HOMOGENEOUS MULTIPLEX SCREENING ASSAYS AND KITS
First Claim
1. A highly multiplexed homogeneous in vitro assay to screen a sample for the presence of any of a minimum of six different nucleic acid targets comprisinga) contacting the sample with a set of at least six fluorescent hybridization probes, each one specific for a different one of said nucleic acid targets, whereini) each probe in said set is subdivided into the same number of portions, said number being in the range of from two to four,ii) each portion of each subdivided probe is labeled with a different emitter fluorophore from a panel of at least four spectrally differentiable fluorophores,iii) each probe in said set has a unique color signal pattern, andiv) hybridization of each probe leads to a detectable fluorescence signal change indicative of the color pattern of the labeling of its portions;
- b) stimulating the fluorophores present in the sample; and
c) detecting changes in emissions signals from said fluorophores without spatially segregating the nucleic acid targets from one another to detect the color pattern of one of said probes so as to identify which target is present.
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Accused Products
Abstract
Highly multiplexed homogeneous in vitro screening assays for numerous possible nucleic acid targets, any of which might be present in a sample, utilize fluorescent hybridization probes that are combinatorially coded from a panel of fluorophores by subdividing each probe into portions and differently labeling each portion such that, when portions are combined, each probe has a unique code. The assays may include target amplification and real-time detection. Probe sets and kits containing additional assay reagents may be used to perform the screening assays.
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Citations
23 Claims
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1. A highly multiplexed homogeneous in vitro assay to screen a sample for the presence of any of a minimum of six different nucleic acid targets comprising
a) contacting the sample with a set of at least six fluorescent hybridization probes, each one specific for a different one of said nucleic acid targets, wherein i) each probe in said set is subdivided into the same number of portions, said number being in the range of from two to four, ii) each portion of each subdivided probe is labeled with a different emitter fluorophore from a panel of at least four spectrally differentiable fluorophores, iii) each probe in said set has a unique color signal pattern, and iv) hybridization of each probe leads to a detectable fluorescence signal change indicative of the color pattern of the labeling of its portions; -
b) stimulating the fluorophores present in the sample; and c) detecting changes in emissions signals from said fluorophores without spatially segregating the nucleic acid targets from one another to detect the color pattern of one of said probes so as to identify which target is present. - View Dependent Claims (2, 3, 5, 6, 7, 8, 9, 10, 11, 12, 23)
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4. (canceled)
- 13. A kit of reagents for use in a highly multiplexed in vitro assay to screen a sample for the presence of any of a minimum of six different nucleic acid targets comprising a set of fluorescent hybridization probes including at least six combinatorially coded fluorescent hybridization probes, each specific for a different nucleic acid target, whose hybridization to their targets can be detected in said assay by a fluorescent signal change indicative of their labeling, wherein the probes in the set are combinatorially coded with up to four different emitter fluorophores from a panel of at least four spectrally differentiable fluorophores by labeling portions of each probe with different emitter fluorophores.
Specification