detection rolling circle replication primer is associated with a peptide nucleic acid quencher, wherein the peptide nucleic acid quencher is associated with the detection rolling circle replication primer via the quencher complement portion, wherein the peptide nucleic acid quencher comprises a quenching moiety, wherein association of the peptide nucleic acid quencher with the detection rolling circle replication primer quenches fluorescence from the fluorescent moiety, wherein the amplification operation results in disassociation of the peptide nucleic acid quenchers from the detection rolling circle replication primers, thereby allowing the fluorescent moiety of the detection rolling circle replication primers to fluoresce.
  - 92. The method of claim 91 wherein the quencher complement portion of each detection rolling circle replication primer is the same, wherein the peptide nucleic acid quencher associated with each detection rolling circle replication primer is the same.
  - 93. The method of claim 91 wherein the quencher complement portion of at least one of the detection rolling circle replication primers is different from the quencher complement portion of at least one of the other detection rolling circle replication primers.
  - 94. The method of claim 91 wherein the quencher complement portion of each detection rolling circle replication primer corresponding to an open circle probe in the set of open circle probes is the same, wherein the peptide nucleic acid quencher associated with each detection rolling circle replication primer is the same.
  - 95. The method of claim 91 wherein the quencher complement portion of at least one of the detection rolling circle replication primers corresponding to an open circle probe in the set of open circle probes is different from the quencher complement portion of at least one of the other detection rolling circle replication primers corresponding to an open circle probe in the set of open circle probes.
  - 96. The method of claim 91 wherein each detection rolling circle replication primer comprises a different fluorescent moiety.
  - 97. The method of claim 90 wherein the detection rolling circle replication primer is a peptide nucleic acid quenched primer, wherein the detection rolling circle replication primer further comprises a quenching moiety and a quencher complement portion, wherein each detection rolling circle replication primer is associated with a peptide nucleic acid fluor, wherein the peptide nucleic acid fluor is associated with the detection rolling circle replication primer via the quencher complement portion, wherein the peptide nucleic acid fluor comprises a fluorescent moiety, wherein association of the peptide nucleic acid fluor with the detection rolling circle replication primer quenches fluorescence from the fluorescent moiety, wherein the amplification operation results in disassociation of the peptide nucleic acid fluors from the detection rolling circle replication primers, thereby allowing the fluorescent moiety of the peptide nucleic acid fluors to fluoresce.
  - 98. The method of claim 97 wherein the quencher complement portion of each detection rolling circle replication primer is the same, wherein the peptide nucleic acid fluor associated with each detection rolling circle replication primer is the same.
  - 99. The method of claim 97 wherein the quencher complement portion of at least one of the detection rolling circle replication primers is different from the quencher complement portion of at least one of the other detection rolling circle replication primers.
  - 100. The method of claim 97 wherein the quencher complement portion of each detection rolling circle replication primer corresponding to an open circle probe in the set of open circle probes is the same, wherein the peptide nucleic acid fluor associated with each detection rolling circle replication primer is the same.
  - 101. The method of claim 97 wherein the quencher complement portion of at least one of the detection rolling circle replication primers corresponding to an open circle probe in the set of open circle probes is different from the quencher complement portion of at least one of the other detection rolling circle replication primers corresponding to an open circle probe in the set of open circle probes.
  - 102. The method of claim 101 wherein each peptide nucleic acid fluor comprises a different fluorescent moiety.
  - 103. The method of claim 90 wherein the detection rolling circle replication primer is a hairpin quenched primer.
  - 104. The method of claim 89 wherein the ligation operation is carried out in the presence of one or more additional sets of open circle probes, wherein each set of open circle probes comprises a plurality of different open circle probes.
  - 105. The method of claim 89 wherein each detection rolling circle replication primer is associated with a peptide nucleic acid quencher, wherein the peptide nucleic acid quencher comprises peptide nucleic acid and a quenching moiety, wherein the detection rolling circle replication primer comprises a fluorescent moiety.
  - 106. The method of claim 89 wherein each detection rolling circle replication primer is associated with a peptide nucleic acid fluor, wherein the peptide nucleic acid fluor comprises peptide nucleic acid and a fluorescent moiety, wherein the detection rolling circle replication primer comprises a quenching moiety.
  - 107. The method of claim 89 wherein two or more of the open circle probes in the set of open circle probes constitute a matched open circle probe set, wherein the open circle probes in the matched open circle probe set are targeted to different forms of the same target sequence.
  - 108. The method of claim 107 wherein each detection rolling circle replication primer comprises a different fluorescent moiety.
  - 109. The method of claim 89 further comprising, following the ligation operation, heating the circularized open circle probes.
  - 110. The method of claim 89 wherein each open circle probe comprises two ends, wherein the open circle probes are each specific for a target sequence, wherein each target sequence comprises a 5′
    - region and a 3′
      
