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

Abstract

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, by one or more of the following steps: 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.

What is claimed is: 
     
         1 . A method of enhancing the sensitivity of detection of a nucleic acid target, in solution, by formation of a duplex between a nucleic acid target and the target binding domain of an oligonucleotide probe, said probe having a target binding domain and a complementary closing domain capable of forming a duplex with the target binding domain, and a joining region between the target binding domain and the closing domain, the method comprising:
 attaching the probe to the outer surface of a bead at a lateral density on the surface exceeding a predetermined minimum, wherein the predetermined minimum is determined such that the distance between the occupied probe site and the nearest unoccupied site permits target release and re-capture on a time-scale less than time for target escape into solution.   
     
     
         2 . The method of  claim 1  wherein the assay is performed under conditions favoring retention of un-duplexed target, or substantially within, a region of polarization associated with the interface between the bead and the solution. 
     
     
         3 . A method of enhancing the sensitivity of detection of a nucleic acid target, in solution, by formation of a duplex between a nucleic acid target and the target binding domain of an oligonucleotide probe, said probe having a target binding domain and a complementary closing domain capable of forming a duplex with the target binding domain, and a joining region between the target binding domain and the closing domain, the method comprising:
 attaching the probe to the outer surface of a bead at a lateral density on the surface exceeding a predetermined minimum, wherein the predetermined minimum is selected so as to provide a large local excess of probes over targets in the vicinity of the bead surface such that there is an effective reduction in the disassociation constant as a result of target successively contacting multiple probes on the bead surface.   
     
     
         4 . 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.   
     
     
         5 . The method of  claim 4  wherein the detection of elongation is by detecting a signal associated with labeled nucleotides incorporated into the elongated probe. 
     
     
         6 . The method of  claim 5  wherein labeled dNTPs or ddNTPs are incorporated into the elongated probe. 
     
     
         7 . The method of  claim 4  wherein detection of elongation is performed by conducting a thermal stability analysis, by cycling to a temperature above the de-annealing temperature of non-elongated duplexes and then monitoring probe fluorescence to determine probe-target-associated fluorescence. 
     
     
         8 . A method of expanding the operating range of stringencies of a multiplexed format of nucleic acid analysis, wherein a 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 are 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 a probe bound to the target and elongated, the method comprising: stabilizing the duplex by elongating the 3′ terminal ends of certain probes which have a nucleotide in the target sequence aligned with a complementary nucleotide in the target binding domain to thereby generating a stable duplex capable of withstanding a wider range of reaction conditions without causing changes in the assay results. 
     
     
         9 . 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. 
   
     
     
         10 . The method of  claim 9  further including increasing the effective concentration of the targets available for binding to the probes by one or more of the following: (i) adjusting the solution's ionic strength to 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. 
     
     
         11 . The method of  claim 8  or  9  wherein the reaction time is selected to reflect the stringency of the imposed conditions, the stringency determining the probability of random formation of a probe-target-enzyme-substrate intermediate state in the formation of elongation product, such that a sufficient number of stable elongated duplex states are capable of being formed. 
     
     
         12 . 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.   
     
     
         13 . The method of any of  claims 1  to  12  wherein the substrate is a microparticle. 
     
     
         14 . The method of any of  claims 1  to  12  wherein the ionic strength of the solution is increased by adding salt. 
     
     
         15 . The method of any of  claims 1  to  12  wherein there are several different types of oligonucleotide probes on each microparticle. 
     
     
         16 . The method of any of  claims 1  to  12  wherein the target binding domain is fully complementary to the target domain. 
     
     
         17 . The method of any of  claims 1  to  12  wherein the oligonucleotide probes and the nucleic acid targets can either be DNA or RNA. 
     
     
         18 . The method of any of  claims 1  to  12  wherein the ionic strength threshold corresponds to a buffer concentration of 50 mM salt.