Expression monitoring by hybridization to high density oligonucleotide arrays

Abstract

The present invention provides methods for comparing and identifying differences in nucleic acid sequences using a plurality of sequence specific recognition reagents (i.e., probes comprising a nucleic acid complementary to a nucleic acid sequence in collections to be compared) bound to a solid surface.

What is claimed is:  
     
         1 . A substrate with a surface having at least 1000 distinct polynucleotide or polypeptide biopolymers per cm 2  surface area, each distinct biopolymer sample (i) being disposed at separtate, defined positions in said array, (ii) having a length of at least 50 subunits, and (iii) being present in an effective amount to be detectable when hybridized to a target by detection of a labeled sample.  
     
     
         2 . A method of detecting differential expression of each of a plurality of genes in a first cell type with respect to expression of the same genes in second cell type, said method comprising: 
 producing labeled mRNA or mRNA products isolated from the two cell types;    adding a mixture of labeled said mRNA or mRNA products from the two cell types to a high density array of polynucleotdes representing a plurality of known genes derived from at least the two cell types, under conditions that result in hybridization to complementary sequence polynucleotides in the array; and    examining the array by fluroescence under fluorescence excitation conditions in which (i) polynucleotides in the array that are hybridized.    
     
     
         3 . The method as recited in  claim 2  wherein the labeled mRNA or mRNA products are fluoroscein labeled.  
     
     
         4 . The method as recited in  claim 2  wherein the labeled mRNA or mRNA products from the two cell types are labeled with labels of first and second fluorescent reporters.  
     
     
         5 . The method as recited in  claim 4  wherein said first and second fluorescent reporters are different colors of fluroescent reporters.  
     
     
         6 . The method as recited in  claim 5  wherein said colors are red and green.  
     
     
         7 . The method as recited in  claim 2  wherein said array is formed by placing biologically prepared DNA or RNA on a solid support.  
     
     
         8 . The method as recited in  claim 2  wherein said array is formed by synthesis of RNA or DNA on a solid support.  
     
     
         9 . A method of performing an analysis on a sample comprising the steps of: 
 placing or forming one or more high density arrays of oligonucleotides on one or more solid supports, said arrays comprising more than 100 oligonucleotides per square centimeter, said oligonucleotides formed on said solid supports or preformed and placed on said solid support at known locations;    extracting messenger RNA sample from at least two cell populations and labeling said messenger RNA or products of said messenger RNA from said at least two cell populations;    exposing products of said extracting and labeling step to said one or more high density arrays;    detecting where said products have hybridized to said high density arrays;    based on said detecting step, determining a level of expression of said messenger RNA in said at least two cell populations.    
     
     
         10 . A method of simultaneously monitoring the expression of a multiplicity of genes, said method comprising: 
 (a) providing a pool of target nucleic acids comprising RNA transcripts of one or more of said genes, or nucleic acids derived from said RNA transcripts;    (b) hybridizing said pool of nucleic acids to an array of oligonucleotide probes immobilized on a surface, said array comprising more than 100 different oligonucleotides wherein: 
 each different oligonucleotide is localized in a predetermined region of said surface;  
 each different oligonucleotide is attached to said surface through a single covalent bond;  
 the density of said different oligonucleotides is greater than about 60 different oligonucleotides per 1 cm 2 ; and  
 said oligonucleotide probes are complementary to a subsequence of said RNA transcripts or said nucleic acids derived from said RNA transcripts; and  
   (c) quantifying the hybridization of said nucleic acids to said array wherein said quantifying provides a measure of the levels of transcription of said genes.    
     
     
         11 . A method of comparing a level of different RNA or DNA sequences in a sample comprising the steps of: 
 labeling a first RNA or DNA sequence in said sample with a first fluorescent dye;    labeling a second RNA or DNA sequence in said sample with a second dye, said second dye emitting light upon excitation at a wavelength different from said first fluorescent dye;    exposing said sample to RNA or DNA probes affixed to a solid support;    determining a relative amount of said first and said second RNA or DNA sequences based upon a level of emission of light from said substrate at said first and said second wavelenghts.    
     
     
         12 . A method for comparing copy number of nucleic acid sequences two or more collections of nucleic acid molecules, the method comprising: 
 providing a plurality of target elements bound to a solid surface, each target element comprising a target nucleic acid;    contacting the target elements with 
 a first collection of labeled nucleic acid comprising a sequence substantially complementary to a target nucleotide sequence, and  
 at least a second labeled nucleic acid comprising a sequence complementary to the target nucleotide sequence; wherein said first and second labels are distinguishable from each other; and  
   detecting the amount of binding of the first and second labeled complementary nucleic acids to the target nucleic acids.    
     
     
         13 . The method of  claim 12  wherein the target nucleic acids are DNA.  
     
     
         14 . The method of  claim 12  wherein the target nucleic acids are cDNA.  
     
     
         15 . The method of  claim 12  wherein the first and second labeled nucleic acids comprise human DNA.  
     
     
         16 . The method of  claim 12  wherein the target nucleic acids are greater than about 25 nucleotides in complexity.  
     
     
         17 . The method of  claim 12  wherein the solid support is glass.  
     
     
         18 . The method of  claim 12  wherein the first and second labels are fluorescent labels.  
     
     
         19 . The method of  claim 12  wherein the first labeled nucleic acids comprise mRNA or cDNA from a test cell and the second labeled nucleic acids comprise mRNA or cDNA from a reference cell.  
     
     
         20 . The method of  claim 12  wherein the first labeled nucleic acids are from a test genome and the second labeled nucleic acids are from a normal reference genome.  
     
     
         21 . The method of  claim 12  wherein the first labeled nucleic acids are from a tumor.  
     
     
         22 . A kit for performing the assay of  claim 12.