Polymerase compositions and methods

• US 9,365,839 B2
• Filed: 05/12/2015
• Issued: 06/14/2016
• Est. Priority Date: 03/27/2009
• Status: Active Grant

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Phelan, Michael, Peng, Xinzhan, Castillo, Amy, Hendricks, Stephen P., Peris, Marian, Chen, Cheng-Yao, Mazur, Daniel
• Primary Examiner(s): HUTSON, RICHARD G
• Application Number:
  US14/710,160
• Publication Number:
  US 20150247136A1
• Time in Patent Office: 399 Days
• Field of Search: None
• US Class Current: 1/1
• CPC Class Codes: C07H 19/20 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C12N 9/1241 Nucleotidyltransferases (2.7.7)
• CPC Class Codes: C12N 9/1252 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
• CPC Class Codes: C12N 9/96 Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Y 207/07 Nucleotidyltransferases (2.7.7)
• CPC Class Codes: C12Y 207/07007 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
• CPC Class Codes: G01N 2021/6432 Quenching
• CPC Class Codes: G01N 21/6428 Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" in vivo A61B5/00; immunoassay G01N33/53
• CPC Class Codes: G01N 33/582 with fluorescent label

Abstract

Disclosed herein are modified polymerase compositions exhibiting altered polymerase activity, which can be useful in a variety of biological applications. Also disclosed herein are methods of making and using such compositions. In some embodiments, the compositions exhibit altered properties that can enhance their utility in a variety of biological applications. Such altered properties, can include, for example, altered nucleotide binding affinities, altered nucleotide incorporation kinetics, altered photostability and/or altered nanoparticle tolerance, as well as a range of other properties as disclosed herein.

Claims

1. A mutant DNA polymerase comprising the amino acid sequence of SEQ ID NO:

7.

2. The mutant DNA polymerase of claim 1, which exhibits reduced 3′

to 5′

exonuclease activity compared to a wild type DNA polymerase which comprises the amino acid sequence of SEQ ID NO;

6.

3. The mutant DNA polymerase of claim 1, further comprising a peptide tag which includes 2-8 consecutive histidine residues, wherein the peptide tag is linked to the N-terminal or C-terminal end of the mutant DNA polymerase.

4. The mutant DNA polymerase of claim 3, wherein the peptide tag further comprises zinc, nickel, cobalt or copper ions.

5. The mutant DNA polymerase of claim 1, further comprising a peptide tag which includes the amino acid sequence MNHLVHHHHHHIEGRHMELGTLEGS (SEQ ID NO:

51), wherein the peptide tag is linked to the N-terminal or C-terminal end of the mutant DNA polymerase.

6. The mutant DNA polymerase of claim 1, further comprising a peptide tag which includes the amino acid sequence MHHHHHHKH (SEQ ID NO:

52), wherein the peptide tag is linked to the N-terminal or C-terminal end of the mutant DNA polymerase.

7. The mutant DNA polymerase of claim 1, further comprising a peptide tag which includes the amino acid sequence LLGAAAKGAAAKGSAA (SEQ ID NO:

16), wherein the peptide tag is linked to the N-terminal or C-terminal end of the mutant DNA polymerase.

8. The mutant DNA polymerase of claim 1, further comprising a peptide tag which includes the amino acid sequence LLGGGGSGGGGSAAAGSAA (SEQ ID NO:

17), wherein the peptide tag is linked to the N-terminal or C-terminal end of the mutant DNA polymerase.

9. The mutant DNA polymerase of claim 1, further comprising a peptide tag which includes the amino acid sequence GLNDIFEAQKIEWHE (SEQ ID NO:

53), wherein the peptide tag is linked to the N-terminal or C-terminal end of the mutant DNA polymerase.