      region, wherein each 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, wherein at least one of the target sequences 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 specific for the target sequence nor the right target probe portion of the open circle probe specific for the target sequence is complementary to the central region of the target sequence.
  - 111. The method of claim 89 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 intramolecular stem structure of at least one of the open circle probes forms a stem and loop structure.
  - 112. The method of claim 89 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 if one or more of the open circle probes that can form an intramolecular stem structure are not circularized, the end of at least one of the uncircularized open circle probes that forms the intramolecular stem structure is extended during the amplification operation using the open circle probe as a template.
  - 113. The method of claim 89 wherein the amplification operation produces tandem sequence DNA, wherein the method further comprises detecting the tandem sequence DNA.
  - 114. The method of claim 113 wherein the tandem sequence DNA is detected via one or more fluorescent change probes.
  - 115. The method of claim 114 wherein the fluorescent change probes are hairpin quenched probes, cleavage quenched probes, cleavage activated probes, fluorescent activated probes, or a combination.
  - 116. The method of claim 113 wherein the tandem sequence DNA is detected via one or more fluorescent change primers.
  - 117. The method of claim 116 wherein the fluorescent change primers are stem quenched primers, hairpin quenched primers, or a combination.
  - 118. The method of claim 89 wherein the amplification operation produces tandem sequence DNA and secondary tandem sequence DNA, wherein the method further comprises detecting the tandem sequence DNA, the secondary tandem sequence DNA, or both.

119. A method of amplifying nucleic acid sequences, the method comprising an amplification operation, wherein the amplification operation is carried out in the presence of a set of amplification target circles, wherein the set of amplification target circles comprises a plurality of different amplification target circles, wherein the amplification operation comprises rolling circle replication of the amplification target circles, wherein the amplification operation is carried out in the presence of a plurality of detection rolling circle replication primers, a secondary DNA strand displacement primer, and a common rolling circle replication primer, wherein each detection rolling circle replication primer corresponds to a different amplification target circle in the set of amplification target circles, wherein the secondary DNA strand displacement primer corresponds to all of the amplification target circles in the set of amplification target circles, wherein the common rolling circle replication primer corresponds to all of the amplification target circles in the set of amplification target circles.
- View Dependent Claims (120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142)
- - 120. The method of claim 119 wherein each detection rolling circle replication primer comprises a complementary portion, wherein each amplification target circle comprises a detection primer complement portion, wherein the complementary portion of the detection rolling circle replication primer is complementary to the detection primer complement portion of the amplification target circle to which the detection rolling circle replication primer corresponds, wherein the complementary portion of the detection rolling circle replication primer is not substantially complementary to an amplification target circle to which the detection rolling circle replication primer does not correspond.
  - 121. The method of claim 120 wherein the detection rolling circle replication primer is a peptide nucleic acid quenched primer, wherein the detection rolling circle replication primer further comprises a fluorescent moiety and a quencher complement portion, wherein each detection rolling circle replication primer is associated with a peptide nucleic acid quencher, wherein the peptide nucleic acid quencher is associated with the detection rolling circle replication primer via the quencher complement portion, wherein the peptide nucleic acid quencher comprises a quenching moiety, wherein association of the peptide nucleic acid quencher with the detection rolling circle replication primer quenches fluorescence from the fluorescent moiety, wherein the amplification operation results in disassociation of the peptide nucleic acid quenchers from the detection rolling circle replication primers, thereby allowing the fluorescent moiety of the detection rolling circle replication primers to fluoresce.
  - 122. The method of claim 121 wherein the quencher complement portion of each detection rolling circle replication primer is the same, wherein the peptide nucleic acid quencher associated with each detection rolling circle replication primer is the same.
  - 123. The method of claim 121 wherein the quencher complement portion of at least one of the detection rolling circle replication primers is different from the quencher complement portion of at least one of the other detection rolling circle replication primers.
  - 124. The method of claim 121 wherein the quencher complement portion of each detection rolling circle replication primer corresponding to an amplification target circle in the set of amplification target circles is the same, wherein the peptide nucleic acid quencher associated with each detection rolling circle replication primer is the same.
  - 125. The method of claim 121 wherein the quencher complement portion of at least one of the detection rolling circle replication primers corresponding to an amplification target circle in the set of amplification target circles is different from the quencher complement portion of at least one of the other detection rolling circle replication primers corresponding to an amplification target circle in the set of amplification target circles.
  - 126. The method of claim 121 wherein each detection rolling circle replication primer comprises a different fluorescent moiety.
  - 127. The method of claim 120 wherein the detection rolling circle replication primer is a peptide nucleic acid quenched primer, wherein the detection rolling circle replication primer further comprises a quenching moiety and a quencher complement portion, wherein each detection rolling circle replication primer is associated with a peptide nucleic acid fluor, wherein the peptide nucleic acid fluor is associated with the detection rolling circle replication primer via the quencher complement portion, wherein the peptide nucleic acid fluor comprises a fluorescent moiety, wherein association of the peptide nucleic acid fluor with the detection rolling circle replication primer quenches fluorescence from the fluorescent moiety, wherein the amplification operation results in disassociation of the peptide nucleic acid fluors from the detection rolling circle replication primers, thereby allowing the fluorescent moiety of the peptide nucleic acid fluors to fluoresce.
  - 128. The method of claim 127 wherein the quencher complement portion of each detection rolling circle replication primer is the same, wherein the peptide nucleic acid fluor associated with each detection rolling circle replication primer is the same.
  - 129. The method of claim 127 wherein the quencher complement portion of at least one of the detection rolling circle replication primers is different from the quencher complement portion of at least one of the other detection rolling circle replication primers.
  - 130. The method of claim 127 wherein the quencher complement portion of each detection rolling circle replication primer corresponding to an amplification target circle in the set of amplification target circles is the same, wherein the peptide nucleic acid fluor associated with each detection rolling circle replication primer is the same.
  - 131. The method of claim 127 wherein the quencher complement portion of at least one of the detection rolling circle replication primers corresponding to an amplification target circle in the set of amplification target circles is different from the quencher complement portion of at least one of the other detection rolling circle replication primers corresponding to an amplification target circle in the set of amplification target circles.
  - 132. The method of claim 131 wherein each peptide nucleic acid fluor comprises a different fluorescent moiety.
  - 133. The method of claim 120 wherein the detection rolling circle replication primer is a hairpin quenched primer.
  - 134. The method of claim 119 wherein the amplification operation is carried out in the presence of one or more additional sets of amplification target circles, wherein each set of amplification target circles comprises a plurality of different amplification target circles.
  - 135. The method of claim 119 wherein each detection rolling circle replication primer is associated with a peptide nucleic acid quencher, wherein the peptide nucleic acid quencher comprises peptide nucleic acid and a quenching moiety, wherein the detection rolling circle replication primer comprises a fluorescent moiety.
  - 136. The method of claim 119 wherein each detection rolling circle replication primer is associated with a peptide nucleic acid fluor, wherein the peptide nucleic acid fluor comprises peptide nucleic acid and a fluorescent moiety, wherein the detection rolling circle replication primer comprises a quenching moiety.
  - 137. The method of claim 119 wherein the amplification operation produces tandem sequence DNA, wherein the method further comprises detecting the tandem sequence DNA.
  - 138. The method of claim 137 wherein the tandem sequence DNA is detected via one or more fluorescent change probes.
  - 139. The method of claim 138 wherein the fluorescent change probes are hairpin quenched probes, cleavage quenched probes, cleavage activated probes, fluorescent activated probes, or a combination.
  - 140. The method of claim 137 wherein the tandem sequence DNA is detected via one or more fluorescent change primers.
  - 141. The method of claim 140 wherein the fluorescent change primers are stem quenched primers, hairpin quenched primers, or a combination.
  - 142. The method of claim 119 wherein the amplification operation produces tandem sequence DNA and secondary tandem sequence DNA, wherein the method further comprises detecting the tandem sequence DNA, the secondary tandem sequence DNA, or both.

