Enzymatically catalysed signal amplification
View Patent ↗The present invention concerns detection of target molecules using methods of enzymatically catalysed amplification of target associated detectable structures.
1. A method of detecting a target molecule, comprising the steps of:
i) contacting a sample with a locator probe comprising a binding moiety specific for the target molecule and an amplification nucleic acid sequence to produce a target molecule-locator probe complex;
ii) producing an amplification structure bound to the complex by performing one or more times an amplification step of treating the sample and locator probe with:
a) a single stranded amplification template comprising:
i) arranged in a 5′ to 3′ direction:
a) an extension nucleic acid sequence;
b) a hybridisation nucleic acid sequence complementary to the amplification nucleic acid sequence having substantially the same sequence as the extension nucleic acid sequence; and
c) an amplification moiety, being limited in all but the final repeat to a nucleic acid sequence; and
ii) optionally comprising at least one signal moiety being other than a nucleic acid sequence;
b) a polymerising agent which extends the 3′ terminus of the amplification nucleic acid sequence by synthesising a complementary strand to the extension nucleic acid sequence of the amplification template;
c) a separating agent which removes sufficient of the extension nucleic acid sequence of the amplification template when hybridised to the complementary strand to allow subsequent hybridisation of the hybridisation nucleic acid sequence of the amplification template to the complementary strand; and
d) the reagents and conditions necessary to effect the action of the polymerising agent and separating agent to allow the extension of the 3′ terminus of the amplification nucleic acid sequence by the synthesis of a plurality of sequences complementary to the extension nucleic acid sequence of the amplification template;
iii) detecting any bound amplification template from the amplification step or steps; and
iv) correlating the results of the detection step with the presence of the target molecule.
2. A method for detecting target molecule according to claim 1 , wherein the separating agent is a 5′ double stranded exonuclease, the activity against which the hybridisation nucleic acid sequence is protected.
3. A method for detecting a target molecule comprising the steps of:
i) contacting a sample with a locator probe comprising a binding moiety specific for the target molecule and an amplification nucleic acid sequence to produce a target molecule-locator probe complex;
ii) producing an amplification structure bound to any complex produced in the preceding step by performing one or more times the amplification step of treating the sample and locator probe with:
a) a single stranded first amplification template comprising:
i) arranged in a 5′ to 3′ direction:
a) an extension nucleic acid sequence;
b) a hybridisation nucleic acid sequence complementary to the amplification nucleic acid sequence having a substantially different sequence to the extension nucleic acid sequence; and
c) an amplification moiety, being limited in all but the final repeat to a nucleic acid sequence; and
ii) optionally comprising at least one signal moiety being other than a nucleic acid sequence;
b) a single stranded second amplification template comprising:
i) arranged in a 5′ to 3′ direction'
a) an extension nucleic acid sequence comprising the hybridisation nucleic acid sequence of the first amplification template;
b) a hybridisation nucleic acid sequence comprising the extension nucleic acid sequence of the first amplification template; and
c) an amplification moiety, being limited in all but the final amplification step to a nucleic acid sequence; and
ii) optionally comprising at least one signal moiety being other than a nucleic acid sequence;
c) a polymerising agent which extends the 3′ terminus of the amplification nucleic acid sequence by synthesising a complementary strand to the extension nucleic acid sequence of the first and second amplification templates;
d) a separating agent which removes sufficient of the extension nucleic acid sequence of the first and second amplification templates when hybridised to the complementary strand to allow subsequent hybridisation of the hybridisation nucleic acid sequence of the first and second amplification templates to the complementary strand; and
e) the reagents and conditions necessary to effect the action of the polymerising agent and separating agent to allow the extension of the 3′ terminus of the amplification nucleic acid sequence by the synthesis of a plurality of sequences complementary to the extension nucleic acid sequences of the first and second amplification templates;
iii) detecting any bound first and/or second amplification template from the amplification step or steps; and
iv) correlating the results of the detection step (iii) with the presence of the target molecule.
4. A method of detecting a target molecule according to claim 3 , wherein the separating agent is a 5′ double-stranded exonuclease, the activity against which the hybridisation nucleic acid sequence of said the first amplification template and the hybridisation nucleic acid sequence of the second amplification template are protected.
