Engineered polymerases with improved thermal stability

Abstract

Provided herein are engineered variants of archaea polymerases, relative to wild-type polymerases, in particular, the engineered variants exhibit exonuclease-minus activity, enhanced thermal stability, enhanced incorporation of 3 '-modified nucleotides, improved uracil tolerance, and / or reduced sequence-specific errors in polymerase catalyzed nucleotide binding and extension reactions, as well as methods and uses thereof. Also provided are uses of the engineered polymerases for forming complex polymerases and forming binding complexes, as well as for performing nucleic acid sequencing reactions.

Description

[0001] Throughout this application, various publications, patents and / or patent applications are cited. The disclosures of these publications, patents and / or patent applications are hereby incorporated by reference in their entirety into this application in order to more fully describe the state of the art to which this disclosure pertains.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to and the benefit of U.S. Provisional Application No. 63 / 351,294, filed on June 10, 2022; U.S. Provisional Application No. 63 / 422,855, filed on November 4, 2023; and U.S. Provisional Application No. 63 / 491,374, filed on March 21, 2023, the entire disclosures of which are hereby incorporated by reference in their entirety.

[0004] Sequence Listing

[0005] This application contains a sequence listing that has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. The XML copy, created on June 7, 2023, is named 55744WO_CRF_sequencelisting and is 5,651 kilobytes in size. Technical Field

[0006] The present disclosure provides mutant polymerases engineered to improve thermal stability, showing improved binding of nucleotide reagents and / or improved binding and incorporation of nucleotide reagents, improved uracil tolerance and / or reduced sequence-specific sequencing errors. Exemplary nucleotide reagents include detectably labeled nucleotides, unlabeled nucleotides, nucleotides including 3' chain termination moieties, nucleotides labeled with phosphate chains, and multivalent molecules. In certain embodiments, the engineered polymerases show a reduced conversion from a nucleotide polymerization conformation to an exonuclease conformation. Compared to wild-type polymerases, the mutant polymerases show an increased incorporation rate. Background Art

[0007] Next generation sequencing (NGS) technology has become a powerful tool for obtaining sequencing data used in molecular biology techniques, taxonomy, agricultural science, medical diagnostics, and the development of new therapeutics. The present disclosure provides engineered polymerases that can be used to perform any nucleic acid sequencing method that employs labeled or unlabeled chain-terminating nucleotides, wherein the chain-terminating nucleotide comprises a 3'-O-azido group (or 3'-O-methylazido group) or any other type of large blocking group at the 3' position of the sugar. For example, the engineered polymerase can be used to perform affinity sequencing methods (SBA) using labeled multivalent molecules and unlabeled chain-terminating nucleotides. In addition, the engineered polymerase can be used to perform sequencing by synthesis (SBS) methods (employing labeled chain-terminating nucleotides) and sequencing by binding methods (SBB) (employing unlabeled chain-terminating nucleotides).

[0008] Adding a single nucleotide to a DNA strand individually does not produce enough signal to be easily detected. Currently available SBS technologies overcome this problem by increasing the signal-to-noise ratio of nucleotide additions, combined with detection methods with sufficient sensitivity to make accurate base calls. The most commercially successful platforms use monoclonal template DNA amplification in a spatially confined matrix to produce discrete DNA islands containing multiple copies of the sequence to be interrogated. The result of this amplification is a "colony" of DNA copies, which allows the detection modality to be focused based on the addition of a single DNA from all copies in a manner sufficient to overcome the signal-to-noise ratio problem. The sequencing of multiple spatially confined copies of the same DNA further increases the reliance on a controlled stepping mechanism to ensure that one and only one nucleotide base can be added, thereby ensuring that all copies within the DNA colony remain in the same position relative to each other (N, N+1, N+2, N+3, etc.).

[0009] The molecular engine required to perform SBS is DNA polymerase. In vivo, this type of enzyme is responsible for DNA replication and maintaining genomic integrity. Under natural conditions, DNA-dependent DNA polymerases (dDdP's) catalyze the addition of deoxynucleotide triphosphates (dNTPs) to DNA in the 5' to 3' direction, generating a phosphodiester bond between the 3' hydroxyl group at the end of the primer DNA and the 5' α phosphate of the incoming nucleotide. Due to hydrogen bonding between the correct incoming dNTP and the templated base, this chemistry occurs with high fidelity for the correct Watson-Crick base pair. This "correct" base pairing induces conformational changes in the enzyme that align the catalytic amino acids to efficiently perform phosphodiester bond formation. The newly added dNTP also has a 3' OH for the next round of catalysis to further extend the DNA chain.

[0010] To ensure that only a single dNTP is added to the growing DNA chain in each SBS cycle, reversibly terminated dNTPs are used. These bases contain a modification to the 3' hydroxyl group of the dNTP that blocks incorporation in subsequent rounds. The most commercially successful reversible terminator is the 3' methylazido group, but other reversible terminators including 3'-aminoallyl and 3' oxoamine have also been used. Each of these reversibly terminated dNTPs works in the same way; once incorporated, the large 3' block inhibits the addition of the next nucleotide because the 3' hydroxyl group is absent. When exposed to a catalyst, the 3' block reacts to regenerate a 3' hydroxyl group that can form a new phosphodiester bond during the next cycle. Although effective, these large 3' modifications pose a challenge to the polymerase.

