Polymerase mutants and use with 3′-OH unblocked reversible terminators
AssigneeAgilent Technologies, Inc.
InventorsMichelle Cayouette, Jeffrey Fox, Connie Hansen, Holly Hogrefe, Weidong Wu
Life of this patent
Estimating the term…
Abstract
Mutant polymerases are provided that have improved ability to incorporate modified nucleotides, including 3′-OH unblocked reversible terminators. The mutant polymerases may be used in a variety of applications, such as for polynucleotide sequencing, primer extension reactions, and template-independent enzymatic oligonucleotide synthesis.
Claims20
A method for incorporating a single nucleotide into a priming strand in a template-independent reaction, the method comprising:
- combining a priming strand with a 3′-OH-unmodified reversible terminator and a mutant polymerase, wherein the mutant polymerase is at least 96% identical to SEQ ID NO:2 and comprises:
- a Y546H mutation at a position functionally equivalent to position 546 in Pfu polymerase;
- a L409Y, L409H or L409F mutation at a position functionally equivalent to position 409 in Pfu polymerase;
- and
- a A486X mutation at a position functionally equivalent to position 486 in Pfu polymerase, wherein X is any amino acid except alanine;
- wherein incorporation of the terminator is at least 2-fold higher than for the mutant DNA polymerase of SEQ ID NO:11.
The method of 1, wherein the 3′-OH-unmodified reversible terminator is a 2-nitrobenzyl-modified nucleotide.
A method of template-independent oligonucleotide synthesis comprising:
- combining a priming strand, an 3′-OH-unmodified reversible terminator, and a mutant DNA polymerase, wherein mutant DNA polymerase comprises:
- an amino acid sequences that is at least 96% identical to SEQ ID NO:2;
- a Y546H mutation to histidine at a position functionally equivalent to position 546 in Pfu polymerase;
- a L409Y, L409H, or L409F mutation at a position functionally equivalent to position 409 in Pfu polymerase;
- and
- a A486X mutation at a position functionally equivalent to position 486 in Pfu polymerase, wherein X is any amino acid except alanine;
- incorporating the 3′-OH-unmodified reversible terminator to the priming strand.
The method of 11, wherein the 3′-OH-unmodified reversible terminator is a 2-nitrobenzyl-modified nucleotide.
Dependencies are inferred from the claim text.