US2023212534A1PendingUtilityA1

Polymerases, compositions, and methods of use

77
Assignee: ILLUMINA INCPriority: Oct 31, 2018Filed: Dec 21, 2022Published: Jul 6, 2023
Est. expiryOct 31, 2038(~12.3 yrs left)· nominal 20-yr term from priority
Inventors:Misha GolynskiySeth McdonaldSaurabh NirantarMatthew KellingerMichael PreviteSergio PeisajovichMolly He
C12N 9/1241C12N 1/20C12Y 207/07007C12N 9/1252C12Q 1/6844C12Q 2521/101C12Q 2563/107C12N 15/52
77
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Claims

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.

Claims

exact text as granted — not AI-modified
1 - 19 . (canceled) 
     
     
         20 . A recombinant DNA polymerase comprising an amino acid sequence that is at least 80% identical to a 9° N DNA polymerase amino acid sequence SEQ ID NO:1, wherein the recombinant DNA polymerase comprises an amino acid substitution mutation at position functionally equivalent to Tyr497 and at least one amino acid substitution mutation at a position functionally equivalent to Lys476, Lys477, Thr514, Ile521, or Thr590 in the 9° N DNA polymerase amino acid sequence. 
     
     
         21 . The polymerase of  claim 20 , wherein the substitution mutation at the position functionally equivalent to Lys476 comprises a mutation to a non-polar or hydrophobic amino acid. 
     
     
         22 . The polymerase of  claim 20 , wherein the substitution mutation at the position functionally equivalent to Lys476 comprises a mutation to Trp. 
     
     
         23 . The polymerase of  claim 20 , wherein the substitution mutation at the position functionally equivalent to Lys477 comprises a mutation to a non-polar or hydrophobic amino acid. 
     
     
         24 . The polymerase of  claim 20 , wherein the substitution mutation at the position functionally equivalent to Lys477 comprises a mutation to Met. 
     
     
         25 . The polymerase of  claim 20 , wherein the substitution mutation at the position functionally equivalent to Thr514 comprises a mutation to a polar or uncharged amino acid. 
     
     
         26 . The polymerase of  claim 20 , wherein the substitution mutation at the position functionally equivalent to Thr514 comprises a mutation to Ser. 
     
     
         27 . The polymerase of  claim 20 , wherein the substitution mutation at the position functionally equivalent to Ile521 comprises a mutation to a non-polar or hydrophobic amino acid. 
     
     
         28 . The polymerase of  claim 20 , wherein the substitution mutation at the position functionally equivalent to Ile521 comprises a mutation to Leu. 
     
     
         29 . The polymerase of  claim 20 , wherein the substitution mutation at the position functionally equivalent to Thr590 comprises a mutation to a non-polar or hydrophobic amino acid. 
     
     
         30 . The polymerase of  claim 20 , wherein the substitution mutation at the position functionally equivalent to Thr590 comprises a mutation to Ile. 
     
     
         31 . The polymerase of  claim 20 , wherein the polymerase comprises at least two, at least three, at least four, or five amino acid substitution mutations at positions functionally equivalent to an amino acid selected from Lys476, Lys477, Thr514, Ile521, and Thr590 in the 9° N DNA polymerase amino acid sequence. 
     
     
         32 . The polymerase of  claim 20 , wherein the polymerase further comprises amino acid substitution mutations at positions functionally equivalent to amino acids Met129, Asp141, Glu143, Cys223, Leu408, Tyr409, Pro410, and Ala485 in the 9° N DNA polymerase amino acid sequence. 
     
     
         33 - 45 . (canceled) 
     
     
         46 . A recombinant DNA polymerase comprising an amino acid sequence that is at least 80% identical to a 9° N DNA polymerase amino acid sequence SEQ ID NO:1, wherein the recombinant DNA polymerase comprises (i) an amino acid substitution mutation at position functionally equivalent to Tyr497; (ii) at least one amino acid substitution mutation at a position functionally equivalent to Phe152, Va1278, Met329, Va1471, Leu631, or Glu734 in the 9° N DNA polymerase amino acid sequence, and (iii) at least one amino acid substitution mutation at a position functionally equivalent to Lys476, Lys477, Thr514, Ile521, or Thr590 in the 9° N DNA polymerase amino acid sequence. 
     
     
         47 . The polymerase of  claim 46 , wherein the polymerase comprises at least two, at least three, at least four, at least five, or six amino acid substitution mutations at positions functionally equivalent to an amino acid selected from Phe152, Va1278, Met329, Va1471, Leu631, and Glu734 in the 9° N DNA polymerase amino acid sequence. 
     
