Probe density self-considerations and elongation of complementary looped probes where probes are attached to a solid phase

A method comprising:

• providing a plurality of oligonucleotide probes bound to a substrate, wherein each probe comprises
• a target binding domain, which is complementary to a target nucleic acid;
• a closing domain with a sequence that is complementary to the sequence of the target binding domain;
• and
• a joining region between the binding domain and the closing domain, which is not complementary to the target nucleic acid;
• wherein the set comprises at least two probes that differ in the sequence of their target binding domain, and wherein the probes are bound to the substrate via the joining region;
• placing the probes in contact with a solution containing target nucleic acids under conditions where a target nucleic acid forms a duplex with the target binding domain of a probe;
• and
• stabilizing the duplex by elongating the 3′ terminal ends of probes that have a nucleotide in the target sequence aligned with a complementary nucleotide in the target binding.

A method comprising:

• providing a plurality of oligonucleotide probes bound to a substrate, wherein each probe comprises
• a target binding domain, which is complementary to a target nucleic acid;
• a closing domain with a sequence that is complementary to the sequence of the target binding domain;
• and
• a joining region between the binding domain and the closing domain, which is not complementary to the target nucleic acid;
• wherein the set comprises at least two probes that differ in the sequence of their target binding domain, and wherein the probes are bound to the substrate via the joining region;
• placing the probes in contact with a solution containing target nucleic acids under conditions that permit binding of the target nucleic acids to a target binding domain so as to form probe-target complexes;
• adjusting assay conditions so as to permit stabilization of probe-target complexes by target-mediated enzymatic elongation;
• and
• detecting the presence of probe-target complexes by monitoring probe fluorescence from the target-associated state of the probe and comparing it to the pre-assay signal.