5. A method for detecting a target molecule comprising the steps of:
i) contacting a sample with a locator probe comprising a binding moiety specific for the target molecule and an amplification nucleic acid sequence to produce a target molecule-locator probe complex, the amplification nucleic acid sequence having one or more restriction sites for a restriction endonuclease when hybridised to a complementary strand;
ii) producing an amplification structure bound to any complex by performing one or more times the amplification step of treating the sample and locator probe with:
a) a single stranded amplification template comprising:
i) arranged in a 5′ to 3′ direction:
a) an extension nucleic acid sequence;
b) a hybridisation nucleic acid sequence complementary to the amplification nucleic acid sequence and having substantially the same sequence as the extension nucleic acid sequence; and
c) an amplification moiety, being limited in all but the final amplification step to a nucleic acid sequence; and
ii) optionally comprising at least one signal moiety being other than a nucleic acid sequence;
b) a polymerising agent which extends the 3′ terminus of the amplification nucleic acid sequence by synthesising a complementary strand to the extension nucleic acid sequence of the amplification template;
c) the restriction endonuclease; and
d) the reagents and conditions necessary to:
i) effect the action of the polymerising agent and a separating agent to allow the extension of the 3′ terminus of the amplification nucleic acid sequence by the synthesis of a plurality of sequences complementary to the extension nucleic acid sequence of the amplification template; and
ii) effect dissociation of fragments of nucleic acid strands which have been cut by the restriction endonuclease activity from uncut complementary strands whilst not effecting dissociation of uncut nucleic acid strands from uncut complementary strands;
iii) detecting any bound amplification template from the amplification step or steps; and
iv) correlating the results of the detection step (iii) with the presence of the target molecule.
6. A method according to claim 5 , the amplification nucleic acid sequence and the hybridisation nucleic acid sequence having nucleotide modifications which prevent cleavage by the restriction endonuclease, and the reagents including at least one modified nucleotide which, when incorporated into the complementary strand by the polymerising agent, prevent cleavage of the complementary strand by the restriction endonuclease.
7. A method according to claim 5 , wherein the hybridisation nucleic acid sequence has at least one nucleotide modification which prevents cleavage by the restriction endonuclease, the restriction endonuclease having single stranded nicking activity only.
8. A method according to claim 5 , wherein the method is performed isothermally.
9. A method according to claim 5 , wherein the method is performed at more than one temperature.
10. A method according to claim 1 , wherein the amplification step of step (ii) is performed two or more times.
11. A method for detecting a target molecule comprising the steps of:
i) contacting a sample with a locator probe comprising:
a) a binding moiety specific for the target molecule;
b) an amplification nucleic acid sequence to produce a target molecule-locator probe complex; and
c) optionally comprising a signal moiety being other than a nucleic acid sequence;
ii) producing an amplification structure bound to the complex by performing one or more times the amplification step of treating the complex with:
a) a single stranded amplification template comprising:
i) arranged in a 5′ to 3′ direction:
a) an extension nucleic acid sequence ( 162 , 182 ); and
b) a hybridisation nucleic acid sequence ( 161 , 181 ) complementary to the amplification nucleic acid sequence having substantially the same sequence as the extension nucleic acid sequence; and
ii) optionally comprising at least one signal moiety being other than a nucleic acid sequence;
b) a polymerising agent which extends the 3′ terminus of the amplification nucleic acid sequence by synthesising a complementary strand to the extension nucleic acid sequence of the amplification template;
c) a separating agent which removes sufficient of the extension nucleic acid sequence of the amplification template when hybridised to the complementary strand to allow subsequent hybridisation of the hybridisation nucleic acid sequence of the amplification template to the complementary strand;
d) the reagents and conditions necessary to effect the action of the polymerising agent and separating agent to allow the extension of the 3′ terminus of the amplification nucleic acid sequence by the synthesis of a plurality of sequences complementary to the extension nucleic acid sequence of the amplification template; and
iii) optionally repeating one or more times the steps of treating the amplification structure with:
a) a separating agent which removes the remainder of the hybridisation nucleic acid sequence of the amplification template when hybridised to the complementary strand;
b) an additional locator probe comprising:
i) a hybridisation nucleic acid probe specific to the complementary strand; and
ii) an amplification moiety, being limited in all but the final repeat to a nucleic acid sequence;
to produce a complex; and
c) performing the amplification step, optionally using an amplification template different to that which was previously used;
iv) detecting any bound additional locator probes or amplification template from the amplification step or steps; and
v) correlating the results of the detection step (iv) with the presence of the target molecule.
