ISOLATION OF POLYMERASE-NUCLEIC ACID COMPLEXES

摘要

Compositions, methods and systems are provided for the isolation of polymerase-nucleic acid complexes. Complexes can be separated from free enzyme by using hook molecules to target single stranded regions on the nucleic acid. Hook oligonucleotides are used to capture polymerase nucleic acid complexes where the nucleic acids comprise circular nucleic acids having a double stranded central region with hairpin regions on each end. Methods for loading such complexes onto substrates and for single molecule sequencing of such complexes are also provided.

主权项

1. A method comprising;
fragmenting a double stranded DNA sample into double stranded fragments; ligating to each end of the double stranded fragments a hairpin to produce 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; thereby isolating complexes from the mixture.