Methods for detection of a target nucleic acid by capture using multi-subunit probes

A method of generating a signal indicative of the presence of a target nucleic acid sequence in a sample, comprising

• a) forming a detection complex by incubating a sample comprising a target nucleic acid sequence, an upstream primer, and a downstream probe, wherein said downstream probe comprises a first and a second subunit, and wherein said first subunit of said probe comprises a binding moiety, and binding said probe to said target nucleic acid sequence,
• b) subjecting said detection complex to a nucleic acid polymerization activity under conditions which permit extension of said upstream primer by polymerization of a nucleic acid strand, and displacement of said first subunit of said probe from said target nucleic acid sequence by said nucleic acid strand, such that said first subunit dissociates from said second subunit of said probe and is released to generate a signal,
• wherein said binding is performed at a binding temperature and said first subunit of said probe does not dissociate from said second subunit of said probe when not bound to said target nucleic acid sequence at or below said binding temperature, and
• wherein said polymerization is performed at a polymerization temperature and said first subunit of said probe does not dissociate from said second subunit of said probe when not displaced by said nucleic acid strand at or below said polymerization temperature, and
• wherein generation of said signal is indicative of the presence of a target nucleic acid sequence in said sample.

A method of detecting or measuring a target nucleic acid sequence comprising the steps of:

• a) forming a detection complex by incubating a sample comprising a target nucleic acid sequence with a probe, wherein said probe comprises at least a first and a second subunit, and wherein said first subunit of said probe comprises a binding moiety, and binding said probe to said target nucleic acid sequence to dissociate said first subunit of said probe from said second subunit of said probe to generate a signal,
• wherein said binding is performed at a binding temperature and said first subunit of said probe does not dissociate from said second subunit of said probe when not bound to said target nucleic acid sequence at or below said binding temperature;
• and
• b) detecting and/or measuring the amount of said released first subunit captured by binding of said binding moiety to a capture element on a solid support as an indication of the presence of the target sequence in the sample.

A method of detecting or measuring a target nucleic acid sequence comprising the steps of:

• a) forming a detection complex by incubating a sample comprising a target nucleic acid sequence, an upstream primer, and a downstream probe, wherein said probe comprises at least a first and a second subunit, and said first subunit of said probe comprises a binding moiety, and binding said probe to said target nucleic acid sequence,
• b) subjecting said detection complex to a nucleic acid polymerization activity under conditions which permit extension of said upstream primer by polymerization of a nucleic acid strand, and displacement of said first subunit of said probe from said target nucleic acid by said nucleic acid strand, such that said first subunit dissociates from said second subunit of said probe and is released to generate a signal,
• wherein said binding is performed at a binding temperature and said first subunit of said probe does not dissociate from said second subunit of said probe when not bound to said target nucleic acid sequence at or below said binding temperature,
• wherein said polymerization is performed at a polymerization temperature and said first subunit of said probe does not dissociate from said second subunit of said probe when not displaced by said nucleic acid strand, at or below said polymerization temperature, and
• detecting and/or measuring the amount of said released first subunit captured by binding of said binding moiety to a capture element on a solid support, as an indication of the presence of the target sequence in the sample.

A polymerase chain reaction process for detecting a target nucleic acid sequence in a sample comprising:

• (a) providing a detection complex comprising a probe, wherein said probe comprises at a first and a second subunit, and wherein said first subunit of said probe comprises a binding moiety, a set of oligonucleotide primers wherein a first primer contains a sequence complementary to a region in one strand of said target nucleic acid sequence and primes the synthesis of a complementary DNA strand, and a second primer contains a sequence complementary to a region in a second strand of the target nucleic acid sequence and primes the synthesis of a complementary DNA strand;
• and
• b) amplifying the target nucleic acid sequence employing a nucleic acid polymerase as a template-dependent polymerizing agent under conditions which are permissive for PCR cycling steps of (i) annealing of primers required for amplification to a template nucleic acid sequence contained within said target nucleic acid sequence, (ii) extending the primers wherein said nucleic acid polymerase synthesizes a primer extension product, and thereby dissociates said first subunit of said probe from at least said second subunit of said probe thereby creating detectable, released labeled first subunits of said probe;
• wherein said amplification is performed at an amplification temperature and said first subunit of said probe does not dissociate from at least said second subunit of said probe when not bound to said target nucleic acid sequence at or below said amplification temperature;
• and
• c) detecting and/or measuring the amount of released, labeled first subunit of said probe captured by binding of said binding moiety to a capture element on a solid support as an indicator of the presence of the target sequence in the sample.

A polymerase chain reaction process for detecting a target nucleic acid sequence in a sample comprising:

• a) providing a detection complex comprising a probe, wherein said probe comprises a first and a second subunit, and wherein said first subunit of said probe comprises a binding moiety, a set of oligonucleotide primers wherein a first primer contains a sequence complementary to a region in one strand of said target nucleic acid sequence and primes the synthesis of a complementary DNA strand, and a second primer contains a sequence complementary to a region in a second strand of the target nucleic acid sequence and primes the synthesis of a complementary DNA strand;
• and
• b) amplifying the target nucleic acid sequence employing a nucleic acid polymerase as a template-dependent polymerizing agent under conditions which are permissive for PCR cycling steps of (i) annealing of primers required for amplification to a template nucleic acid sequence contained within said target nucleic acid sequence, (ii) extending the primers wherein said nucleic acid polymerase synthesizes a primer extension product, and (iii) displacement of said first subunit of said probe from said target nucleic acid sequence by said primer extension product, such that said first subunit dissociates from said second subunit of said probe thereby creating detectable, released labeled first subunits of said probe;
• wherein said amplification is performed at an amplification temperature and said first subunit of said probe does not dissociate from said second subunit of said probe when not bound to said target nucleic acid sequence, or when not displaced from said target nucleic acid by said primer extension product, at or below said amplification temperature;
• and
• c) detecting and/or measuring the amount of released, labeled first subunit of said probe captured by binding of said binding moiety to a capture element on a solid support as an indicator of the presence of the target sequence in the sample.

A method of forming a detection complex comprising the steps of:

• (a) providing a target nucleic acid sequence,
• (b) providing a probe, wherein said probe comprises a first and a second subunit, and wherein said first subunit of said probe comprises a binding moiety, and
• (c) binding said target nucleic acid sequence, and said probe and;
• wherein said binding is performed at a binding temperature and said first subunit of said probe does not dissociate from said second subunit of said probe when not bound to said target nucleic acid sequence at or below said binding temperature.

A method of forming a detection complex comprising the steps of:

• (a) providing a target nucleic acid sequence,
• (b) providing an upstream primer complementary to said target nucleic acid sequence,
• (c) providing a probe, wherein said probe comprises a first and a second subunit, and wherein said first subunit of said probe comprises a binding moiety, and
• (d) binding said target nucleic acid sequence, said upstream primer and said probe and;
• wherein said binding is performed at a binding temperature and said first subunit of said probe does not dissociate from said second subunit of said probe when not bound to said target nucleic acid sequence at or below said binding temperature.