Combined polynucleotide sequences as discrete assay endpoints

Abstract

Methods are provide for determining the relative amounts of individual polynucleotides in a complex mixture. The polynucleotides, after fluorescent labeling, are contacted under hybridization conditions with an array having element disposed at discrete locations on a substrate. The elements comprise two or more distinct polynucleotides that are combined prior to arraying. The level of fluorescence associated with each element provides a measure of its relative amount in the mixture.

1 . A microarray of non-contiguous polynucleotides, the array comprising: 
 a solid support having a surface; and    a plurality of elements arranged in a defined manner and stably associated with the surface, wherein each element comprises two or more combined, distinct polynucleotides having distinct sequences.    
     
     
         2 . The array of  claim 1 , wherein the solid support is selected from the group consisting of glass, silica, plastic, ceramic, beads, and nylon, and combinations thereof.  
     
     
         3 . The array of  claim 1 , wherein each element comprises about two to about ten combined, distinct polynucleotides having distinct sequences.  
     
     
         4 . The array of  claim 1 , wherein each element comprises about two to about five combined, distinct polynucleotides having distinct sequences.  
     
     
         5 . The array of  claim 1 , wherein the polynucleotide sequences are complementary to a single target polynucleotide.  
     
     
         6 . The array of  claim 1 , wherein the polynucleotides independently comprise from about 5 to about 10000 nucleotides.  
     
     
         7 . The array of  claim 6 , wherein the polynucleotides independently comprise from about 20 to about 300 nucleotides.  
     
     
         8 . The array of  claim 7 , wherein the polynucleotides independently comprise from about 50 to about 65 nucleotides.  
     
     
         9 . The array of  claim 1 , wherein the polynucleotides are double stranded.  
     
     
         11 . The array of  claim 1 , wherein the elements are associated with the surface by non-covalent means.  
     
     
         12 . The array of  claim 1 , wherein the elements are associated with the surface by covalent means.  
     
     
         13 . The array of  claim 12 , wherein the covalent means comprises a linker.  
     
     
         14 . The array of  claim 1 , wherein the elements are associated with the surface by non-covalent and covalent means.  
     
     
         15 . The array of  claim 1 , wherein the array is a microarray made by high density contact deposition printing.  
     
     
         16 . A hybridization assay comprising the steps of: 
 contacting the array of  claim 1  with detectable nucleic acid probes under hybridization conditions to produce a hybridization pattern; and    detecting the hybridization pattern.    
     
     
         17 . The assay of  claim 16 , wherein the contacting step is preceded by the step of making the array by high density contact deposition printing.  
     
     
         18 . A method for analyzing polynucleotide sequences, the method comprising the steps of: 
 making an array according to  claim 1  by high deposition contact printing;    contacting the array with detectable polynucleotide probes under hybridization conditions to produce a hybridization pattern;    detecting the hybridization pattern.    
     
     
         19 . The method of  claim 18  wherein the detectable probes are fluorescently labeled and the detection step comprises detecting fluorescent emissions from immobilized probes.  
     
     
         20 . A kit for use in measuring hybridization, comprising: 
 an array according to  claim 1;  and    instructional material for measuring hybridization with the array.