Purified extended polymerase/template complex for sequencing

摘要

Methods, Compositions, and Systems are provided for obtaining polymerase-template complex mixtures with improved levels of active polymerase. In some aspects, methods are described in which a polymerase-template complex is exposed to reaction conditions in which a complementary strand to the template is produced. The extended reaction mixture is purified, for example by gel filtration chromatography to produce a mixture of polymerase-template complex having a higher active fraction. This purified mixture can be used for further analyses including single molecule sequencing.

主权项

1. A method comprising:
producing a reaction mixture comprising a polymerase enzyme complex having a polymerase enzyme and a circular template nucleic acid, the reaction mixture comprising reagents for template directed nucleic acid synthesis including nucleotides, nucleotide analogs or both; extending a complementary strand such that a portion of the polymerase enzyme complexes have complementary strands within a desired size range; purifying the reaction mixture based on the size of the complexes to enhance the relative amount of polymerase enzyme complex having complementary strands within the desired size range; loading the purified reaction mixture onto a substrate.