Method for nucleic acid sequencing

• US 10,577,651 B2
• Filed: 05/10/2018
• Issued: 03/03/2020
• Est. Priority Date: 04/30/2004
• Status: Active Grant

• Current Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Original Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Inventors: Williams, John G. K.
• Primary Examiner(s): Bertagna, Angela M.
• Application Number:
  US15/976,701
• Publication Number:
  US 20180258481A1
• Time in Patent Office: 663 Days
• Field of Search: None
• CPC Class Codes: B01J 2219/00497 Features relating to the solid phase supports
• CPC Class Codes: B01J 2219/005 Beads
• CPC Class Codes: B01J 2219/00576 fluorophore
• CPC Class Codes: B01J 2219/00648 by the use of solid beads
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: C07H 21/04 with deoxyribosyl as saccharide radical
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 1/6825 Nucleic acid detection involving sensors
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2521/101 DNA polymerase
• CPC Class Codes: C12Q 2521/107 RNA dependent DNA polymerase,(i.e. reverse transcriptase)
• CPC Class Codes: C12Q 2523/307 Denaturation or renaturation by electric current/voltage
• CPC Class Codes: C12Q 2525/113 incorporating modified backbone
• CPC Class Codes: C12Q 2525/117 incorporating modified base
• CPC Class Codes: C12Q 2525/307 Circular oligonucleotides
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: C12Q 2563/149 Particles, e.g. beads
• CPC Class Codes: C12Q 2565/518 characterised by the immobilisation of the nucleic acid sample or target

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.

Claims

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 and a primer 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

extending the primer strand by 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 is selected from the group consisting of Klenow, Taq, phi29, RB-69, T4, and T7 polymerase.

13. The method of claim 12, wherein the polymerase is phi29.

14. 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.

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

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

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

18. The method of claim 1, wherein the nucleotides comprise at least one detectably labeled nucleoside triphosphate.