Purified extended polymerase/template complex for sequencing
AssigneePacific Biosciences of California, Inc.
InventorsLei Sun, Insil Park
Life of this patent
Estimating the term…
Abstract
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.
Claims28
A method comprising:
- producing a reaction mixture comprising a polymerase enzyme complex having a polymerase enzyme and a 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 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.
(canceled)
-22. (canceled)
A composition comprising:
- a purified solution of polymerase enzyme complex, the enzyme complex comprising polymerase enzyme, template nucleic acid, and complementary strand, wherein the median size of the complementary strand is from about 10 percent to about 300 percent the median size of the median template nucleic acid.
-27. (canceled)
(canceled)
-33. (canceled)
A method of obtaining an array of zero mode waveguides with greater than 37 percent of the zero mode waveguides having a single active polymerase comprising:
- producing a reaction mixture comprising a polymerase enzyme complex having a polymerase enzyme and a 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 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 comprising an array of zero mode waveguides such that greater than 36 percent of the zero mode waveguides have a single active polymerase.
(canceled)
A method for isolating active polymerase enzyme nucleic acid complexes comprising:
- contacting a polymerase enzyme with a template nucleic acid to produce a population of polymerase enzyme nucleic acid complexes in solution, at least some of which are active;
- exposing the population of polymerase enzyme nucleic acid complexes with a resin having nucleoside phosphate moities attached thereto under conditions whereby the active polymerase enzyme nucleic acid complexes bind to the resin;
- washing the resin to remove components not bound to the resin;
- and
- eluting under conditions whereby the active polymerase enzyme nucleic acid complex releases from the resin, thereby isolating the active polymerase enzyme nucleic acid complex.
-42. (canceled)
Dependencies are inferred from the claim text.