Patent ID: 7943307

Claim:
A method of sequencing a target nucleic acid molecule, comprising: (a) providing a complex comprising a polymerase enzyme, a target nucleic acid molecule, and a primer, wherein the complex is immobilized on a support, whereby the complex is within an observation volume of a detector; (b) exposing said complex to a plurality of types of labeled nucleotides or labeled nucleotide analogs for incorporation into a growing nucleic acid strand complementary to the target nucleic acid, each type of labeled nucleotide or labeled nucleotide analog comprising a fluorescent label that is distinguishable from the other types of said plurality, wherein the fluorescent label is attached to a terminal phosphate group of the nucleotide or nucleotide analog; and (c) detecting with the detector the label on the nucleotide or nucleotide analog while the labeled nucleotide or nucleotide analog is retained in the complex for a period of time sufficient for incorporating into the growing strand, thereby identifying the nucleotide or nucleotide analog incorporated by the polymerase enzyme into the growing strand, whereby the time duration of the signal from labeled nucleotides or nucleotide analogs that become incorporated is distinguished from freely diffusing labels by a longer retention in the observation volume for the nucleotides or nucleotide analogs that become incorporated than for the freely diffusing labels.