SUBSTRATES COMPRISING POLYNUCLEOTIDE MICROARRAYS

• US 20030012695A1
• Filed: 11/24/1998
• Published: 01/16/2003
• Est. Priority Date: 06/17/1994
• Status: Active Grant

• Current Assignee: Patrick O. Brown, Tidhar Dari Shalon
• Original Assignee: Patrick O. Brown, Tidhar Dari Shalon
• Inventors: SHALON, TIDHAR DARI, BROWN, PATRICK O.
• Granted Patent: US 7,442,499 B2
• Time in Patent Office: Days
• US Class Current: 422/68.1
• 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 2219/00317 Microwell devices, i.e. having large numbers of wells
• CPC Class Codes: B01J 2219/00367 capillary
• CPC Class Codes: B01J 2219/00475 Sheets
• CPC Class Codes: B01J 2219/00527 Sheets
• 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 2219/00612 the surface being inorganic
• 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 2219/00637 by coating it with another layer
• CPC Class Codes: B01J 2219/00659 Two-dimensional arrays
• CPC Class Codes: B01J 2219/00689 using computers
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: B01J 2219/00725 Peptides
• CPC Class Codes: B01J 4/02 for feeding measured , i.e. prescribed quantities of reagents
• CPC Class Codes: B01L 3/0244 using pins
• CPC Class Codes: B01L 3/0248 Prongs, quill pen type dispenser
• CPC Class Codes: B01L 3/5088 : confining liquids at a location by surface tension, e.g. virtual wells on plates, wires B01L3/50857 takes precedence
• CPC Class Codes: C12Q 1/6809 : Methods for determination or identification of nucleic acids involving differential detection
• CPC Class Codes: C12Q 1/6837 : using probe arrays or probe chips C12Q1/6874 takes precedence
• 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: G01N 2001/282 : with mapping; Identification of areas; Spatial correlated pattern
• CPC Class Codes: G01N 2035/00237 : Handling microquantities of analyte, e.g. microvalves, capillary networks
• CPC Class Codes: G01N 2035/1037 : Using surface tension, e.g. pins or wires
• CPC Class Codes: G01N 33/54366 : Apparatus specially adapted for solid-phase testing

Abstract

A method of determining the relative amounts of individual polynucleotides in a complex mixture of different-sequence polynucleotides is disclosed. The polynucleotides, after fluorescent labeling, are contacted under hybridization conditions with an array of different DNA sequences disposed at discrete locations on a non-porous surface, at an array density of at least about 100 sequences/cm², where the different DNA sequences in the array are effective to hybridize to individual polynucleotides in the mixture. The level of fluorescence associated with each array sequence provides a measure of its relative amount in the mixture.

Claims

1. A method of determining the relative amounts of individual polynucleotides in a mixture of different-sequence polynucleotides, comprising

labeling the polynucleotides with a fluorescent reporter, to form a mixture of labeled polynucleotides,

contacting the labeled polynucleotides, under hybridization conditions, with a microarray of different DNA sequences disposed at discrete locations a non-porous surface, at a density of at least about 100 locations/cm², where the different DNA sequences in the array are each (i) present in multiple copies, and (ii) effective to hybridize to individual polynucleotides in the mixture, and

determining the level of fluorescence at each position in the microarray.

2. The method of claim 1, wherein said contacting includes covering the array surface with a solution of the mixture of labeled polynucleotides, to a solution depth of less than 500 microns.

3. The method of claim 1, wherein the DNA sequences in the array are at least about 50 bases in length.

4. The method of claim 1, wherein the labeled polynucleotides represent at least 1 million unique base sequences.

5. The method of claim 1, wherein the density of array elements corresponding to different-sequence DNA locations in the array is at least 1,000/cm².

6. The method of claim 1, for use in determining the relative amounts of each polynucleotide from first and second different sources, wherein (i) the polynucleotides from the first and second sources are labeled with independently detectable first and second fluorescent reporters, respectively, (ii) said contacting of labeled polynucleotides from first and second sources is carried out simultaneously under competitive hybridization conditions, and (iii) said determining includes measuring the levels of the two reporters at each position in the array.