Method, kits and compositions pertaining to detection complexes

• US 6,361,942 B1
• Filed: 03/24/1999
• Issued: 03/26/2002
• Est. Priority Date: 03/24/1998
• Status: Expired due to Term

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Boston Probes, Inc. (Thermo Fisher Scientific Incorporated)
• Inventors: Coull, James M., Gildea, Brian D., Hyldig-Nielsen, Jens J.
• Primary Examiner(s): Marschel, Ardin H.
• Application Number:
  US09/275,848
• Time in Patent Office: 1,098 Days
• Field of Search: 435/6, 435/7.1, 435/7.2, 530/300, 530/350, 536/22.1, 536/23.1, 536/24.1, 536/24-- 2, 422/50, 422/68.1
• US Class Current: 435/6.11
• CPC Class Codes: C07K 14/003 Peptide-nucleic acids (PNAs)
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 1/6851 Quantitative amplification
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2525/107 incorporating a peptide nucleic acid
• CPC Class Codes: C12Q 2561/113 Real time assay
• CPC Class Codes: C12Q 2565/101 Interaction between at least two labels

Abstract

This invention is directed to methods, kits and compositions which utilize Detection Complexes to detect or identify the presence, absence or quantity of a target molecule in a sample of interest. A Detection Complex comprises at least two component polymers and at least one set of donor and accepter moieties. To each of at least two component polymers is linked at least one moiety of a set of donor and accepter moieties, such that formation of the complex facilitates transfer of energy between donor and acceptor moieties of each set in a manner which, in an assay, produces changes in detectable signal which can be correlated with the presence absence of quantity of target sequence and/or target molecule of interest in the sample.

Claims

1. A composition comprising;

a) at least one component probing polymer wherein the probing polymer comprises;

     1) a probing segment that is complementary or substantially complementary to a target sequence; and

     2) one or more interacting groups suitable for the formation of a complex with at least one other component polymer that is not the target sequence wherein the probing polymer can perform as a primer in an extension reaction upon hybridization of the probing segment to the target sequence;

b) at least one component annealing polymer that is not the target sequence and which, at a minimum, comprises one or more interacting groups wherein the interacting groups of the component polymers form and stabilize the complex; and

c) at least one set of donor and acceptor moieties wherein to each of at least two of the component polymers, that are not the target sequence, is linked at least one moiety from the set such that formation of the complex facilitates transfer of energy between donor and acceptor moieties of the set;

provided however, that at least one of the component polymers of the composition is a non-nucleic acid polymer.

2. The composition of claim 1, wherein all the component polymers are non-nucleic acid polymers.

3. The composition of claim 1, wherein at least one of the component polymers is a PNA.

4. The composition of claim 1, wherein all of the component polymers are PNA.

5. The composition of claim 3, wherein PNA subunits of a component polymer have the formula:

wherein,

each J is the same or different and is selected from the group consisting of;

H, R¹, OR¹, SR¹, NHR¹, NR¹₂, F, Cl Br and I;

each K is the same or different and is selected from the group consisting of;

O, S, NH and NR¹;

each R¹ is the same or different and is an alkyl group having one to five carbon atoms which may optionally contain a heteroatom or a substituted or unsubstituted aryl group;

each A is selected from the group consisting of a single bond, a group of the formula;

—

(CJ₂)_s— and

a group of the formula;

—

(CJ₂)_sC(O)—

wherein, J is defined above and each s is an integer from one to five;

each t is 1 or 2;

each u is 1 or 2; and

each L is the same or different and is independently selected from the group consisting of J, adenine, cytosine, guanine, thymine, uridine, 5-methylcytosine, 2-aminopurine, 2-amino-6-chloropurine, 2,6-diaminopurine, hypoxanthine, pseudoisocytosine, 2-thiouracil, 2-thiothymidine, other naturally occurring nucleobase analogs, other non-naturally occurring nucleobases, substituted and unsubstituted aromatic moieties, biotin, fluorescein and dabcyl.