[0011] The evolutionary requirement for high-fidelity genome replication and stability arises every 10 4 -10 7 Polymerases that only incorporate non-Watson-Crick base pairs in an incorporation event. Polymerases are often also required to discriminate between nucleotides that are in large excess in the cellular environment. Discrimination between nucleotides is typically accomplished via a steric gate, where the presence of a 2' hydroxyl group sterically clashes with the amino acid side chain at the nucleotide binding site to select for nucleotide binding and catalysis. Additionally, the enzyme may also sense damage or modification of the nucleotide 3' hydroxyl group, as bases containing an inactive 3' hydroxyl group can act as chain terminators that inhibit DNA synthesis. Discrimination of these unwanted bases occurs via a kinetic pathway, where incorrect nucleotide substrates bind with a weaker overall affinity, and phosphodiester bond formation is accelerated by 10 2 -10 4 This occurs orders of magnitude more slowly. This is due to the lack of induced fit that can correctly align the catalytic amino acids to form the bond. As a result, naturally evolved polymerases rarely incorporate reversible chain terminator nucleotides. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] The novel advantages and features of the compositions and methods disclosed herein are particularly set forth in the appended claims. A better understanding of the features and advantages of the compositions and methods disclosed herein will be obtained by reference to the following detailed description and accompanying drawings which set forth illustrative embodiments, in which:

[0013] Figure 1 is a schematic diagram of an exemplary low-binding support comprising a glass substrate and alternating layers of a hydrophilic coating covalently or non-covalently adhered to the glass, and further comprising chemically reactive functional groups that serve as attachment sites for oligonucleotide primers (e.g., capture oligonucleotides). In an alternative embodiment, the support can be made of any material such as glass, plastic, or a polymeric material.

[0014] Figure 2 Schematic diagrams of various exemplary configurations of multivalent molecules. Left (Class I): Schematic diagram of a multivalent molecule with a "starburst" or "scrambled" configuration. Middle (Class II): Schematic diagram of a multivalent molecule with a dendrimer configuration. Right (Class III): Schematic diagram of various multivalent molecules formed by reacting streptavidin with a 4-arm or 8-arm PEG-NHS with biotin and dNTPs. The nucleotide unit is designated 'N', biotin is designated 'B', and streptavidin is designated 'SA'.

[0015] Figure 3 is a schematic diagram of an exemplary multivalent molecule comprising a universal core attached to multiple nucleotide arms.

[0016] Figure 4 is a schematic diagram of an exemplary multivalent molecule comprising a dendrimer core to which are attached multiple nucleotide arms.

[0017] Figure 5 A schematic diagram of an exemplary multivalent molecule comprising a core attached to multiple nucleotide arms, wherein the nucleotide arms comprise biotin, a spacer, a linker, and nucleotide units is shown.

[0018] Figure 6 is a schematic diagram of an exemplary nucleotide arm including a core attachment moiety, a spacer, a linker, and a nucleotide unit.

[0019] Figure 7 Shown are the chemical structures of exemplary spacers (top), as well as the chemical structures of various exemplary linkers, including an 11-atom linker, a 16-atom linker, a 23-atom linker, and an N3 linker (bottom).

[0020] Figure 8 The chemical structures of various exemplary linkers (including linkers 1-9) are shown.

[0021] Figure 9A The chemical structures of various exemplary linkers joined / attached to the nucleotide units are shown.

[0022] Figure 9B The chemical structures of various exemplary linkers joined / attached to the nucleotide units are shown.

[0023] Figure 9C The chemical structures of various exemplary linkers joined / attached to the nucleotide units are shown.

[0024] Figure 9D The chemical structures of various exemplary linkers joined / attached to the nucleotide units are shown.

[0025] Figure 10The chemical structure of an exemplary biotinylated nucleotide arm is shown. In this example, the nucleotide unit is linked to the linker via propargylamine attachment at the 5-position of a pyrimidine base or the 7-position of a purine base.

[0026] Figure 11 is the amino acid sequence of a wild-type DNA polymerase having a backbone sequence from RLF 89458.1 (SEQ ID NO: 1).

[0027] Figure 12 is the amino acid sequence of a wild-type DNA polymerase having a backbone sequence from RLF 78286.1 (SEQ ID NO: 2).

[0028] Figure 13 is the amino acid sequence of a wild-type DNA polymerase having a backbone sequence from NOZ 58130.1 (SEQ ID NO: 1714).

[0029] Figure 14 is the amino acid sequence of a wild-type DNA polymerase having a backbone sequence from RMF 90817.1 (SEQ ID NO: 2789).

[0030] Figure 15 is the amino acid sequence of a wild-type DNA polymerase having a backbone sequence from MBC 7218772.1 (SEQ ID NO: 2790).

[0031] Figure 16 is the amino acid sequence of a wild-type DNA polymerase having a backbone sequence from WP 175059460.1 (SEQ ID NO: 2791).

[0032] Figure 17 is the amino acid sequence of a wild-type DNA polymerase having a backbone sequence from KUO 42443.1 (SEQ ID NO: 2792).

[0033] Figure 18 is the amino acid sequence of a wild-type DNA polymerase having a backbone sequence from NOZ 77387.1 (SEQ ID NO: 2793).

[0034] Figure 19 is the amino acid sequence of a wild-type DNA polymerase having a backbone sequence from Geobacillus stearothermophilus (Bst polymerase) (SEQ ID NO: 2794).

