Polymerase enzyme from 9°n

Abstract

The present invention relates to a polymerase enzyme from 9°N with improved ability to incorporate reversibly terminating nucleotides. The enzyme comprising mutations in the motif A region. The invention also relates to methods of using such enzymes as well as a kit with such polymerases.

1 . A polymerase enzyme according to SEQ ID NO. 1 or any polymerase that shares at least 70% amino acid sequence identity thereto, comprising the following mutation(s):
 i. at position 408 of SEQ ID NO. 1 a mutation selected from:
 1. (H—histidine) (L408H), 
 2. (M—methionine) (L408M), 
 3. (T—threonine) (L408T), 
 4. (G—glycine) (L408M), 
 5. (N—asparagine) (L408N), and 
 6. (S—serine) (L408S) 
   ii. at position 409 of SEQ ID NO. 1 a mutation selected from:
 1. (T—threonine) (Y409T), 
 2. (V—valine) (Y409V), and 
 3. (G—glycine) (Y409G) 
   iii. at position 410 of SEQ ID NO. 1 a mutation selected from:
 1. (C—cysteine) (P410C), 
 2. (N—asparagine) (P410N), 
 3. (D—aspartic acid) (P410D), 
 4. (Q—glutamine) (P410Q), 
 5. (F—phenylalanine) (P410F), and 
 6. (S—serine) (P410S) 
   wherein the enzyme has little or no 3′-5′ exonuclease activity.   
     
     
         2 . The polymerase enzyme of  claim 1 , wherein the polymerase is from an organism belonging to the family of Thermococcaceae, preferably from the genera of  Pyrococcus , preferably 9°N and the mutations are: 
       
         
           
                 
                 
                 
                 
                 
               
                     
                     
                 
                     
                   Name 
                   408 
                   409 
                   410 
                 
                     
                     
                 
                     
                   JPol105 
                   H 
                   T 
                   C 
                 
                     
                   JPol106 
                   M 
                   T 
                   C 
                 
                     
                   JPol107 
                   G 
                   T 
                   F 
                 
                     
                   JPol108 
                   G 
                   T 
                   C 
                 
                     
                   JPol109 
                   N 
                   T 
                   C 
                 
                     
                   JPol110 
                   G 
                   T 
                   N 
                 
                     
                   JPol111 
                   T 
                   T 
                   C 
                 
                     
                   JPol112 
                   H 
                   V 
                   C 
                 
                     
                   JPol113 
                   M 
                   V 
                   C 
                 
                     
                   JPol114 
                   T 
                   V 
                   C 
                 
                     
                   JPol115 
                   H 
                   V 
                   D 
                 
                     
                   JPol116 
                   M 
                   V 
                   D 
                 
                     
                   JPol117 
                   T 
                   V 
                   D 
                 
                     
                   JPol118 
                   H 
                   T 
                   Q 
                 
                     
                   JPol119 
                   M 
                   T 
                   Q 
                 
                     
                   JPol120 
                   T 
                   T 
                   Q 
                 
                     
                   JPol121 
                   H 
                   T 
                   N. 
                 
                     
                     
                 
             
                
                
                
               
               
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
               
            
           
         
       
     
     
         3 . The polymerase enzyme according to  claim 1 , wherein the polymerase further comprises a D141A mutation, a E143A mutation, or both. 
     
     
         4 . The polymerase enzyme according to  claim 1 , wherein the polymerase further comprises a A485L mutation. 
     
     
         5 . The polymerase enzyme according to  claim 1 , wherein the polymerase enzyme shares 95% or 98% sequence identity with SEQ ID NO. 1 and comprises the following mutations, (i) L408S, Y409G, P410S and (ii) A485L. 
     
     
         6 . The polymerase enzyme according to  claim 1 , wherein the polymerase enzyme has an amino acid sequence according to SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20. 
     
     
         7 . The polymerase enzyme according to  claim 1 , wherein the polymerase enzyme exhibits an increased rate of incorporation of nucleotides which have been modified at the 3′ sugar hydroxyl such that the substituent is larger in size than the naturally occurring 3′ hydroxyl group, compared to the control polymerase. 
     
     
         8 . A nucleic acid molecule encoding a polymerase enzyme according to  claim 1 , wherein the polymerase enzyme has a sequence according to SEQ ID NO. 3. 
     
     
         9 . An expression vector comprising the nucleic acid molecule of  claim 8 . 
     
     
         10 . A method for incorporating nucleotides which have been modified at the 3′ sugar hydroxyl such that the substituent is larger in size than the naturally occurring 3′ hydroxyl group into DNA comprising the following substances (i) a polymerase enzyme according to  claim 1 , (ii) template DNA, (iii) one or more nucleotides, which have been modified at the 3′ sugar hydroxyl such that the substituent is larger in size than the naturally occurring 3′ hydroxyl group. 
     
     
         11 . Use of a polymerase enzyme according to  claim 1  for DNA sequencing, DNA labeling, primer extension, amplification or the like. 
     
     
         12 . A kit comprising a polymerase enzyme according to  claim 1 .