6. The composition of claim 5, wherein each PNA subunit consists of a naturally occurring nucleobase attached to the aza nitrogen of the N-[2-(aminoethyl)]glycine backbone through a methylene carbonyl linkage.

7. The composition of claim 1, wherein the donor moiety is a fluorophore.

8. The composition of claim 7, wherein the fluorophore is selected from the group consisting of:

5(6)-carboxyfluorescein, 6-((7-amino-4-methylcoumarin-3-acetyl)amino)hexanoic acid, 5(and

     6)-carboxy-X-rhodamine, Cyanine 2 Dye, Cyanine 3 Dye, Cyanine 3.5 Dye, Cyanine 5 Dye, Cyanine 5.5 Dye, Cyanine 7 Dye and Cyanine 9 Dye.

9. The composition of claim 1, wherein the acceptor moiety is a quencher moiety.

10. The composition of claim 9, wherein the quencher moiety is 4-((-4-(dimethylamino)phenyl)azo)benzoic acid (dabcyl).

11. The composition of claim 1, wherein a spacer moiety tethers one or more of the donor and acceptor moieties to the component polymer.

12. The composition of claim 1, wherein at least one linker is used to link together two segments of a component polymer.

13. The composition of claim 12, wherein the annealing polymer comprises two segments of interacting groups linked by one or more linkers.

14. The composition of claim 1, wherein at least one component polymer of the complex is immobilized or tethered to a surface.

15. The composition of claim 1, wherein the interacting groups are selected from the group consisting of hydrophobic moieties, ionic moieties and hydrogen bonding moieties.

16. The composition of claim 15, wherein the ionic moieties comprise salt pairs.

17. The composition of claim 15, wherein the hydrogen bonding moieties comprise complementary nucleobases linked to the subunits of the two or more component polymers.

18. The composition of claim 1, wherein the interacting groups are located at one termini of one or more of the component polymers.

19. The composition of claim 1, wherein the interacting groups are located at both termini of one or more of the component polymers.

20. The composition of claim 1, wherein the interacting groups are centered within one or more of the component polymers.

21. The composition of claim 1, wherein the complex comprises only one probing polymer and one annealing polymer.

22. The composition of claim 1, wherein the complex comprises at least three component polymers.

23. The composition of claim 1, wherein the complex comprises at least two Beacon Sets.

24. The composition of claim 23, wherein each Beacon Set comprises independently detectable moieties.

25. The composition of claim 24, wherein the independently detectable moieties of each Beacon Set are independently detectable donor fluorophores and the acceptor moieties of each Beacon Set are the same or different quencher moiety.

26. The composition of claim 25, wherein all acceptor moieties are the same quencher moiety.

27. The composition of claim 26, wherein the quencher moiety is dabcyl.

28. The composition of claim 1, wherein at least one of the component polymers is a PNA polymer labeled with a quencher moiety.

29. The composition of claim 28, wherein the PNA polymer is C-terminally labeled with dabcyl.

30. The composition of claim 1, wherein the probing polymer is selected from the group consisting of a nucleic acid and a PNA that has been modified to thereby facilitate polymerase extension.

31. The composition of claim 30, wherein:

(i.) the probing polymer comprises nucleobase containing interacting groups that do not sequence specifically interact with the target sequence; and

wherein

(ii.) the annealing polymer comprises nucleobase containing interacting groups that are the exact complement to the interacting groups of the probing polymer.

32. The composition of claim 31, wherein the probing polymer is a nucleic acid bearing a 5′

-fluorophore and the annealing polymer is a PNA polymer bearing a C-terminal quencher moiety.

33. The composition of claim 32, wherein at least one of the component polymers of the complex is immobilized or tethered to a surface.

