High throughput assay system

• US 20050026193A1
• Filed: 06/14/2004
• Published: 02/03/2005
• Est. Priority Date: 07/02/1998
• Status: Active Grant

• Current Assignee: HTG Molecular Diagnostics, Inc.
• Original Assignee: High Throughput Genomics, Inc.
• Inventors: Kris, Richard M., Felder, Stephen
• Granted Patent: US 7,659,063 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: B01J 2219/00315 Microtiter plates
• CPC Class Codes: B01J 2219/00527 Sheets
• CPC Class Codes: B01J 2219/00547 Bar codes
• CPC Class Codes: B01J 2219/00585 Parallel processes
• CPC Class Codes: B01J 2219/00596 Solid-phase processes
• CPC Class Codes: B01J 2219/00605 the compounds being directly bound or immobilised to solid supports
• CPC Class Codes: B01J 2219/00608 DNA chips
• CPC Class Codes: B01J 2219/0061 The surface being organic
• CPC Class Codes: B01J 2219/00612 the surface being inorganic
• CPC Class Codes: B01J 2219/00621 by physical means, e.g. trenches, raised areas
• CPC Class Codes: B01J 2219/00626 Covalent
• CPC Class Codes: B01J 2219/0063 Other, e.g. van der Waals forces, hydrogen bonding
• CPC Class Codes: B01J 2219/00644 the porous medium being present in discrete locations, e.g. gel pads
• CPC Class Codes: B01J 2219/00659 Two-dimensional arrays
• CPC Class Codes: B01J 2219/00662 Two-dimensional arrays within two-dimensional arrays
• CPC Class Codes: B01J 2219/00702 Processes involving means for analysing and characterising the products
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 2521/307 Single strand endonuclease
• CPC Class Codes: C12Q 2527/119 : pH
• CPC Class Codes: C12Q 2537/125 : Sandwich assay format
• CPC Class Codes: C12Q 2561/108 : Hybridisation protection assay [HPA]
• CPC Class Codes: C12Q 2563/107 : fluorescence
• CPC Class Codes: C12Q 2565/513 : characterised by the pattern of the arrayed oligonucleotides
• CPC Class Codes: C12Q 2565/519 : characterised by the capture moiety being a single stranded oligonucleotide
• CPC Class Codes: C40B 40/06 : Libraries containing nucleotides or polynucleotides, or derivatives thereof
• CPC Class Codes: C40B 60/14 : for creating libraries
• CPC Class Codes: C40B 70/00 : Tags or labels specially adapted for combinatorial chemistry or libraries, e.g. fluorescent tags or bar codes
• CPC Class Codes: G16B 25/00 : ICT specially adapted for hybridisation; ICT specially adapted for gene or protein expression

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.

Claims

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.