[0035] Figure 20is the amino acid sequence of 9°N polymerase (SEQ ID NO: 2795).

[0036] Figure 21 It is the amino acid sequence of 9°N polymerase UniProt Q56366 (SEQ ID NO: 2796).

[0037] Figure 22 is the amino acid sequence of THERMINATOR polymerase (SEQ ID NO: 2797).

[0038] Figure 23 is the amino acid sequence of VENT polymerase UniProt P30317 (SEQ ID NO: 2798).

[0039] Figure 24 It is the amino acid sequence of DEEP VENT polymerase UniProt Q51334 (SEQ ID NO: 2799).

[0040] Figure 25 It is the amino acid sequence of Pfu polymerase UniProt P61875 (SEQ ID NO: 2800).

[0041] Figure 26 It is the amino acid sequence of the Pyrococcus abyssi polymerase UniProt P0CL77 (SEQ ID NO: 2801).

[0042] Figure 27 is the amino acid sequence of RB69 polymerase (SEQ ID NO: 2802).

[0043] Figure 28 is the amino acid sequence of Phi29 polymerase (SEQ ID NO: 2803).

[0044] Figure 29 The amino acid sequence of the structural domain of the RLF 89458.1 polymerase is shown.

[0045] Figure 30 The amino acid sequence of the domains of the NOZ 58130.1 polymerase is shown.

[0046] Figure 31(115 sheets) is Table 1, which lists the protein unfolding transition temperatures (in ° C) for Tm1 and Tm2, ​​and the activities of mutant variants (SEQ ID NO: 3-1713) having the backbone sequence of RLF 89458.1 and carrying mutant substitution sites. In Table 1, truncations are designated by "^". In Table 1, deleted amino acids are designated by "X". In Table 1, inserted amino acids are designated by "[X inserted after P411]", where X is the single-letter amino acid code.

[0047] Figure 32 (68 sheets) is Table 2, which lists the protein unfolding transition temperatures (in ° C.) of Tm1 and Tm2, ​​as well as the activities of mutant variants (SEQ ID NOs: 1715-2787) having the backbone sequence of NOZ 58130.1 and carrying mutant substitution sites. In Table 2, truncations are designated with "^". In Table 2, deleted amino acids are designated with "X". In Table 2, deleted amino acid portions are designated with "[deletion ...]", where the deleted portion is indicated by a single-letter amino acid code.

[0048] Figure 33 (5 sheets) shows an alignment of amino acid sequences from the following DNA polymerases: RLF 89458.1 (SEQ ID NO: 1); WP 175059460 (SEQ ID NO: 2791); MBC 7218772 (SEQ ID NO: 2790); KUO 42443 (SEQ ID NO: 2792); NOZ 58130 (SEQ ID NO: 1714); RMF 90817 (SEQ ID NO: 2789); and NOZ 77387 (SEQ ID NO: 2793).

[0049] Figure 34 (5 sheets) shows an amino acid sequence alignment of DNA polymerases from: RLF 89458.1 (SEQ ID NO: 1); Geobacillus stearothermophilus (Bst polymerase) (SEQ ID NO: 2794); 9°N (SEQ ID NO: 2795); Pfu polymerase (SEQ ID NO: 2800); and Pyrococcus pluvialis polymerase (SEQ ID NO: 2801).

[0050] Figure 35 (5 sheets) shows an amino acid sequence alignment of DNA polymerases from: NOZ 58130 (SEQ ID NO: 1714); Geobacillus stearothermophilus (Bst polymerase) (SEQ ID NO: 2794); 9°N (SEQ ID NO: 2795); Pfu polymerase (SEQ ID NO: 2800); and Pyrococcus pluvialis polymerase (SEQ ID NO: 2801).

[0051] Figure 36 (5 sheets) shows an amino acid sequence alignment of DNA polymerases from: RMF 90817 (SEQ ID NO: 2789); Geobacillus stearothermophilus (Bst polymerase) (SEQ ID NO: 2794); 9°N (SEQ ID NO: 2795); Pfu polymerase (SEQ ID NO: 2800); and Pyrococcus pluvialis polymerase (SEQ ID NO: 2801).

[0052] Figure 37 (5 sheets) shows an alignment of the amino acid sequences of DNA polymerases from: MBC 7218772 (SEQ ID NO: 2790); Geobacillus stearothermophilus (Bst polymerase) (SEQ ID NO: 2794); 9°N (SEQ ID NO: 2795); Pfu polymerase (SEQ ID NO: 2800); and Pyrococcus pluvialis polymerase (SEQ ID NO: 2801).

[0053] Figure 38 (5 sheets) shows an amino acid sequence alignment of DNA polymerases from: WP 175059460 (SEQ ID NO: 2791); Geobacillus stearothermophilus (Bst polymerase) (SEQ ID NO: 2794); 9°N (SEQ ID NO: 2795); Pfu polymerase (SEQ ID NO: 2800); and Pyrococcus pluvialis polymerase (SEQ ID NO: 2801).

[0054] Figure 39 (5 ​​sheets) shows the amino acid sequence alignment of DNA polymerases from: KUO 42443 (SEQ ID NO: 2792); Geobacillus stearothermophilus (Bst polymerase) (SEQ ID NO: 2794); 9°N (SEQ ID NO: 2795); Pfu polymerase (SEQ ID NO: 2800); and Pyrococcus pluvialis polymerase (SEQ ID NO: 2801).