34. An array of two or more different complexes wherein at least two complexes of the array each comprise;

a) at least one component probing polymer wherein the probing polymer comprises;

     1) a probing segment that is complementary or substantially complementary to a unique target sequence; and

     2) one or more interacting groups suitable for the formation of a complex with at least one other component polymer that is not the target sequence;

b) at least one component annealing polymer that is common to the at least two complexes of the array, that is not the target sequence and that, at a minimum, comprises one or more interacting groups wherein the interacting groups of the component polymers form and stabilize the complex; and

c) at least one set of donor and acceptor moieties wherein to at least two of the component polymers is linked at least one moiety from the set such that formation of the complex facilitates transfer of energy between donor and acceptor moieties of the set;

provided however, that at least one of the component polymers of each different complex is immobilized or tethered to the surface of the array.

35. The array of claim 34, wherein all the component polymers are non-nucleic acid polymers.

36. The array of claim 34, wherein at least one of the component polymers is a PNA.

37. The array of claim 34, wherein all of the component polymers are PNA.

38. The array of any of claims 36 or 37, wherein PNA subunits of a component polymer or polymers have the formula:

wherein,

each J is the same or different and is selected from the group consisting of;

H, R¹, OR¹, SR¹, NHR¹, NR¹₂, F, Cl, Br and I;

each K is the same or different and is selected from the group consisting of;

O, S, NH and NR¹;

each R¹ is the same or different and is an alkyl group having one to five carbon atoms which may optionally contain a heteroatom or a substituted or unsubstituted aryl group;

each A is selected from the group consisting of a single bond, a group of the formula;

—

(CJ₂)_s— and

a group of the formula;

—

(CJ₂)_sC(O)—

wherein, J is defined above and each s is an integer from one to five;

each t is 1 or 2;

each u is 1 or 2; and

each L is the same or different and is independently selected from the group consisting of J, adenine, cytosine, guanine, thymine, uridine, 5-methylcytosine, 2-aminopurine, 2-amino-6-chloropurine, 2,6-diaminopurine, hypoxanthine, pseudoisocytosine, 2-thiouracil, 2-thiothymidine, other naturally occurring nucleobase analogs, other non-naturally occurring nucleobases, substituted and unsubstituted aromatic moieties, biotin, fluorescein and dabcyl.

39. The array of claim 38, wherein each PNA subunit consists of a naturally occurring nucleobase attached to the aza nitrogen of a N-[2-(aminoethyl)]glycine backbone through a methylene carbonyl linkage.

40. The array of claim 34, wherein at least one complex of the array comprises at least three polymers.

41. The array of claim 34, wherein the common annealing polymer of the array is a PNA polymer comprising a quencher moiety.

42. An array of two or more different complexes wherein at least two complexes of the array each comprise;

a) at least one component probing polymer wherein the probing polymer comprises;

     1) a probing segment that is complementary or substantially complementary to a unique target sequence; and

     2) one or more interacting groups suitable for the formation of a complex with at least one other component polymer that is not the target sequence wherein the probing polymer of at least one complex can perform as a primer in an amplification reaction upon hybridization of the probing segment to a target sequence;

b) at least one component annealing polymer that is not the target sequence and which, at a minimum, comprises one or more interacting groups wherein the interacting groups of the component polymers form and stabilize the complex; and

c) at least one set of donor and acceptor moieties wherein to at least two of the component polymers is linked at least one moiety from the set such that formation of the complex facilitates transfer of energy between donor and acceptor moieties of the set;

provided however that;

(i) at least one of the component polymers of the complex is a non-nucleic acid polymer; and

(ii) at least one of the component polymers of each different complex is immobilized or tethered to the surface of the array.

43. The array of claim 42, wherein the probing polymer is selected from the group consisting of a nucleic acid and a PNA that has been modified to thereby facilitate polymerase extension.

44. The array of claim 43, wherein:

(i) the probing polymer comprises nucleobase containing interacting groups that do not sequence specifically interact with the target sequence; and

wherein

(ii) the annealing polymer comprises nucleobase containing interacting groups that are the exact complement to the interacting groups of the probing polymer.