12. A method for detecting a target molecule comprising the steps of:
i) contacting a sample with a locator probe comprising a binding moiety specific for the target molecule and an amplification nucleic acid sequence to produce a target molecule-locator probe complex;
ii) producing an amplification structure bound to the complex by performing one or more times the amplification step of treating the complex with:
a) a single stranded first amplification template comprising:
i) arranged in a 5′ to 3′ direction:
a) an extension nucleic acid sequence; and
b) a hybridisation nucleic acid sequence complementary to the amplification nucleic acid sequence having substantially the same sequence as the extension nucleic acid sequence; and
ii) optionally comprising at least one signal moiety being other than a nucleic acid sequence;
b) a single stranded second amplification template comprising:
i) arranged in a 5′ to 3′ direction:
a) an extension nucleic acid sequence comprising the first amplification template hybridisation nucleic acid sequence; and
b) a hybridisation nucleic acid sequence comprising the first amplification template extension nucleic acid sequence; and
ii) optionally comprising at least one signal moiety being other than a nucleic acid sequence;
c) a polymerising agent which extends the 3′ terminus of the amplification nucleic acid sequence by synthesising a complementary strand to the extension nucleic acid sequence of the amplification template;
d) a separating agent which removes sufficient of the extension nucleic aid sequence of the first and second amplification templates when hybridised to the complementary strand to allow subsequent hybridisation of the hybridisation nucleic acid sequence of the first and second amplification templates to the complementary strand;
e) the reagents and conditions necessary to effect the action of the polymerising agent and separating agent to allow the extension of the 3′ terminus of the amplification nucleic acid sequence by the synthesis of a plurality of sequences complementary to the extension nucleic acid sequence of the amplification template;
iii) optionally repeating one or more times the steps of treating the amplification structure with:
a) a separating agent which removes the remainder of the hybridisation nucleic acid sequences of the first and second amplification templates when hybridised to the complementary strand;
b) an additional locator probe comprising:
i) a hybridisation nucleic acid probe specific to the complementary strand; and
ii) an amplification moiety, being limited in all but the final repeat to a nucleic acid sequence to produce a complex;
c) performing the amplification step, optionally using an amplification template other that the first or second amplification templates;
iv) detecting any bound additional locator probes or amplification template from the amplification step or steps; and
v) correlating the results of the detection step (iv) with the presence of the target molecule.
13. A method for detecting a target molecule according to claim 11 , wherein the removal of the amplification template is achieved by the use of a 5′ double strand specific exonuclease.
14. A method for detecting a target molecule according to claim 11 , wherein the removal of the amplification template is achieved through the use of elevated temperature.
15. A method for detecting a target molecule according to claim 14 , wherein the locator probe is covalently attached to the target molecule prior to the removal of the amplification template.
16. A method for detecting a target molecule according to claim 1 , prior to said detection step further comprising the amplification step according to steps (ii) and (iii) of claim 11 .
17. A method for detecting a target molecule according to claim 1 , wherein the amplification moiety of the amplification template from the final amplification step comprising a nucleic acid sequence, and prior to the detection step further comprising the amplification step according to steps (ii)–(iii) of a claim 11 .
18. A method for detecting a target molecule according to claim 11 , prior to the detection step further comprising the amplification step according to step (ii) of claim 1 .
19. A method for detecting a target molecule according to claim 11 , the amplification moiety of the locator probe or additional locator probe from the final amplification step comprising a nucleic acid sequence, and prior to the detection step further comprising an amplification step according to step (ii) of a claim 1 .
20. A method for detecting a target molecule according to claim 1 , wherein the step of detecting any bound amplification template further comprises the steps of:
i) treating the sample, locator probe and amplification template or amplification templates with a detection probe which binds specifically to the amplification moiety of the last of the amplification templates; and
ii) detecting any bound detection probe.
21. A method for detecting a target molecule according to claim 12 , wherein the step of detecting any bound amplification template further comprises the steps of:
i) treating the sample, locator probe and amplification template with a detection probe which binds specifically to the amplification moiety of the last of the amplification templates; and
ii) detecting any bound detection probe.
22. A method according to claim 20 , wherein the detection probe has a label which is detected by any one of the group of luminometry, fluorometry, spectrophotometry, and radiometry.
23. A method according to claim 22 , wherein the detection probe is labelled with any one of the group of, FAM (carboxyfluorescein), HEX (hexachlorofluorescein), TET (tetrachlorofluorescein), ROX (carboxy-X-rhodamine), TAMRA (carboxytetramethylrhodamine), JOE (carboxy-4′,5′-dichloro-2′,7′-dimethoxyfluorescein), or with biotin.
24. A method according to claim 1 , wherein the amplification step is performed two or more times, each amplification step being performed using an amplification template having a different extension nucleic acid sequence, hybridisation nucleic acid sequence and amplification moiety to that of the amplification template used in the previous amplification step.
