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.
Claims31
-85. (canceled)
An altered family B archaeal DNA polymerase comprising a 3-amino acid region, motif A, wherein the first amino acid of the motif A region is an amino acid selected from the group consisting of isoleucine (I), alanine (A), valine (V), and serine (S);
- the second amino acid of the motif A region is an amino acid selected from the group consisting of alanine (A) and glycine (G);
- and the third amino acid of the motif A region is an amino acid selected from the group consisting of isoleucine (I), valine (V), leucine (L), threonine (T), and proline (P);
- wherein the altered family B archaeal DNA polymerase is capable of incorporating a 3′ O-azidomethyl-modified nucleotide.
The polymerase of claim 0, further comprising a second 3-amino acid region, motif B, wherein the second amino acid of the motif B region is a leucine (L).
An altered family B archaeal DNA polymerase comprising a 3-amino acid region, motif A, wherein the first amino acid of the motif A region is an amino acid selected from the group consisting of isoleucine (I), alanine (A), valine (V), and serine (S);
- the second amino acid of the motif A region is an amino acid selected from the group consisting of alanine (A) and glycine (G);
- and the third amino acid of the motif A region is an amino acid selected from the group consisting of isoleucine (I), valine (V), leucine (L), threonine (T), and proline (P);
- wherein the altered family B archaeal DNA polymerase is capable of incorporating a nucleotide that has been modified at the 3′ sugar hydroxyl such that the substituent is larger in size than the naturally occurring 3′ hydroxyl group.
Dependencies are inferred from the claim text.