     
         48 . The polymerase of  claim 46 , wherein the polymerase comprises at least two, at least three, at least four, or five amino acid substitution mutations at positions functionally equivalent to an amino acid selected from Lys476, Lys477, Thr514, Ile521, and Thr590 in the 9° N DNA polymerase amino acid sequence. 
     
     
         49 . The polymerase of  claim 46 , wherein the polymerase further comprises amino acid substitution mutations at positions functionally equivalent to amino acids Met129, Asp141, Glu143, Cys223, Leu408, Tyr409, Pro410, and Ala485 in the 9° N DNA polymerase amino acid sequence. 
     
     
         50 - 53 . (canceled) 
     
     
         54 . A recombinant DNA polymerase comprising an amino acid sequence that is at least 80% identical to a 9° N DNA polymerase amino acid sequence SEQ ID NO:1, wherein the recombinant DNA polymerase comprises (i) an amino acid substitution mutation at position functionally equivalent to Tyr497; (ii) at least one amino acid substitution mutation at a position functionally equivalent to Lys476, Lys477, Thr514, Ile521, or Thr590 in the 9° N DNA polymerase amino acid sequence, and (iii) at least one amino acid substitution mutation at a position functionally equivalent to Arg247, Glu599, Lys620, His633, or Va1661 in the 9° N DNA polymerase amino acid sequence. 
     
     
         55 . The polymerase of  claim 54 , wherein the polymerase comprises at least two, at least three, at least four, or five amino acid substitution mutations at positions functionally equivalent to an amino acid selected from Lys476, Lys477, Thr514, Ile521, or Thr590 in the 9° N DNA polymerase amino acid sequence. 
     
     
         56 . The polymerase of  claim 54 , wherein the polymerase comprises at least two, at least three, at least four, or five amino acid substitution mutations at positions functionally equivalent to an amino acid selected from Arg247, Glu599, Lys620, His633, or Va1661 in the 9° N DNA polymerase amino acid sequence. 
     
     
         57 . The polymerase of  claim 54 , wherein the polymerase further comprises amino acid substitution mutations at positions functionally equivalent to amino acids Met129, Asp141, Glu143, Cys223, Leu408, Tyr409, Pro410, and Ala485 in the 9° N DNA polymerase amino acid sequence. 
     
     
         58 . A recombinant DNA polymerase comprising an amino acid sequence that is at least 80% identical to a 9° N DNA polymerase amino acid sequence SEQ ID NO:1, wherein the recombinant DNA polymerase comprises (i) an amino acid substitution mutation at position functionally equivalent to Tyr497; (ii) at least one amino acid substitution mutation at a position functionally equivalent to Phe152, Va1278, Met329, Va1471, Leu631, or Glu734 in the 9° N DNA polymerase amino acid sequence, (iii) at least one amino acid substitution mutation at a position functionally equivalent to Lys476, Lys477, Thr514, Ile521, or Thr590 in the 9° N DNA polymerase amino acid sequence, and (iv) at least one amino acid substitution mutation at a position functionally equivalent to Arg247, Glu599, Lys620, His633, or Va1661 in the 9° N DNA polymerase amino acid sequence. 
     
     
         59 . The polymerase of  claim 58 , wherein the polymerase comprises at least two, at least three, at least four, at least five, or six amino acid substitution mutations at positions functionally equivalent to an amino acid selected from Phe152, Va1278, Met329, Va1471, Leu631, and Glu734 in the 9° N DNA polymerase amino acid sequence. 
     
     
         60 . The polymerase of  claim 58 , wherein the polymerase comprises at least two, at least three, at least four, or five amino acid substitution mutations at positions functionally equivalent to an amino acid selected from Lys476, Lys477, Thr514, Ile521, or Thr590 in the 9° N DNA polymerase amino acid sequence. 
     
     
         61 . The polymerase of  claim 58 , wherein the polymerase comprises at least two, at least three, at least four, or five amino acid substitution mutations at positions functionally equivalent to an amino acid selected from Arg247, Glu599, Lys620, His633, or Va1661 in the 9° N DNA polymerase amino acid sequence. 
     
     
         62 . The polymerase of  claim 58 , wherein the polymerase further comprises amino acid substitution mutations at positions functionally equivalent to amino acids Met129, Asp141, Glu143, Cys223, Leu408, Tyr409, Pro410, and Ala485 in the 9° N DNA polymerase amino acid sequence. 
     
     
         63 - 98 . (canceled)

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