Isolation of polymerase-nucleic acid complexes

A method for DNA sequencing comprising isolating active complexes comprising:

• obtaining a population of circular DNA templates having a central double stranded region and hairpin regions on each end;
• exposing the population of circular DNA templates to a DNA polymerase enzyme having strand displacement activity under conditions in which a mixture comprising a population of polymerase-template complexes is formed;
• adding to the population of complexes a hook oligonucleotide comprising a capture region complementary to a portion of the hairpin region;
• using the hook oligonucleotide to isolate the complexes to which it hybridized;
• loading the active complexes onto a substrate such that single complexes can be individually optically resolved;
• exposing the active complexes to a plurality of differentially labeled nucleotide analogs whose labels are cleaved upon incorporation, and
• initiating DNA synthesis and observing each complex to determine the time sequence of nucleotides that are incorporated.