143. 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 the ligation operation is carried out in the presence of a set of open circle probes, wherein the set of open circle probes comprises a plurality of different open circle probes, wherein the amplification operation comprises rolling circle replication of the circularized open circle probes, wherein two or more of the open circle probes in the set of open circle probes constitute a matched open circle probe set.
- View Dependent Claims (144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172)
- - 144. The method of claim 143 wherein the amplification operation is carried out in the presence of a plurality of detection rolling circle replication primers, a secondary DNA strand displacement primer, and a common rolling circle replication primer, wherein each detection rolling circle replication primer comprises a complementary portion, wherein each open circle probe comprises a detection primer complement portion, wherein each detection rolling circle replication primer corresponds to a different open circle probe in the set of open circle probes, wherein the complementary portion of the detection rolling circle replication primer is complementary to the detection primer complement portion of the open circle probe to which the detection rolling circle replication primer corresponds, wherein the complementary portion of the detection rolling circle replication primer is not substantially complementary to an open circle probe to which the detection rolling circle replication primer does not correspond.
  - 145. The method of claim 144 wherein the detection rolling circle replication primer is a peptide nucleic acid quenched primer, wherein the detection rolling circle replication primer further comprises a fluorescent moiety and a quencher complement portion, wherein each detection rolling circle replication primer is associated with a peptide nucleic acid quencher, wherein the peptide nucleic acid quencher is associated with the detection rolling circle replication primer via the quencher complement portion, wherein the peptide nucleic acid quencher comprises a quenching moiety, wherein association of the peptide nucleic acid quencher with the detection rolling circle replication primer quenches fluorescence from the fluorescent moiety, wherein the amplification operation results in disassociation of the peptide nucleic acid quenchers from the detection rolling circle replication primers, thereby allowing the fluorescent moiety of the detection rolling circle replication primers to fluoresce.
  - 146. The method of claim 145 wherein the quencher complement portion of each detection rolling circle replication primer is the same, wherein the peptide nucleic acid quencher associated with each detection rolling circle replication primer is the same.
  - 147. The method of claim 145 wherein the quencher complement portion of at least one of the detection rolling circle replication primers is different from the quencher complement portion of at least one of the other detection rolling circle replication primers.
  - 148. The method of claim 145 wherein the quencher complement portion of each detection rolling circle replication primer corresponding to an open circle probe in the set of open circle probes is the same, wherein the peptide nucleic acid quencher associated with each detection rolling circle replication primer is the same.
  - 149. The method of claim 145 wherein the quencher complement portion of at least one of the detection rolling circle replication primers corresponding to an open circle probe in the set of open circle probes is different from the quencher complement portion of at least one of the other detection rolling circle replication primers corresponding to an open circle probe in the set of open circle probes.
  - 150. The method of claim 145 wherein each detection rolling circle replication primer comprises a different fluorescent moiety.
  - 151. The method of claim 144 wherein the detection rolling circle replication primer is a peptide nucleic acid quenched primer, wherein the detection rolling circle replication primer further comprises a quenching moiety and a quencher complement portion, wherein each detection rolling circle replication primer is associated with a peptide nucleic acid fluor, wherein the peptide nucleic acid fluor is associated with the detection rolling circle replication primer via the quencher complement portion, wherein the peptide nucleic acid fluor comprises a fluorescent moiety, wherein association of the peptide nucleic acid fluor with the detection rolling circle replication primer quenches fluorescence from the fluorescent moiety, wherein the amplification operation results in disassociation of the peptide nucleic acid fluors from the detection rolling circle replication primers, thereby allowing the fluorescent moiety of the peptide nucleic acid fluors to fluoresce.
  - 152. The method of claim 151 wherein the quencher complement portion of each detection rolling circle replication primer is the same, wherein the peptide nucleic acid fluor associated with each detection rolling circle replication primer is the same.
  - 153. The method of claim 151 wherein the quencher complement portion of at least one of the detection rolling circle replication primers is different from the quencher complement portion of at least one of the other detection rolling circle replication primers.
  - 154. The method of claim 151 wherein the quencher complement portion of each detection rolling circle replication primer corresponding to an open circle probe in the set of open circle probes is the same, wherein the peptide nucleic acid fluor associated with each detection rolling circle replication primer is the same.
  - 155. The method of claim 151 wherein the quencher complement portion of at least one of the detection rolling circle replication primers corresponding to an open circle probe in the set of open circle probes is different from the quencher complement portion of at least one of the other detection rolling circle replication primers corresponding to an open circle probe in the set of open circle probes.
  - 156. The method of claim 155 wherein each peptide nucleic acid fluor comprises a different fluorescent moiety.
  - 157. The method of claim 144 wherein the detection rolling circle replication primer is a hairpin quenched primer.
  - 158. The method of claim 143 wherein the ligation operation is carried out in the presence of one or more additional sets of open circle probes, wherein each set of open circle probes comprises a plurality of different open circle probes.
  - 159. The method of claim 143 wherein the amplification operation is carried out in the presence of one or more detection rolling circle replication primers, wherein each detection rolling circle replication primer is associated with a peptide nucleic acid quencher, wherein the peptide nucleic acid quencher comprises peptide nucleic acid and a quenching moiety, wherein the detection rolling circle replication primer comprises a fluorescent moiety.
  - 160. The method of claim 143 wherein the amplification operation is carried out in the presence of one or more detection rolling circle replication primers, wherein each detection rolling circle replication primer is associated with a peptide nucleic acid fluor, wherein the peptide nucleic acid fluor comprises peptide nucleic acid and a fluorescent moiety, wherein the detection rolling circle replication primer comprises a quenching moiety.
  - 161. The method of claim 143 wherein the open circle probes in the matched open circle probe set are targeted to different forms of the same target sequence.
  - 162. The method of claim 161 wherein the amplification operation is carried out in the presence of one or more detection rolling circle replication primers, wherein each detection rolling circle replication primer comprises a different fluorescent moiety.
  - 163. The method of claim 143 further comprising, following the ligation operation, heating the circularized open circle probes.
  - 164. The method of claim 143 wherein each open circle probe comprises two ends, wherein the open circle probes are each specific for a target sequence, wherein each target sequence comprises a 5′
    - region and a 3′
      