[0055] Figure 40 (5 sheets) shows an amino acid sequence alignment of DNA polymerases from NOZ 77387 (SEQ ID NO: 2793); Geobacillus stearothermophilus (Bst polymerase) (SEQ ID NO: 2794); 9°N (SEQ ID NO: 2795); Pfu polymerase (SEQ ID NO: 2800); and Pyrococcus pluvialis polymerase (SEQ ID NO: 2801).

[0056] Figure 41Schematic diagram showing the predicted three-dimensional ribbon model of wild-type polymerase RLF 89458.1 (SEQ ID NO: 1) with an N-terminal His tag. The ribbon model includes the predicted positions of post-translational modifications at certain amino acid residues. The ribbon model is based on mass spectrometry data.

[0057] Figure 42 Schematic diagram showing the predicted three-dimensional ribbon model of wild-type polymerase NOZ 58130.1 (SEQ ID NO: 1714) with an N-terminal His tag. The ribbon model includes the predicted positions of post-translational modifications at certain amino acid residues. The ribbon model is based on mass spectrometry data.

[0058] Figure 43 is a graph showing the % error for a 150 cycle sequencing run of a nucleic acid library prepared from E. coli DNA.

[0059] Figure 44 is a schematic diagram of an exemplary immobilized nucleic acid template molecule hybridized to a first nucleic acid primer and a second nucleic acid primer. Figure 44 The nucleic acid template molecule shown in comprises a concatemer hybridized to a plurality of nucleic acid primers.

[0060] Figure 45 is a schematic diagram of an exemplary composite polymerase indicated by dashed circles, wherein the individual composite polymerases include a DNA polymerase bound to nucleic acid duplexes, wherein each duplex includes a nucleic acid template hybridized to a nucleic acid primer.

[0061] Figure 46 is a schematic diagram of an exemplary first binding complex (e.g., indicated by a dashed circle) that includes a first nucleic acid primer, a first DNA polymerase, and a first multivalent molecule that bind to a first portion of a concatemer template molecule, thereby forming the first binding complex. Figure 46 Also shown are multiple multivalent molecules that are not part of the first binding complex.

[0062] Figure 47is a schematic diagram of an exemplary affinity complex (e.g., indicated by a dashed circle) comprising (i) a first binding complex comprising a first nucleic acid primer, a first DNA polymerase, and a first multivalent molecule, the first nucleic acid primer, the first DNA polymerase, and the first multivalent molecule binding to a first portion of a concatemer template molecule, thereby forming the first binding complex, wherein a first nucleotide unit of the multivalent molecule binds to the first DNA polymerase, and (ii) a second binding complex comprising a second nucleic acid primer, a second DNA polymerase, and the same first multivalent molecule, the second nucleic acid primer, the second DNA polymerase, and the same first multivalent molecule binding to a second portion of the same concatemer template molecule, thereby forming a second binding complex, wherein a second nucleotide unit of the multivalent molecule binds to the second DNA polymerase, and wherein the first binding complex comprising the same multivalent molecule and the second binding complex form an affinity complex. DETAILED DESCRIPTION

[0063] definition:

[0064] The headings provided herein are not limitations of the various aspects of the disclosure, which can be understood by reference to the specification as a whole.

[0065] Unless otherwise defined, the technology and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art. Generally speaking, the terms related to the technology of molecular biology, nucleic acid chemistry, protein chemistry, genetics, microbiology, transgenic cell production and hybridization described herein are well-known and commonly used terms in the art. The technology and procedures described herein are generally performed according to conventional methods well known in the art and as described in the various general and more specific references cited and discussed throughout this specification. For example, referring to Sambrook et al., Molecular Cloning: A Laboratory Manual (Molecular Cloning: A Laboratory Manual) (3rd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY) 2000). See also Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992). The nomenclature used in conjunction with laboratory procedures and techniques described herein is well known and commonly used in the art.

[0066] Unless the context otherwise requires, singular terms shall include pluralities and plural terms shall include the singular. The singular forms "a / an" and "the" and any word used in the singular include plural referents unless expressly and unequivocally limited to one referent.

[0067] It will be understood that use of alternative terms such as "or" is taken to mean one or both of the alternatives, or any combination thereof.

[0068] As used herein, the term "and / or" should be taken to mean a specific disclosure of each of the specified features or components with or without the other. For example, the term "and / or" as used in phrases such as "A and / or B" is intended to encompass: "A and B"; "A or B"; "A" (A alone); and "B" (B alone). In a similar manner, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to encompass each of the following: "A, B, and C"; "A, B, or C"; "A or C"; "A or B"; "B or C"; "A and B"; "B and C"; "A and C"; "A" (A alone); "B" (B alone); and "C" (C alone).

[0069] As used herein and in the appended claims, the terms "comprising," "including," "having," and "containing," and grammatical variations thereof, as used herein, are intended to be non-limiting, such that the inclusion of one or more items in a list is not to the exclusion of other items that can be substituted or added to the listed items. It should be understood that whenever aspects are described herein with the language "comprising," other similar aspects described with "consisting of" and / or "consisting essentially of" are also provided.

