Modified polymerases for improved incorporation of nucleotide analogues
AssigneeIllumina Cambridge Limited
InventorsGeoffrey Paul Smith, David Mark Dunstan Bailey, Raquel Maria Sanches-Kuiper, Harold Swerdlow, David James Earnshaw
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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.
Claims21
-85. (canceled)
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.
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.
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.
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.
Dependencies are inferred from the claim text.