      region, wherein each 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, wherein at least one of the target sequences 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 specific for the target sequence nor the right target probe portion of the open circle probe specific for the target sequence is complementary to the central region of the target sequence.
  - 165. The method of claim 143 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 intramolecular stem structure of at least one of the open circle probes forms a stem and loop structure.
  - 166. The method of claim 143 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 if one or more of the open circle probes that can form an intramolecular stem structure are not circularized, the end of at least one of the uncircularized open circle probes that forms the intramolecular stem structure is extended during the amplification operation using the open circle probe as a template.
  - 167. The method of claim 143 wherein the amplification operation produces tandem sequence DNA, wherein the method further comprises detecting the tandem sequence DNA.
  - 168. The method of claim 167 wherein the tandem sequence DNA is detected via one or more fluorescent change probes.
  - 169. The method of claim 168 wherein the fluorescent change probes are hairpin quenched probes, cleavage quenched probes, cleavage activated probes, fluorescent activated probes, or a combination.
  - 170. The method of claim 167 wherein the tandem sequence DNA is detected via one or more fluorescent change primers.
  - 171. The method of claim 170 wherein the fluorescent change primers are stem quenched primers, hairpin quenched primers, or a combination.
  - 172. The method of claim 143 wherein the amplification operation produces tandem sequence DNA and secondary tandem sequence DNA, wherein the method further comprises detecting the tandem sequence DNA, the secondary tandem sequence DNA, or both.

