Unimolecular segment amplification and sequencing

• US 6,143,495 A
• Filed: 11/21/1996
• Issued: 11/07/2000
• Est. Priority Date: 11/21/1995
• Status: Expired due to Term

• Current Assignee: Yale University
• Original Assignee: Yale University
• Inventors: Caplan, Michael, Lizardi, Paul M.
• Primary Examiner(s): Jones, W. Gary
• Assistant Examiner(s): WHISENANT, ETHAN C
• Application Number:
  US08/754,681
• Time in Patent Office: 1,447 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 536/23.1, 536/24.3, 935/76, 935/77, 935/78
• US Class Current: 435/6.12
• CPC Class Codes: C12Q 1/6804 Nucleic acid analysis using immunogens immunoassay G01N33/53
• CPC Class Codes: C12Q 1/6809 Methods for determination or identification of nucleic acids involving differential detection
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 1/6853 using modified primers or templates
• CPC Class Codes: C12Q 1/6862 Ligase chain reaction [LCR]
• CPC Class Codes: C12Q 2525/161 incorporating target specific and non-target specific sites
• CPC Class Codes: C12Q 2525/307 Circular oligonucleotides
• CPC Class Codes: C12Q 2531/119 Strand displacement amplification [SDA]
• CPC Class Codes: C12Q 2531/125 Rolling circle
• CPC Class Codes: C12Q 2531/143 Promoter based amplification, e.g. NASBA, 3SR, TAS
• CPC Class Codes: C12Q 2537/125 Sandwich assay format
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: C12Q 2539/113 Differential Display Analysis [DDA]
• CPC Class Codes: C12Q 2563/131 the label being a member of a cognate binding pair, i.e. extends to antibodies, haptens, avidin
• CPC Class Codes: C12Q 2563/179 the label being a nucleic acid
• CPC Class Codes: C12Q 2565/102 Multiple non-interacting labels

Abstract

Disclosed are compositions and a method for amplification of and multiplex detection of molecules of interest involving rolling circle replication. The method is useful for simultaneously detecting multiple specific nucleic acids in a sample with high specificity and sensitivity. The method also has an inherently low level of background signal. A preferred form of the method consists of an association operation, an amplification operation, and a detection operation. The association operation involves association of one or more specially designed probe molecules, either wholly or partly nucleic acid, to target molecules of interest. This operation associates the probe molecules to a target molecules present in a sample. The amplification operation is rolling circle replication of circular nucleic acid molecules, termed amplification target circles, that are either a part of, or hybridized to, the probe molecules. A single round of amplification using rolling circle replication results in a large amplification of the amplification target circles. Following rolling circle replication, the amplified sequences are detected using combinatorial multicolor coding probes that allow separate, simultaneous, and quantitative detection of multiple different amplified target circles representing multiple different target molecules. Since the amplified product is directly proportional to the amount of target sequence present in a sample, quantitative measurements reliably represent the amount of a target sequence in a sample. Major advantages of this method are that a large number of distinct target molecules can be detected simultaneously, and that differences in the amounts of the various target molecules in a sample can be accurately quantified. It is also advantageous that the DNA replication step is isothermal, and that signals are strictly quantitative because the amplification reaction is linear and is catalyzed by a highly processive enzyme.

Claims

1. A method of amplifying nucleic acid sequences, the method comprising,

(a) mixing a specific binding molecule with a target sample comprising a target molecule wherein a rolling circle replication primer is coupled to the specific binding molecule, wherein the specific binding molecule is bound to the target molecule,

(b) mixing the rolling circle replication primer with an amplification target circle, to produce a primer-ATC mixture, and incubating the primer-ATC mixture under conditions that promote hybridization between the amplification target circle and the rolling circle replication primer in the primer-ATC mixture,

wherein the amplification target circle comprises a single-stranded, circular DNA molecule comprising a primer complement portion, wherein the primer complement portion is complementary to the rolling circle replication primer, and

(c) 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 circle,

wherein replication of the amplification target circle results in the formation of tandem sequence DNA.

