SEQUENCING METHODS USING ENZYME CONFORMATION

• US 20110160077A1
• Filed: 12/14/2010
• Published: 06/30/2011
• Est. Priority Date: 12/16/2009
• Status: Active Grant

• Current Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Original Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Inventors: Bjornson, Keith, Chaisson, Mark, Jia, Lei, Silk, Christopher, Lyle, John, Clark, Sonya
• Granted Patent: US 8,911,972 B2
• Time in Patent Office: Days
• US Class Current: 506/9
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2521/101 DNA polymerase
• CPC Class Codes: C12Q 2561/113 Real time assay
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: C12Q 2565/133 conformational analysis

Abstract

Systems and methods are provided for single-molecule sequencing of template nucleic acids in which the signal from a label attached to a polymerase enzyme is used to monitor conformational changes in the polymerase which occur while labeled nucleotides or nucleotide analogs are added to a growing nucleic acid chain which is complementary to the template nucleic acid. The signal indicative of the conformational state of the enzyme is used to determine with higher confidence when true nucleotide or nucleotide analog incorporation events occur, allowing for the improved quality of base calls and sequence determination.

Claims

1. A method of sequencing comprising;

providing a single polytherase enzyme complex comprising a polymerase enzyme, a nucleic acid template, and a primer;

wherein the polymerase enzyme comprises a label which has a signal that changes when the enzyme undergoes a conformational change;

contacting the complex with sequencing reagents including two or more types of nucleotides or nucleotide analogs, each labeled with a different label, whereby enzyme mediated addition of nucleotides or nucleotide analogs to the primer to produce a growing strand complementary to the template occurs;

observing signals from the labeled nucleotides or nucleotide analogs and signals from the labeled enzyme during the enzyme mediated addition; and

determining the type of the nucleotides or nucleotide analog that is added to the growing strand using the observed signal from the label of that nucleotide or nucleotide analog;

whereby observed signals from the labeled polymerase enzyme are used to indicate whether a type of nucleotide or nucleotide analog is incorporated into the growing strand.

2. The method of claim 1 wherein the labels for the enzyme and the nucleotides or nucleotide analogs comprise fluorescent labels.

3. The method of claim 1 wherein the signal that changes when the enzyme undergoes a conformational change comprises a FRET signal from a donor and an acceptor.

4. The method of claim 3 wherein the donor and acceptor are both attached to the polymerase enzyme.

5. The method of claim 4 wherein the donor and acceptor are attached to portions of the enzyme which move relative to one another during a conformational change.

6. The method of claim 1 wherein the label whose signal changes when the enzyme undergoes a conformational change comprises a fluorescent label whose fluorescence is sensitive to changes in its local environment.

7. The method of claim 1 wherein the signal that changes when the enzyme undergoes a conformational change comprises quenching of a fluorescent label.

8. The method of claim 7 wherein the polymerase enzyme has both a fluorescent label and a quencher attached to different portions of the enzyme, and the quenching results from relative motions of the different portions of enzyme from a conformational change.

9. The method of claim 1, wherein the polymerase enzyme comprises a DNA polymerase or an RNA polymerase.

10. The method of claim 9 wherein the template comprises DNA or RNA.

11. The method of claim 9 wherein the polymerase is a modified Type B polymerase.

12. The method of claim 1 wherein the conformational change comprises translocation, or opening/closing of the fingers domain.

13. The method of claim 1 wherein at least one label is attached to the fingers, palm, thumb, or exo domains.

14. A method for nucleic acid sequencing comprising:

providing a reaction mixture for nucleic acid polymerization comprising four types of nucleotides or nucleotide analogs, each having a different label;

contacting the reaction mixture with a polymerase enzyme complex comprising a polymerase enzyme, a template nucleic acid, and a primer;

under conditions whereby enzyme mediated addition of nucleotides to produce a growing nucleic acid strand from the primer occurs, wherein the enzyme comprises a label which exhibits a change in optical properties when the enzyme undergoes a conformational change;

observing optical signals from the nucleotides or nucleotide analog and the enzyme while the nucleotides or nucleotide analogs are incorporated into the growing nucleic acid strand;

determining, using, the observed optical signal from the nucleotides or nucleotide analogs, when a particular type of nucleotide or nucleotide analog is associated with the enzyme;

determining, using the observed optical signal, when the enzyme undergoes a conformational change;

using the determination of when a particular nucleotide or nucleotide analog is associated with the enzyme in combination with the determination of when the enzyme undergoes conformational change to determine a nucleic acid sequence of the template.

15. The method of claim 14 wherein the labels for the enzyme and for the nucleotides &

nucleotide analogs comprise fluorescent labels.

16. The method of claim 14 wherein the signal that changes when the enzyme undergoes a conformational change comprises a FRET signal from a donor and an acceptor.

17. The method of claim 16 wherein the donor and acceptor are both attached to the polymerase enzyme.

18. The method of claim 17 wherein the donor and acceptor are attached to portions of the enzyme which move relative to one another during a conformational change.

19. The method of claim 14 wherein the label whose signal changes when the enzyme undergoes a conformational change comprises a fluorescent label whose fluorescence is sensitive to changes in its local environment.

20-24. -24. (canceled)

25. A system for sequencing comprising:

a substrate comprising a plurality of single polymerase enzyme complexes each comprising a polymerase enzyme, a nucleic acid template, and a primer;

wherein the polymerase enzyme comprises a label which has a signal that changes when the enzyme undergoes a conformational change, wherein the substrate comprising the complexes is in contact with sequencing reagents including two or more types of nucleotides or nucleotide analogs, each labeled with a different label, whereby enzyme mediated addition of nucleotides or nucleotide analogs to the primer to produce a growing strand complementary to the template occurs;

illumination optics for illuminating the enzyme complexes;

detection optics for observing signals from the labeled nucleotides or. nucleotide analogs and signals from the labeled enzyme during the enzyme mediated addition; and

a computer configured to determine the type of the nucleotides or nucleotide analog that is added to the growing strand using the observed signal from the label of the nucleotide or nucleotide analogs;

whereby observed signals from the labeled polymerase enzyme are used to indicate whether a type of nucleotide or nucleotide analog is incorporated into the growing strand.

32. The system of claim 25 wherein the label whose signal changes when the enzyme undergoes a conformational change comprises a fluorescent label whose fluorescence is sensitive to changes in its local environment.

33. The system of claim 25 wherein the signal that changes when the enzyme undergoes a conformational change comprises quenching of a fluorescent label.

34. The system of claim 33 wherein the polymerase enzyme has both a fluorescent label and a quencher attached to different portions of the enzyme, and the quenching results from relative motions of the different portions of the enzyme from a conformational change.

26-31. -31. (canceled)

35-39. -39. (canceled)