Multipart reagents having increased avidity for polymerase binding

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.

What is claimed is: 
     
         1 . A method of determining the identity of a nucleotide in a target nucleic acid comprising:
 a. providing a composition comprising:
 i. a target nucleic acid comprising two or more repeats of an identical sequence; 
 ii. two or more primer nucleic acids complementary to one or more regions of said target nucleic acid; and 
 iii. two or more polymerase molecules; 
   b. contacting said composition with binding composition comprising a nucleotide moiety under conditions sufficient to allow a binding complex to be formed between said nucleotide moiety and the composition of step (a); and   c. detecting said binding complex, thereby establishing the identity of said nucleotide in the target nucleic acid.   
     
     
         2 . The method of  claim 1 , wherein the target nucleic acid is DNA. 
     
     
         3 . The method of  claim 1  wherein the detection of the binding complex is performed in the absence of unbound or solution-borne nucleotide moieties. 
     
     
         4 . The method of  claim 1  wherein the target nucleic acid has been replicated or amplified or has been produced by replication or amplification. 
     
     
         5 . The method of  claim 1  wherein the detectable label is a fluorescent label. 
     
     
         6 . The method of  claim 1  wherein detecting the complex comprises a fluorescence measurement. 
     
     
         7 . The method of  claim 1  wherein the binding composition comprises one type of nucleotide moieties. 
     
     
         8 . The method of  claim 1  wherein the binding composition comprises two or more types of nucleotide moieties. 
     
     
         9 . The method of  claim 8 , wherein each type of the two or more types of nucleotide moieties comprises a different type of nucleotide. 
     
     
         10 . The method of  claim 9  wherein the binding composition consists of three types of nucleotide moieties and wherein each type of the three types of nucleotide moiety comprises a different type of nucleotide. 
     
     
         11 . The method of  claim 1  wherein the binding complex further comprises a blocked nucleotide. 
     
     
         12 . The method of  claim 11  wherein the blocked nucleotide is a 3′-O-azidomethyl, 3′-O-methyl nucleotide, or 3′-O-alkyl hydroxylamine. 
     
     
         13 . The method of  claim 1  wherein said contacting occurs in the presence of an ion that stabilizes said binding complex, said complex comprising a nucleotide moiety, two or more polymerase molecules, and two or more binding sites within the target nucleic acid. 
     
     
         14 . The method of  claim 1  wherein the contacting is done in the presence of strontium, magnesium, calcium ions, or any combination thereof. 
     
     
         15 . The method of  claim 1  wherein the polymerase molecule is catalytically inactive. 
     
     
         16 . The method of  claim 1  wherein the binding complex has a persistence time of greater than 2 seconds. 
     
     
         17 . The method of  claim 1 , further comprising hybridizing the two or more primer nucleic acids to the one or more regions of said target nucleic acid by bringing the two or more primer nucleic acids into contact with a hybridizing composition comprising said target nucleic acid at a concentration of 1 nanomolar or less under conditions sufficient for said target nucleic acid to hybridize to the two or more primer nucleic acids in 30 minutes or less. 
     
     
         18 . The method of  claim 17 , wherein the two or more primer nucleic acids are coupled to a hydrophilic polymer surface having a water contact angle of less than 45 degrees. 
     
     
         19 . The method of  claim 17 , wherein the hybridization composition further comprises:
 (a) at least one organic solvent having a dielectric constant of no greater than about 115 as measured at 68 degrees Fahrenheit; and   (b) a pH buffer.   
     
     
         20 . The method of  claim 17 , wherein the hybridization composition further comprises:
 (c) at least one organic solvent that is polar and aprotic; and   (d) a pH buffer.