2. The method of claim 1 wherein the method includes at least one of the following:

(1) the use of a solid-state sample wherein the solid-state sample comprises the target molecule, (2) a nucleic acid collapse operation, (3) a multiplex detection operation comprising separately and simultaneously detecting a plurality of different sequences present in the tandem sequence DNA, (4) differential amplification of at least two of the amplification target circles, and (5) primer-extension sequencing.

3. A method of amplifying nucleic acid sequences, the method comprising,

(a) mixing one or more rolling circle replication primers with one or more amplification target circles, to produce a primer-ATC mixture, and incubating the primer-ATC mixture under conditions that promote hybridization between the amplification target circles and the rolling circle replication primers in the primer-ATC mixture,

wherein the amplification target circles each comprise a single-stranded, circular DNA molecule comprising a primer complement portion, wherein the primer complement portion is complementary to at least one of the rolling circle replication primers,

(b) mixing DNA polymerase with the primer-ATC mixture, to produce a polymerase-ATC mixture, and incubating the polymerase-ATC mixture under conditions that promote replication of the amplification target circles,

wherein replication of the amplification target circles results in the formation of tandem sequence DNA, and, simultaneous with, or following, step (b),

(c) mixing RNA polymerase with the polymerase-ATC mixture, and incubating the polymerase-ATC mixture under conditions that promote transcription of the tandem sequence DNA, wherein transcription of the tandem sequence DNA results in the formation of tandem sequence RNA.

4. A method of amplifying nucleic acid sequences, the method comprising,

(a) mixing one or more rolling circle replication primers with one or more amplification target circles, to produce a primer-ATC mixture, and incubating the primer-ATC mixture under conditions that promote hybridization between the amplification target circles and the rolling circle replication primers in the primer-ATC mixture,

wherein the amplification target circles each comprise a single-stranded, circular DNA molecule comprising a primer complement portion, wherein the primer complement portion is complementary to at least one of the rolling circle replication primers,

(b) mixing DNA polymerase with the primer-ATC mixture, to produce a polymerase-ATC mixture, and incubating the polymerase-ATC mixture under conditions that promote replication of the amplification target circles,

wherein replication of the amplification target circles results in the formation of tandem sequence DNA,

(c) forming an interrogation mixture, wherein one or more interrogation primers are hybridized to the tandem sequence DNA,

(d) simultaneous with, or following, step (c), mixing at least two different tagged chain terminating nucleotides and DNA polymerase with the interrogation mixture, wherein each different tagged chain terminating nucleotide comprises a different chain terminating nucleotide triphosphate coupled to a different tag molecule,

(e) incubating the interrogation mixture under conditions that promote template-based addition of the tagged chain terminating nucleotides to the interrogation primers, wherein addition of the tagged chain terminating nucleotides to the interrogation primers results in association of the tagged chain terminating nucleotides with the tandem sequence DNA, and

(f) detecting the association of the tagged chain terminating nucleotides with the tandem sequence DNA.

5. The method of claim 4 wherein formation of the interrogation mixture comprises

(c)(1) mixing an interrogation probe and a plurality of degenerate probes with the tandem sequence DNA to produce a probe mixture, under conditions that promote hybridization between the tandem sequence DNA and the interrogation probe and degenerate probes, wherein each degenerate probe has a 3'"'"' blocking group,

(c)(2) mixing ligase with the probe mixture, to produce a degenerate ligation mixture, and incubating the degenerate ligation mixture under conditions that promote ligation of the interrogation probe to one of the degenerate probes hybridized to the tandem sequence DNA, wherein the degenerate probe that is ligated to the interrogation probe is a ligated degenerate probe,

(c)(3) removing the 3'"'"' blocking group of the ligated degenerate probe,

wherein ligation of the interrogation probe to one or more degenerate probes results in the formation of the interrogation primer, wherein the formation of the interrogation primer results in formation of the interrogation mixture.

