Nucleotide compositions and uses thereof

Abstract

The present invention relates to preparation of nucleotide compositions and uses thereof for conducting nucleic acid analyses. The compositions and methods embodied in the present invention are particularly useful for nucleic acid analyses that require high-resolution detection of labeled nucleotides or labeled nucleic acid targets.

1 - 14 . (canceled) 
     
     
         15 . A method of sequencing a nucleic acid, comprising:
 providing a reaction mixture comprising a template nucleic acid, a primer sequence complementary to the template nucleic acid sequence, a polymerase enzyme, at least one accessory protein, and a nucleotide composition comprising a labeled nucleotide or nucleotide analog capable of incorporation into a nascent nucleic acid strand and said composition being substantially free of unlabeled nucleotides or nucleotide analogs capable of incorporation into the nascent nucleic acid strand, wherein the template nucleic acid, primer sequence, and polymerase enzyme are in an individually resolvable complex; and   detecting incorporation of an individual base of the labeled nucleotide or nucleotide analog by the polymerase into the nascent nucleic acid strand.   
     
     
         16 . (canceled) 
     
     
         17 . The method of  claim 15 , wherein within the reaction mixture, less than 5% of the nucleotides or nucleotide analogs are unlabeled nucleotides or nucleotide analogs that are capable of participating in a sequencing reaction in which a labeled nucleotide or nucleotide analog participates. 
     
     
         18 . The method of  claim 15 , wherein within the reaction mixture, less than 1% of the nucleotides or nucleotide analogs are unlabeled nucleotides or nucleotide analogs that are capable of participating in a sequencing reaction in which a labeled nucleotide or nucleotide analog participates. 
     
     
         19 . The method of  claim 15 , wherein within the reaction mixture, less than 0.1% of the nucleotides or nucleotide analogs are unlabeled nucleotides or nucleotide analogs that are capable of participating in a sequencing reaction in which a labeled nucleotide or nucleotide analog participates. 
     
     
         20 . The method of  claim 15 , wherein within the reaction mixture, less than 0.01% of the nucleotides or nucleotide analogs are unlabeled nucleotides or nucleotide analogs that are capable of participating in a sequencing reaction in which a labeled nucleotide or nucleotide analog participates. 
     
     
         21 . The method of  claim 15 , wherein the reaction mixture comprises at least two different labeled nucleotides or nucleotide analogs. 
     
     
         22 . The method of  claim 15 , wherein the reaction mixture comprises at least four different labeled nucleotides or nucleotide analogs. 
     
     
         23 . The method of  15 , wherein the detecting step is performed during incorporation of the labeled nucleotide or nucleotide analog by the polymerase into the nascent nucleic acid strand. 
     
     
         24 . The method of  claim 15 , wherein the detecting step involves detecting an optical signal from the labeled nucleotide or nucleotide analog. 
     
     
         25 . The method of  claim 15 , wherein the template nucleic acid and/or the polymerase is immobilized to a support. 
     
     
         26 . The method of  claim 15 , wherein the reaction mixture comprises a plurality of single complexes of a template nucleic acid, a polymerase enzyme, and a nascent strand that is complementary to the template nucleic acid wherein each of the single complexes is individually monitored to detect incorporation of individual bases into each nascent strand. 
     
     
         27 . (canceled) 
     
     
         28 . A method for identifying a nucleotide base incorporated into a nascent strand during template-directed synthesis, said method comprising:
 (a) providing a complex comprising a polymerase and a template nucleic acid such that said polymerase is capable of incorporating a nucleotide base complementary to a nucleotide base on the template nucleic acid, wherein the complex is immobilized to a solid support;   (b) providing a solution comprising at least one incorporatable labeled nucleotide or nucleotide analog to the complex to effect polymerizing a nucleotide base into a nascent strand that is complementary to the target nucleic acid, wherein the solution is substantially free of incorporatable unlabeled nucleotides or nucleotides analogs, and further wherein the solution comprises at least one accessory protein; and   (c) optically detecting the incorporated nucleotide base by sensing its detectable label, thereby identifying the incorporated nucleotide base.   
     
     
         29 . The method of  claim 28 , wherein said detecting is performed by passing radiation through the complex at a spatial location of the complex where said label is expected to occur. 
     
     
         30 . The method of  claim 28 , further comprising repeating, with the complex immobilized on the support, steps (b) through (c) so that a plurality of incorporated nucleotides is identified and, by complementarity, a sequence of the template nucleic acid is determined. 
     
     
         31 . The method of  claim 28 , wherein the solid support comprises an array of individually optically resolvable complexes. 
     
     
         32 . The method of  claim 28 , wherein the template nucleic acid is selected from the group consisting of a circular DNA, a linear DNA, and a DNA containing repeated sequences. 
     
     
         33 . The method of  claim 28 , wherein the accessory protein is selected from the group consisting of a single-stranded binding protein, a primase, and a helicase. 
     
     
         34 . The method of  claim 15 , wherein the detecting further comprises detecting additional individual bases of labeled nucleotides or nucleotide analogs as they are sequentially incorporated into the nascent nucleic acid strand by the polymerase enzyme. 
     
     
         35 . The method of  claim 34 , wherein the detecting of the additional individual bases is performed without adding any reactants to the reaction mixture or removing any by-products from the reaction mixture. 
     
     
         36 . The method of  claim 15 , wherein the template nucleic acid is selected from the group consisting of a circular DNA, a linear DNA, and a DNA containing repeated sequences. 
     
     
         37 . The method of  claim 15 , wherein the accessory protein is selected from the group consisting of a single-stranded binding protein, a primase, and a helicase.