Polymerases for nucleotide analogue incorporation

Abstract

Compositions that include polymerases with features for improving entry of nucleotide analogues into active site regions and for coordinating with the nucleotide analogues in the active site region are provided. Methods of making the polymerases and of using the polymerases in sequencing and DNA replication and amplification as well as kinetic models of polymerase activity and computer-implemented methods of using the models are also provided.

1 - 55 . (canceled) 
     
     
         56 . A recombinant nucleic acid polymerase comprising at least a first exogenous tag at the C-terminal region of the polymerase and at least a second exogenous tag at the N-terminal region of the polymerase. 
     
     
         57 . The polymerase of  claim 56 , wherein the first tag is selected from: a polyhistidine tag, a HIS-6 tag, a GST tag, a BiTag, an S Tag, a SNAP-tag, an HA tag, a plurality of HIS-6 tags, a plurality of GST tags, a plurality BiTags, a plurality of S Tags, a plurality of SNAP-tags, a plurality of HA tags, biotin, avidin, one or more. enterokinase sites, thrombin sites, antibodies or antibody domains, antibody fragments, antigens, receptors, receptor domains, receptor fragments, ligands, dyes, acceptors, quenchers, or combinations thereof. 
     
     
         58 . The polymerase of  claim 56 , wherein the second tag is selected from: a polyhistidine tag, a HIS-6 tag, a GST tag, a BiTag, an S Tag, a SNAP-tag, an HA tag, a plurality of HIS-6 tags, a plurality of GST tags, a plurality BiTags, a plurality of S Tags, a plurality of SNAP-tags, a plurality of HA tags, biotin, avidin, one or more enterokinase sites, thrombin sites, antibodies or antibody domains, antibody fragments, antigens, receptors, receptor domains, receptor fragments, ligands, dyes, acceptors, quenchers, or combinations thereof. 
     
     
         59 . The polymerase of  claim 56 , wherein the first tag and the second tag are the same. 
     
     
         60 . The polymerase of  claim 59 , wherein the first and second tags are polyhistidine tags. 
     
     
         61 . The polymerase of  claim 56 , wherein the first and second tags are different. 
     
     
         62 . The polymerase of  claim 56 , comprising a plurality of tags at the C-terminal region or a plurality of tags at the N-terminal region, or both. 
     
     
         63 . The polymerase of  claim 56 , wherein the polymerase is homologous to a 129 DNA polymerase. 
     
     
         64 . The polymerase of  claim 56 , wherein the tag is selected from: an affinity tag, a purification tag and a substrate binding tag. 
     
     
         65 . The polymerase of  claim 64 , wherein the substrate binding tag is capable of binding biotin or avidin.