6. The method of claim 5 wherein formation of the interrogation mixture further comprises, following step (c)(3),

(c)(4) mixing the plurality of degenerate probes with the ligation mixture, to produce a secondary probe mixture, under conditions that promote hybridization between the tandem sequence DNA and the degenerate probes,

(c)(5) mixing ligase with the secondary probe mixture, to produce a secondary degenerate ligation mixture, and incubating the secondary degenerate ligation mixture under conditions that promote ligation of the ligated degenerate probe to one of the degenerate probes hybridized to the tandem sequence DNA, wherein the degenerate probe that is ligated to the ligated degenerate probe is a secondary ligated degenerate probe,

(c)(6) removing the 3'"'"' blocking group of the secondary degenerate probe,

wherein steps (c)(4), (c)(5), and (c)(6) are performed, in order, one or more times.

7. The method of claim 4 wherein formation of the interrogation mixture comprises mixing an interrogation primer with the tandem sequence DNA under conditions that promote hybridization between the tandem sequence DNA and the interrogation primer.

8. A method of amplifying nucleic acid sequences, the method comprising,

(a) mixing one or more rolling circle replication primers with one or more amplification target circles, to produce a primer-ATC mixture, and incubating the primer-ATC mixture under conditions that promote hybridization between the amplification target circles and the rolling circle replication primers in the primer-ATC mixture,

wherein the amplification target circles each comprise a single-stranded, circular DNA molecule comprising a primer complement portion, wherein the primer complement portion is complementary to at least one of the rolling circle replication primers,

(b) mixing DNA polymerase with the primer-ATC mixture, to produce a polymerase-ATC mixture, and incubating the polymerase-ATC mixture under conditions that promote replication of the amplification target circles,

wherein replication of the amplification target circles results in the formation of tandem sequence DNA,

(c) mixing a set of detection probes with the tandem sequence DNA under conditions that promote hybridization between the tandem sequence DNA and the detection probes, wherein the set of detection probes is labeled using combinatorial multicolor coding.

9. A method of amplifying nucleic acid sequences, the method comprising,

(a) mixing one or more different open circle probes and one or more gap oligonucleotides with a target sample comprising one or more target sequences, to produce an OCP-target sample mixture,

wherein the target sequences each comprise 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, and wherein the left target probe portion and the right target probe portion of the same open circle probe are each complementary to the 3'"'"' region and the 5'"'"' region, respectively, of the same 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 nor the right target probe portion of any of the open circle probes is complementary to the central region of the target sequences,

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 at least one of the target sequences, and

incubating the OCP-target sample mixture under conditions that promote hybridization between the open circle probes and the gap oligonucleotides 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 circle results in the formation of tandem sequence DNA.

10. The method of claim 9 further comprising, simultaneous with, or following, step (d),

(e) mixing RNA polymerase with the polymerase-ATC mixture, and incubating the polymerase-ATC mixture under conditions that promote transcription of the tandem sequence DNA, wherein transcription of the tandem sequence DNA results in the formation of tandem sequence RNA, or

(e) mixing a secondary DNA strand displacement primer with the polymerase-ATC mixture, and incubating the polymerase-ATC mixture under conditions that promote (i) hybridization between the tandem sequence DNA and the secondary DNA strand displacement primer, and (ii) replication of the tandem sequence DNA in the polymerase-ATC mixture, wherein replication of the tandem sequence DNA results in the formation of secondary tandem sequence DNA.

11. The method of claim 10 wherein the method includes at least one of the following:

(1) the use of a solid-state sample wherein the solid-state sample comprises the target molecule, (2) a nucleic acid collapse operation, (3) a multiplex detection operation comprising separately and simultaneously detecting a plurality of different sequences present in the tandem sequence DNA, (4) differential amplification of at least two of the amplification target circles, and (5) primer-extension sequencing.

12. The method of claim 9 wherein at least one of the target sequences is coupled to a specific binding molecule, wherein the specific binding molecule binds to a target molecule.

13. The method of claim 12 wherein the method includes at least one of the following:

(1) the use of a solid-state sample wherein the solid-state sample comprises the target molecule, (2) a step of bringing the specific binding molecule into contact with the target molecule, (3) a nucleic acid collapse operation, (4) a multiplex detection operation comprising separately and simultaneously detecting a plurality of different sequences present in the tandem sequence DNA, (5) differential amplification of at least two of the amplification target circles, and (6) primer-extension sequencing.

