Multivalent binding composition for nucleic acid analysis

Abstract

Multivalent binding compositions including a particle-nucleotide conjugate having a plurality of copies of a nucleotide attached to the particle are described. The multivalent binding compositions allow one to localize detectable signals to active regions of biochemical interaction, e.g., sites of protein-protein interaction, protein-nucleic acid interaction, nucleic acid hybridization, or enzymatic reaction, and can be used to identify sites of base incorporation in elongating nucleic acid chains during polymerase reactions and to provide improved base discrimination for sequencing and array based applications.

1 . A method for detecting a target nucleic acid, comprising:
 a) providing a composition comprising (i) a plurality of target nucleic acids, and (ii) a plurality of nucleic acid sequencing primers hybridized to the plurality of target nucleic acids;   b) contacting the composition with a detection reagent comprising two or more types of labeled probes under a condition suitable to form a plurality of binding complexes between a labeled probe and a target nucleic acid of the plurality of target nucleic acids, wherein individual labeled probes comprise a plurality of oligonucleotides;   c) detecting the plurality of binding complexes; and   d) repeating steps (b)-(c) at least once, wherein the total concentration of the two or more types of labeled probes in any two successive repeat cycles of step (b)-(c) is less than 1.5 uM.   
     
     
         2 . The method of  claim 1 , wherein the detecting of step (c) comprises fluorescence imaging. 
     
     
         3 . The method of  claim 1 , wherein the detection reagent comprises four types of labeled probes each type labeled with a distinct fluorophore. 
     
     
         4 . The method of  claim 1 , wherein the detection reagent comprises three types of labeled probes each type labeled with a distinct fluorophore and a fourth type of unlabeled probes. 
     
     
         5 . The method of  claim 1 , wherein the detection reagent comprises two types of labeled probes each type labeled with a distinct fluorophore and two types of unlabeled probes. 
     
     
         6 . The method of  claim 1 , wherein the target nucleic acid comprise a clonally amplified target nucleic acid immobilized to a support. 
     
     
         7 . The method of  claim 6 , wherein the clonally amplified target nucleic acid is generated by bridge amplification thereby forming a cluster of target nucleic acids immobilized to a support. 
     
     
         8 . The method of  claim 7 , wherein the bridge amplification comprises isothermal bridge amplification. 
     
     
         9 . The method of  claim 6 , wherein the clonally amplified target nucleic acids are generated by rolling circle amplification thereby forming a concatemer immobilized to a support. 
     
     
         10 . The method of  claim 1 , wherein the support comprises at least one hydrophilic polymer coating layer. 
     
     
         11 . The method of  claim 10 , wherein the hydrophilic polymer coating layer has a water contact angle of no more than 45 degrees.