Method for nucleic acid sequencing

Patent metadata

Field	Value
Publication number	US-2018258481-A1
Application number	US-201815976701-A
Country	US
Kind code	A1
Filing date	May 10, 2018
Priority date	Apr 30, 2004
Publication date	Sep 13, 2018
Grant date	—

Abstract

The present invention provides novel compositions, methods and apparatus for DNA sequencing that can be performed, e.g., in a two-electrode chamber. The present invention also provides a method for sequencing a nucleic acid comprising immobilizing a plurality of complexes comprising a target nucleic acid, a primer nucleic acid, and a polymerase onto a surface, contacting the surface with a plurality of charged particles comprising a nucleotide phosphate by applying an electric field, reversing the electric field to transport unbound charged particles away from the surface, and detecting the incorporation of a nucleotide phosphate into a single molecule of the primer nucleic acid.

First claim

What is claimed is:

1 . A method of incorporating nucleotides with a polymerase comprising;
providing a polymerase-nucleic acid complex, the polymerase-nucleic acid complex comprising: a nucleic acid comprising a circular template strand, a polymerase comprising a nucleic acid binding cleft, and a topological tether, wherein the topological tether is attached to the polymerase through at least two anchors that straddle the nucleic acid binding cleft, whereby the topological tether entraps the template strand in the nucleic acid binding cleft of the polymerase, and
incorporating nucleotides to form a product strand complementary to the template strand of the nucleic acid.

2 . The method of claim 1 , wherein at least one of the anchors comprises at least one amino acid of the polymerase or an epitope of the polymerase.

3 . The method of claim 2 , wherein the at least one amino acid is selected from the group consisting of a cysteine, a phenylalanine derivative and a histidine

4 . The method of claim 3 , wherein the at least one amino acid is a cysteine.

5 . The method of claim 1 , wherein the nucleic acid binding cleft is a DNA binding cleft.

6 . The method of claim 1 , wherein the at least two anchors comprise cysteines of the polymerase.

7 . The method of claim 1 , wherein the at least two anchors each comprise a first member of a complementary binding pair that is bound to a second member of the complementary binding pair, which second member is located on the topological tether, whereby the topological tether is attached to the polymerase.

8 . The method of claim 7 , wherein the binding pair is biotin-streptavidin or biotin-avidin.

9 . The method of claim 1 , wherein the topological tether is covalently attached to the polymerase.

10 . The method of claim 1 , wherein the topological tether irreversibly associates the nucleic acid with the polymerase.

11 . The method of claim 1 , wherein the topological tether is directly anchored to a solid support.

12 . The method of claim 1 , wherein the polymerase-nucleic acid complex further comprises a primer nucleic acid which complements a region of the nucleic acid.

13 . The method of claim 1 , wherein the polymerase is selected from the group consisting of Klenow, Taq, phi29, RB-69, T4, and T7 polymerase.

14 . The method of claim 14 , wherein the polymerase is phi29.

15 . The method of claim 1 , wherein the polymerase-nucleic acid complex shows a higher processivity index than a polymerase-nucleic acid complex without said topological tether.

16 . The method of claim 1 , wherein the polymerase is a DNA polymerase from Thermus flavus, Pyrococcus furiosus, Thermotoga neapolitana, Thermococcus litoralis, Sulfolobus solfataricus, Thermatoga maritima , or E. coli.

17 . The method of claim 1 , wherein the polymerase is a reverse transcriptase.

18 . The method of claim 1 , wherein the polymerase is an HIV reverse transcriptase.

19 . The method of claim 1 , wherein the nucleotides comprise detectably labeled nucleotides.

20 . The method of claim 1 , wherein the nucleotides comprise detectably labeled nucleoside triphosphate.

Assignees

• Pacific Biosciences California Inc

Inventors

• Williams John G K

Classifications

• C12Q2563/149

  Particles, e.g. beads · CPC title

• B01J2219/00497

  Features relating to the solid phase supports · CPC title

• C12Q1/6818

  involving interaction of two or more labels, e.g. resonant energy transfer · CPC title

• C07H21/04

  with deoxyribosyl as saccharide radical · CPC title

• B01J2219/005

  Beads · CPC title