FIELD-SWITCH SEQUENCING

• US 20150086994A1
• Filed: 10/07/2014
• Published: 03/26/2015
• 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., Anderson, Jon P.
• Granted Patent: US 10,000,798 B2
• Time in Patent Office: Days
• US Class Current: 435/6.12
• 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 polymerase-nucleic acid complex, the polymerase-nucleic acid complex comprising:

a target nucleic acid, a polymerase, and an attachment complex, wherein the attachment complex comprises a topological tether attached to the polymerase through at least two anchors; and

wherein the attachment complex entraps the target nucleic acid.

2. The polymerase-nucleic acid complex 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 polymerase-nucleic acid complex of claim 2, wherein the at least one amino acid is a member selected from the group consisting of a cysteine, a phenylalanine derivative and a histidine.

4. The polymerase-nucleic acid complex of claim 3, wherein the at least one amino acid is a cysteine.

5. The polymerase-nucleic acid complex of claim 1, wherein the attachment complex straddles the DNA binding cleft.

6. The polymerase-nucleic acid complex of claim 1, wherein the at least two anchors comprise cysteines of the polymerase.

7. The polymerase-nucleic acid complex of claim 1, wherein the topological tether is attached to the polymerase via complementary binding pairs.

8. The polymerase-nucleic acid complex of claim 7, wherein the binding pair is biotin-streptavidin or biotin-avidin.

9. The polymerase-nucleic acid complex of claim 1, wherein the attachment complex is covalently attached to the polymerase.

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

11. The polymerase-nucleic acid complex of claim 1, wherein the topological tether is anchored to a solid support.

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

13. The polymerase-nucleic acid complex of claim 1, wherein the target nucleic acid is a circular DNA.

14. The polymerase-nucleic acid complex of claim 1, wherein the polymerase is a member selected from the group consisting of Klenow, Taq, and T7 polymerase.

15. The polymerase-nucleic acid complex of claim 1, wherein the polymerase is a member selected from the group consisting of a therminator polymerase, phi29, RB-69 and T4 polymerase.

16. The polymerase-nucleic acid complex of claim 15, wherein the polymerase is phi29.

17. A method for single molecule sequencing comprising observing a polymerase-nucleic acid complex of claim 1 in real-time while it incorporates detectably labeled nucleic acid triphosphates onto the growing end of a primer nucleic acid molecule.

18. A polymerase-nucleic acid complex, the polymerase-nucleic acid complex comprising:

a target nucleic acid, a polymerase, and an attachment complex, wherein the attachment complex comprises a topological tether attached to the polymerase through at least two anchors; and

wherein said polymerase-nucleic acid complex shows a higher processivity index than a polymerase-nucleic acid complex without said, attachment complex.