[0070] As used herein, the terms "about" and "approximately" refer to values ​​or compositions within an acceptable error range for a particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system. For example, according to practice in the art, "about" or "approximately" may mean within one or more standard deviations. Alternatively, "about" or "approximately" may mean a range of up to 10% (i.e., ±10%) or greater, depending on the limitations of the measurement system. For example, about 5 mg may include any number between 4.5 mg and 5.5 mg. In addition, with specific reference to a biological system or process, the term may mean up to an order of magnitude or up to 5 times the value. When a specific value or composition is provided in the present disclosure, unless otherwise stated, the meaning of "about" or "approximately" should be assumed to be within an acceptable error range for the specific value or composition. In addition, where a range and / or subrange of a value is provided, the range and / or subrange may include the endpoints of the range and / or subrange.

[0071] As used herein, the terms "peptide," "polypeptide," and "protein," and other related terms, are used interchangeably and refer to polymers of amino acids and are not limited to any particular length. Polypeptides can include natural and non-natural amino acids. Polypeptides include recombinant or chemically synthesized forms. Polypeptides also include precursor molecules that have not undergone post-translational modifications, such as proteolytic cleavage, cleavage due to ribosome skipping, hydroxylation, methylation, lipidation, acetylation, sumoylation, ubiquitination, glycosylation, phosphorylation, and / or disulfide bond formation. These terms encompass natural and artificial proteins, protein fragments, and polypeptide analogs (such as mutants, variants, chimeric proteins, and fusion proteins) of protein sequences, as well as proteins that have been post-translationally or otherwise covalently or non-covalently modified.

[0072] As used herein, the term "polymerase" and its variants include any enzyme that can catalyze the polymerization of nucleotides (including their analogs) into nucleic acid chains. Typically, but not necessarily, such nucleotide polymerization can occur in a template-dependent manner. Typically, a polymerase includes one or more active sites at which nucleotide binding and / or nucleotide polymerization catalysis can occur. In some embodiments, the polymerase includes other enzymatic activities, such as 3' to 5' exonuclease activity or 5' to 3' exonuclease activity. In some embodiments, the polymerase has chain displacement activity. The polymerase may include, but is not limited to, naturally occurring polymerases and any subunits and truncations thereof, mutant polymerases, variant polymerases, recombinant, fused or otherwise engineered polymerases, chemically modified polymerases, synthetic molecules or assemblies, and any analogs, derivatives, or fragments thereof (e.g., catalytically active fragments) that retain the ability to catalyze nucleotide polymerization. In some embodiments, the polymerase can be isolated from a cell or produced using recombinant DNA technology or chemical synthesis methods. In some embodiments, the polymerase can be expressed in prokaryotes, eukaryotes, viruses, or phage organisms. In some embodiments, the polymerase can be a post-translationally modified protein or fragment thereof. Polymerases can be derived from prokaryotes, eukaryotes, viruses or bacteriophages. Polymerases include DNA-guided DNA polymerases and RNA-guided DNA polymerases.

[0073] As used herein, the term "fidelity" refers to the accuracy of DNA polymerization by a template-dependent DNA polymerase. The fidelity of a DNA polymerase is typically measured by the error rate (the frequency of incorporation of inaccurate nucleotides (i.e., nucleotides that are not complementary to the template nucleotides). The accuracy or fidelity of DNA polymerization is maintained by the polymerase activity and 3'-5' exonuclease activity of the DNA polymerase.

[0074] As used herein, the term "bound complex" refers to a complex formed by binding together a nucleic acid duplex, a polymerase, and free nucleotides or nucleotide units of a multivalent molecule, wherein the nucleic acid duplex includes a nucleic acid template molecule hybridized to a nucleic acid primer. In the bound complex, the free nucleotides or nucleotide units may or may not bind to the 3' end of the nucleic acid primer at a position opposite to the complementary nucleotide in the nucleic acid template molecule. A "ternary complex" is an example of a bound complex formed by binding together a nucleic acid duplex, a polymerase, and free nucleotides or nucleotide units of a multivalent molecule, wherein the free nucleotides or nucleotide units bind to the 3' end of the nucleic acid primer (as part of the nucleic acid duplex) at a position opposite to the complementary nucleotide in the nucleic acid template molecule.

[0075] The term "residence time" and related terms refer to the length of time that a binding complex remains stable in the absence of any component dissociation, wherein the components of the binding complex include nucleic acid templates and nucleic acid primers, polymerases, nucleotide units of multivalent molecules or free (e.g., unconjugated) nucleotides. Nucleotide units or free nucleotides can be complementary or non-complementary to the nucleotide residues in the template molecule. Nucleotide units or free nucleotides can be combined with the 3' end of the nucleic acid primer at a position relative to the complementary nucleotide residues in the nucleic acid template molecule. The residence time indicates the stability of the binding complex and the intensity of the binding interaction. The residence time can be measured by observing the start and / or duration of the binding complex (such as by observing the signal of the labeled component from the binding complex). For example, labeled nucleotides or labeled reagents including one or more nucleotides can be present in the binding complex, thereby allowing the signal from the label to be detected during the residence time of the binding complex. An exemplary label is a fluorescent label. Binding complex (e.g., a ternary complex) remains stable before being subjected to conditions that result in the dissociation of the interaction between any one of polymerase, template molecule, primer and / or nucleotide units or nucleotides. For example, dissociation conditions include contacting the bound complex with any one of detergent, EDTA, and / or water, or any combination thereof.

