COGNATE SAMPLING KINETICS

• US 20110244447A1
• Filed: 02/22/2011
• Published: 10/06/2011
• Est. Priority Date: 02/12/2008
• Status: Active Grant

• Current Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Original Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Inventors: Korlach, Jonas
• Granted Patent: US 8,652,781 B2
• Time in Patent Office: Days
• US Class Current: 435/6.1
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C07K 2319/22 containing a Strep-tag
• CPC Class Codes: C07K 2319/23 containing a GST-tag
• CPC Class Codes: C07K 2319/30 Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
• CPC Class Codes: C07K 2319/41 containing a Myc-tag
• CPC Class Codes: C07K 2319/42 containing a HA(hemagglutinin)-tag
• CPC Class Codes: C07K 2319/50 containing protease site
• 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 2521/101 DNA polymerase
• CPC Class Codes: C12Q 2525/101 incorporating non-naturally occurring nucleotides, e.g. inosine
• CPC Class Codes: C12Q 2527/113 Time
• CPC Class Codes: C12Q 2527/137 Concentration of a component of medium
• CPC Class Codes: C12Q 2537/161 A competitive reaction step
• CPC Class Codes: C12Q 2563/107 fluorescence

Abstract

Provided are methods and compositions for measuring the transient binding of nucleotides and nucleotide analogs under conditions where the nucleotides or nucleotide analogs are unincorporable. The transient binding can be determined under single molecule observation conditions providing information about the kinetics of nucleotide analog sampling of the active site of the enzyme. The methods can be used for polymerase enzyme development, mechanistic understanding, and drug discovery.

Claims

1. A method comprising:

providing a polymerase enzyme complex comprising a template nucleic acid, a primer, and a polymerase enzyme under conditions wherein the interactions between a single polymerase and a labeled nucleotide analog can be monitored;

exposing the polymerase enzyme complex to one or more labeled nucleotide analogs in solution under conditions wherein the nucleotide analogs are unincorporable;

observing the transient binding of the one or more nucleotide analogs over time; and

using the observed transient binding to provide information about the enzyme, template nucleic acid, primer, or nucleotide analog or analogs.

2. The method of claim 1 wherein the primer is terminated, rendering the nucleotide analogs unincorporable.

3. The method of claim 1 wherein the method is carried out with a plurality of nucleotide analogs, and the transient binding over time is used to determine the effect of nucleotide structure on enzyme performance.

4. A method for identifying a base in a template nucleic acid comprising:

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

exposing the polymerase enzyme complex to one or more labeled nucleotide analogs in solution under conditions wherein the nucleotide analogs are unincorporatable, wherein one of the nucleotide analogs is a cognate nucleotide analog; and

detecting multiple transient binding events corresponding to the binding of a cognate nucleotide analog to the polymerase complex, thereby identifying the base corresponding to the cognate nucleotide analog in the template nucleic acid.

5. The method of claim 4 wherein the polymerase enzyme complex comprises a single polymerase molecule.

6. The method of claim 4 wherein the polymerase enzyme complex is immobilized on a substrate.

7. The method of claim 4 wherein the polymerase enzyme complex is immobilized in an optical confinement.

8. The method of claim 4 wherein at least a first and second primer are provided, wherein each of the primers are selected to bind to a known nucleotide sequence in which the identity of the base in the nucleotide sequence corresponding to the next cognate nucleotide is known and is different for the different primers;

and wherein the polymerase enzyme complex is exposed to two or more unincorporatable nucleotide analogs, wherein the two or more unincorporable nucleotides comprise the cognate nucleotides for the two primers.