14. The method of claim 9 wherein at least one of the rolling circle replication primers is coupled to a specific binding molecule, wherein the specific binding molecule is bound to a target molecule.

15. The method of claim 14 wherein the method includes at least one of the following:

(1) the use of a solid-state sample wherein the solid-state sample comprises the target molecule, (2) a step of bringing the specific binding molecule into contact with the target molecule, (3) a nucleic acid collapse operation, (4) a multiplex detection operation comprising separately and simultaneously detecting a plurality of different sequences present in the tandem sequence DNA, (5) differential amplification of at least two of the amplification target circles, and (6) primer-extension sequencing.

16. The method of claim 9 wherein the method further comprises at least one of the following:

(1) a nucleic acid collapse operation, (2) a multiplex detection operation comprising separately and simultaneously detecting a plurality of different sequences present in the tandem sequence DNA, (3) differential amplification of at least two of the amplification target circles, and (4) primer-extension sequencing.

17. The method of claim 9 further comprising

mixing a set of detection probes with the tandem sequence DNA under conditions that promote hybridization between the tandem sequence DNA and the detection probes, wherein the set of detection probes is labeled using combinatorial multicolor coding, and

detecting a plurality of different sequences present in the tandem sequence DNA.

18. The method of claim 9 wherein the target molecule is part of a solid-state sample.

19. The method of claim 18 wherein the method includes at least one of the following:

(1) a nucleic acid collapse operation, (2) a multiplex detection operation comprising separately and simultaneously detecting a plurality of different sequences present in the tandem sequence DNA, (3) differential amplification of at least two of the amplification target circles, and (4) primer-extension sequencing.

20. A kit for selectively detecting one or more target molecules, the kit comprising,

(a) one or more amplification target circles,

wherein the amplification target circles each comprise a single-stranded, circular DNA molecule comprising a primer complement portion, and

(b) a rolling circle replication primer comprising 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 amplification target circles,

wherein each amplification target circle is tethered to a specific binding molecule,

wherein the specific binding molecule binds to at least one of the target molecules.

21. The kit of claim 20 further comprising a secondary DNA strand displacement primer comprising a single-stranded, linear nucleic acid molecule comprising a matching portion that matches a portion of one or more of the amplification target circles.

22. The kit of claim 20 further comprising an interrogation probe and a plurality of degenerate probes.

23. The kit of claim 20 further comprising an interrogation primer.

24. A kit for selectively amplifying nucleic acid sequences related to one or more target sequences, each comprising a 5'"'"' region and a 3'"'"' region, the kit comprising,

(a) one or more open circle probes each comprising 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, and

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,

(b) a rolling circle replication primer comprising 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, and

(c) one or both of (1) a secondary DNA strand displacement primer comprising a single-stranded, linear nucleic acid molecule comprising a matching portion that matches a portion of one or more of the open circle probes, and (2) one or more reporter binding agents each comprising an affinity portion and an oligonucleotide portion, wherein the oligonucleotide portion comprises one of the target sequences.

25. A kit for selectively amplifying nucleic acid sequences related to one or more target sequences, each comprising a 5'"'"' region and a 3'"'"' region, the kit comprising,

(a) one or more open circle probes each comprising 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, and

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,

(b) a rolling circle replication primer comprising 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,

(c) one or more gap oligonucleotides, and

(d) a plurality of degenerate probes,

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 nor the right target probe portion of the open circle probe is complementary to the central region, and

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 at least one of the target sequences.

26. The kit of claim 25 further comprising an interrogation probe.

27. The kit of claim 25 further comprising an interrogation primer.

28. A method of amplifying nucleic acid sequences, the method comprising,

(a) mixing one or more rolling circle replication primers with one or more amplification target circles, to produce a primer-ATC mixture, and incubating the primer-ATC mixture under conditions that promote hybridization between the amplification target circles and the rolling circle replication primers in the primer-ATC mixture,

wherein the amplification target circles each comprise a single-stranded, circular DNA molecule comprising a primer complement portion, wherein the primer complement portion is complementary to at least one of the rolling circle replication primers, and

(b) mixing DNA polymerase with the primer-ATC mixture, to produce a polymerase-ATC mixture, and incubating the polymerase-ATC mixture under conditions that promote replication of the amplification target circles,

wherein replication of the amplification target circles results in the formation of tandem sequence DNA,

wherein the tandem sequence DNA is collapsed using collapsing probes.