45. The array of claim 44, wherein the probing polymer is a nucleic acid polymer bearing a 5′

-fluorophore and the annealing polymer is a PNA polymer bearing a C-terminal quencher moiety.

46. A method for determining the presence, absence or quantity of a target molecule, comprising at least one target sequence, in a sample;

said method comprising;

a) contacting the sample with at least one complex, said complex comprising;

(i) at least one component probing polymer wherein the probing polymer comprises;

     1) a probing segment that hybridizes to said target sequence; and

     2) one or more interacting groups suitable for the formation of a complex with at least one other component polymer that is not the target sequence;

(ii) at least one component annealing polymer that is not the target sequence and which, at a minimum, comprises one or more interacting groups wherein the interaction of the interacting groups of the component polymers form and stabilize the complex; and

(iii) at least one set of donor and acceptor moieties wherein to each of at least two component polymers is linked at least one moiety from the set such that formation of the complex facilitates transfer of energy between donor and acceptor moieties of the set in a manner that is detectably different as compared to when the component polymers are not complexed or when the probing segment is hybridized to the target sequence;

provided however, that at least one of the component polymers of the complex is a non-nucleic acid polymer; and

b) detecting, identifying or quantitating changes in detectable signal attributable to the changes in the transfer of energy between the donor and acceptor moieties of a set upon dissociation of the complex or upon the hybridization of the probing segment to the target sequence and correlating the change in signal with the presence, absence or quantity of the target molecule in the sample wherein the detectable signal is produced as a result of hybridization of the probing segment of the probing polymer to the target sequence of the target molecule which thereby facilitates a secondary triggering event that causes indirect dissociation of the complex.

47. The method of claim 46, wherein all the component polymers are non-nucleic acid polymers.

48. The method of claim 46, wherein at least one of the component polymers is a PNA.

49. The method of claim 46, wherein all of the component polymers are PNA.

50. The method of claim 46, wherein the method is used to detect a nucleic acid target molecule of interest which may be present or produced in a closed tube assay.

51. The method of claim 50, wherein the target molecule to be determined by the method is produced by a process selected from the group consisting of Polymerase Chain Reaction (PCR), Ligase Chain Reaction (LCR), Strand Displacement Amplification (SDA), Transcription-Mediated Amplification (TMA), Q-beta replicase amplification (Q-beta) and Rolling Circle Amplification (RCA).

52. The method of claim 50, wherein the nucleic acid target molecule of interest is an amplicon that has been produced as a result of performing a nucleic acid amplification reaction.

53. The method of claim 46, wherein the target molecule is immobilized to a surface.

54. The method of claim 46, wherein at least one of the component probing polymers of the complex is immobilized or tethered to a surface with which the sample of interest is contacted.

55. The method of claim 54, wherein the complex dissociates to release the common annealing component polymer into solution.

56. The method of claim 46, wherein the detectable signal is produced as a result of hybridization of the probing segment of the probing polymer to the target sequence of the target molecule without direct or indirect dissociation of the complex.

57. The method of claim 46, wherein the detectable signal is produced as a result of hybridization of the probing segment of the probing polymer to the target sequence of the target molecule which thereby causes direct dissociation of the complex.

58. A method for detecting, identifying or quantitating a nucleic acid target molecule of interest in a sample wherein said target molecule comprises at least one priming site and said method comprises:

a) contacting the sample with;

I) at least one complex formed from two or more component polymers that are not the target sequence, the complex comprising;

(i) at least one component probing polymer that can perform as a primer in an primer extension reaction and wherein the probing polymer, at a minimum comprises;

1) a probing segment that hybridizes to a first priming site; and

2) one or more interacting groups suitable for the formation of a complex with at least one other component polymer;

(ii) at least one component annealing polymer which, at a minimum, comprises one or more interacting groups wherein the interacting groups of the component polymers form and stabilize the complex; and

