Methods for single molecule sequencing

A method for sequencing a nucleic acid template comprising:

• providing a sequencing mixture comprising (i) a polymerase enzyme, (ii) a template nucleic acid, and (iii) a polymerase reagent solution having the components for carrying out template directed synthesis of a growing nucleic acid strand, wherein said polymerase reagent solution includes a component for a requenching reaction and a plurality of types of nucleotide analogs;
• wherein each type of nucleotide analog has a labeled leaving group that is cleavable by the polymerase, and each type of nucleotide analog has a different label, wherein the labeled leaving group is cleaved upon polymerase-dependent binding of a respective nucleotide analog to the template strand;
• carrying out nucleic acid synthesis such that a plurality of nucleotide analogs are added sequentially to the template whereby:
• a) a nucleotide analog associates with the polymerase, b) the nucleotide analog is incorporated on the template strand by the polymerase when the labeled leaving group on that nucleotide analog is cleaved by the polymerase, wherein the labeled leaving group emits light upon cleavage, then c) the labeled leaving group on the nucleotide analog is quenched by the requenching reaction;
• and
• detecting light from the labels while nucleic acid synthesis is occurring, and using light detected in the time between step b) when the labelled leaving group is cleaved, and step c) in which the labeled leaving group is quenched, to determine a sequence of the template nucleic acid.