Substrates comprising polynucleotide microarrays

• US 7,442,499 B2
• Filed: 11/24/1998
• Issued: 10/28/2008
• Est. Priority Date: 06/17/1994
• Status: Expired due to Fees

• Current Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford University)
• Original Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford University)
• Inventors: Brown, Patrick O., Shalon, Tidhar Dari
• Primary Examiner(s): FORMAN, BETTY J
• Application Number:
  US09/356,322
• Publication Number:
  US 20030012695A1
• Time in Patent Office: 3,626 Days
• Field of Search: 435/6, 435/69.1, 435/91.1, 435/91.2, 422/50, 422/68.1
• US Class Current: 435/6.16
• 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 substrate with a surface consisting of a hydrophobic surface formed by the surface material or by a coating applied to the surface, comprising a microarray of DNA sequences, wherein (i) the microarray has a density of 1,000 or more discrete regions of DNA sequences per cm² of substrate surface, (ii) the DNA sequences are isolated polynucleotides, (iii) the microarray comprises 1,000 or more regions, and (iv) the DNA sequences contained in each discrete region are at least 50 subunits in length, which are individually applied to each region in the microarray, wherein each said region is formed by applying to said surface a volume of aqueous reagent solution comprising a DNA sequence selected for said region, wherein said hydrophobic surface prevents spreading of said volume applied to said surface via reagent bead formation.

2. The substrate of claim 1, wherein the density of discrete regions of DNA sequences is at least 2500/cm².

3. The substrate of claim 1 or 2, wherein the substrate is glass.

4. The substrate of claim 1 or 2, wherein the substrate is non-porous.

5. The substrate of claim 1 or 2, wherein the DNA sequences are non-covalently bound to the surface of the substrate.

6. The substrate of claim 5, wherein the DNA sequences are non-covalently bound to a polycationic polymer on the surface of the substrate.

7. The substrate of claim 1 or 2, wherein the DNA sequences are selected from the group of polynucleotides consisting of mRNA-derived sequences, genomic DNA sequences and fragments thereof.

8. The substrate of claim 1 or 2, wherein the microarray comprises 2,500 or more regions.

20. The substrate of claim 1, 9, 16 or 18, wherein the DNA sequences are distinct gene sequences whose expression levels are specifically related to the differences between test cells relative to control cells.

9. A substrate with a surface consisting of a hydrophobic surface formed by the surface material or by a coating applied to the surface, comprising a microarray of DNA sequences, wherein the DNA sequences are polynucleotides of at least 50 subunits in length, produced by a method comprising the steps of

(a) depositing a selected volume between about 0.002 nl and about 2 nl of a solution comprising a selected, isolated polynucleotide at a discrete region on the hydrophobic surface of the substrate, wherein said hydrophobic surface prevents spreading of said volume via reagent bead formation;

and

(b) repeating step (a) at other locations on the surface of the substrate until a microarray of 1,000 or more regions is formed, wherein the regions are at a density of at least 1,000 regions/cm².

10. The substrate of claim 9, wherein the density of discrete regions in the microarray is 2,500 regions/cm² or more.

11. The substrate of claim 9, wherein the substrate is glass.

12. The substrate of claim 9, wherein the substrate is non-porous.

13. The substrate of claim 9, wherein the DNA sequences are non-covalently bound to the surface of the substrate.

14. The substrate of claim 9, wherein the DNA sequences are selected from the group of polynucleotides consisting of mRNA-derived sequences, genomic DNA sequences, and fragments thereof.

15. The substrate of claim 9 or 10, wherein the microarray comprises 2,500 or more regions.

16. A substrate with a surface consisting of a hydrophobic surface formed by the surface material or by a coating applied to the surface, comprising a microarray of DNA sequences and suitable for analysis of a polynucleotide mixture, wherein (i) the microarray has a density of 1,000 or more discrete regions of DNA sequences per cm² of substrate surface (ii) each of said regions contains, as an isolated polynucleotide, a unique DNA sequence having at least 50 subunits, (iii) the microarray comprises at least 1,000 regions having individually applied DNA sequences unique to others of said 1,000 regions, such that the DNA sequences in said regions are selective in hybridizing with corresponding members of said mixture, wherein each said region is formed by applying to said surface a volume of aqueous reagent solution comprising a DNA sequence unique for said region, wherein said hydrophobic surface prevents spreading of said volume applied to said surface via reagent bead formation.

17. The substrate of claim 16, wherein the microarray permits detection of a two-fold change in the relative abundance of polynucleotides in mixtures subjected to analysis.

18. A substrate with a surface consisting of a hydrophobic surface formed by the surface material or by a coating applied to the surface, comprising a microarray of DNA sequences where (i) the microarray has a density of 1,000 or more discrete regions of DNA sequences per cm² of substrate surface, (ii) the DNA sequences are purified polynucleotides having at least 50 subunits, and (iii) the DNA sequences are non-covalently bound to a polycationic polymer on the surface of the substrate, wherein each said region is formed by applying to said surface a volume of aqueous reagent solution comprising a DNA sequence unique for said region, wherein said hydrophobic surface prevents spreading of said volume applied to said surface via reagent bead formation.

19. The substrate according to claim 18, wherein the DNA sequences are cDNAs.