(iii) at least one set of donor and acceptor moieties wherein to each of at least two component polymers is linked at least one moiety from the set such that formation of the complex facilitates transfer of energy between donor and acceptor moieties of the set in a manner that is detectably different from when the component polymers exist independently or unassociated; and

II) all other components necessary to perform a primer extension reaction; and

b) performing a primer extension reaction which thereby causes the complex to dissociate in proportion to the quantity of nucleic acid produced by the primer extension reaction; and

c) detecting, identifying or quantitating changes in detectable signal attributable to the transfer of energy between the donor and acceptor moieties of a set upon dissociation of the complex and correlating the changes in detectable signal with the presence, absence or quantity of the nucleic acid target molecule of interest in the sample.

59. The method of claim 58, wherein all component polymers of the complex are labeled nucleic acids.

60. The method of claim 58, wherein at least one component polymer of the complex is a non-nucleic acid polymer.

61. The method of claim 58, wherein at least one component polymer of the complex is a PNA.

62. The method of claim 58, wherein the primer extension reaction is part of a nucleic acid amplification process.

63. The method of claim 62, wherein the method is used to detect a nucleic acid target molecule of interest that is a template for PCR.

64. The method of claim 63, wherein complex dissociation occurs as a consequence of the activity of the amplification process and the signal produced by dissociation of the complex is monitored in a closed tube assay format that facilitates both end-point or real-time assay measurements.

65. The method of claim 64, wherein the amplification process is PCR.

66. The method of claim 65, wherein other assay components include buffers, inorganic salt, magnesium, nucleoside triphosphates, a polymerase enzyme, and a second primer wherein the second primer and the complex comprise the forward and reverse primers of the PCR reaction.

67. The method of claim 66, wherein the second primer is a second complex that hybridizes to a second primer site of the target molecule;

said second complex comprising;

(a) at least one second component probing polymer which, at a minimum, comprises;

1) a probing segment, wherein said probing segment performs as the second primer in the PCR process; and

2) one or more interacting groups suitable for the formation of the second complex with at least one other component polymer that is not the target sequence;

(b) at least one second component annealing polymer that is not the target sequence and which, at a minimum, comprises one or more interacting groups wherein the interacting groups of the component polymers form and stabilize the second complex; and

(c) at least one second set of donor and acceptor moieties wherein to each of at least two component polymers of the second complex is linked at least one moiety from the second set such that formation of the second complex facilitates transfer of energy between donor and acceptor moieties of the set in a manner that is detectably different from when the component polymers of the second complex exist independently or unassociated.

68. The method of claim 58, wherein the probing polymer is selected from the group consisting of a nucleic acid and a PNA that has been modified to thereby facilitate polymerase extension.

69. The method of claim 68, wherein:

(i) the probing polymer comprises nucleobase containing interacting groups that do not sequence specifically interact with the first primer site; and

wherein

(ii) the annealing polymer comprises nucleobase containing interacting groups that are the exact complement to the interacting groups of the probing polymer.

70. The method of claim 58, wherein at least one of the component polymers of the complex is immobilized or tethered to a surface.

71. The method of claim 58, wherein the extension reaction is a component of a process selected from the group consisting of Polymerase Chain Reaction (PCR), Strand Displacement Amplification (SDA), Transcription-Mediated Amplification (TMA), Q-beta replicase amplification (Q-beta) and Rolling Circle Amplification (RCA).

72. The method of claim 67, wherein all component polymers of the second complex are appropriately labeled nucleic acids.

73. The method of claim 67, wherein at least one component polymer of the complex is a non-nucleic acid polymer.

74. The method of claim 67, wherein at least one component polymer of the complex is a PNA.

75. The method of claim 67, wherein the annealing polymer of the second complex is a PNA polymer bearing a quencher moiety.

76. The method of claim 67, wherein the same annealing polymer is common to both the first complex and the second complex.

