Taq polymerase sequences useful for incorporating analogs of nucleoside triphosphates

• US 9,428,737 B1
• Filed: 01/11/2010
• Issued: 08/30/2016
• Est. Priority Date: 01/11/2010
• Status: Active Grant

• Current Assignee: Eric Gaucher
• Original Assignee: Eric Gaucher
• Inventors: Gaucher, Eric
• Primary Examiner(s): HUTSON, RICHARD G
• Application Number:
  US12/655,931
• Time in Patent Office: 2,423 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

Abstract

This patent applications concerns compositions of matter that are DNA polymerases, where those polymerases have had one or more of their amino acids replaced at sites chosen by an analysis of patterns of conservation and replacement within homologous protein sequences. Disclosed here are sites within Family A DNA polymerases where amino acid replacement creates polymerases having utility, in an example where DNA nucleotides are incorporated having modified or unnatural nucleobases, and/or nucleotides whose sugar is unnatural or derivatized, including 3′-O-amino-2′-deoxyribonucleoside triphosphates. The claimed compositions include polymerases that hold amino acid replacements at claimed sites in Taq polymerase, and are prepared by site-directed mutagenesis that modifies a gene encoding a parent Taq gene (natural or already mutated) to change the codon encoding the amino acid at the claimed site, giving a variant gene that encodes a Taq polymerase protein that has a different amino acid at the claimed site.

Claims

1. A DNA polymerase from Thermus aquaticus wherein the amino acid at position 616, as defined by SEQ ID NO:

1, is replaced by an amino acid selected from the group consisting of Ala, Arg, Cys, Glu, Gln, Gly, His, Ile, Lys, Met, Phe, Ser, Thr, Trp, Tyr, Val and Asp.