Patent ID: 8936926
Filing Date: 2015-01-20
Classification: C07K,C12N,C12Q

Abstract:
1. An isolated or recombinant DNA polymerase bound to a solid surface, the polymerase comprising: an active site; and, an artificial or recombinant surface coupling domain, wherein the surface coupling domain is distal to the active site; wherein the polymerase comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 1 and that comprises at least one mutation selected from the group consisting of an E375H mutation, an E375S mutation, an E375K mutation, an E375R mutation, an E375A mutation, an E375Q mutation, an E375W mutation, an E375Y mutation, an E375F mutation, and a K512A mutation, wherein identification of positions is relative to SEQ ID NO:1; wherein the surface coupling domain comprises: a biotin, a biotinylatable polypeptide sequence, an avidin sequence, a AviTag sequence, a recombinant dimer domain of the protein, a large extraneous polypeptide domain, a GST sequence, a glutathione, an S tag, a recombinant polymerase domain, or a combination thereof; wherein the surface coupling domain is bound to a binding partner that is coupled or bound to the solid surface, whereby the polymerase is bound to the solid surface; wherein the polymerase is capable of catalyzing a polymerization reaction while bound to the solid surface; and wherein the polymerase, when bound to the surface, displays a kcat/Km that is at least 10% as high as a corresponding DNA polymerase in solution; wherein the surface comprises or is proximal to a zero mode waveguide.