77. The method of claim 76, wherein the common annealing polymer is a PNA bearing a quencher acceptor moiety.

78. The method of claim 67, wherein the first and second complexes comprise the same donor fluorophore and acceptor quencher.

79. The method of any of claims 58 or 67, wherein the sample is additionally contacted with one or more blocking probes to thereby suppress the binding of a probing segment of a probing polymer of one or more of the complexes to a priming site on the target molecule.

80. The method of any of claims 58 or 67, wherein PCR clamping is used to modify the assay so that it is suitable for performing a closed tube point mutation analysis that can be monitored in real-time or at the end-point.

81. The method of claim 80, wherein at least one independently detectable unimolecular “

beacon”

probe is also included in a PCR assay and hybridizes to a hybridization site within the amplicon to thereby generate an independently detectable signal that can be used to either monitor proper operation of the PCR amplification or be used to independently identify a feature of the amplicon generated in the assay.

82. The method of claim 81, wherein the specific feature to be determined by the assay is whether wild type, mutant or both wild type and mutant target molecules were present in the sample.

83. The method of claim 82, wherein the hybridization site of the wild type and mutant target molecules are related as a point mutation.

84. The method of claim 82, wherein the specific feature can be independently detected in a closed tube format in either real-time or at the end-point of the assay.

85. The method of claim 84, wherein signal from the one or more complexes and the one or more unimolecular “

Beacon”

probes present in the assay is independently detectable in a closed tube format in either real-time or at the end-point of the assay.

86. The method of any of claims 58 or 67, wherein at least two or more independently detectable complexes are present in a single multiplex assay that is used to simultaneously detect, identify or quantitate two or more target molecules of interest in the same sample and in the same assay.

87. The method of claim 86, wherein the independently detectable complexes are used in a closed tube format and the detectable change in signal from each independently detectable complex is monitored in either real-time or at the end-point of the assay.

88. A method for the formation of two or more different complexes each comprising a common annealing polymer, said method comprising mixing together two or more different component probing polymers with a common component annealing polymer under conditions that facilitate complex formation, wherein each complex comprises:

a) at least one component probing polymer wherein the probing polymer comprises;

     1) a probing segment that is complementary or substantially complementary to a target sequence; and

     2) one or more interacting groups suitable for the formation of a complex with at least one other component polymer that is not the target sequence;

b) at least one common component annealing polymer that is not the target sequence and which, at a minimum, comprises one or more interacting groups wherein the interaction of the interacting groups of the component polymers form and stabilize the different complexes; and

c) at least one set of donor and acceptor moieties wherein to each of at least two component polymers of a particular complex is linked at least one moiety from the set such that formation of the complex facilitates transfer of energy between donor and acceptor moieties of the set in a manner that is detectably different as compared to when the component polymers are not complexed or when the probing segment is hybridized to the target sequence.

89. The method of claim 88, wherein all the component polymers are non-nucleic acid polymers.

90. The method of claim 88, wherein at least one of the component polymers is a PNA.

91. The method of claim 88, wherein at least one component polymer is support bound.

92. A kit comprising three or more different component polymers suitable for the formation of at least two different complexes, said kit comprising;

a) at least two different component probing polymers wherein the probing polymers each comprise;

     1) a probing segment that is complementary or substantially complementary to a target sequence; and

     2) one or more interacting groups suitable for the formation of a complex with at least one other component polymer that is not the target sequence;

b) at least one common component annealing polymer that is not the target sequence and which, at a minimum, comprises one or more interacting groups wherein the interacting groups of the component polymers form and stabilize the two or more different complexes; and

c) at least one set of donor and acceptor moieties wherein to each of at least two component polymers of a particular complex is linked at least one moiety from the set such that formation of the complex facilitates transfer of energy between donor and acceptor moieties of the set in a manner that is detectably different as compared to when the component polymers are not complexed or when the probing segment is hybridized to the target sequence.