29. The method of claim 28 wherein at least one of the collapsing probes is a collapsing detection probe.

30. The method of claim 28 wherein the tandem sequence DNA is collapsed by mixing the collapsing probes with the tandem sequence DNA, and incubating under conditions that promote hybridization between the collapsing probes and the tandem sequence DNA.

31. The method of claim 30 further comprising, prior to or simultaneous with the mixing of the collapsing probes with the tandem sequence DNA, mixing detection probes with the tandem sequence DNA, and incubating under conditions that promote hybridization between the detection probes and the tandem sequence DNA.

32. The method of claim 31 wherein the detection probes are labeled using combinatorial multicolor coding.

33. The method of claim 28 wherein the collapsing probes comprise ligands, haptens, or both coupled to or incorporated into oligonucleotides.

34. A method of amplifying nucleic acid sequences, the method comprising,

(a) mixing one or more rolling circle replication primers with one or more amplification target circles, to produce a primer-ATC mixture, and incubating the primer-ATC mixture under conditions that promote hybridization between the amplification target circles and the rolling circle replication primers in the primer-ATC mixture,

wherein the amplification target circles each comprise a single-stranded, circular DNA molecule comprising a primer complement portion, wherein the primer complement portion is complementary to at least one of the rolling circle replication primers, and

(b) mixing DNA polymerase with the primer-ATC mixture, to produce a polymerase-ATC mixture, and incubating the polymerase-ATC mixture under conditions that promote replication of the amplification target circles,

wherein replication of the amplification target circles results in the formation of tandem sequence DNA,

wherein the tandem sequence DNA is collapsed using ligand/ligand binding pairs, hapten/antibody pairs, or both.

35. The method of claim 34 wherein ligands, haptens, or both are coupled to or incorporated into the tandem sequence DNA.

36. The method of claim 8 wherein at least one of the detection probes is a collapsing detection probe.

37. The method of claim 8 further comprising, simultaneous with or following the mixing of the detection probes with the tandem sequence DNA, mixing collapsing probes with the tandem sequence DNA, and incubating under conditions that promote hybridization between the collapsing probes and the tandem sequence DNA.

38. The method of claim 36 wherein the collapsing detection probe comprises ligands, haptens, or both coupled to or incorporated into an oligonucleotide.

39. The method of claim 8 wherein the tandem sequence DNA is collapsed using ligand/ligand binding pairs, hapten/antibody pairs, or both.

40. The method of claim 39 wherein ligands, haptens, or both are coupled to or incorporated into the tandem sequence DNA.

41. A method of amplifying nucleic acid sequences, the method comprising,

(a) mixing one or more rolling circle replication primers with one or more amplification target circles, to produce a primer-ATC mixture, and incubating the primer-ATC mixture under conditions that promote hybridization between the amplification target circles and the rolling circle replication primers in the primer-ATC mixture,

wherein the amplification target circles each comprise a single-stranded, circular DNA molecule comprising a primer complement portion, wherein the primer complement portion is complementary to at least one of the rolling circle replication primers, and

(b) mixing DNA polymerase with the primer-ATC mixture, to produce a polymerase-ATC mixture, and incubating the polymerase-ATC mixture under conditions that promote replication of the amplification target circles,

wherein replication of the amplification target circles results in the formation of tandem sequence DNA, and simultaneous with, or following, step (b),

(c) mixing a secondary DNA strand displacement primer with the polymerase-ATC mixture, and incubating the polymerase-ATC mixture under conditions that promote (i) hybridization between the tandem sequence DNA and the secondary DNA strand displacement primer, and (ii) replication of the tandem sequence DNA in the polymerase-ATC mixture, wherein replication of the tandem sequence DNA results in the formation of secondary tandem sequence DNA.

