High throughput assay system

Abstract

The present invention relates to compositions, apparatus and methods useful for concurrently performing multiple, high throughput, biological or chemical assays, using repeated arrays of probes. A combination of the invention comprises a surface, which comprises a plurality of test regions, at least two of which, and in a preferred embodiment, at least twenty of which, are substantially identical, wherein each of the test regions comprises an array of generic anchor molecules. The anchors are associated with bifunctional linker molecules, each containing a portion which is specific for at least one of the anchors and a portion which is a probe specific for a target of interest. The resulting array of probes is used to analyze the presence or test the activity of one or more target molecules which specifically interact with the probes. In a preferred embodiment, a sample to be tested is subjected to a nuclease protection procedure before it is contacted with a combination of the invention.

What is claimed is:  
     
         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 target specificities,  
   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 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 detection linkers with a specific reporter reagent which comprises an enzyme that generates a chemiluminescent signal.