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US 7,001,726 B1Granted
Enzymatically catalysed signal amplification
AssigneeTepnel Medical Limited
InventorsConor Mulrooney, John Douglas Oultram
Life of this patent
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Abstract
The present invention concerns detection of target molecules using methods of enzymatically catalysed amplification of target associated detectable structures.
Claims79
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.
14 more elements
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.
22 more elements
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.
17 more elements
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.
26 more elements
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.
26 more elements
Dependencies are inferred from the claim text.