Methods and systems for direct sequencing of single DNA molecules

• US 9,382,584 B2
• Filed: 03/28/2014
• Issued: 07/05/2016
• Est. Priority Date: 12/11/2008
• Status: Active Grant

• Current Assignee: Regents of the University of California (University of California)
• Original Assignee: Regents of the University of California (University of California)
• Inventors: Huang, Xiaohua
• Primary Examiner(s): HUTSON, RICHARD G
• Application Number:
  US14/229,621
• Publication Number:
  US 20140295412A1
• Time in Patent Office: 830 Days
• Field of Search: None
• US Class Current: 1/1
• CPC Class Codes: C12N 9/1252 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2565/101 Interaction between at least two labels
• CPC Class Codes: C12Q 2565/133 conformational analysis

Abstract

The invention provides improved methods for sequencing nucleic acids, e.g., for medical applications and biomedical research. The disclosed methods can be applied to rapid personalized medicine, genetic diagnosis, pathogen identification, and sequencing species genomes.

Claims

1. A labeled DNA polymerase, wherein said DNA polymerase comprises at least one FRET donor and at least one FRET acceptor, wherein said FRET donor and FRET acceptor are positioned on the DNA polymerase so that when the polymerase adds a nucleotide to the nascent strand of DNA, a distinct FRET signal is generated at least depending on which base (A, C, G, T) is incorporated, wherein:

the FRET acceptor is positioned on the finger domain of the DNA polymerase and the FRET donor is positioned on the thumb domain of the DNA polymerase;

or

wherein the FRET donor is positioned on the finger domain of the DNA polymerase and the FRET acceptor is positioned on the thumb domain of the DNA polymerase,

the labeled DNA polymerase is a phi-29 DNA polymerase, and wherein the phi-29 DNA polymerase comprises E375C and K240C mutations.

2. The labeled DNA polymerase of claim 1, wherein the FRET donor is within 10 angstroms of the Fö

rster radius (R₀) from the FRET acceptor when the DNA polymerase is in the open position.

3. The labeled DNA polymerase of claim 1, wherein the Fö

rster radius between the FRET donor and the FRET acceptor changes at least 2.5 angstroms from the open position to the closed position of the DNA polymerase.

4. The labeled DNA polymerase of claim 1, further comprising at least a second FRET donor and at least a second FRET acceptor.

5. The labeled DNA polymerase of claim 1, wherein the FRET donor and FRET acceptor are positioned so that a distinct FRET signal is generated when the DNA polymerase reads a methylated nucleotide on the template DNA.

6. A system for real time sequencing of a DNA molecule comprising the labeled DNA polymerase of claim 1 and optical instrumentation capable of detecting a FRET signal from a single molecule.

7. The labeled DNA polymerase of claim 1, wherein the FRET donor and the FRET acceptor are separately linked to different amino acid residues selected from:

E375C and K240C.

8. The labeled DNA polymerase of claim 1, wherein the labeled DNA polymerase is labeled with more than one FRET pair, wherein the FRET donor in each FRET pair is within 10 angstroms of the Fö

rster radius (R_o) from the FRET acceptor in each FRET pair when the DNA polymerase is in the open position.

9. The labeled DNA polymerase of claim 1, wherein the FRET acceptor is positioned on the finger domain of the DNA polymerase and the FRET donor is positioned on the thumb domain of the DNA polymerase.

10. The labeled DNA polymerase of claim 1, wherein the FRET acceptor is positioned on the thumb domain of the DNA polymerase and the FRET donor is positioned on the finger domain of the DNA polymerase.

11. The system of claim 6, comprising a plurality of DNA molecules immobilized onto a substrate.

12. The system of claim 11, wherein the DNA molecules are attached to the substrate at more than one site.

13. The labeled DNA polymerase of claim 1, wherein the Fö

rster radius between the FRET donor and the FRET acceptor changes 1-10 angstroms from the open position to the closed position of the DNA polymerase.