42. The method of claim 41 further comprising

(d) mixing RNA polymerase with the polymerase-ATC mixture, and incubating the polymerase-ATC mixture under conditions that promote transcription of the secondary tandem sequence DNA, wherein transcription of the secondary tandem sequence DNA results in the formation of tandem sequence RNA.

43. The method of claim 42 wherein the method includes at least one of the following:

(1) a nucleic acid collapse operation, (2) a multiplex detection operation comprising separately and simultaneously detecting a plurality of different sequences present in the tandem sequence DNA, (3) differential amplification of at least two of the amplification target circles, and (4) primer-extension sequencing.

44. A method of amplifying nucleic acid sequences, the method comprising,

(a) mixing a specific binding molecule with a target sample comprising a target molecule wherein an amplification target circle is tethered to the specific binding molecule, wherein the specific binding molecule binds to the target molecule,

(b) mixing a rolling circle replication primer with the amplification target circle, to produce a primer-ATC mixture, and incubating the primer-ATC mixture under conditions that promote hybridization between the amplification target circle and the rolling circle replication primer in the primer-ATC mixture,

wherein the amplification target circle comprises a single-stranded, circular DNA molecule comprising a primer complement portion, wherein the primer complement portion is complementary to the rolling circle replication primer, and

(c) 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 circle,

wherein replication of the amplification target circle results in the formation of tandem sequence DNA.

45. A kit for selectively detecting one or more target molecules, the kit comprising,

(a) one or more amplification target circles,

wherein the amplification target circles each comprise a single-stranded, circular DNA molecule comprising a primer complement portion, and

(b) a rolling circle replication primer comprising 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 amplification target circles,

wherein the rolling circle replication primer is coupled to a specific binding molecule,

wherein the specific binding molecule binds to at least one of the target molecules.

46. The kit of claim 45 further comprising a secondary DNA strand displacement primer comprising a single-stranded, linear nucleic acid molecule comprising a matching portion that matches a portion of one or more of the amplification target circles.

47. The method of claim 9 further comprising

(e) forming an interrogation mixture, wherein one or more interrogation primers are hybridized to the tandem sequence DNA,

(f) simultaneous with, or following, step (e), mixing at least two different tagged chain terminating nucleotides and DNA polymerase with the interrogation mixture, wherein each different tagged chain terminating nucleotide comprises a different chain terminating nucleotide triphosphate coupled to a different tag molecule,

(g) incubating the interrogation mixture under conditions that promote template-based addition of the tagged chain terminating nucleotides to the interrogation primers, wherein addition of the tagged chain terminating nucleotides to the interrogation primers results in association of the tagged chain terminating nucleotides with the tandem sequence DNA, and

(h) detecting the association of the tagged chain terminating nucleotides with the tandem sequence DNA.

48. The method of claim 47 wherein formation of the interrogation mixture comprises

(e)(1) mixing an interrogation probe and a plurality of degenerate probes with the tandem sequence DNA to produce a probe mixture, under conditions that promote hybridization between the tandem sequence DNA and the interrogation probe and degenerate probes, wherein each degenerate probe has a 3'"'"' blocking group;

(e)(2) mixing ligase with the probe mixture, to produce a degenerate ligation mixture, and incubating the degenerate ligation mixture under conditions that promote ligation of the interrogation probe to one of the degenerate probes hybridized to the tandem sequence DNA, wherein the degenerate probe that is ligated to the interrogation probe is a ligated degenerate probe; and

(e)(3) removing the 3'"'"' blocking group of the ligated degenerate probe;

wherein ligation of the interrogation probe to one or more degenerate probes results in the formation of the interrogation primer, wherein the formation of the interrogation primer results in formation of the interrogation mixture.

