Modified polymerases for improved incorporation of nucleotide analogues

Abstract

The invention relates to modified polymerase enzymes which exhibit improved incorporation of nucleotide analogues bearing substituents at the 3′ position of the sugar moiety that are larger in size than the naturally occurring 3′hydroxyl group. Also described are methods of using the polymerases to incorporate nucleotides into polynucleotides, particularly in the context of DNA sequencing.

1 .- 85 . (canceled) 
     
     
         86 . A polynucleotide extension composition comprising:
 one or more 3′-O-azidomethyl nucleotide triphosphates, and   a protein means for incorporating said 3′-O-azidomethyl nucleotide triphosphates into a polynucleotide, wherein said protein means lacks 3′-5′ exonuclease activity and comprises at least two mutations in a motif A region.   
     
     
         87 . The polynucleotide extension composition of  claim 86 , wherein said protein means comprises three mutations in the motif A region. 
     
     
         88 . The polynucleotide extension composition of  claim 86 , wherein said protein means is selected from the group consisting of selected from Vent polymerase, Deep Vent polymerase, 9°N polymerase,  Thermococcus  sp. JDF-3 polymerase, and Pfu polymerase. 
     
     
         89 . The polynucleotide extension composition of  claim 86 , wherein said protein means comprises a first amino acid of the motif A region that is an amino acid selected from the group consisting of isoleucine (I), alanine (A), valine (V), and serine (S); a second amino acid of the motif A region that is an amino acid selected from the group consisting of alanine (A) and glycine (G); and a third amino acid of the motif A region that is an amino acid selected from the group consisting of isoleucine (I), valine (V), leucine (L), threonine (T), and proline (P). 
     
     
         90 . The polynucleotide extension composition of  claim 89 , wherein said first amino acid of the motif A region is an amino acid selected from the group consisting of isoleucine (I), alanine (A), and valine (V). 
     
     
         91 . The polynucleotide extension composition of  claim 89 , wherein said second amino acid of the motif A region is an alanine (A). 
     
     
         92 . The polynucleotide extension composition of  claim 89 , wherein said third amino acid of the motif A region is an amino acid selected from the group consisting of isoleucine (I), valine (V), leucine (L), and threonine (T). 
     
     
         93 . The polynucleotide extension composition of  claim 89 , wherein said first amino acid of the motif A region is an amino acid selected from the group consisting of alanine (A) and serine (S). 
     
     
         94 . The polynucleotide extension composition of  claim 86 , wherein said protein means comprises at least one mutation in a motif B region, wherein a second amino acid of the motif B region is an amino acid selected from the group consisting of leucine (L), valine (V), serine (S), lysine (K), arginine (R), and histidine (H). 
     
     
         95 . The polynucleotide extension composition of  claim 86 , wherein said protein means comprises at least one mutation in a motif B region, wherein a second amino acid of the motif B region is an amino acid selected from the group consisting of leucine (L) and valine (V). 
     
     
         96 . A polynucleotide extension composition comprising:
 a protein means for incorporating nucleotide triphosphates into a polynucleotide, wherein said protein means lacks 3′-5′ exonuclease activity and comprises at least two mutations in a motif A region, and   a buffer solution suitable for protein-mediated incorporation of nucleotide triphosphates into a polynucleotide.   
     
     
         97 . The polynucleotide extension composition of  claim 96 , wherein said composition further comprises one or more nucleotide triphosphate molecules that have been modified at the 3′ sugar hydroxyl such that the substituent is larger in size than the naturally occurring 3′ hydroxyl group. 
     
     
         98 . The polynucleotide extension composition of  claim 96 , wherein said protein means is selected from the group consisting of selected from Vent polymerase, Deep Vent polymerase, 9°N polymerase,  Thermococcus  sp. JDF-3 polymerase, and Pfu polymerase. 
     
     
         99 . The polynucleotide extension composition of  claim 96 , wherein said protein means comprises a first amino acid of the motif A region that is an amino acid selected from the group consisting of isoleucine (I), alanine (A), valine (V), and serine (S); a second amino acid of the motif A region that is an amino acid selected from the group consisting of alanine (A) and glycine (G); and a third amino acid of the motif A region that is an amino acid selected from the group consisting of isoleucine (I), valine (V), leucine (L), threonine (T), and proline (P). 
     
     
         100 . The polynucleotide extension composition of  claim 96 , wherein said protein means comprises at least one mutation in a motif B region, wherein a second amino acid of the motif B region is an amino acid selected from the group consisting of leucine (L) and valine (V). 
     
     
         101 . A polynucleotide extension composition comprising:
 one or more 3′-O-azidomethyl nucleotide triphosphates, and   a protein means for incorporating said 3′-O-azidomethyl nucleotide triphosphates into a polynucleotide, wherein said protein means lacks 3′-5′ exonuclease activity and said protein means incorporates 3′-O-azidomethyl nucleotide triphosphates into a polynucleotide faster than a 9°N DNA polymerase that contains mutations D141A, D143A, Y409V, and A485L (SEQ ID NO: 16) incorporates 3′-O-azidomethyl nucleotide triphosphates into a polynucleotide.   
     
     
         102 . A polynucleotide extension composition comprising:
 a protein means for incorporating nucleotide triphosphates into a polynucleotide, wherein said protein means lacks 3′-5′ exonuclease activity and said protein means incorporates 3′-O-azidomethyl nucleotide triphosphates into a polynucleotide faster than a 9°N DNA polymerase that contains mutations D141A, D143A, Y409V, and A485L (SEQ ID NO: 16) incorporates 3′-O-azidomethyl nucleotide triphosphates into a polynucleotide, and   a buffer solution suitable for protein-mediated incorporation of nucleotide triphosphates into a polynucleotide.   
     
     
         103 . The polynucleotide extension composition of  claim 102 , wherein said composition further comprises one or more nucleotide triphosphate molecules that have been modified at the 3′ sugar hydroxyl such that the substituent is larger in size than the naturally occurring 3′ hydroxyl group. 
     
     
         104 . The polynucleotide extension composition of  claim 103 , wherein said nucleotide triphosphate molecules comprise a purine or pyrimidine base and a deoxyribose sugar moiety having a 3′carbon atom attached to a group of the structure —O—Z, wherein Z is an azidomethyl group. 
     
     
         105 . The polynucleotide extension composition of  claim 102 , wherein said protein means exhibits a time to 50% nucleotide triphosphate incorporation on the first incorporation cycle of 20 seconds or less.