High throughput assay system

A method of detecting at least two nucleic acid targets, comprising

• a) contacting a sample which may comprise said targets with nuclease protection fragments specific for and which bind to said targets, exposing the sample to a nuclease effective to digest remaining single strand nucleic acid, and then contacting the resultant sample with a combination which comprises, before the addition of said sample,
• i) a surface, comprising multiple spatially discrete regions, at least two of which are substantially identical, each region comprising
• ii) at least two different anchors, each in association with
• iii) a bifunctional linker which has a first portion that is specific for the anchor, and a second portion that comprises a probe which is specific for one of said nuclease protection fragments,
• under conditions effective for said nuclease protection fragments to bind to said combination,
• b) contacting said combination and any bound nuclease protection fragments with at least two detection linkers, each of which comprises a first moiety specific for one of said nuclease protection fragments and a second moiety specific for a common reporter reagent, and
• c) detecting said detection linkers.

A method of detecting at least two nucleic acid targets of interest in a sample which may comprise said targets, comprising

• a) incubating said sample with two or more protection fragments under conditions which are effective for hybridization of said protection fragments to said nucleic acids of interest in said sample, wherein each of said protection fragments comprises a common 3′ overhanging sequence which is not specific for said nucleic acids of interest,
• b) subjecting said incubated sample to treatment with one or more nucleases effective for digesting nucleic acid other than the portions of said protection fragments which have hybridized to the nucleic acids of interest and, optionally, the portions of said nucleic acids of interest which have been hybridized,
• c) removing nucleic acid material other than said protection fragments which have hybridized to said nucleic acids of interest, to provide a sample containing the protection fragments, then
• d) contacting said sample containing the protection fragments with a combination which comprises, before the addition of said sample,
• i) a surface, comprising multiple spatially discrete regions, at least two of which are substantially identical, each region comprising
• ii) at least two different anchors, each in association with
• iii) a bifunctional linker which has a first portion that is specific for the anchor, and a second portion that comprises a probe which is specific for one of said protection fragments,
• under conditions effective for said protection fragments to bind to said combination, and
• e) contacting said combination and any bound protection fragments with at least two detection linkers, each of which comprises a first moiety specific for one of said protection fragments and a second moiety specific for said common 3′ overhanging sequence.

A kit useful for the detection of at least one nucleic acid target in a sample, which comprises

• a) at least one nuclease protection fragment specific for at least one of said targets, but not for any of the oligonucleotide anchors in said kit,
• b) a surface, comprising multiple spatially discrete regions, at least two of which are substantially identical, each region comprising at least two different oligonucleotide anchors,
• c) a container comprising at least one bifunctional linker molecule, which has a first portion specific for at least one of said oligonucleotide anchors and a second portion that comprises a probe which is specific for, and in said detection binds to, at least one of said nuclease protection fragments, and
• d) at least one detection linker, which has a first moiety specific for one of said nuclease protection fragments and a second moiety specific for a reporter reagent.

A method of detecting at least one nucleic acid target, comprising contacting a sample which may comprise said target(s) with a nuclease protection fragment(s) specific for said target(s) and exposing the resultant product to a nuclease effective to digest single strand nucleic acid, and then contacting the resultant sample with a combination which comprises, before the addition of said sample,

• i) a surface comprising multiple spatially discrete regions, at least two of which are substantially identical, each region comprising
• ii) at least two different anchors, each in association with
• iii) a bifunctional linker which has a first portion that is specific for the anchor, and a second portion that comprises a probe which is specific for portions of said nucleic acid target(s) which are protected by said nuclease protection fragments,
• under conditions effective for said protected portions(s) to bind to said combination,
• b) contacting said combination and any bound protected portion(s) with at least one detection linker, which comprises a first moiety specific for one of said bound protected portion (s) and a second moiety specific for a reporter reagent, and
• c) detecting said detection linker.

A method of detecting at least one target, comprising

• a) contacting a sample which may comprise said target(s) with a combination which comprises, before the addition of said sample,
• i) a surface, comprising multiple spatially discrete regions, at least two of which are substantially identical, each region comprising
• ii) at least two different loci of anchors, the anchors at each locus each in association with
• iii) a bifunctional linker which has a first portion that is specific for the anchor, and a second portion that comprises a probe which is specific for said target(s),
• under conditions effective for said target(s) to bind to said combination, and
• wherein two or more of the anchors located at at least one locus of a region are in association with different bifunctional linkers, having different target specificities.

A kit useful for the detection of at least one nucleic acid target, comprising

• a) at least one nuclease protection fragment specific for said target(s), but not for any of the oligonucleotide anchors in said kit,
• b) a surface, comprising multiple spatially discrete regions, at least two of which are substantially identical, each region comprising at least two different oligonucleotide anchors,
• c) a container comprising at least one bifunctional linker molecule, which has a first portion specific for at least one of said oligonucleotide anchors and a second portion that comprises a probe which is specific for, and in said detection binds to, at least one of said nuclease protection fragments,
• d) at least one detection linker, which has a first moiety specific for one of said nuclease protection fragments and a second moiety specific for a reporter reagent, and
• e) one or more nucleases effective for digesting single strand nucleic acid and/or the RNA strand of a DNA/RNA duplex.