Detection of nucleic acid reactions on bead arrays

Abstract

The present invention is directed to methods and compositions for the use of microsphere arrays to detect and quantify a number of nucleic acid reactions. The invention finds use in genotyping, i.e. the determination of the sequence of nucleic acids, particularly alterations such as nucleotide substitutions (mismatches) and single nucleotide polymorphisms (SNPs). Similarly, the invention finds use in the detection and quantification of a nucleic acid target using a variety of amplification techniques, including both signal amplification and target amplification. The methods and compositions of the invention can be used in nucleic acid sequencing reactions as well. All applications can include the use of adapter sequences to allow for universal arrays.

1 . A method of detecting a plurality of target nucleic acid sequences, comprising: 
 a) hybridizing a plurality of different first primers to first portions of a plurality of target sequences, wherein said different first primers each comprise a different adapter sequence;    b) hybridizing a plurality of different second primers to second portions of said plurality of target sequences, thereby forming a plurality of hybridization complexes, 
 wherein said hybridization complexes are immobilized on one or more solid phase surfaces;  
   c) extending said first or said second primers, and ligating said first and second primers together to form a plurality of different modified primers;    d) hybridizing said adapter sequences of said modified primers or their complements to an array of capture probes; and    e) detecting the presence of said modified primers, thereby detecting said plurality of target nucleic acid sequences.    
   
   
       2 . The method of  claim 1 , further comprising amplifying said different modified primers, thereby forming amplified products comprising said adapter sequences or their complements.  
   
   
       3 . The method of  claim 2 , further comprising hybridizing said adapter sequences of said amplified products or their complements to said array of capture probes.  
   
   
       4 . The method of  claim 2 , comprising detecting a label attached to said amplified products.  
   
   
       5 . The method of  claim 2 , wherein said amplifying comprises a polymerase chain reaction.  
   
   
       6 . The method of  claim 1 , further comprising identifying a nucleotide at a detection position for each of said target nucleic acid sequences, wherein a primer of said plurality of first primers or said plurality of second primers is complementary to said detection position.  
   
   
       7 . The method of  claim 1 , wherein said target sequences are attached to said one or more solid phase surfaces.  
   
   
       8 . The method of  claim 1 , wherein said array comprises a population of beads comprising said capture probes.  
   
   
       9 . The method of  claim 8 , wherein said beads are associated with individual sites of a substrate.  
   
   
       10 . The method of  claim 9 , wherein each of said sites is configured to have a single associated bead.  
   
   
       11 . The method of  claim 9 , wherein said substrate comprises a fiber optic bundle.  
   
   
       12 . The method of  claim 1 , wherein said array is made by a method selected from the group consisting of a spotting technique, photolithographic technique, and printing technique.  
   
   
       13 . The method of  claim 1 , comprising detecting a label attached to said modified primers.  
   
   
       14 . The method of  claim 1 , comprising detecting capture probes modified by polymerase extension.  
   
   
       15 . The method of  claim 1 , comprising detecting a fluorescent label.