Polymerase compositions and methods

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.

1 . A DNA polymerase having a photostability that is at least about 80% under standard photostability assay conditions. 
     
     
         2 . The DNA polymerase of  claim 1 , wherein the polymerase comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 7. 
     
     
         3 . The DNA polymerase of  claim 2 , further comprising one or more amino acid substitutions selected from the group consisting of: D9A, E11A, E11I, T12I, H58R, N59D, D63A, Y162F, Y162C, D166A, Q377A, S385G, H370G, H370T, H370S, H370K, H370R, H370A, H370Q, H370 W, H370Y, H370F, E371G, E371H, E371T, E371S, E371K, E371R, E371A, E371Q, E371W, E371Y, E371F, K372G, K372E, K372T, K372S, K372R, K372A, K372Q, K372W, K372Y, K372F, K380E, K380T, K380S, K380R, K380A, K380Q, K380W, K380Y, K380F, D507H, D507G, D507E, D507T, D5075, D507R, D507A, D507R, D507Q, D507W, D507Y, D507F, K509H, K509G, K509D, K509R, K509E, K509T, K5095, K509R, K509A, K509Q, K509W, K509Y and K509F, wherein the numbering is relative to the amino acid sequence of SEQ ID NO: 7. 
     
     
         4 . The DNA polymerase of  claim 1 , wherein the polymerase further comprises a mutation reducing 3′ to 5′ exonuclease activity. 
     
     
         5 . The DNA polymerase of  claim 4 , wherein the mutation comprises one or more amino acid substitutions at positions selected from the group consisting of: 2, 9, 11, 12, 58, 59, 63, 162, 166, 377 and 385. 
     
     
         6 . The DNA polymerase of  claim 1 , wherein the polymerase further comprises a mutation increasing the branching ratio of the enzyme with a labeled nucleotide. 
     
     
         7 . The DNA polymerase of  claim 6 , wherein the mutation comprises one or more amino acid substitutions at positions selected from the group consisting of: 370, 371, 372, 373, 374, 375, 376 and 377. 
     
     
         8 . The DNA polymerase of  claim 1 , wherein the polymerase further comprises a mutation increasing the primer extension activity. 
     
     
         9 . The DNA polymerase of  claim 8 , wherein the mutation comprises one or more amino acid substitutions selected from the group consisting of: 129 and 339. 
     
     
         10 . The DNA polymerase of  claim 1 , wherein the polymerase further comprises a mutation increasing the affinity of the polymerase for a particular labeled nucleotide. 
     
     
         11 . The DNA polymerase of  claim 10 , wherein the mutation comprises one or more amino acid substitutions selected from the group consisting of: 370, 371, 372, 373, 374, 375, 376, 507 and 509. 
     
     
         12 . A nucleic acid molecule encoding the DNA polymerase of  claim 1 , wherein the nucleic acid molecule is DNA or RNA. 
     
     
         13 . A vector comprising a DNA encoding the polymerase of  claim 2 . 
     
     
         14 . An isolated host cell comprising the nucleic acid molecule of  claim 12 . 
     
     
         15 . An isolated host cell comprising the vector of  claim 13 . 
     
     
         16 . A method for obtaining a polymerase comprising: purifying the polymerase from the isolated host cell of  claim 15 . 
     
     
         17 . A method for performing a primer extension reaction, comprising: contacting a modified polymerase comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 7 with a nucleic acid molecule and a nucleotide under conditions where the nucleotide is incorporated into the nucleic acid molecule by the polymerase. 
     
     
         18 . The method of  claim 17 , wherein the at least one nucleotide is a labeled nucleotide, and the label of the nucleotide emits a signal during incorporation of the nucleotide. 
     
     
         19 . The method of  claim 18 , further comprising detecting the signal emitted by the nucleotide label. 
     
     
         20 . The method of  claim 19 , further comprising analyzing the detected signal to determine the identity of the incorporated nucleotide. 
     
     
         21 . The method of  claim 17 , wherein the modified polymerase exhibits a t −1  value for a labeled nucleotide that is equal to or greater than the t −1  value of a reference Phi-29 polymerase comprising the amino acid sequence of SEQ ID NO: 1 for the same nucleotide. 
     
     
         22 . The method of  claim 17 , wherein the modified polymerase exhibits a t pol  value for a labeled nucleotide that is equal to or greater than the t pol  value of a reference Phi-29 polymerase comprising the amino acid sequence of SEQ ID NO: 1 for the same nucleotide. 
     
     
         23 . The method of  claim 17 , wherein the modified polymerase exhibits a residence time for a labeled nucleotide that is equal to or greater than the residence time of a reference Phi-29 polymerase comprising the amino acid sequence of SEQ ID NO: 1 for the same nucleotide. 
     
     
         24 . The method of  claim 17 , wherein the modified polymerase has a photostability of at least about 80% under standard photostability assay conditions. 
     
     
         25 . The method of  claim 17 , wherein the modified polymerase has a nanoparticle tolerance of at least about 80% under standard tolerance assay conditions. 
     
     
         26 . The method of  claim 18 , wherein the labeled nucleotide comprises a nucleotide label linked to the base, sugar or phosphate group of the nucleotide. 
     
     
         27 . The method of  claim 18 , wherein the labeled nucleotide is a reversible terminator. 
     
     
         28 . The method of  claim 18 , wherein the modified polymerase is linked to a label. 
     
     
         29 . The method of  claim 27 , wherein the label of the modified polymerase and the nucleotide label are capable of undergoing FRET with each other. 
     
     
         30 . A modified DNA polymerase having an increased branching ratio in the presence of labeled nucleotides relative to a B103 polymerase comprising the amino acid sequence of SEQ ID NO: 7. 
     
     
         31 . The modified DNA polymerase of  claim 30 , wherein the polymerase comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 7. 
     
     
         32 . The modified DNA polymerase of  claim 31 , further comprising one or more amino acid mutations selected from the group consisting of: T365G, T365F, T365G, T365S, T365K, T365R, T365A, T365Q, T365W, T365Y, T365H, H370G, H370T, H370S, H370K, H370R, H370A, H370Q, H370 W, H370Y, H370F, E371G, E371H, E371T, E371S, E371K, E371R, E371A, E371Q, E371W, E371Y, E371F, K372G, K372E, K372T, K372S, K372R, K372A, K372Q, K372W, K372Y, K372F, K380E, K380T, K380S, K380R, K380A, K380Q, K380W, K380Y, K380F, A481E, A481F, A481G, A481S, A481R, A481K, A481A, A481T, A481Q, A481W, A481Y, D507H, D507G, D507E, D507T, D507S, D507R, D507A, D507R, D507Q, D507W, D507Y, D507F, K509H, K509G, K509D, K509R, K509E, K509T, K509S, K509R, K509A, K509Q, K509W, K509Y and K509F, wherein the numbering is relative to the amino acid sequence of SEQ ID NO: 7. 
     
     
         33 . The modified DNA polymerase of  claim 32 , further comprising the amino acid mutation D166A and having reduced exonuclease activity relative to a DNA polymerase having the amino acid sequence of SEQ ID NO: 7.