173. 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 the amplification operation comprises rolling circle replication of the circularized open circle probes, wherein the amplification operation is carried out in the presence of one or more rolling circle replication primers, wherein at least one of the rolling circle replication primers is associated with a peptide nucleic acid quencher or a peptide nucleic acid fluor.
- View Dependent Claims (174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202)
- - 174. The method of claim 173 wherein each detection rolling circle replication primer comprises a complementary portion, wherein each open circle probe comprises a detection primer complement portion, wherein the ligation operation is carried out in the presence of a set of open circle probes, wherein the set of open circle probes comprises a plurality of different open circle probes, wherein the complementary portion of the detection rolling circle replication primer is complementary to the detection primer complement portion of the open circle probe to which the detection rolling circle replication primer corresponds, wherein the complementary portion of the detection rolling circle replication primer is not substantially complementary to an open circle probe to which the detection rolling circle replication primer does not correspond.
  - 175. The method of claim 174 wherein the detection rolling circle replication primer is a peptide nucleic acid quenched primer, wherein the detection rolling circle replication primer further comprises a fluorescent moiety and a quencher complement portion, wherein each detection rolling circle replication primer is associated with a peptide nucleic acid quencher, wherein the peptide nucleic acid quencher is associated with the detection rolling circle replication primer via the quencher complement portion, wherein the peptide nucleic acid quencher comprises a quenching moiety, wherein association of the peptide nucleic acid quencher with the detection rolling circle replication primer quenches fluorescence from the fluorescent moiety, wherein the amplification operation results in disassociation of the peptide nucleic acid quenchers from the detection rolling circle replication primers, thereby allowing the fluorescent moiety of the detection rolling circle replication primers to fluoresce.
  - 176. The method of claim 175 wherein the quencher complement portion of each detection rolling circle replication primer is the same, wherein the peptide nucleic acid quencher associated with each detection rolling circle replication primer is the same.
  - 177. The method of claim 175 wherein the quencher complement portion of at least one of the detection rolling circle replication primers is different from the quencher complement portion of at least one of the other detection rolling circle replication primers.
  - 178. The method of claim 175 wherein the quencher complement portion of each detection rolling circle replication primer corresponding to an open circle probe in the set of open circle probes is the same, wherein the peptide nucleic acid quencher associated with each detection rolling circle replication primer is the same.
  - 179. The method of claim 175 wherein the quencher complement portion of at least one of the detection rolling circle replication primers corresponding to an open circle probe in the set of open circle probes is different from the quencher complement portion of at least one of the other detection rolling circle replication primers corresponding to an open circle probe in the set of open circle probes.
  - 180. The method of claim 175 wherein each detection rolling circle replication primer comprises a different fluorescent moiety.
  - 181. The method of claim 174 wherein the detection rolling circle replication primer is a peptide nucleic acid quenched primer, wherein the detection rolling circle replication primer further comprises a quenching moiety and a quencher complement portion, wherein each detection rolling circle replication primer is associated with a peptide nucleic acid fluor, wherein the peptide nucleic acid fluor is associated with the detection rolling circle replication primer via the quencher complement portion, wherein the peptide nucleic acid fluor comprises a fluorescent moiety, wherein association of the peptide nucleic acid fluor with the detection rolling circle replication primer quenches fluorescence from the fluorescent moiety, wherein the amplification operation results in disassociation of the peptide nucleic acid fluors from the detection rolling circle replication primers, thereby allowing the fluorescent moiety of the peptide nucleic acid fluors to fluoresce.
  - 182. The method of claim 181 wherein the quencher complement portion of each detection rolling circle replication primer is the same, wherein the peptide nucleic acid fluor associated with each detection rolling circle replication primer is the same.
  - 183. The method of claim 181 wherein the quencher complement portion of at least one of the detection rolling circle replication primers is different from the quencher complement portion of at least one of the other detection rolling circle replication primers.
  - 184. The method of claim 181 wherein the quencher complement portion of each detection rolling circle replication primer corresponding to an open circle probe in the set of open circle probes is the same, wherein the peptide nucleic acid fluor associated with each detection rolling circle replication primer is the same.
  - 185. The method of claim 181 wherein the quencher complement portion of at least one of the detection rolling circle replication primers corresponding to an open circle probe in the set of open circle probes is different from the quencher complement portion of at least one of the other detection rolling circle replication primers corresponding to an open circle probe in the set of open circle probes.
  - 186. The method of claim 185 wherein each peptide nucleic acid fluor comprises a different fluorescent moiety.
  - 187. The method of claim 174 wherein the detection rolling circle replication primer is a hairpin quenched primer.
  - 188. The method of claim 173 wherein the ligation operation is carried out in the presence of a plurality of sets of open circle probes, wherein each set of open circle probes comprises a plurality of different open circle probes.
  - 189. The method of claim 173 wherein the peptide nucleic acid quencher comprises peptide nucleic acid and a quenching moiety, wherein the detection rolling circle replication primer comprises a fluorescent moiety.
  - 190. The method of claim 173 wherein the peptide nucleic acid fluor comprises peptide nucleic acid and a fluorescent moiety, wherein the detection rolling circle replication primer comprises a quenching moiety.
  - 191. The method of claim 173 wherein the ligation operation is carried out in the presence of a set of open circle probes, wherein the set of open circle probes comprises a plurality of different open circle probes, wherein two or more of the open circle probes in the set of open circle probes constitute a matched open circle probe set, wherein the open circle probes in the matched open circle probe set are targeted to different forms of the same target sequence.
  - 192. The method of claim 191 wherein each detection rolling circle replication primer comprises a different fluorescent moiety.
  - 193. The method of claim 173 further comprising, following the ligation operation, heating the circularized open circle probes.
  - 194. The method of claim 173 wherein each open circle probe comprises two ends, wherein the open circle probes are each specific for a target sequence, wherein each target sequence comprises a 5′
    - region and a 3′
      