[0076] As used herein, the terms "nucleic acid," "polynucleotide," and "oligonucleotide," as well as other related terms, are used interchangeably and refer to polymers of nucleotides and are not limited to any specific length. Nucleic acids include recombinant and chemically synthesized forms. Nucleic acids include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), analogs of DNA or RNA produced using nucleotide analogs (e.g., peptide nucleic acids and non-naturally occurring nucleotide analogs), and chimeric forms containing DNA and RNA. Nucleic acids can be single-stranded or double-stranded. Nucleic acids include polymers of nucleotides, wherein the nucleotides include natural or non-natural bases and / or sugars. Nucleic acids include naturally occurring internucleoside bonds, such as phosphodiester bonds. Nucleic acids include non-natural internucleoside bonds, including phosphorothioate, sulfur-containing phosphate, or peptide nucleic acid (PNA) bonds. In certain embodiments, nucleic acids include a mixture of one type of polynucleotide or two or more different types of polynucleotides.

[0077] The term "primer" and related terms used herein refer to natural or synthetic oligonucleotides that can hybridize with DNA and / or RNA polynucleotide templates to form duplex molecules. Primers can have any length, but generally range from 4 to 50 nucleotides. Typical primers include 5' ends and 3' ends. The 3' end of a primer can include a 3'OH portion that serves as a nucleotide polymerization initiation site in a polymerase-mediated primer extension reaction. Alternatively, the 3' end of a primer can lack a 3'OH portion or can include an end 3' blocking group that inhibits nucleotide polymerization in a polymerase-mediated reaction. Any one nucleotide along the length of the primer or more than one nucleotide can be labeled with a detectable reporter gene portion. Primers can be in solution (e.g., soluble primers) or can be fixed to a carrier (e.g., capture primers).

[0078] The terms "template nucleic acid," "template polynucleotide," "target nucleic acid," "target polynucleotide," "template strand," and other variants refer to a nucleic acid strand that serves as a base nucleic acid molecule for generating a complementary nucleic acid strand. The sequence of the template nucleic acid may be partially or completely complementary to the sequence of the complementary strand. The template nucleic acid may be obtained from naturally occurring sources, recombinant forms, or chemically synthesized to include any type of nucleic acid analog. The template nucleic acid may be linear, circular, or in other forms. The template nucleic acid may be isolated in any form, including chromosomes, genomes, organelles (e.g., mitochondria, chloroplasts, or ribosomes), recombinant molecules, cloned, amplified cDNA, RNA (e.g., pre-mRNA or mRNA), oligonucleotides, whole genomic DNA obtained from fresh frozen paraffin-embedded tissue, needle biopsies, cell-free circulating DNA, or any type of nucleic acid library. Template nucleic acid molecules can be isolated from any source, including organisms such as prokaryotes, eukaryotes (e.g., humans, plants, and animals), fungi, and viruses; cells; tissues; normal or diseased cells or tissues, body fluids, including blood, urine, serum, lymph, tumors, saliva, anal and vaginal secretions, amniotic fluid, sweat, and semen; environmental samples; culture samples; or synthetic nucleic acid molecules prepared using recombinant molecular biology or chemical synthesis methods. The template nucleic acid can be subjected to nucleic acid analysis (including sequencing and composition analysis).

[0079] When used to refer to nucleic acid molecules, the term "hybridize" or "hybridizing" or "hybridization" or other related terms refer to hydrogen bonding between two different nucleic acids to form a duplex nucleic acid. Hybridization also includes hydrogen bonding between two different regions of a single nucleic acid molecule to form a self-hybridizing molecule with a duplex region. Hybridization can include Watson-Crick or Hoogstein binding to form a duplex double-stranded nucleic acid or a double-stranded region within a nucleic acid molecule. The two different regions of a double-stranded nucleic acid or a single nucleic acid can be fully complementary or partially complementary. The complementary nucleic acid chains do not need to hybridize to each other across their entire length. Complementary base pairing can be standard AT or CG base pairing or can be other forms of base pairing interactions. The duplex nucleic acid can include mismatched base-paired nucleotides.

[0080] The term "nucleotide" and related terms refer to molecules comprising aromatic bases, five-carbon sugars (e.g., ribose or deoxyribose) and at least one phosphate group. Typical or atypical nucleotides are consistent with the use of the term. In certain embodiments, phosphate comprises monophosphate, diphosphate or triphosphate or corresponding phosphate analogs. In certain embodiments, nucleotide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 phosphate groups. The term "nucleoside" refers to a molecule comprising an aromatic base and a sugar.

[0081] Nucleotides (and nucleosides) generally include heterocyclic bases, comprising substituted or unsubstituted nitrogen-containing parent heteroaromatic rings (commonly present in nucleic acids), including naturally occurring, substituted, modified or engineered variants or analogs thereof. The bases of nucleotides (or nucleosides) are capable of forming Watson-Crick and / or Hoostein hydrogen bonds with appropriate complementary bases. Exemplary bases include, but are not limited to, purines and pyrimidines, such as: 2-aminopurine, 2,6-diaminopurine, adenine (A), ethyleneadenine, N 6 -Δ 2 -Isopentenyl adenine (6iA), N 6 -Δ 2 -Isopentenyl-2-methylthioadenine (2ms6iA), N 6 -methyladenine, guanine (G), isoguanine, N 2 -dimethylguanine (dmG), 7-methylguanine (7mG), 2-thiopyrimidine, 6-thioguanine (6sG), hypoxanthine and O 6-methylguanine; 7-deaza-purines, such as 7-deazaadenine (7-deaza-A) and 7-deazaguanine (7-deaza-G); pyrimidines, such as cytosine (C), 5-propynylcytosine, isocytosine, thymine (T), 4-thiothymine (4sT), 5,6-dihydrothymine, O 4 -methylthymine, uracil (U), 4-thiouracil (4sU) and 5,6-dihydrouracil (dihydrouracil; D); indoles such as nitroindole and 4-methylindole; pyrroles such as nitropyrrole; muscimol; inosine; hydroxymethylcytosine; 5-methylcytosine; base (Y); and methylated, glycosylated and acylated base moieties; etc. Additional exemplary bases can be found in Fasman, 1989, "Practical Handbook of Biochemistry and Molecular Biology", pp. 385-394, CRC Press, Boca Raton, Fla.

