# US 11,299,720 B2 · Polymerases for nucleotide analogue incorporation > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 11,299,720 B2 - Document ID: US11299720B2 - Status: Granted - Kind: B2 - Application number: 16/928,940 - Filed: 2020-07-14 - Published: 2021-04-08 - Granted: 2022-04-12 - Earliest claimed: 2005-12-22 - Assignees: Pacific Biosciences of California, Inc. - Applicants: Pacific Biosciences of California, Inc. - Inventors: David K. Hanzel; Geoff Otto; Paul S. Peluso; Thang Pham; David R. Rank; Fred Christians; Arekadiusz Bibillo; Insil Park; Sonya Clark; John Lyle - Web page: https://patentgrep.com/patent/US11299720B2 ## CPC - C12N 9/1252: {DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase} - C12P 19/34: Polynucleotides, e.g. nucleic acids, oligoribonucleotides - C12Q 1/6827: for detection of mutation or polymorphism - C12Q 1/6869: Methods for sequencing - C12Y 207/07007: DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase - C07K 2319/20: containing a tag with affinity for a non-protein ligand - Y02P 20/52: using catalysts, e.g. selective catalysts - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)} - C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES - C12Y: ENZYMES - C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00) - Y02P: CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS ## Abstract Compositions that include polymerases with features for improving entry of nucleotide analogues into active site regions and for coordinating with the nucleotide analogues in the active site region are provided. Methods of making the polymerases and of using the polymerases in sequencing and DNA replication and amplification as well as kinetic models of polymerase activity and computer-implemented methods of using the models are also provided. ## Claims ### Claim 1 A composition comprising a recombinant DNA polymerase, which recombinant polymerase comprises an amino acid sequence that is at least 80% identical to SEQ ID NO:1, which recombinant polymerase comprises one or more mutations selected from the group consisting of an amino acid substitution at position M8, an amino acid substitution at position M97, and an amino acid substitution at position E408, wherein identification of positions is relative to SEQ ID NO:1, and which recombinant polymerase exhibits polymerase activity. ### Claim 2 (depends on 1) The composition of claim 1, wherein the recombinant polymerase comprises one or more mutations selected from the group consisting of an M8A substitution, an M8S substitution, an M97A substitution, an M97S substitution, an E408K substitution, and an E408R substitution, wherein identification of positions is relative to SEO ID NO:1. ### Claim 3 (depends on 1) The composition of claim 1, wherein the recombinant polymerase comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:1. ### Claim 4 (depends on 1) The composition of claim 1, wherein the recombinant polymerase comprises a mutation that inhibits exonuclease activity of the polymerase. ### Claim 5 (depends on 1) The composition of claim 1, wherein the recombinant polymerase comprises one or more exogenous affinity tag sequence. ### Claim 6 (depends on 5) The composition of claim 5, wherein the affinity tag sequence is selected from: a 6 His tag sequence, a GST tag, an HA tag sequence, a plurality of 6 His tag sequences, a plurality of GST tags, a plurality of HA tag sequences and combinations thereof. ### Claim 7 (depends on 1) The composition of claim 1, comprising a phosphate-labeled nucleotide analog. ### Claim 8 (depends on 7) The composition of claim 7, wherein the nucleotide analog comprises a fluorophore. ### Claim 9 (depends on 1) The composition of claim 1, comprising a phosphate-labeled nucleotide analog and a DNA template, wherein the recombinant polymerase incorporates the nucleotide analog into a copy nucleic acid in response to the DNA template. ### Claim 10 (depends on 9) The composition of claim 9, wherein the template is a circular template. ### Claim 11 (depends on 1) The composition of claim 1, wherein the composition is present in a DNA sequencing system. ### Claim 12 (depends on 11) The composition of claim 11, wherein the sequencing system comprises a zero-mode waveguide. ### Claim 13 (depends on 12) The composition of claim 12, wherein the recombinant polymerase is immobilized on a surface of the zero-mode waveguide in an active form. ### Claim 14 (depends on 1) A method of sequencing a DNA template, the method comprising: a) providing a reaction mixture comprising: the DNA template, a replication initiating moiety that complexes with or is integral to the template, the recombinant DNA polymerase of claim 1, wherein the polymerase is capable of replicating at least a portion of the template using the moiety in a template-dependent polymerization reaction, and one or more nucleotides and/or nucleotide analogs; b) subjecting the reaction mixture to a polymerization reaction in which the recombinant polymerase replicates at least a portion of the template in a template-dependent manner, whereby the one or more nucleotides and/or nucleotide analogs are incorporated into the resulting DNA; and c) identifying a time sequence of incorporation of the one or more nucleotides and/or nucleotide analogs into the resulting DNA. ### Claim 15 (depends on 14) The method of claim 14, wherein the subjecting and identifying steps are performed in a zero mode waveguide. ### Claim 16 (depends on 1) A method of making a DNA, the method comprising: (a) providing a reaction mixture comprising: a template, a replication initiating moiety that complexes with or is integral to the template, the recombinant DNA polymerase of claim 1, which polymerase is capable of replicating at least a portion of the template using the moiety in a template-dependent polymerase reaction, and one or more nucleotides and/or nucleotide analogs; and (b) reacting the mixture such that the polymerase replicates at least a portion of the template in a template-dependent manner, whereby the one or more nucleotides and/or nucleotide analogs are incorporated into the resulting DNA. ### Claim 17 (depends on 16) The method of claim 16, wherein the mixture is reacted in a zero mode waveguide. ### Claim 18 (depends on 16) The method of claim 16, the method comprising detecting incorporation of at least one of the nucleotides and/or nucleotide analogs.