      region, wherein each 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, wherein at least one of the target sequences 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 specific for the target sequence nor the right target probe portion of the open circle probe specific for the target sequence is complementary to the central region of the target sequence.
  - 195. The method of claim 173 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 intramolecular stem structure of at least one of the open circle probes forms a stem and loop structure.
  - 196. The method of claim 173 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 if one or more of the open circle probes that can form an intramolecular stem structure are not circularized, the end of at least one of the uncircularized open circle probes that forms the intramolecular stem structure is extended during the amplification operation using the open circle probe as a template.
  - 197. The method of claim 173 wherein the amplification operation produces tandem sequence DNA, wherein the method further comprises detecting the tandem sequence DNA.
  - 198. The method of claim 197 wherein the tandem sequence DNA is detected via one or more fluorescent change probes.
  - 199. The method of claim 198 wherein the fluorescent change probes are hairpin quenched probes, cleavage quenched probes, cleavage activated probes, fluorescent activated probes, or a combination.
  - 200. The method of claim 197 wherein the tandem sequence DNA is detected via one or more fluorescent change primers.
  - 201. The method of claim 200 wherein the fluorescent change primers are stem quenched primers, hairpin quenched primers, or a combination.
  - 202. The method of claim 173 wherein the amplification operation produces tandem sequence DNA and secondary tandem sequence DNA, wherein the method further comprises detecting the tandem sequence DNA, the secondary tandem sequence DNA, or both.

