US 12,006,519 B2
Polymerase mutants and use with 3′-OH unblocked reversible terminators
Michelle Cayouette, San Diego, CA (US); Jeffrey Fox, Escondido, CA (US); Connie Hansen, San Diego, CA (US); Holly Hogrefe, San Diego, CA (US); and Weidong Wu, Houston, TX (US)
Assigned to Agilent Technologies, Inc., Santa Clara, CA (US)
Filed by Agilent Technologies, Inc., Santa Clara, CA (US)
Filed on Sep. 11, 2023, as Appl. No. 18/464,955.
Application 18/464,955 is a continuation of application No. 17/894,854, filed on Aug. 24, 2022, granted, now 11,773,380.
Application 17/894,854 is a continuation of application No. PCT/US2021/070785, filed on Jun. 29, 2021.
Prior Publication US 2024/0018490 A1, Jan. 18, 2024
This patent is subject to a terminal disclaimer.
Int. Cl. C12N 9/12 (2006.01); C12P 19/34 (2006.01); C12Q 1/6869 (2018.01)
CPC C12N 9/1252 (2013.01) [C12P 19/34 (2013.01); C12Q 1/6869 (2013.01); C12Y 207/07007 (2013.01)] 20 Claims
 
1. 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.