Methods and compositions for enhancing detection in determinations employing cleavable electrophoretic tag reagents
First Claim
1. A method for isolating a signal produced by a detectable moiety of a released predetermined portion of a probe from signal obtained from intact probe and from other fragments thereof, the method comprising:
- (a) combining a probe which comprises a first predetermined portion which is releasable and includes a detectable moiety, and a second portion which includes at least one interactive functionality, with a reagent that potentiates the release of the first portion from the probe;
(b) subjecting the resulting mixture to conditions under which the probe is cleaved, such that the first portion is released from the probe;
(c) contacting the mixture with a capture reagent which binds the interactive functionality, and (d) separating the capture agent or the released predetermined first portion from the mixture.
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Accused Products
Abstract
Probe sets for the multiplexed detection of the binding of, or interaction between, one or more ligands and target antiligands are provided. Detection involves the release of identifying tags as a consequence of target recognition. The probe sets include electrophoretic tag probes or e-tag probes, comprising a detection region and a mobility-defining region, both linked to a target-binding moiety. In a multiplexed assay, different released e-tag reporters may be separated and detected providing for target identification. The probes comprise interactive functionalities adjacent the cleaved portion positioned in the probes such that the interactive functionality does not form part of the e-tag reporters. Also described are biopolymers and nucleosides containing such interactive functionalities.
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Citations
48 Claims
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1. A method for isolating a signal produced by a detectable moiety of a released predetermined portion of a probe from signal obtained from intact probe and from other fragments thereof, the method comprising:
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(a) combining a probe which comprises a first predetermined portion which is releasable and includes a detectable moiety, and a second portion which includes at least one interactive functionality, with a reagent that potentiates the release of the first portion from the probe;
(b) subjecting the resulting mixture to conditions under which the probe is cleaved, such that the first portion is released from the probe;
(c) contacting the mixture with a capture reagent which binds the interactive functionality, and (d) separating the capture agent or the released predetermined first portion from the mixture. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9)
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10. A method for detecting binding of or interaction between a target binding agent and any of a plurality of probes, comprising:
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(a) subjecting a mixture comprising the target binding agent and the probes to conditions for interaction therebetween, wherein each probe comprises (i) a cleavable reporter group, comprising a detectable moiety and having a mobility, when cleaved, that is unique to the probe among the plurality of probes, and (ii) at least one interactive functionality adjacent the cleavable reporter group;
(b) subjecting the mixture to conditions under which the cleavable moiety is cleaved to release the reporter groups, wherein said cleavage is dependent on said interaction between the target binding agent and any of the plurality of probes;
and wherein the interactive functionality enables the isolation of signal produced by the detectable moieties of the released reporter groups from signal obtained from intact probes and from other fragments thereof, (c) separating the released reporter groups by their differences in mobility; and
(d) detecting the detectable moieties of released reporter groups, to determine binding of or interaction between the target binding agent and each probe. - View Dependent Claims (11, 12, 13, 14, 15, 16, 17, 18)
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19. A kit for use in detecting the presence and/or amount of each of a plurality of target molecules, comprising in packaged combination:
a plurality of probes, each capable of target-specific binding to a binding site on a selected target molecule, and each having (i) a unique cleavable reporter group, having a detectable reporter moiety and a unique electrophoretic mobility which allows the reporter group to be uniquely identified among all other reporter groups associated with the plurality of probes, and (ii) at least one interactive functionality adjacent the reporter group, wherein the interactive functionality is effective in isolating the signal produced by released reporter groups from the signal produced by intact probes and other fragments thereof. - View Dependent Claims (20, 21, 22, 23)
- 24. A synthetic biopolymer or nucleoside having conjugated thereto a moiety having the formula Ya:
- 34. A compound of the formula:
Specification