203. A method of amplifying nucleic acid sequences, the method comprising an amplification operation, wherein the amplification operation comprises rolling circle replication of the amplification target circles, wherein the amplification operation is carried out in the presence of one or more rolling circle replication primers, wherein at least one of the rolling circle replication primers is associated with a peptide nucleic acid quencher or a peptide nucleic acid fluor.
- View Dependent Claims (204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227)
- - 204. The method of claim 203 wherein each detection rolling circle replication primer comprises a complementary portion, wherein each amplification target circle comprises a detection primer complement portion, wherein the amplification operation is carried out in the presence of a set of amplification target circles, wherein the set of amplification target circles comprises a plurality of different amplification target circles, wherein the complementary portion of the detection rolling circle replication primer is complementary to the detection primer complement portion of the amplification target circle to which the detection rolling circle replication primer corresponds, wherein the complementary portion of the detection rolling circle replication primer is not substantially complementary to an amplification target circle to which the detection rolling circle replication primer does not correspond.
  - 205. The method of claim 204 wherein the detection rolling circle replication primer is a peptide nucleic acid quenched primer, wherein the detection rolling circle replication primer further comprises a fluorescent moiety and a quencher complement portion, wherein each detection rolling circle replication primer is associated with a peptide nucleic acid quencher, wherein the peptide nucleic acid quencher is associated with the detection rolling circle replication primer via the quencher complement portion, wherein the peptide nucleic acid quencher comprises a quenching moiety, wherein association of the peptide nucleic acid quencher with the detection rolling circle replication primer quenches fluorescence from the fluorescent moiety, wherein the amplification operation results in disassociation of the peptide nucleic acid quenchers from the detection rolling circle replication primers, thereby allowing the fluorescent moiety of the detection rolling circle replication primers to fluoresce.
  - 206. The method of claim 205 wherein the quencher complement portion of each detection rolling circle replication primer is the same, wherein the peptide nucleic acid quencher associated with each detection rolling circle replication primer is the same.
  - 207. The method of claim 205 wherein the quencher complement portion of at least one of the detection rolling circle replication primers is different from the quencher complement portion of at least one of the other detection rolling circle replication primers.
  - 208. The method of claim 205 wherein the quencher complement portion of each detection rolling circle replication primer corresponding to an amplification target circle in the set of amplification target circles is the same, wherein the peptide nucleic acid quencher associated with each detection rolling circle replication primer is the same.
  - 209. The method of claim 205 wherein the quencher complement portion of at least one of the detection rolling circle replication primers corresponding to an amplification target circle in the set of amplification target circles is different from the quencher complement portion of at least one of the other detection rolling circle replication primers corresponding to an amplification target circle in the set of amplification target circles.
  - 210. The method of claim 205 wherein each detection rolling circle replication primer comprises a different fluorescent moiety.
  - 211. The method of claim 204 wherein the detection rolling circle replication primer is a peptide nucleic acid quenched primer, wherein the detection rolling circle replication primer further comprises a quenching moiety and a quencher complement portion, wherein each detection rolling circle replication primer is associated with a peptide nucleic acid fluor, wherein the peptide nucleic acid fluor is associated with the detection rolling circle replication primer via the quencher complement portion, wherein the peptide nucleic acid fluor comprises a fluorescent moiety, wherein association of the peptide nucleic acid fluor with the detection rolling circle replication primer quenches fluorescence from the fluorescent moiety, wherein the amplification operation results in disassociation of the peptide nucleic acid fluors from the detection rolling circle replication primers, thereby allowing the fluorescent moiety of the peptide nucleic acid fluors to fluoresce.
  - 212. The method of claim 211 wherein the quencher complement portion of each detection rolling circle replication primer is the same, wherein the peptide nucleic acid fluor associated with each detection rolling circle replication primer is the same.
  - 213. The method of claim 211 wherein the quencher complement portion of at least one of the detection rolling circle replication primers is different from the quencher complement portion of at least one of the other detection rolling circle replication primers.
  - 214. The method of claim 211 wherein the quencher complement portion of each detection rolling circle replication primer corresponding to an amplification target circle in the set of amplification target circles is the same, wherein the peptide nucleic acid fluor associated with each detection rolling circle replication primer is the same.
  - 215. The method of claim 211 wherein the quencher complement portion of at least one of the detection rolling circle replication primers corresponding to an amplification target circle in the set of amplification target circles is different from the quencher complement portion of at least one of the other detection rolling circle replication primers corresponding to an amplification target circle in the set of amplification target circles.
  - 216. The method of claim 215 wherein each peptide nucleic acid fluor comprises a different fluorescent moiety.
  - 217. The method of claim 204 wherein the detection rolling circle replication primer is a hairpin quenched primer.
  - 218. The method of claim 203 wherein the amplification operation is carried out in the presence of a set of amplification target circles, wherein the set of amplification target circles comprises a plurality of different amplification target circles.
  - 219. The method of claim 218 wherein the amplification operation is carried out in the presence of one or more additional sets of amplification target circles, wherein each set of amplification target circles comprises a plurality of different amplification target circles.
  - 220. The method of claim 203 wherein the peptide nucleic acid quencher comprises peptide nucleic acid and a quenching moiety, wherein the detection rolling circle replication primer comprises a fluorescent moiety.
  - 221. The method of claim 203 wherein the peptide nucleic acid fluor comprises peptide nucleic acid and a fluorescent moiety, wherein the detection rolling circle replication primer comprises a quenching moiety.
  - 222. The method of claim 203 wherein the amplification operation produces tandem sequence DNA, wherein the method further comprises detecting the tandem sequence DNA.
  - 223. The method of claim 222 wherein the tandem sequence DNA is detected via one or more fluorescent change probes.
  - 224. The method of claim 223 wherein the fluorescent change probes are hairpin quenched probes, cleavage quenched probes, cleavage activated probes, fluorescent activated probes, or a combination.
  - 225. The method of claim 222 wherein the tandem sequence DNA is detected via one or more fluorescent change primers.
  - 226. The method of claim 225 wherein the fluorescent change primers are stem quenched primers, hairpin quenched primers, or a combination.
  - 227. The method of claim 203 wherein the amplification operation produces tandem sequence DNA and secondary tandem sequence DNA, wherein the method further comprises detecting the tandem sequence DNA, the secondary tandem sequence DNA, or both.

