Methods for determining receptor-ligand binding using probe arrays

• US 6,124,102 A
• Filed: 04/21/1998
• Issued: 09/26/2000
• Est. Priority Date: 06/07/1989
• Status: Expired due to Fees

• Current Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Original Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Inventors: Solas, Dennis W., Holmes, Christopher P., Winkler, James L., Fodor, Stephen P. A., Stryer, Lubert
• Primary Examiner(s): Horlick, Kenneth R.
• Assistant Examiner(s): Siew, Jeffrey
• Application Number:
  US09/063,936
• Time in Patent Office: 889 Days
• Field of Search: 435/6, 435/5, 435/7.1, 536/23.1, 536/22.1, 536/24.3, 536/24.31, 536/24.32, 536/24.33, 345/1, 345/47, 345/185
• US Class Current: 506/9
• CPC Class Codes: B01J 19/0046 Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays synthesis methods per se C40B50/00
• CPC Class Codes: B01J 2219/00315 Microtiter plates
• CPC Class Codes: B01J 2219/00432 Photolithographic masks
• CPC Class Codes: B01J 2219/00434 Liquid crystal masks
• CPC Class Codes: B01J 2219/00436 Maskless processes
• CPC Class Codes: B01J 2219/00459 Beads
• CPC Class Codes: B01J 2219/00468 by manipulation of individual beads
• CPC Class Codes: B01J 2219/00475 Sheets
• CPC Class Codes: B01J 2219/005 Beads
• CPC Class Codes: B01J 2219/00527 Sheets
• CPC Class Codes: B01J 2219/00529 DNA chips
• CPC Class Codes: B01J 2219/00531 essentially square
• CPC Class Codes: B01J 2219/00585 Parallel processes
• CPC Class Codes: B01J 2219/0059 Sequential 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 2219/00612 the surface being inorganic
• CPC Class Codes: B01J 2219/00617 by chemical means
• CPC Class Codes: B01J 2219/00626 Covalent
• CPC Class Codes: B01J 2219/00637 : by coating it with another layer
• CPC Class Codes: B01J 2219/00648 : by the use of solid beads
• CPC Class Codes: B01J 2219/00659 : Two-dimensional arrays
• CPC Class Codes: B01J 2219/00689 : using computers
• CPC Class Codes: B01J 2219/00695 : Synthesis control routines, e.g. using computer programs
• CPC Class Codes: B01J 2219/00711 : Light-directed synthesis
• CPC Class Codes: B01J 2219/00722 : Nucleotides
• CPC Class Codes: B01J 2219/00725 : Peptides
• CPC Class Codes: B82Y 10/00 : Nanotechnology for information processing, storage or transmission, e.g. quantum computing or single electron logic
• CPC Class Codes: B82Y 30/00 : Nanotechnology for materials or surface science, e.g. nanocomposites
• CPC Class Codes: C07B 2200/11 : Compounds covalently bound to a solid support
• CPC Class Codes: C07C 229/14 : to carbon atoms of carbon skeletons containing rings
• CPC Class Codes: C07C 229/16 : to carbon atoms of hydrocarbon radicals substituted by amino or carboxyl groups, e.g. ethylenediamine-tetra-acetic acid, iminodiacetic acids
• CPC Class Codes: C07D 263/44 : Two oxygen atoms
• CPC Class Codes: C07D 317/62 : with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to atoms of the carbocyclic ring
• CPC Class Codes: C07H 19/073 : with 2-deoxyribosyl as the saccharide radical
• CPC Class Codes: C07H 19/173 : with 2-deoxyribosyl as the saccharide radical
• CPC Class Codes: C07H 21/04 : with deoxyribosyl as saccharide radical
• CPC Class Codes: C07K 1/042 : characterised by the nature of the carrier
• CPC Class Codes: C07K 1/045 : using devices to improve synthesis, e.g. reactors, special vessels
• CPC Class Codes: C07K 1/047 : Simultaneous synthesis of different peptide species; Peptide libraries
• CPC Class Codes: C07K 1/062 : for alpha- or omega-carboxy functions
• CPC Class Codes: C07K 17/06 : attached to the carrier via a bridging agent
• CPC Class Codes: C07K 17/14 : Peptides being immobilised on, or in, an inorganic carrier
• CPC Class Codes: C07K 7/06 : having 5 to 11 amino acids
• CPC Class Codes: C12Q 1/6809 : Methods for determination or identification of nucleic acids involving differential detection
• 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 1/6874 : involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C40B 40/06 : Libraries containing nucleotides or polynucleotides, or derivatives thereof
• CPC Class Codes: C40B 40/10 : Libraries containing peptides or polypeptides, or derivatives thereof
• CPC Class Codes: C40B 60/14 : for creating libraries
• CPC Class Codes: C40B 80/00 : Linkers or spacers specially adapted for combinatorial chemistry or libraries, e.g. traceless linkers or safety-catch linkers
• CPC Class Codes: G01N 15/1433 : using image recognition
• CPC Class Codes: G01N 21/253 : for batch operation, i.e. multisample apparatus analytical automats G01N35/00
• CPC Class Codes: G01N 21/6428 : Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" in vivo A61B5/00; immunoassay G01N33/53
• CPC Class Codes: G01N 21/6452 : Individual samples arranged in a regular 2D-array, e.g. multiwell plates
• CPC Class Codes: G01N 21/6458 : Fluorescence microscopy fluorescence microscopes per se G02B21/0076 and G02B21/16
• CPC Class Codes: G03F 7/00 : Photomechanical, e.g. photolithographic, production of textured or patterned surfaces, e.g. printing surfaces; Materials therefor, e.g. comprising photoresists; Apparatus specially adapted therefor using photoresist structures for special production processes, see the relevant places, e.g. B44C, H01L, e.g. H01L21/00, H05K
• CPC Class Codes: G03F 7/0045 : with organic non-macromolecular light-sensitive compounds not otherwise provided for, e.g. dissolution inhibitors
• CPC Class Codes: G03F 7/265 : Selective reaction with inorganic or organometallic reagents after image-wise exposure, e.g. silylation
• CPC Class Codes: G03F 7/38 : Treatment before imagewise removal, e.g. prebaking G03F7/265 takes precedence
• CPC Class Codes: G11C 13/0014 : comprising cells based on organic memory material
• CPC Class Codes: G11C 13/0019 : comprising bio-molecules
• CPC Class Codes: Y02P 20/55 : Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
• CPC Class Codes: Y10S 435/969 : Multiple layering of reactants
• CPC Class Codes: Y10S 435/973 : Simultaneous determination of more than one analyte
• CPC Class Codes: Y10S 436/807 : Apparatus included in process claim, e.g. physical support structures
• CPC Class Codes: Y10S 436/809 : Multifield plates or multicontainer arrays