93. The kit of claim 92, wherein all the component polymers are non-nucleic acid polymers.

94. The kit of claim 92, wherein at least one of the component polymers is a PNA.

95. A composition comprising:

a) at least two component polymers that are not a target sequence to be detected in an assay and wherein the component polymers each comprise interacting groups that form and stabilize a complex; and

;

b) at least one set of donor and acceptor moieties wherein to at least two component polymers is linked at least one moiety from the set such that formation of the composition facilitates transfer of energy between donor and acceptor moieties of the set in a manner that is detectably different as compared to when the component polymers are not complexed; and

wherein the composition is further characterized in that the composition forms a substrate that causes the complex to dissociate when acted upon by an enzyme; and

further provided that at least one of the component polymers is a non-nucleic acid polymer.

96. The composition of claim 95, wherein at least one of the non-nucleic acid polymers is a PNA.

97. A method for determining the presence, absence or quantity of a target molecule, comprising at least one target sequence, in a sample, said method comprising:

a) immobilizing an enzyme to the target sequence of the target molecule; and

b) contacting the sample with at least one complex, said complex comprising;

(i) at least two component polymers wherein the component polymers each comprise interacting groups that form and stabilize the complex; and

(ii) at least one set of donor and acceptor moieties wherein to at least two component polymers is linked at least one moiety from the set such that formation of the composition facilitates transfer of energy between donor and acceptor moieties of the set in a manner that is detectably different as compared to when the component polymers are not complexed; and

wherein the complex is further characterized in that the complex forms a substrate that dissociates when acted upon by the enzyme; and

c) detecting, identifying or quantitating changes in detectable signal attributable to the transfer of energy between the donor and acceptor moieties of a set upon dissociation of the complex and correlating the changes in detectable signal with the presence, absence or quantity of the target molecule of interest in the sample.

98. The method of claim 97 wherein at least one of the component polymers of the complex is non-nucleic acid polymer.

99. The method of claim 98, wherein at least one of the non-nucleic acid polymers of the complex is a PNA.

100. A method for determining the presence, absence or quantity of a target molecule, comprising at least one target sequence, in a sample using an array and then regenerating an array;

said method comprising;

a) contacting the sample with an array containing at least one complex, said complex comprising;

(i) at least one component probing polymer wherein the probing polymer comprises;

     1) a probing segment that hybridizes to said target sequence; and

     2) one or more interacting groups suitable for the formation of a complex with at least one other component polymer that is not the target sequence;

(ii) at least one component annealing polymer that is not the target sequence and which, at a minimum, comprises one or more interacting groups wherein the interaction of the interacting groups of the component polymers form and stabilize the complex; and

(iii) at least one set of donor and acceptor moieties wherein to each of at least two component polymers is linked at least one moiety from the set such that formation of the complex facilitates transfer of energy between donor and acceptor moieties of the set in a manner that is detectably different as compared to when the component polymers are not complexed or when the probing segment is hybridized to the target sequence;

provided however, that at least one of the component polymers of the complex is a non-nucleic acid polymer; and

b) detecting, identifying or quantitating changes in detectable signal attributable to the changes in the transfer of energy between the donor and acceptor moieties of a set upon dissociation of the complex or upon the hybridization of the probing segment to the target sequence and correlating the change in signal with the presence, absence or quantity of the target molecule in the sample, wherein;

(i) at least one of the component polymers of the complex is one of at least two immobilized or tethered complexes on the array with which the sample of interest is contacted;

(ii) the complex dissociates to release at least one common labeled component polymer into solution;

(iii) the common labeled component polymer is released from two or more of the different immobilized or tethered complexes of an array; and

(c) regenerating complexes on the surface by;

a) removing any hybridized target molecules from the surface; and

b) contacting the surface with a quantity of the common labeled component polymer as is necessary to regenerate all the complexes of the array.

101. The method of claim 100, wherein the common labeled component polymer is a PNA polymer labeled with a quencher moiety.

102. The method of claim 100, wherein at least two of the immobilized or tethered component polymers are PNA polymers.