Modified polymerases for improved incorporation of nucleotide analogues

Patent metadata

Field	Value
Publication number	US-9677057-B2
Application number	US-201514869792-A
Country	US
Kind code	B2
Filing date	Sep 29, 2015
Priority date	Sep 30, 2014
Publication date	Jun 13, 2017
Grant date	Jun 13, 2017

Abstract

Presented herein are polymerase enzymes for improved incorporation of nucleotide analogs, in particular nucleotides which are modified at the 3′ sugar hydroxyl, as well as methods and kits using the same.

First claim

What is claimed is:

1. A recombinant DNA polymerase comprising an amino acid sequence that is at least 80% identical to SEQ ID NO:10, which recombinant DNA polymerase comprises at least one amino acid substitution mutation at one or more positions functionally equivalent to Thr144, Gly153, Lys476, Leu478, Thr590, Ala639 or Asp718 in the 9° N DNA polymerase amino acid sequence, wherein the mutation at the position functionally equivalent to Gly153 comprises a mutation to a polar amino acid, wherein the mutation at the position functionally equivalent to Lys476 comprises a mutation to a hydrophobic amino acid, wherein the mutation at the position functionally equivalent to Leu478 comprises a mutation to a polar amino acid, wherein the mutation at the position functionally equivalent to Asp718 comprises a mutation homologous to Asp718Asn, and wherein the DNA polymerase is a family B DNA polymerase.

2. The altered polymerase of claim 1 , wherein said substitution mutation at position Thr144 comprises a mutation to a nonpolar amino acid.

3. The altered polymerase of claim 1 , wherein said substitution mutation at position Thr144 comprises a mutation homologous to Thr144Ala, Thr144Gly, or Thr144Leu.

4. The altered polymerase of claim 1 , wherein said substitution mutation at position Gly153 comprises a mutation homologous to Gly153Asp.

5. The altered polymerase of claim 1 , wherein said substitution mutation at position Lys476 comprises a mutation homologous to Lys476Trp.

6. The altered polymerase of claim 1 , wherein said substitution mutation at position Leu478 comprises a mutation homologous to Leu478Ser, Leu478Arg, or Leu478Thr.

7. The altered polymerase of claim 1 , wherein said substitution mutation at position Thr590 comprises a mutation to a non-polar amino acid.

8. The altered polymerase of claim 1 , wherein said substitution mutation at position Thr590 comprises a mutation homologous to Thr590Ile, or Thr590Gly.

9. The altered polymerase of claim 1 , wherein said substitution mutation at position Ala639 comprises a mutation homologous to Ala639Val, or Ala639Phe.

10. The altered polymerase of claim 1 , wherein the altered polymerase further comprises substitution mutations at positions functionally equivalent to Leu408, Tyr409, Pro410, or a combination thereof, in the 9° N DNA polymerase amino acid sequence.

11. The altered polymerase of claim 1 , wherein the altered polymerase comprises substitution mutations at positions functionally equivalent to Asp141, Glu143, or a combination thereof, in the 9° N DNA polymerase amino acid sequence.

12. The altered polymerase of claim 1 , wherein the altered polymerase further comprises substitution mutations at positions functionally equivalent to Ala485 in the 9° N DNA polymerase amino acid sequence.

13. The recombinant DNA polymerase of claim 1 , wherein said substitution mutation comprises a mutation homologous to Thr144Ala, Thr144Gly, Thr144Leu, Gly153Asp, Lys476Trp, Leu478Ser, Leu478Arg, Leu478Thr, Thr590Ile, Thr590Gly, Ala639Val, Ala639Phe or Asp718Asn, relative to SEQ ID NO: 10.

14. The altered polymerase of claim 1 , wherein amino acid sequence is at least 90% identical to SEQ ID NO: 10.

15. The altered polymerase of claim 1 , wherein amino acid sequence is at least 95% identical to SEQ ID NO: 10.

16. The altered polymerase of claim 1 , wherein amino acid sequence is at least 99% identical to SEQ ID NO: 10.

Assignees

• Illumina Inc

Inventors

• Bomati Erin
• Previte Michael
• Kellinger Matthew William
• Chen Cheng-Yao
• He Molly

Classifications

• C12Y207/07007

  DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase · CPC title

• C12Q1/6844

  Nucleic acid amplification reactions · CPC title

• C12N9/1252 Primary

  DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase · CPC title

• C12Q1/686

  Polymerase chain reaction [PCR] · CPC title

• C12Q2521/101

  DNA polymerase · CPC title