49. The method of claim 48 wherein formation of the interrogation mixture further comprises, following step (e)(3),

(e)(4) mixing the plurality of degenerate probes with the ligation mixture, to produce a secondary probe mixture, under conditions that promote hybridization between the tandem sequence DNA and the degenerate probes;

(e)(5) mixing ligase with the secondary probe mixture, to produce a secondary degenerate ligation mixture, and incubating the secondary degenerate ligation mixture under conditions that promote ligation of the ligated degenerate probe to one of the degenerate probes hybridized to the tandem sequence DNA, wherein the degenerate probe that is ligated to the ligated degenerate probe is a secondary ligated degenerate probe; and

(e)(6) removing the 3'"'"' blocking group of the secondary degenerate probe;

wherein steps (e)(4), (e)(5), and (e)(6) are performed, in order, one or more times.

50. A method of amplifying nucleic acid sequences, the method comprising,

(a) mixing one or more different open circle probes with a target sample comprising one or more target sequences, to produce an OCP-target sample mixture,

wherein the target sequences each comprise 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, and wherein the left target probe portion and the right target probe portion of the same open circle probe are each complementary to the 3'"'"' region and the 5'"'"' region, respectively, of the same 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 nor the right target probe portion of any of the open circle probes is complementary to the central region of the target sequences, 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 and DNA polymerase 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 during incubation the DNA polymerase fills in the central region of the target sequences,

(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 circle results in the formation of tandem sequence DNA.

51. The method of claim 1 further comprising, prior to step (a) binding the specific binding molecule to the target molecule.

52. The method of claim 14 further comprising, prior to step (c) binding the specific binding molecule to the target molecule.

53. A method of amplifying nucleic acid sequences, the method comprising,

(a) mixing one or more different open circle probes with a target sample comprising one or more target sequences, to produce an OCP-target sample mixture,

(b) 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,

(c) 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,

(d) 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

(e) mixing DNA polymerase and a secondary DNA strand displacement primer with the primer-ATC mixture, to produce a polymerase-ATC mixture, and incubating the polymerase-ATC mixture under conditions that promote

(i) replication of the amplification target circles, wherein replication of the amplification target circle results in the formation of tandem sequence DNA,

(ii) hybridization between the tandem sequence DNA and the secondary DNA strand displacement primer,

(iii) replication of the tandem sequence DNA, wherein replication of the tandem sequence DNA results in the formation of secondary tandem sequence DNA,

(iv) hybridization between the secondary tandem sequence DNA and the rolling circle replication primer, and

(v) replication of the secondary tandem sequence DNA, wherein replication of the tandem sequence DNA results in the formation of tertiary tandem sequence DNA.

54. A method of amplifying nucleic acid sequences, the method comprising,

(a) mixing one or more rolling circle replication primers with one or more amplification target circles, to produce a primer-ATC mixture, and incubating the primer-ATC mixture under conditions that promote hybridization between the amplification target circles and the rolling circle replication primers in the primer-ATC mixture,

wherein the amplification target circles each comprise a single-stranded, circular DNA molecule comprising a primer complement portion, wherein the primer complement portion is complementary to at least one of the rolling circle replication primers, and

(b) mixing DNA polymerase and a secondary DNA strand displacement primer with the primer-ATC mixture, to produce a polymerase-ATC mixture, and incubating the polymerase-ATC mixture under conditions that promote

(i) replication of the amplification target circles, wherein replication of the amplification target circle results in the formation of tandem sequence DNA,

(ii) hybridization between the tandem sequence DNA and the secondary DNA strand displacement primer,

(iii) replication of the tandem sequence DNA, wherein replication of the tandem sequence DNA results in the formation of secondary tandem sequence DNA,

(iv) hybridization between the secondary tandem sequence DNA and the rolling circle replication primer, and

(v) replication of the secondary tandem sequence DNA, wherein replication of the tandem sequence DNA results in the formation of tertiary tandem sequence DNA.