25. A method according to claim 1 , wherein the target molecule to be detected is a nucleic acid sequence and the binding moiety of the locator probe comprising a nucleic acid sequence complementary to the target molecule nucleic acid sequence.
26. A method according to claim 1 , wherein the method is performed using more than one locator probe, each locator probe having the same amplification nucleic acid sequence.
27. A method according to claim 1 , wherein the method further comprises two repeats.
28. A method according to claim 1 , wherein unreacted reagents are removed by washing.
29. A method according to claim 28 , wherein the unreacted reagents are selected from the group of locator probe, amplification template, and detection probe.
30. A method according to claim 3 , wherein the amplification step is performed two or more times.
31. A method according to claim 5 , wherein the amplification step is performed two or more times.
32. A method for detecting a target molecule according to claim 12 , wherein the removal of the amplification template is achieved by the use of a 5′ double strand specific exonuclease.
33. A method for detecting a target molecule according to claim 12 , wherein the removal of the amplification template is achieved through the use of elevated temperature.
34. A method according to claim 21 , wherein the detection probe having a label is detected by any one of the group of luminometry, fluorometry, spectrophotometry, and radiometry.
35. A method according to claim 34 , wherein the detection probe is labelled with any one of the group of, FAM (carboxyfluorescein), HEX (hexachlorofluorescein), TET (tetrachlorofluorescein), ROX (carboxy-X-rhodamine), TAMRA (carboxytetramethylrhodamine), JOE (carboxy-4′,5′-dichloro-2′,7′-dimethoxyfluorescein), or with biotin.
36. A method for detecting a target molecule according to claim 3 , wherein prior to the detection step further comprising the amplification steps (ii) and (iii) of claim 11 .
37. A method for detecting a target molecule according to claim 5 , wherein prior to the detection step further comprising the amplification steps (ii) and (iii) of claim 11 .
38. A method for detecting a target molecule according to claim 1 , wherein prior to the detection step further comprising the amplification steps (ii) and (iii) of claim 12 .
39. A method for detecting a target molecule according to claim 3 , wherein prior to the detection step further comprising the amplification steps (ii) and (iii) of claim 12 .
40. A method for detecting a target molecule according to claim 5 , wherein prior to the detection step further comprising the amplification steps (ii) and (iii) of claim 12 .
41. A method for detecting a target molecule according to claim 12 , wherein prior to the detection step further comprising the amplification step (ii) of claim 1 .
42. A method for detecting a target molecule according to claim 11 , wherein prior to the detection step further comprising the amplification step (ii) of claim 3 .
43. A method for detecting a target molecule according to claim 12 , wherein prior to the detection step further comprising the amplification step (ii) of claim 3 .
44. A method for detecting a target molecule according to claim 11 , wherein prior to the detection step further comprising the amplification step (ii) of claim 5 .
45. A method for detecting a target molecule according to claim 12 , wherein prior to the detection step further comprising the amplification step (ii) of claim 5 .
46. A method for detecting a target molecule according to claim 12 , wherein the amplification moiety of the locator probe or additional locator probe from the final amplification step comprising a nucleic acid sequence, and prior to the detection step further comprising the amplification step (ii) of claim 1 .
47. A method for detecting a target molecule according to claim 11 , wherein the amplification moiety of the locator probe or additional locator probe from the final amplification step comprising a nucleic acid sequence, and prior to the detection step further comprising the amplification step (ii) of claim 3 .
48. A method for detecting a target molecule according to claim 12 , wherein the amplification moiety of the locator probe or additional locator probe from the final amplification step comprising a nucleic acid sequence, and prior to the detection step further comprising the amplification step (ii) of claim 3 .
49. A method for detecting a target molecule according to claim 11 , wherein the amplification moiety of the locator probe or additional locator probe from the final amplification step comprising a nucleic acid sequence, and prior to the detection step further comprising the amplification step (ii) of claim 5 .
50. A method for detecting a target molecule according to claim 12 , wherein the amplification moiety of the locator probe or additional locator probe from the final amplification step comprising a nucleic acid sequence, and prior to the detection step further comprising the amplification step (ii) of claim 5 .
51. A method for detecting a target molecule according to claim 3 , the step of detecting any bound amplification template comprising the steps of:
i) treating the sample, locator probe and amplification template or amplification templates with a detection probe which binds specifically to the amplification moiety of the amplification templates; and
ii) detecting any bound detection probe.
52. A method for detecting a target molecule according to claim 5 , the step of detecting any bound amplification template comprising the steps of:
i) treating the sample, locator probe and amplification template or amplification templates with a detection probe which binds specifically to the amplification moiety of the amplification templates; and
ii) detecting any bound detection probe.
