Modified polymerases for improved incorporation of nucleotide analogues

• US 9,447,389 B2
• Filed: 09/03/2014
• Issued: 09/20/2016
• Est. Priority Date: 09/11/2003
• Status: Active Grant

• Current Assignee: Illumina Cambridge Limited (Illumina Incorporated)
• Original Assignee: Illumina Cambridge Limited (Illumina Incorporated)
• Inventors: Smith, Geoffrey Paul, Bailey, David Mark Dunstan, Sanches-Kuiper, Raquel Maria, Swerdlow, Harold, Earnshaw, David James
• Primary Examiner(s): HUTSON, RICHARD G
• Application Number:
  US14/476,387
• Publication Number:
  US 20150024463A1
• Time in Patent Office: 748 Days
• Field of Search: None
• US Class Current: 1/1
• CPC Class Codes: C12N 9/1252 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Y 207/07007 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase

Abstract

The invention relates to modified polymerase enzymes which exhibit improved incorporation of nucleotide analogs 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.

Claims

1. An altered Vent DNA polymerase, wherein the second amino acid of the motif A region is mutated to alanine (A);

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 Vent DNA polymerase is capable of incorporating a 3′

O-azidomethyl-modified nucleotide.

2. The polymerase according to claim 1, which exhibits an increased rate of incorporation of the modified nucleotides, compared to a wild type Vent DNA polymerase, an exo-variant of wild type Vent DNA polymerase, or a wild type or exo-variant of wild type having one or more of the following additional mutations:

Y412V, Y412L, Y412F, Q486E, Q486L, R487K, A488C, A488S, A488L, A488I, A488F, A488V, K490A, K490R, K490N, N494D, S495A, Y496F, Y496L, Y497S, Y497F, Y499L, Y499F, A488C/Y499F or A488L/Y499L.

3. The polymerase according to claim 1, wherein the third amino acid of the motif A region is mutated to isoleucine (I), threonine (T), valine (V) or leucine (L).

4. The polymerase according to claim 1, wherein the third amino acid of the motif A region is mutated to serine (S), alanine (A) or glycine (G).

5. The polymerase according to claim 1, further comprising an amino acid substitution mutation at the first amino acid of the motif A region selected from tyrosine (Y), phenylalanine (F), isoleucine (I), alanine (A), valine (V), glutamine (Q), cysteine (C) or serine (S).

6. The polymerase according to claim 1, wherein the polymerase lacks 3′

-5′

exonuclease activity.

7. The polymerase according to claim 1, further comprising at least one amino acid substitution mutation in the motif B region, whereby the activity of the polymerase is enhanced.

8. The polymerase according to claim 7, wherein the substitution mutation in the motif B region is to the second amino acid.

9. The polymerase according to claim 8, wherein the substitution mutation is to an amino acid selected from the group consisting of F, I, S, V and C.

10. The polymerase according to claim 8, wherein the substitution mutation is to L.

11. The polymerase according to claim 7, wherein the motif B substitution mutations are to G at the first amino acid position and L at the third.

12. The polymerase according to claim 7, wherein the motif B substitution mutations are to N at the first amino acid position and Q at the third.

13. The polymerase according to claim 7, wherein the motif B substitution mutations are to those substitutions selected from the group consisting of SKN, GRD, KHN, ISN and THH.

14. The polymerase according to claim 1, further comprising at least one substitution mutation in the motif C region.

15. The polymerase according to claim 14, wherein the motif C region comprises at least one substitution mutation.