Modified polymerases for improved incorporation of nucleotide analogues

• US 10,696,955 B2
• Filed: 11/28/2018
• Issued: 06/30/2020
• Est. Priority Date: 09/30/2014
• Status: Active Grant

• Current Assignee: Illumina Incorporated
• Original Assignee: Illumina Incorporated
• Inventors: Bomati, Erin, Previte, Michael, Kellinger, Matthew William, Chen, Cheng-Yao, He, Molly
• Primary Examiner(s): Raghu, Ganapathirama
• Application Number:
  US16/202,787
• Publication Number:
  US 20190144839A1
• Time in Patent Office: 580 Days
• CPC Class Codes: C12N 9/1252 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2521/101 DNA polymerase
• CPC Class Codes: C12Y 207/07007 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase

Abstract

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

Claims

1. A method for incorporating modified nucleotides into DNA comprising allowing the following components to interact:

(i) a DNA template;

(ii) a nucleotide solution; and

(iii) 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 method of claim 1, wherein the DNA template comprises a clustered array.

3. The method of claim 1, wherein the nucleotide solution comprises nucleotides modified at the 3′

sugar hydroxyl.

4. A kit for performing a nucleotide incorporation reaction comprising:

a nucleotide solution and 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.

5. The kit of claim 4, wherein the nucleotide solution comprises labelled nucleotides.

6. The kit of claim 4, wherein the nucleotides comprise synthetic nucleotides.

7. The kit of claim 4, wherein the nucleotides comprise modified nucleotides.

8. The kit of claim 4, wherein the modified nucleotides have been modified at the 3′

sugar hydroxyl such that the substituent is larger in size than the naturally occurring 3′

hydroxyl group.

9. The kit of claim 7, wherein modified nucleotides comprise a modified nucleotide or nucleoside molecule comprising 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 any of —

C(R′

)₂—

O—

R″

, —

C(R′

)₂—

N(R″

)₂, —

C(R′

)₂—

N(H)R″

, —

C(R′

)₂—

S—

R″ and

—

C(R′

)₂—

F,

wherein each R″

is or is part of a removable protecting group;

each R′

is independently a hydrogen atom, an alkyl, substituted alkyl, arylalkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclic, acyl, cyano, alkoxy, aryloxy, heteroaryloxy or amido group, or a detectable label attached through a linking group;

or (R′

)₂ represents an alkylidene group of formula ═

C(R′

″

)₂ wherein each R′

″

may be the same or different and is selected from the group comprising hydrogen and halogen atoms and alkyl groups; and

wherein said molecule may be reacted to yield an intermediate in which each R″

is exchanged for H or, where Z is —

C(R′

)₂—

F, the F is exchanged for OH, SH or NH₂, preferably OH, which intermediate dissociates under aqueous conditions to afford a molecule with a free 3′

OH;

with the proviso that where Z is —

C(R′

)₂—

S—

R″

, both R′

groups are not H.

10. The kit of claim 9, wherein R′

of the modified nucleotide or nucleoside is an alkyl or substituted alkyl.

11. The kit of claim 9, wherein —

Z of the modified nucleotide or nucleoside is of formula —

C(R′

)₂—

N₃.

12. The kit of claim 9, wherein Z is an azidomethyl group.

13. The kit of claim 7, wherein the modified nucleotides are fluorescently labelled to allow their detection.

14. The kit of claim 7, wherein the modified nucleotides comprise a nucleotide or nucleoside having a base attached to a detectable label via a cleavable linker.

15. The kit of claim 14, wherein the detectable label comprises a fluorescent label.

16. The kit of claim 4, further comprising one or more DNA template molecules, primers, or a combination thereof.