53. A method for detecting a target molecule according to claim 18 , the step of detecting any bound amplification template comprising the steps of:
i) treating the sample, locator probe and amplification template or amplification templates with a detection probe which binds specifically to the amplification moiety of the amplification templates; and
ii) detecting any bound detection probe.
54. A method for detecting a target molecule according to claim 19 , the step of detecting any bound amplification template comprising the steps of:
i) treating the sample, locator probe and amplification template or amplification templates with a detection probe which binds specifically to the amplification moiety of the last of said amplification templates; and
ii) detecting any bound detection probe.
55. A method for detecting a target molecule according to claim 12 , the step of detecting any bound amplification template comprising the steps of:
i) treating the sample, locator probe and amplification template with a detection probe which binds specifically to the amplification moiety of the amplification templates; and
ii) detecting any bound detection probe.
56. A method according to claim 3 , wherein the amplification step is performed two or more times, each amplification step being performed using an amplification template having a different extension nucleic acid sequence, hybridisation nucleic acid sequence and amplification moiety to that of the amplification template used in the previous amplification step.
57. A method according to claim 5 , wherein the amplification step is performed two or more times, each amplification step being performed using an amplification template having a different extension nucleic acid sequence, hybridisation nucleic acid sequence and amplification moiety to that of the amplification template used in the previous amplification step.
58. A method according to claim 11 , wherein the amplification step is performed two or more times, each amplification step being performed using an amplification template having a different extension nucleic acid sequence, hybridisation nucleic acid sequence and amplification moiety to that of the amplification template used in the previous amplification step.
59. A method according to claim 12 , wherein the amplification step is performed two or more times, each amplification step being performed using an amplification template having a different extension nucleic acid sequence, hybridisation nucleic acid sequence and amplification moiety to that of the amplification template used in the previous amplification step.
60. A method according to claim 3 , wherein the target molecule to be detected is a nucleic acid sequence and the binding moiety of the locator probe comprising a nucleic acid sequence complementary to the target molecule nucleic acid sequence.
61. A method according to claim 5 , wherein the target molecule to be detected is a nucleic acid sequence and the binding moiety of the locator probe comprises a nucleic acid sequence complementary to the target molecule nucleic acid sequence.
62. A method according to claim 11 , wherein the target molecule to be detected is a nucleic acid sequence and the binding moiety of the locator probe comprises a nucleic acid sequence complementary to the target molecule nucleic acid sequence.
63. A method according to claim 12 , wherein the target molecule to be detected is a nucleic acid sequence and the binding moiety of the locator probe comprises a nucleic acid sequence complementary to the target molecule nucleic acid sequence.
64. A method according to claim 3 , wherein the method is performed using more than one locator probe, each locator probe having the same amplification nucleic acid sequence.
65. A method according to claim 5 , wherein the method is performed using more than one locator probe, each locator probe having the same amplification nucleic acid sequence.
66. A method according to claim 11 , wherein the method is performed using more than one locator probe, each locator probe having the same amplification nucleic acid sequence.
67. A method according to claim 12 , wherein the method is performed using more than one locator probe, each locator probe having the same amplification nucleic acid sequence.
68. A method according to claim 3 , further comprising two repeats of the amplication step.
69. A method according to claim 5 , further comprising two repeats of the amplication step.
70. A method according to claim 11 , further comprising two repeats of the amplication step.
71. A method according to claim 12 , further comprising two repeats of the amplication step.
72. A method according to claim 3 , wherein unreacted reagents are removed at the end of step (i), each repeat, of the amplication step or detection step by washing.
73. A method according to claim 72 , wherein the unreacted reagents are selected from the group of locator probe, amplification template, and detection probe.
74. A method according to claim 5 , wherein unreacted reagents are removed at the end of step (i), each repeat, of the amplication step or detection step by washing.
75. A method according to claim 74 , wherein the unreacted reagents are selected from the group of locator probe, amplification template, and detection probe.
76. A method according to claim 11 , wherein unreacted reagents are removed at the end step (i), each repeat, of the amplication step or detection step by washing.
77. A method according to claim 76 , wherein the unreacted reagents are selected from the group of locator probe, amplification template, and detection probe.
78. A method according to claim 12 , wherein unreacted reagents are removed at the end of step (i), each repeat, of the amplication step or detection step by washing.
79. A method according to claim 78 , wherein the unreacted reagents are selected from the group of locator probe, amplification template, and detection probe.