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 .- 34 . (canceled) 
     
     
         35 . A system for detecting a plurality of target nucleic acid sequences, comprising:
 a) an array of capture probes attached to discrete sites on a solid support adapted to hybridize to adapter sequences of modified primers or their complements;   b) a solution comprising modified primers, wherein the solution is in contact with the array and is prepared by a method comprising:
 i) hybridizing a plurality of different first primers to first portions of a plurality of target sequences, wherein each of said different first primers comprises an adapter sequence exogenous to said target sequences, 
 ii) hybridizing a plurality of different second primers to second portions of said plurality of target sequences, thereby forming a plurality of hybridization complexes, 
 iii) extending said first or said second primers, and ligating said first and second primers together to form a plurality of different modified primers, and 
 iv) removing unextended first or second primers from said modified primers; 
   c) a polymerase adapted to extend said capture probes; and   d) a detector adapted to detect extended capture probes hybridized to said modified primers.   
     
     
         36 . The system of  claim 35 , wherein the modified primers are hybridized to the capture probes. 
     
     
         37 . The system of  claim 36 , wherein the capture probes have been extended by the polymerase. 
     
     
         38 . The system of  claim 36 , wherein the capture probes have been amplified by the polymerase. 
     
     
         39 . The system of  claim 35 , wherein the method of preparing the solution comprises amplifying the plurality of different modified primers. 
     
     
         40 . The system of  claim 39 , wherein the amplified modified primers are hybridized to the capture probes. 
     
     
         41 . The system of  claim 39 , wherein the amplifying the plurality of different modified primers comprises hybridizing said plurality of different modified primers with a plurality of amplifier probes complementary to said plurality of first and second primers and amplifying said different modified primers. 
     
     
         42 . The system of  claim 35 , wherein the plurality of target sequences are immobilized to one or more beads. 
     
     
         43 . The system of  claim 35 , wherein the plurality of target nucleic acid sequences comprises deoxyribonucleic acids. 
     
     
         44 . The system of  claim 35 , wherein the removing the unextended first or second primers comprises removing the unextended first or second primers affinity chromatography. 
     
     
         45 . The system of  claim 35 , wherein the detector is adapted to identify 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. 
     
     
         46 . The system of  claim 35 , wherein said array comprises a population of beads comprising said capture probes. 
     
     
         47 . The system of  claim 46 , wherein said beads are associated with individual sites of said solid support. 
     
     
         48 . The system of  claim 47 , wherein each of said sites is configured to have a single associated bead. 
     
     
         49 . The system of  claim 35 , wherein the detector is adapted to detect a label attached to said modified capture probes. 
     
     
         50 . The system of  claim 49 , wherein the label comprises a fluorescent label. 
     
     
         51 . The system of  claim 35 , wherein said target sequences comprise loci having a single nucleotide polymorphism (SNP) allele. 
     
     
         52 . The system of  claim 51 , wherein said plurality of said first primers comprise allele specific primers and said plurality of second primers comprise locus specific primers. 
     
     
         53 . The system of  claim 52 , wherein a terminal base of said allele specific primers correspond to said SNP allele.