[0082] Nucleotides (and nucleosides) generally include sugar moieties such as carbocyclic moieties (Ferraro and Gotor 2000 Chem. Rev. 100:4319-48), acyclic moieties (Martinez et al. 1999 Nucleic Acids Research 27:1271-1274; Martinez et al. 1997 Bioorganic & Medicinal Chemistry Letters 7:3013-3016), and other sugar moieties (Joeng et al. 1993 J. Med. Chem. 36:2627-2638; Kim et al. 1993 J. Med. Chem. 36:30-7; Eschenmosser et al. 1993 J. Med. Chem. 36:31-32; Kim et al. 1993 J. Med. Chem. 36:32-33).

[0015] Science, 284: 2118-2124, 1999; and U.S. Patent No. 5,558,991. Sugar moieties include: ribosyl; 2'-deoxyribosyl; 3'-deoxyribosyl; 2',3'-dideoxyribosyl; 2',3'-didehydrodideoxyribosyl; 2'-alkoxyribosyl; 2'-azidoribosyl; 2'-aminoribosyl; 2'-fluororibosyl; 2'-thiolribosyl; 2'-alkylthioribosyl; 3'-alkoxyribosyl; 3'-azidoribosyl; 3'-aminoribosyl; 3'-fluororibosyl; 3'-thiolribosyl; 3'-alkylthioribosyl; carbocyclic; acyclic or other modified sugars.

[0083] In some embodiments, the nucleotide comprises a chain of one, two, or three phosphorus atoms, wherein the chain is typically attached to the 5' carbon of the sugar moiety via an ester or phosphoramide bond. In some embodiments, the nucleotide is an analog having a phosphorus chain in which the phosphorus atoms are linked together with an intervening O, S, NH, methylene, or ethylene group. In some embodiments, the phosphorus atoms in the chain comprise substituted side groups (comprising O, S, or BH3). In some embodiments, the chain comprises a phosphate group substituted with an analog comprising a phosphoramide, phosphorothioate, phosphorodithioate, and O-methylphosphoramidite group.

[0084] When used in reference to nucleic acids, the terms "extend," "extending," "extension," and other variants refer to the incorporation of one or more nucleotides into a nucleic acid molecule. Nucleotide incorporation comprises the polymerization of one or more nucleotides into the terminal 3' OH end of a nucleic acid chain, resulting in extension of the nucleic acid chain. Nucleotide incorporation can be performed using natural nucleotides and / or nucleotide analogs. Typically, but not necessarily, nucleotide incorporation occurs in a template-dependent manner. Any suitable method for extending nucleic acid molecules can be used, including primer extension catalyzed by a DNA polymerase or an RNA polymerase.

[0085] The term "reporter moiety," "reporter moieties," or related terms refers to a compound that produces or causes a detectable signal. A reporter moiety is sometimes referred to as a "label." Any suitable reporter moiety can be used, including luminescence, photoluminescence, electroluminescence, bioluminescence, chemiluminescence, fluorescence, phosphorescence, chromophores, radioisotopes, electrochemistry, mass spectrometry, Raman, haptens, affinity tags, atoms, or enzymes. The reporter moiety produces a detectable signal caused by a chemical or physical change (e.g., heat, light, electricity, pH, salt concentration, enzymatic activity, or a neighboring event). A neighboring event comprises two reporter moieties that are close to, associate with, or bind to each other. It is well known to those skilled in the art that the reporter moiety is selected so that each reporter moiety absorbs excitation radiation and / or fluoresces at a wavelength that is distinguishable from other reporter moieties, to allow monitoring of the presence of different reporter moieties in the same reaction or in different reactions. Two or more different reporter moieties can be selected that have spectrally distinct emission curves or have minimal overlapping spectral emission curves. The reporter moiety can be linked (eg, operably linked) to a nucleotide, nucleoside, nucleic acid, enzyme (eg, polymerase or reverse transcriptase), or carrier (eg, surface).