Abstract

The invention provides a method for analyzing the binding between a receptor and ligand using a probe array. The method generally involves contacting a labeled receptor with a substrate that includes a plurality of known locations to permit labeled receptor/ligand complexes to form. Intensity values based upon signal generated from bound labeled receptor are determined for a plurality of pixels within each of the known locations. A subset of pixels within each known location that fall within a statistical norm of intensity values for all the pixels within the known location is identified using a computer and then utilized to determine relative binding.

Claims

1. A method of determining the tendency of a receptor to bind to a ligand comprising:

(a) exposing a labelled receptor to a substrate, said substrate comprising a plurality of ligands at known locations;

(b) at a plurality of pixels within each of the known locations, determining an intensity value for the amount of signal generated from within each pixel; and

(c) for each known location, identifying a subset of pixels that fall within a statistical norm of intensity values for all pixels within the known location; and

(d) determining relative binding of the receptor to the ligands from the subset of pixels

wherein steps (b)-(d) are performed by a digital computer.

2. The method of claim 1, wherein the ligands are nucleic acids.

3. The method of claim 1, wherein the ligands are peptides.

4. The method of claim 1, wherein the receptor is a nucleic acid.

5. The method of claim 1, wherein the receptor is a peptide.

6. The method of claim 4, wherein the nucleic acid is RNA.

7. The method of claim 1, wherein each pixel occupies less than half of the area of the known location in which the pixel is located.

8. The method of claim 1, wherein each pixel occupies less than 10 percent of the area of the known location in which the pixel is located.

9. The method of claim 1, wherein each pixel occupies less than 1 percent of the area of the known location in which the pixel is located.

10. The method of claim 1, further comprising plotting number of pixels versus signal intensity for a known location.

11. The method of claim 1, wherein the subset lacks those pixels having the highest intensity values.

12. The method of claim 10, further comprising displaying the plot on a video display.

13. The method of claim 1, wherein the known locations have dimensions of 500 microns by 500 microns.

14. The method of claim 1, wherein the label is a fluorophore and the signal detected is fluorescence.

15. The method of claim 1, wherein relative binding is determined from an average of the pixels in the subset.

16. The method of claim 10, further comprising:

determining from the plot the number of pixels within a plurality of intensity bands, each intensity band having the same bandwidth but being centered at a different intensity, and wherein the subset includes those pixels in the intensity band having the highest number of pixels.