Purification of polymerase complexes

Patent metadata

Field	Value
Publication number	US-11802276-B2
Application number	US-201815989109-A
Country	US
Kind code	B2
Filing date	May 24, 2018
Priority date	Nov 25, 2015
Publication date	Oct 31, 2023
Grant date	Oct 31, 2023

Abstract

Disclosed are methods for isolating polymerase complexes from a mixture of polymerase complex components. The polymerase complexes can comprise a nanopore to provide isolated nanopore sequencing complexes. The methods relate to the positive and negative isolation of the polymerase complexes and/or nanopore sequencing complexes. Also disclosed is a nucleic acid adaptor for isolating active polymerase complexes, polymerase complexes comprising the nucleic acid adaptor, and methods for isolating active polymerase complexes using the nucleic acid adaptor.

First claim

The invention claimed is:

1. A method for isolating active polymerase complexes, wherein the method comprises the following steps:
(a) providing a reaction mixture comprising: (i) a polymerase enzyme, wherein the polymerase enzyme is either active or inactive, (ii) a single-stranded polynucleotide template, and (iii) a capture oligonucleotide conjugated to a purification moiety, wherein the capture oligonucleotide hybridizes to a portion of the polynucleotide template, wherein the polynucleotide template is a self-priming template that generates a self-complementary sequence that acts as a primer for template-dependent extension by the polymerase enzyme, thereby forming polymerase complexes, wherein the polymerase complex comprises a complex of: (i) a polymerase enzyme, and (ii) a single-stranded polynucleotide template;
(b) binding the polymerase complexes to a solid phase support, wherein the solid phase support comprises a purification moiety-binding compound, by binding of the purification moiety-binding compound of the solid phase support with the purification moiety of the capture oligonucleotide;
(c) providing reagents comprising nucleotides for enabling template-dependent extension by an active polymerase enzyme, wherein the self-priming template of the polynucleotide template generates a self-complementary sequence that acts as a primer for template-dependent extension by the active polymerase, thereby generating a plurality of the active polymerase complexes comprising extended complementary sequences; and
(d) isolating (i) the active polymerase complexes having extended complementary sequences from (ii) the inactive polymerase complexes comprising unextended complementary sequences, thereby isolating said active polymerase complexes from said polymerase complex components,
wherein the inactive polymerase complexes having unextended complementary sequences remain bound to said solid phase support, and the active polymerase complexes having extended complementary sequences are released from the solid phase support by the activity of the active polymerase enzyme.

2. The method of claim 1 , wherein the polynucleotide template is a linear or a circular template, and wherein the reaction mixtures of step (a) each further comprise an oligonucleotide primer.

3. The method of claim 1 , further comprising removing active polymerase complexes that are not bound to the solid phase support from the inactive polymerase complexes that are bound to the solid phase support.

4. The method of claim 1 , wherein the purification moiety is a biotin or modified biotin, and the purification moiety-binding compound is streptavidin or modified streptavidin.

5. The method of claim 4 , wherein the biotin or modified biotin is desthiobiotin or a derivative thereof.

6. The method of claim 1 , wherein the polymerase complex further comprises a nanopore.

7. A method for positively isolating a polymerase complex, wherein the polymerase complex comprises a complex of: (i) a polymerase enzyme, (ii) a polynucleotide template, and (iii) a capture oligonucleotide, wherein each capture oligonucleotide comprises a purification moiety, wherein the capture oligonucleotide hybridizes to a portion of the polynucleotide template due to complementarity, and wherein method comprises the following steps:
(a) providing a reaction mixture comprising a plurality of polymerase enzymes, a plurality of polynucleotide templates, and a plurality of capture oligonucleotides, wherein each capture oligonucleotide comprising a purification moiety, thereby enabling the formation of a plurality of polymerase complexes, wherein a polymerase complex is formed only when a polymerase enzyme associates with a capture oligonucleotide that is hybridized to a portion of the polynucleotide template due to complementarity;
(b) binding the plurality of polymerase complexes formed in step (a) to a solid phase support, wherein the solid phase support comprises a purification moiety-binding compound; and
(c) removing the polymerase enzymes, polynucleotide templates, and capture oligonucleotides that are not bound to the solid phase support from the polymerase complexes that are bound to the solid phase support, thereby positively isolating polymerase complexes.

8. The method of claim 7 , wherein the polynucleotide template is a linear or a continuous template, and wherein the capture oligonucleotide is a capture oligonucleotide primer.

9. The method of claim 7 , further comprising a step of step (d) releasing polymerase complexes from the solid phase support.

10. The method of claim 7 , wherein the purification moiety is a biotin or modified biotin, and the purification moiety-binding compound is streptavidin or modified streptavidin.

11. The method of claim 10 , wherein the biotin or modified biotin is desthiobiotin or a derivative thereof.

12. The method of claim 7 , wherein the isolated polymerase complex further comprises a nanopore.

Assignees

• Roche Sequencing Solutions Inc

Inventors

• Franklin Helen
• Cech Cynthia
• Craig Timothy Kellogg
• AYER Aruna
• Dhiman Kirti
• Chechelski Johnston Natalie B
• Mabry Joshua N
• Bibillo Arkadiusz
• Crisalli Peter
• Davis Randall W

Classifications

• C12N9/1252 Primary

  DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase · CPC title

• C12Q1/6869

  Methods for sequencing · CPC title

• B01L9/527

  for microfluidic devices, e.g. used for lab-on-a-chip · CPC title

• C12N9/96

  Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates · CPC title

• C12Q1/6806

  Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence) · CPC title