Purification of polymerase complexes

Patent metadata

Field	Value
Publication number	US-11525124-B2
Application number	US-202016871732-A
Country	US
Kind code	B2
Filing date	May 11, 2020
Priority date	Nov 25, 2015
Publication date	Dec 13, 2022
Grant date	Dec 13, 2022

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, said method comprising:
providing a reaction mixture comprising:
(a1) a polymerase complex comprising:
(a1a) a nucleic acid adaptor for isolating active polymerase complexes, said adaptor having a single-stranded region comprising a primer recognition sequence, a runway sequence located 5′ to the primer recognition sequence, and a polymerase termination sequence located 5′ to the runway sequence, wherein:
said runway sequence comprises a nucleotide sequence having between 2 and 50 contiguous nucleotide bases selected from no more than three of the four nucleotide bases of adenine, cytosine, guanine, and thymine,
the nucleotide base that is not contained in the runaway sequence is designated as a stop base,
said runway sequence functions as a template for polymerase-driven primer extension, and
said polymerase termination sequence comprises at least one stop base that is effective to terminate any such polymerase-driven primer extension;

(a1b) a primer specific to the primer recognition sequence of the adaptor; and
(a1c) a polymerase enzyme; and

(a2) a nucleic acid sample, wherein the adaptor of the polymerase complex is ligated to said nucleic acid sample;

(b) providing a deoxynucleotide triphosphate (dNTP) mixture comprising only those dNTPs that are complementary to the nucleotide bases contained in the runway sequence of the adaptor, wherein one or more of the dNTPs is modified to include a capture moiety having affinity to a binding partner;
(c) combining the reaction mixture and the dNTP mixture: enable synthesis of a polynucleotide sequence complementary to the runway sequence by the activity of the polymerase to obtain a plurality of active polymerase complexes comprising extended runway complementary sequences having modified dNTPs incorporated therein;
(d) binding said active polymerase complexes to a solid phase support, wherein the capture moieties of the modified dNTPs are bound to binding partners on the solid phase support; and
(e) isolating said active polymerase complexes having the extended runway complementary sequences from inactive polymerase complexes comprising unextended runway complementary sequences.

2. The method of claim 1 , wherein said isolating comprises washing away the inactive polymerase complexes to yield active polymerase complexes bound to the solid phase support.

3. The method of claim 2 , further comprising eluting the active polymerase complexes from the solid phase support.

4. The method of claim 1 , wherein said single-stranded region of the adaptor is a linear or a circular template.

5. The method of claim 1 , wherein said binding of said active polymerase complex to said solid phase support is reversible.

6. The method of claim 1 , wherein said capture moiety is a biotin or modified biotin and said binding partner is streptavidin or modified streptavidin.

7. The method of claim 6 , where said biotin compound or said modified biotin compound comprises desthiobiotin or a derivative thereof and said binding partner comprises streptavidin or a derivative thereof.

8. The method of claim 1 , wherein the isolated active polymerase complexes each comprise 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

• C12Q2565/50

  Detection characterised by immobilisation to a surface · CPC title

• C12N9/96

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

• C12Q2565/631

  being a biochannel or pore · CPC title

• C12Q1/6806

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

• C12Q1/6869

  Methods for sequencing · CPC title