55. A method of amplifying nucleic acid sequences, the method comprising,

(a) mixing one or more different open circle probes with a target sample comprising one or more target sequences, to produce an OCP-target sample mixture,

(b) 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,

(c) 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,

(d) 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

(e) mixing DNA polymerase, a secondary DNA strand displacement primer, and a tertiary DNA strand displacement primer with the primer-ATC mixture, to produce a polymerase-ATC mixture, and incubating the polymerase-ATC mixture under conditions that promote

(i) replication of the amplification target circles, wherein replication of the amplification target circle results in the formation of tandem sequence DNA,

(ii) hybridization between the tandem sequence DNA and the secondary DNA strand displacement primer,

(iii) replication of the tandem sequence DNA, wherein replication of the tandem sequence DNA results in the formation of secondary tandem sequence DNA,

(iv) hybridization between the secondary tandem sequence DNA and the tertiary DNA strand displacement primer, and

(v) replication of the secondary tandem sequence DNA, wherein replication of the tandem sequence DNA results in the formation of tertiary tandem sequence DNA.

56. A method of amplifying nucleic acid sequences, the method comprising,

(a) mixing one or more rolling circle replication primers with one or more amplification target circles, to produce a primer-ATC mixture, and incubating the primer-ATC mixture under conditions that promote hybridization between the amplification target circles and the rolling circle replication primers in the primer-ATC mixture,

wherein the amplification target circles each comprise a single-stranded, circular DNA molecule comprising a primer complement portion, wherein the primer complement portion is complementary to at least one of the rolling circle replication primers, and

(b) mixing DNA polymerase, a secondary DNA strand displacement primer, and a tertiary DNA strand displacement primer with the primer-ATC mixture, to produce a polymerase-ATC mixture, and incubating the polymerase-ATC mixture under conditions that promote

(i) replication of the amplification target circles, wherein replication of the amplification target circle results in the formation of tandem sequence DNA,

(ii) hybridization between the tandem sequence DNA and the secondary DNA strand displacement primer,

(iii) replication of the tandem sequence DNA, wherein replication of the tandem sequence DNA results in the formation of secondary tandem sequence DNA,

(iv) hybridization between the secondary tandem sequence DNA and the tertiary DNA strand displacement primer, and

(v) replication of the secondary tandem sequence DNA, wherein replication of the tandem sequence DNA results in the formation of tertiary tandem sequence DNA.

57. A method of amplifying nucleic acid sequences, the method comprising,

(a) mixing one or more different open circle probes with a target sample comprising one or more target sequences, to produce an OCP-target sample mixture,

(b) 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,

(c) 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,

(d) 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

(e) mixing DNA polymerase and a secondary DNA strand displacement primer with the primer-ATC mixture, to produce a polymerase-ATC mixture, and incubating the polymerase-ATC mixture under conditions that promote

(i) replication of the amplification target circles, wherein replication of the amplification target circle results in the formation of tandem sequence DNA,

(ii) hybridization between the tandem sequence DNA and the secondary DNA strand displacement primer, and

(iii) replication of the tandem sequence DNA, wherein replication of the tandem sequence DNA results in the formation of secondary tandem sequence DNA,

wherein the rolling circle replication primer is prevented from hybridizing to the secondary tandem sequence DNA and the secondary tandem sequence DNA is not replicated.

58. A method of amplifying nucleic acid sequences, the method comprising,

(a) mixing one or more rolling circle replication primers with one or more amplification target circles, to produce a primer-ATC mixture, and incubating the primer-ATC mixture under conditions that promote hybridization between the amplification target circles and the rolling circle replication primers in the primer-ATC mixture,

wherein the amplification target circles each comprise a single-stranded, circular DNA molecule comprising a primer complement portion, wherein the primer complement portion is complementary to at least one of the rolling circle replication primers, and

(b) mixing DNA polymerase and a secondary DNA strand displacement primer with the primer-ATC mixture, to produce a polymerase-ATC mixture, and incubating the polymerase-ATC mixture under conditions that promote

(i) replication of the amplification target circles, wherein replication of the amplification target circle results in the formation of tandem sequence DNA,

(ii) hybridization between the tandem sequence DNA and the secondary DNA strand displacement primer, and

(iii) replication of the tandem sequence DNA, wherein replication of the tandem sequence DNA results in the formation of secondary tandem sequence DNA,

wherein the rolling circle replication primer is prevented from hybridizing to the secondary tandem sequence DNA and the secondary tandem sequence DNA is not replicated.