US 11,913,038 B2
Engineered polymerases for improved sequencing
Pinar Iyidogan, San Diego, CA (US)
Assigned to PACIFIC BIOSCIENCES OF CALIFORNIA, INC., Menlo Park, CA (US)
Filed by Pacific Bioscience of California, Inc., Menlo Park, CA (US)
Filed on Dec. 29, 2021, as Appl. No. 17/564,500.
Application 17/564,500 is a continuation of application No. 16/567,476, filed on Sep. 11, 2019, granted, now 11,242,512.
Claims priority of provisional application 62/732,510, filed on Sep. 17, 2018.
Prior Publication US 2022/0235336 A1, Jul. 28, 2022
This patent is subject to a terminal disclaimer.
Int. Cl. C12N 9/12 (2006.01); C12Q 1/6874 (2018.01); C12P 19/34 (2006.01)
CPC C12N 9/1252 (2013.01) [C12N 9/1276 (2013.01); C12P 19/34 (2013.01); C12Q 1/6874 (2013.01); C12Y 207/07007 (2013.01); C12Y 207/07049 (2013.01)] 19 Claims
 
1. A method of identifying a base in a primed template nucleic acid, comprising
(a) forming a ternary complex by contacting a primer-template nucleic acid hybrid with an engineered DNA polymerase and a test nucleotide comprising a fluorescent label, wherein the polymerase comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:1 and comprises amino acid substitutions L408F, Y409F, and P410T or amino acid substitutions L408F, Y409H, and P410V, wherein the polymerase has increased accuracy in pairing nucleotides to template bases as compared to a control polymerase having the amino acid sequence of SEQ ID NO:1, and wherein the test nucleotide is complementary to a next template base;
(b) detecting the ternary complex by detecting the fluorescent label without incorporation of the test nucleotide into the primer, thereby identifying the next template base; and
(c) incorporating into the primer an unlabeled nucleotide that is complementary to the next template base.