Methods for detecting nucleic acid analyte

A method of detecting and characterizing multiple analytes in a sample, comprising the steps of:

• (a) anchoring to each analyte a specific template nucleic acid sequence with a unique base at the site opposite to the complementary nucleotide being added;
• (b) conducting a DNA polymerase reaction to produce uniquely labeled polyphosphates;
• said reaction comprising the reaction of said templates, nuclease resistant primers complementary to said specific target sequence of each of said multiple analytes, two or more terminal phosphate-labeled nucleotides having 2 or more phosphate groups in the polyphosphate chain and each bearing a different label, a DNA polymerase and an enzyme having 3′-5′ exonuclease activity;
• and (c) detecting the labeled polyphosphates.

A method of detecting and characterizing multiple analytes in a reaction compartment, comprising the steps of:

• (a) anchoring a unique template nucleic acid sequence to each of said analytes;
• (b) anchoring said analytes to the surface of said reaction compartment;
• (c) conducting a DNA polymerase reaction to produce labeled polyphosphate;
• said reaction comprising the reaction of the unique template sequence of one of said analytes, a nuclease resistant primer complementary to said unique template sequence, at least one terminal phosphate-labeled nucleotides having 2 or more phosphate groups in the polyphosphate chain, a DNA polymerase and an enzyme having 3′-5′ exonuclease activity;
• (d) detecting said labeled polyphosphate;
• (e) washing off the unanchored components;
• and (f) repeating steps (a) to (d) with a nuclease resistant primer complementary to another unique template sequence of a different analyte until all the analytes are analyzed.

A method of detecting and characterizing multiple analytes in a sample, comprising the steps of:

• (a) anchoring to each analyte a specific template nucleic acid sequence with a unique base at the site opposite to the complementary nucleotide being added;
• (b) conducting a DNA polymerase reaction to produce labeled polyphosphates, said reaction comprising the reaction of said templates, primers complementary to said specific template sequence, two or more terminal phosphate-labeled nucleotides with different labels, a DNA polymerase and an enzyme having 3′-5′ exonuclease activity;
• (c) permitting said labeled polyphosphates to react with one or more additional enzymes to produce detectable species unique to each of said analytes;
• and (d) detecting said detectable species.

A method of sequencing a target nucleic acid, comprising:

• providing a reaction mixture comprising the target nucleic acid, a primer, a polymerase enzyme, and one or more labeled nucleotides or nucleotide analogs, wherein the target nucleic acid and/or the primer is immobilized upon a substrate;
• sequentially optically detecting incorporation of at least first and second labeled nucleotides or nucleotide analogs in a nascent nucleic acid strand by an individual polymerase enzyme;
• and
• identifying the first and second nucleotides or nucleotide analogs incorporated into the nascent nucleic acid strand.

A method of sequencing a target nucleic acid, comprising:

• providing a reaction mixture comprising the target nucleic acid, a primer, a polymerase enzyme, and one or more labeled nucleotides or nucleotide analogs, wherein the target nucleic acid and/or the primer is immobilized upon a substrate;
• sequentially optically detecting incorporation of at least first and second labeled nucleotides or nucleotide analogs into an individual nascent nucleic acid strand by the polymerase enzyme;
• and
• identifying the first and second nucleotides or nucleotide analogs incorporated into the nascent nucleic acid strand.

A method for sequencing a target nucleic acid, comprising:

• (a) providing a complex comprising a polymerase and a target nucleic acid such that said polymerase is capable of incorporating a nucleotide or nucleotide analog complementary to a nucleotide on the target nucleic acid, wherein the complex is immobilized on a substrate;
• (b) providing a plurality of labeled nucleotides or nucleotide analogs to the complex to effect incorporation of a labeled nucleotide or analog into a nascent strand that is complementary to the target nucleic acid;
• (c) optically detecting the incorporated nucleotide or analog by sensing its detectable label, wherein said detecting is performed by applying radiation through the complex at a spatial location of the complex where said label is expected to occur;
• (d) repeating, with the complex immobilized on the substrate, steps (b) through (c) so that a plurality of nucleotides incorporated is identified and, as a result, a sequence of the target nucleic acid is determined.