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High throughput assay system

  • US 20050026193A1
  • Filed: 06/14/2004
  • Published: 02/03/2005
  • Est. Priority Date: 07/02/1998
  • Status: Active Grant
First Claim
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1. 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 and which binds to said target(s), exposing the sample to a nuclease effective to digest single stranded nucleic acid, and 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 loci of oligonucleotide anchors, each anchor in association with iii) a bifunctional linker which has a first portion that is specific for the oligonucleotide anchor, and a second portion that comprises a probe which is specific for said nuclease protection fragment(s), under conditions effective for said nuclease protection fragment(s) to bind to said combination, wherein at least one locus in at least one of said regions is a “

  • mixed locus,”

    which comprises about 2 to about 4 different anchors, each having a specificity for a different bifunctional linker, and wherein two or more of said different anchors located in said mixed locus are each in association with about 2 to about 4 different bifunctional linkers, having different nuclease protection fragment, and thus different target, specificities, hybridizing said bound nuclease protection fragments with specific detection linkers, at least one of which is a “

    blocked”

    detection linker and wherein at least one of said detection linkers is specific for a first protection fragment which is associated with a first of said about 2 to about 4 different bifunctional linkers, detecting said detection linkers with a specific reporter reagent which comprises an enzyme that generates a chemiluminescent signal;

    stopping said signal by adjusting the pH of the reaction mixture, hybridizing said bound protection fragments with specific detection linkers, at least one of which is a “

    blocked”

    detection linker and wherein at least one of said detection linkers is specific for a second protection fragment which is associated with a second of said about 2 to about 4 different bifunctional linkers, and detecting said desection linkers with a specific reporter reagent which comprises an enzyme that generates a chemiluminescent signal.

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