Purification of polymerase complexes

Patent metadata

Field	Value
Publication number	US-11746337-B2
Application number	US-202016871973-A
Country	US
Kind code	B2
Filing date	May 11, 2020
Priority date	Nov 25, 2015
Publication date	Sep 5, 2023
Grant date	Sep 5, 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 a polymerase complex comprising
(a4) isolating active polymerase complexes by a method comprising:
(a4a) providing a reaction mixture comprising:
(i) a polymerase complex comprising:
(1) 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 runway 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;
(2) a primer specific to the primer recognition sequence of the adaptor; and
(3) a polymerase enzyme; and

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

(a4b) 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;
(a4c) combining the reaction mixture and the dNTP mixture to 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;
(a4d) 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
(a4e) isolating said active polymerase complexes having extended runway complementary sequences from inactive polymerase complexes comprising unextended runway complementary sequences;

wherein said polymerase complex isolated according to (a4) optionally further comprises a nanopore to provide a nanopore-polymerase complex; and
(b) preparing a biochip by a method comprising either:
(b1) attaching said polymerase complex to a nanopore pre-formed in the membrane of said biochip to provide a nanopore-polymerase complex; or
(b2) inserting said nanopore-polymerase complexes obtained from (a) into the membrane of said biochip;

(c) optionally, sequencing a polynucleotide template by a method comprising:
(c1) providing tagged nucleotides or nucleotide analogs to said nanopore sequencing complex, wherein the tag of said tagged nucleotide is detectable with the aid of said nanopore;
(c2) carrying out a polymerization reaction with the aid of said polymerase coupled to said nanopore in said nanopore-sequencing complex, thereby incorporating an individual tagged nucleotide of said tagged nucleotides into a growing strand complementary to a sample polynucleotide template; and
(c3) detecting, with the aid of said nanopore, a tag associated with said individual tagged nucleotide during incorporation of said individual tagged nucleotide, wherein said tag is detected with the aid of said nanopore while said nucleotide is associated with said polymerase, thereby providing a sequence of said nucleic acid sample.

2. The method of claim 1 , wherein step (a4e) further 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 the single-stranded region of the adaptor of step (a4a) is a linear or a circular template.

5. The method of claim 1 , wherein the binding of the active polymerase complex to the solid phase support in step (a4d) is reversible.

6. The method of claim 1 , wherein the capture moiety of step (a4b) is a biotin or modified biotin, and the binding partner is streptavidin or modified streptavidin.

7. The method of claim 6 , wherein the biotin compound or said modified biotin compound comprises desthiobiotin or a derivative thereof.

8. The method of claim 1 , wherein the isolated active polymerase complexes of step (a4e) 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

• 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