Probe Density Considerations and Elongation of Self-Complementary Looped Probes Where Probes Are Attached to a Solid Phase

In a multiplexed assay method carried out in solution, wherein the solution contains nucleic acid targets and, wherein several different types of oligonucleotide probes, each type having a different sequence in a target binding domain, are bound to a substrate and used to detect the nucleic acid targets, and wherein said probes have a target binding domain and a complementary closing domain capable of forming a duplex with the target binding domain (and when the duplex is formed, no signal is emitted by the probe), and a joining region between the target binding domain and the closing domain, and wherein the same signal is generated by a probe in a non-duplex as by a probe bound to the target or by an elongated probe bound to the target, the method comprising:

• placing the probes in contact with the targets under conditions suitable for capture of the target and formation of a probe-target duplex,
• generating conditions suitable for enzyme-mediated probe elongation wherein the 3′ terminal end of the probe is elongated if a nucleotide in the target sequence which is aligned with the 3′ terminal end of the target binding domain is complementary;
• and
• detecting the increase in cumulative signal associated with each type of probe, resulting from probe elongation.

A method of conducting a multiplexed format of nucleic acid analysis, wherein a solution containing nucleic acid targets is placed in contact with several different types of oligonucleotide probes, each different type having a different sequence in a region designated as a target binding domain, (and when the duplex is formed, no signal is emitted by the probe), and a joining region between the target binding domain and the closing domain, and wherein the same signal is generated by a probe in a non-duplex as by a probe bound to the target, the method comprising:

• said target binding domain joined to a complementary closing domain through a joining region, the method comprising:
• adjusting assay conditions so as to permit stabilization of probe-target complexes by target-mediated enzymatic elongation;
• and
• detecting capture by monitoring probe fluorescence from the target-associated state of the probe and comparing it to the pre-assay signal.

In a multiplexed assay method carried out in solution, wherein the solution contains nucleic acid targets and, wherein several different types of oligonucleotide probes, each type having a different sequence in a region designated as a target binding domain, are used to detect the nucleic acid targets, said assay method including a method for increasing the effective concentration of the nucleic acid targets at the surface of a bead to which the oligonucleotide probes are bound, comprising:

• adjusting assay conditions so as to increase the effective concentration of the targets available for binding to the probes, by one or more of the following:
• (i) selecting a particular probe density on the surface of the bead;
• (ii) selecting a solution having an ionic strength greater than a threshold;
• (ii) selecting a target domain of a size less than a threshold;
• or (iii) selecting target domains within a specified proximity to a terminal end of the targets.