Modified polymerases for improved incorporation of nucleotide analogues
First Claim
Patent Images
1. An altered 9°
- N DNA polymerase, comprising the sequence of SEQ ID NO;
14, whereinthe first amino acid of the motif A region is mutated to an amino acid selected from the group consisting of the aromatic amino acids, the amino acids with aliphatic side chains, glutamine (Q), cysteine (C), and serine (S);
the second amino acid of the motif A region is mutated to an amino acid selected from the group consisting of alanine (A), serine (S), and glycine (G); and
the third amino acid of the motif A region is mutated to an amino acid selected from the group consisting of serine (S), alanine (A), glycine (G), and the amino acids having beta-branched side chains,whereby the altered 9°
N DNA polymerase is capable of incorporating a 3′
O-azidomethyl-modified nucleotide.
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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.
33 Citations
33 Claims
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1. An altered 9°
- N DNA polymerase, comprising the sequence of SEQ ID NO;
14, whereinthe first amino acid of the motif A region is mutated to an amino acid selected from the group consisting of the aromatic amino acids, the amino acids with aliphatic side chains, glutamine (Q), cysteine (C), and serine (S); the second amino acid of the motif A region is mutated to an amino acid selected from the group consisting of alanine (A), serine (S), and glycine (G); and the third amino acid of the motif A region is mutated to an amino acid selected from the group consisting of serine (S), alanine (A), glycine (G), and the amino acids having beta-branched side chains, whereby the altered 9°
N DNA polymerase is capable of incorporating a 3′
O-azidomethyl-modified nucleotide.- View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 10, 11, 12, 26, 27, 28, 29, 30, 31, 32)
- N DNA polymerase, comprising the sequence of SEQ ID NO;
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9. An altered 9°
- N DNA polymerase, comprising the sequence of SEQ ID NO;
14, whereinthe first amino acid of the motif A region is mutated to an amino acid selected from the group consisting of the aromatic amino acids, the amino acids with aliphatic side chains, glutamine (Q), cysteine (C), and serine (S); the second amino acid of the motif A region is mutated to an amino acid selected from the group consisting of alanine (A), serine (S), and glycine (G); the third amino acid of the motif A region is mutated to an amino acid selected from the group consisting of serine (S), alanine (A), glycine (G), and the amino acids having beta-branched side chains, and comprises at least one amino acid substitution mutation in the motif B region, whereby the activity of the polymerase is enhanced, with the proviso that said at least one amino acid substitution mutation does not include the exact combination of 409V and 485L, whereby the altered 9°
N DNA polymerase is capable of incorporating a 3′
O-azidomethyl-modified nucleotide.- View Dependent Claims (13, 14, 15, 16, 17, 18)
- N DNA polymerase, comprising the sequence of SEQ ID NO;
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19. An altered 9°
- N DNA polymerase, comprising the sequence of SEQ ID NO;
14, whereinthe first amino acid of the motif A region is mutated to an amino acid selected from the group consisting of the aromatic amino acids, the amino acids with aliphatic side chains, glutamine (Q), cysteine (C), and serine (S); the second amino acid of the motif A region is mutated to an amino acid selected from the group consisting of alanine (A), serine (S), and glycine (G); the third amino acid of the motif A region is mutated to an amino acid selected from the group consisting of serine (S), alanine (A), glycine (G), and the amino acids having beta-branched side chains, and wherein the motif C region comprises at least one substitution mutation, whereby the altered 9°
N DNA polymerase is capable of incorporating a 3′
O-azidomethyl-modified nucleotide.
- N DNA polymerase, comprising the sequence of SEQ ID NO;
-
20. An altered 9°
- N DNA polymerase, comprising the sequence of SEQ ID NO;
14, whereinthe first amino acid of the motif A region is mutated to an amino acid selected from the group consisting of the aromatic amino acids, the amino acids with aliphatic side chains, glutamine (Q), cysteine (C), and serine (S); the second amino acid of the motif A region is mutated to an amino acid selected from the group consisting of alanine (A), serine (S), and glycine (G); the third amino acid of the motif A region is mutated to an amino acid selected from the group consisting of serine (S), alanine (A), glycine (G), and the amino acids having beta-branched side chains, and which comprises at least one substitution mutation from amino acid 141 or 143 to an amino acid that is not negatively charged, whereby exonuclease activity is removed, whereby the altered 9°
N DNA polymerase is capable of incorporating a 3′
O-azidomethyl-modified nucleotide.- View Dependent Claims (21)
- N DNA polymerase, comprising the sequence of SEQ ID NO;
-
22. An altered 9°
- N DNA polymerase comprising the sequence of SEQ ID NO;
12.
- N DNA polymerase comprising the sequence of SEQ ID NO;
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23. An altered 9°
- N DNA polymerase comprising the sequence of SEQ ID NO;
18.
- N DNA polymerase comprising the sequence of SEQ ID NO;
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24. An altered 9°
- N DNA polymerase comprising the sequence of SEQ ID NO;
20.
- N DNA polymerase comprising the sequence of SEQ ID NO;
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25. An altered 9°
- N DNA polymerase comprising the amino acid sequence SEQ ID NO;
22.
- N DNA polymerase comprising the amino acid sequence SEQ ID NO;
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33. An altered 9°
- N DNA polymerase, comprising the sequence of SEQ ID NO;
14, whereinthe first amino acid of the motif A region is mutated to an amino acid selected from the group consisting of the aromatic amino acids, the amino acids with aliphatic side chains, glutamine (Q), cysteine (C), and serine (S); the second amino acid of the motif A region is mutated to an amino acid selected from the group consisting of alanine (A), serine (S), and glycine (G); the third amino acid of the motif A region is mutated to an amino acid selected from the group consisting of serine (S), alanine (A), glycine (G), and the amino acids having beta-branched side chains, comprises at least one amino acid substitution mutation in the motif B region, whereby the activity of the polymerase is enhanced, with the proviso that said at least one amino acid substitution mutation does not include the exact combination of 409V and 485L, and which comprises at least one substitution mutation from amino acid 141 or 143 to an amino acid that is not negatively charged, whereby exonuclease activity is removed, whereby the altered 9°
N DNA polymerase is capable of incorporating a 3′
O-azidomethyl-modified nucleotide.
- N DNA polymerase, comprising the sequence of SEQ ID NO;
Specification