US 12,139,727 B2
Engineered polymerases
Mark Ambroso, Escondido, CA (US); Tyler Lopez, Winchester, CA (US); Michael Klein, Encinitas, CA (US); Virginia Saade, San Diego, CA (US); and Matthew Kellinger, San Diego, CA (US)
Assigned to ELEMENT BIOSCIENCES, INC., San Diego, CA (US)
Filed by Element Biosciences, Inc., San Diego, CA (US)
Filed on Mar. 25, 2022, as Appl. No. 17/705,043.
Claims priority of provisional application 63/212,540, filed on Jun. 18, 2021.
Prior Publication US 2022/0403353 A1, Dec. 22, 2022
This patent is subject to a terminal disclaimer.
Int. Cl. C12N 9/12 (2006.01); C12P 19/34 (2006.01); C12Q 1/6806 (2018.01); C12Q 1/6834 (2018.01); C12Q 1/6874 (2018.01)
CPC C12N 9/1252 (2013.01) [C12P 19/34 (2013.01); C12Q 1/6806 (2013.01); C12Q 1/6834 (2013.01); C12Q 1/6874 (2013.01); C12Y 207/07007 (2013.01)] 27 Claims
 
1. A method for performing nucleic acid sequencing, comprising:
(a) contacting an engineered polymerase with (i) a nucleic acid template molecule and (ii) a nucleic acid primer,
wherein said contacting is conducted under a condition suitable for the engineered polymerase to bind to the nucleic acid template molecule and the nucleic acid primer, thereby forming a complexed polymerase, wherein the complexed polymerase comprises an engineered polymerase bound to a nucleic acid duplex,
wherein the nucleic acid duplex comprises the nucleic acid template molecule hybridized to the nucleic acid primer,
wherein the engineered polymerase comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:1 and has amino acid substitutions Asp141Ala and Glu143Ala;
(b) contacting the complexed polymerase with a multivalent polymer-nucleotide conjugate to form a multivalent-binding complex,
wherein the multivalent polymer-nucleotide conjugate comprises a core attached to multiple nucleotide arms,
wherein at least one of the nucleotide arms is attached to a nucleotide unit,
wherein said contacting is conducted under a condition suitable for
binding the nucleotide unit of at least one of the nucleotide arms of the multivalent polymer-nucleotide conjugate to a corresponding complementary nucleotide base of the nucleic acid template molecule, and
inhibiting polymerase-catalyzed extension of the nucleic acid duplex;
(c) detecting the multivalent-binding complex; and
(d) determining the sequence of the nucleic acid template molecule.