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.

Description

BACKGROUND [0001] Molecular Stringency in Multiplexed Assays—A self-complementary oligonucleotide capture probe in a “looped” configuration may be used to adjust molecular stringency in an assay. Assay stringency relates to the positive results produced by an assay, such that high stringency conditions generate relatively fewer positive results than lower stringency conditions. Looped probes are described in WO 01 / 98765, entitled: “Multianalyte Molecular Analysis Using Application-Specific Random Particle Arrays” and U.S. Pat. No. 6,361,945 (assigned to Gen Probe, Inc.). Such a probe consists of a 5′-terminal subsequence and a complementary 3′-terminal subsequence, tethered by an unrelated subsequence, the two terminal subsequences capable of forming a duplex (“stem”), and the tether forming a loop, and either the 5′-terminal subsequence of the 3′-terminal subsequence capable of forming a duplex with a target nucleic acid. The probe may be attached to a solid phase such as an encode...

AI Technical Summary

Benefits of technology

[0004] Disclosed are methods of enhancing detection sensitivity and expanding the range of stringencies compatible with detection of specific targets, especially where there is a low target concentration, as typically encountered in, e.g., the detection of genomic material from infectious agents (see e.g., Chen, Martinez & Mulchandani, “Molecular Beacons: A Real-Time Polymerase Chain Reaction Assay for Detecting Salmonella,” Analytical Biochemistry 280, 166-172 (2000)). Also disclosed is a method of enhancing detection sensitivity by providing for target capture to a self complementary (“looped”) probe, anchored, preferably by its loop subsequence, at a lateral density of at least a certain preset minimum, on a solid phase carrier, preferably a microparticle (“bead”).

[0007] (i) enhance the sensitivity of target detection—by converting the C state of the probe into the eOT state; even under conditions of extreme stringency, selected, for example, to ensure enzymatic efficiency particularly in homogeneous assay designs ((see e. g. “Transcription Amplification System with Integrated Multiplex Detection; Functional Integration of Capture, Amplification and Multiplex Detection” filed Sep. 2, 2005; Ser. No. 11 / 218838, incorporated by reference), this conversion ensures high detection sensitivity by accumulation of elongation product, over an extended period of time, by way of random fluctuations of the closed into the open (or related reactive intermediate, see below) state permitting target capture and enzyme-catalyzed elongation; to the extent that the eOT state is irreversible under prevailing assay conditions, this conversion is akin to a digital “ON” signal;

Problems solved by technology

In this competitive equilibrium, low stringency, favoring the closed state, will reduce the likelihood of formation of the open (or other intermediate state, see Detailed Description, below) required for probe-target duplex formation, thereby diminishing the detection sensitivity.

Conversely, high stringency, favoring the open state, will likewise reduce the likelihood of target capture—by reducing the stability of any probe-target duplex—while producing indiscriminate fluorescence, independent of captured target, thereby reducing specificity.

Thus, identifying the optimal stringency range in a multiplexed assay thus becomes increasingly difficult with each different probe added, given the dispersion of the melting curve profiles of a set of different probe-target complexes under given assay conditions.

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