Polymerases, compositions, and methods of use

Patent metadata

Field	Value
Publication number	US-12104182-B2
Application number	US-202318120639-A
Country	US
Kind code	B2
Filing date	Mar 13, 2023
Priority date	Dec 5, 2018
Publication date	Oct 1, 2024
Grant date	Oct 1, 2024

Abstract

Presented herein are altered polymerase enzymes for improved incorporation of nucleotides and nucleotide analogues, in particular altered polymerases that maintain high fidelity under reduced incorporation times, as well as methods and kits using the same.

First claim

The invention claimed is:

1. A kit for performing a nucleotide incorporation reaction comprising: a recombinant altered DNA polymerase comprising an amino acid sequence that is at least 80% identical to the 9°N DNA polymerase amino acid sequence comprising SEQ ID NO:1, wherein the polymerase comprises an amino acid substitution mutation at a position functionally equivalent to Ala281, Phe283, Thr349, or Trp397 in the 9°N DNA polymerase amino acid sequence, and a nucleotide solution.

2. The kit of claim 1 , wherein the nucleotide solution comprises modified nucleotides, wherein the modified nucleotides comprise a purine or pyrimidine base and a ribose or deoxyribose sugar moiety having a removable 3′-OH blocking group covalently attached thereto, such that the 3′ carbon atom has attached a group of the structure —O—Z, wherein Z is an azidomethyl group.

3. The kit of claim 2 , wherein the modified nucleotides comprise a nucleotide or nucleoside having a base attached to a detectable label via a cleavable linker.

4. The kit of claim 1 , the polymerase further comprising an amino acid substitution mutation at a position functionally equivalent to Tyr497 and at least one amino acid substitution mutation at a position functionally equivalent to Arg247, Glu599, or His633 in the 9°N DNA polymerase amino acid sequence.

5. The kit of claim 4 , wherein the substitution mutation at the position functionally equivalent to Tyr497 comprises a mutation to Gly.

6. The kit of claim 4 , wherein the substitution mutation at the position functionally equivalent to Arg247 comprises a mutation to Tyr.

7. The kit of claim 4 , wherein the substitution mutation at the position functionally equivalent to Glu599 comprises a mutation to Asp.

8. The kit of claim 4 , wherein the substitution mutation at the position functionally equivalent to His633 comprises a mutation to Gly.

9. The kit of claim 4 , wherein the polymerase comprises at least two or three amino acid substitution mutations at positions functionally equivalent to an amino acid selected from Arg247, Glu599, or His633 in the 9°N DNA polymerase amino acid sequence.

10. The kit of claim 4 , wherein the polymerase further comprises at least one amino acid substitution mutation at a position functionally equivalent to Lys620 or Val661 in the 9°N DNA polymerase amino acid sequence.

11. The kit of claim 10 , wherein the substitution mutation at the position functionally equivalent to Lys620 comprises a mutation to Arg.

12. The kit of claim 10 , wherein the substitution mutation at the position functionally equivalent to Val661 comprises a mutation to Asp.

13. The kit of claim 4 , wherein the polymerase further comprises amino acid substitution mutations at positions functionally equivalent to amino acids Met129, Asp141, Glu143, Cys223, Leu408, Tyr409, Pro410, or Ala485 in the 9°N DNA polymerase amino acid sequence.

14. The kit of claim 1 , wherein the polymerase comprises the amino acid sequence of any one of SEQ ID NOs:9-17.

15. The kit of claim 1 , wherein the substitution mutation at the position functionally equivalent to Ala281 comprises a mutation to Gly or Phe.

16. The kit of claim 1 , wherein the substitution mutation at the position functionally equivalent to Phe283 comprises a mutation to Ser.

17. The kit of claim 1 , wherein the substitution mutation at the position functionally equivalent to Thr349 comprises a mutation to Ser or Asn.

18. The kit of claim 1 , wherein the substitution mutation at the position functionally equivalent to Thr349 comprises a mutation to Lys.

19. The kit of claim 1 , wherein the substitution mutation at the position functionally equivalent to Trp397 comprises a mutation to Cys.

20. The kit of claim 1 , wherein the substitution mutation at the position functionally equivalent to Trp397 comprises a mutation to Phe.

21. The kit of claim 1 , wherein the polymerase comprises at least two, at least three, or four amino acid substitution mutations at positions functionally equivalent to an amino acid selected from Ala281, Phe283, Thr349, or Trp397 in the 9°N DNA polymerase amino acid sequence.

22. The kit of claim 1 , wherein the polymerase comprises the substitution mutation at the position functionally equivalent to Thr349, and further comprises amino acid substitution mutations at positions functionally equivalent to amino acids Met129, Asp141, Glu143, Cys223, Leu408, Tyr409, Pro410, Ala485, Tyr497, Arg247, Glu599, and His633 in the 9°N DNA polymerase amino acid sequence.

23. The kit of claim 22 , wherein the substitution mutation at the position functionally equivalent to Thr349 comprises a mutation to Lys.

Assignees

• Illumina Inc
• Illumina Cambridge Ltd
• Illumina Singapore Pte Ltd

Inventors

• Klausing Kay
• Ghomi Hamed Tabatabaei
• Golynskiy Misha
• Nirantar Saurabh
• Mcdonald Seth
• Peisajovich Sergio

Classifications

• C12Y207/07007

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

• C12Q2563/107

  fluorescence · CPC title

• C12Q1/68

  involving nucleic acids · CPC title

• C12N15/52

  Genes encoding for enzymes or proenzymes · CPC title

• C12N9/1252 Primary

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