228. A method of selectively amplifying nucleic acid sequences related to one or more target sequences, the method comprising, (a) mixing a set of 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, wherein the set of open circle probes comprises a plurality of different 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 two or more of the open circle probes in the set of open circle probes constitute a matched open circle probe set, (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, wherein the amplification target circles formed from the open circle probes in the set of open circle probes comprise a set of amplification target circles, (c) mixing a plurality of detection rolling circle replication primers, a secondary DNA strand displacement primer, and a common 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 primers in the primer-ATC mixture, wherein each detection rolling circle replication primer is associated with a peptide nucleic acid quencher, wherein each detection rolling circle replication primer corresponds to a different open circle probe in the set of open circle probes, wherein the secondary DNA strand displacement primer corresponds to all of the open circle probes in the set of open circle probes, wherein the common rolling circle replication primer corresponds to all of the open circle probes in the set of open circle probes, 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.

229. 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 set 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, a plurality of detection rolling circle replication primers, wherein all or a portion of each detection rolling circle replication primer is complementary to a portion of one or more of the open circle probes, one or more secondary DNA strand displacement primers, wherein all or a portion of each secondary DNA strand displacement primer matches a portion of one or more of the open circle probes, and one or more common rolling circle replication primers, wherein all or a portion of each common rolling circle replication primer is complementary to a portion of one or more of the open circle probes.
- View Dependent Claims (230, 231, 232, 233, 234, 235, 236, 237)
- - 230. The kit of claim 229 wherein all or a portion of each detection rolling circle replication primer is complementary to a portion of a different one or more of the open circle probes in the set of open circle probes, wherein all or a portion of each secondary DNA strand displacement primer matches a portion of all of the open circle probes in the set of open circle probes, and wherein all or a portion of each common rolling circle replication primer is complementary to a portion of all of the open circle probes in the set of open circle probes.
  - 231. The kit of claim 229 wherein the end of the open circle probe that can form an intramolecular stem structure is a 3′
    - end.
  - 232. The kit of claim 229 wherein each target sequence comprises a 5′
    - region and a 3′
      
      region, wherein the open circle probes each comprise 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 spacer portion comprises a primer complement portion, wherein the left target probe portion is complementary to the 3′
      
      region of at least one of the target sequences and the right target probe portion is complementary to the 5′
      
      region of the same target sequence, wherein the rolling circle replication primer comprises a single-stranded, linear nucleic acid molecule comprising a complementary portion that is complementary to the primer complement portion of one or more of the open circle probes.
  - 233. The kit of claim 232 wherein at least one target sequence further comprises a central region located between the 5′
    - region and the 3′
      
      region, wherein neither the left target probe portion nor the right target probe portion of the open circle probe complementary to the target sequence is complementary to the central region of the target sequence.
  - 234. The kit of claim 233 further comprising one or more gap oligonucleotides, wherein the gap oligonucleotides are complementary to all or a portion of the central region of the target sequence.
  - 235. The kit of claim 232 the target probe portions of the open circle probes are complementary to a different target sequence for each of a plurality of the open circle probes.
  - 236. The kit of claim 229 further comprising one or more reporter binding agents each comprising a specific binding molecule and an oligonucleotide portion, wherein the oligonucleotide portion comprises one of the target sequences.
  - 237. The kit of claim 229 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.

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Litigation Campaign Assessment

Current Assignee
Qiagen GmbH (Qiagen NV)
Original Assignee
Qiagen GmbH (Qiagen NV)
Inventors
Egholm, Michael, Abarzua, Patricio, Driscoll, Mark D., Alsmadi, Osama A.

Application Number

US10/325,490
Publication Number

US 20030165948A1
Time in Patent Office

Days
Field of Search
US Class Current

435/6
CPC Class Codes

C12Q 1/6844   Nucleic acid amplification ...

C12Q 2525/301   Hairpin oligonucleotides

C12Q 2525/307   Circular oligonucleotides

C12Q 2531/125   Rolling circle

Method and compositions for efficient and specific rolling circle amplification

First Claim

2 Assignments

0 Petitions

Accused Products

Abstract

Citations

237 Claims

Specification

Solutions

Use Cases

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Method and compositions for efficient and specific rolling circle amplification

First Claim

2 Assignments

Subscription Required

Subscription Required

0 Petitions

Subscription Required

Accused Products

Subscription Required

Abstract

Citations

237 Claims

Specification

Subscription Required

Solutions

Use Cases

Quick Links