[0086] The reporter moiety (or label) includes a fluorescent marker or fluorophore. Exemplary fluorescent moieties that can serve as fluorescent markers or fluorophores include, but are not limited to, fluorescein and fluorescein derivatives (such as carboxyfluorescein, tetrachlorofluorescein, hexachlorofluorescein, carboxynaphthofluorescein, fluorescein isothiocyanate, NHS-fluorescein, iodoacetamidofluorescein, fluorescein maleimide, SAMSA-fluorescein, fluorescein thiosemicarbazide, carbohydrazinomethylthioacetamidofluorescein), rhodamine and rhodamine derivatives (such as TRITC, TMR, lissamine rhodamine, fluorescein thiocarbazide ... rhodamine), Texas Red, Rhodamine B, Rhodamine 6G, Rhodamine 10, NHS-rhodamine, TMR-iodoacetamide, lissamine rhodamine B sulfonyl chloride, lissamine rhodamine B sulfonyl hydrazide, Texas Red sulfonyl chloride, Texas Red hydrazide), coumarin and coumarin derivatives (e.g., AMCA, AMCA-NHS, AMCA-sulfo-NHS, AMCA-HPDP, DCIA, AMCE-hydrazide), BODIPY and derivatives (e.g., BODIPY FL C3-SE, BODIPY 530 / 550 C3, BODIPY 530 / 550 C3-SE, BODIPY 530 / 550 C3 hydrazide, BODIPY 493 / 503 C3 hydrazide, BODIPY FL C3 hydrazide, BODIPY FL IA, BODIPY 530 / 551 IA, Br-BODIPY 493 / 503), Cascade Blue and derivatives (such as Cascade Blue acetyl azide, Cascade Blue cadaverine, Cascade Blue ethylenediamine, Cascade Blue hydrazide), Fluorescent Yellow and derivatives (such as Fluorescent Yellow iodoacetamide, Fluorescent Yellow CH), Cyanine and derivatives (such as indole-based cyanine dyes, benzindolium-based cyanine dyes, pyridinium-based cyanine dyes, thiazolium-based cyanine dyes, quinolinium-based cyanine dyes, imidazolium-based cyanine dyes, Cy 3, Cy 5), lanthanide chelates and derivatives (such as BCPDA, TBP, TMT, BHHCT, BCOT, europium chelates, terbium chelates, Alexa Fluor dyes, DyLight dyes, Atto dyes, LightCycler Red dyes, CAL Flour dyes), JOE and its derivatives, Oregon Green dye ( dyes), WellRED dyes, IRD dyes, phycoerythrin and phycobilin dyes, malachite green, stilbene, DEG dyes, NR dyes, near-infrared dyes, and other fluorescent moieties known in the art, such as Haugland, Molecular Probes Handbook, 6th ed. (Eugene, Oreg.);A fluorescent moiety as described in Lakowicz, Principles of Fluorescence Spectroscopy, 2nd ed., Plenum Press New York (1999) or Hermanson, Bioconjugate Techniques, 2nd ed., or a derivative thereof;Cyanine dyes may exist in sulfonated or non-sulfonated form and consist of two indolenine, benzindolium, pyridinium, thiazolium and / or quinolinium groups separated by a polymethine bridge between the two nitrogen atoms. Commercially available cyanine fluorophores include, for example, Cy3 (which may include 1-[6-(2,5-dioxopyrrolidin-1-yloxy)-6-oxohexyl]-2-(3-{1-[6-(2,5-dioxopyrrolidin-1-yloxy)-6-oxohexyl]-3,3-dimethyl-1,3-dihydro-2H-indol-2-ylidene}prop-1-en-1-yl)-3,3-dimethyl-3H-indolium or 1-[6-(2,5-dioxopyrrolidin-1-yloxy)-6-oxohexyl]-2-(3-{1-[6-(2,5-dioxopyrrolidin-1-yloxy)-6-oxohexyl]-3,3-dimethyl-1,3-dihydro-2H-indol-2-ylidene}prop-1-en-1-yl)-3,3-dimethyl-3H-indolium oxopyrrolidin-1-yloxy)-6-oxohexyl]-3,3-dimethyl-5-sulfo-1,3-dihydro-2H-indol-2-ylidene}prop-1-en-1-yl)-3,3-dimethyl-3H-indolium-5-sulfonate), Cy5 (which may include 1-(6-((2,5-dioxopyrrolidin-1-yl)oxy)-6-oxohexyl)-2-((1E,3E)-5-((E)-1-(6-((2,5-dioxopyrrolidin-1-yl)oxy)-6-oxohexyl)-3,3-dimethyl-5-indolin-2-ylidene 3-dimethyl-3H-indol-1-ium or 1-(6-((2,5-dioxopyrrolidin-1-yl)oxy)-6-oxohexyl)-2-((1E,3E)-5-((E)-1-(6-((2,5-dioxopyrrolidin-1-yl)oxy)-6-oxohexyl)-3,3-dimethyl-5-sulfoindolinoline-2-ylidene)penta-1,3-dien-1-yl)-3,3-dimethyl-3H-indol-1-ium-5-sulfonate) and Cy7 (which may include 1-(5-carboxypentyl) 1-(5-Carboxypentyl)-2-[(1E,3E,5E,7Z)-7-(1-ethyl-1,3-dihydro-2H-indol-2-ylidene)hepta-1,3,5-trien-1-yl]-3H-indolium or 1-(5-carboxypentyl)-2-[(1E,3E,5E,7Z)-7-(1-ethyl-5-sulfo-1,3-dihydro-2H-indol-2-ylidene)hepta-1,3,5-trien-1-yl]-3H-indolium-5-sulfonate), where "Cy" stands for 'cyanine' and the first digit indicates the number of carbon atoms between the two indolenine groups. Cy2 is an oxazole derivative rather than an indolenine, and the benzo-derived Cy3.5, Cy5.5, and Cy7.5 are exceptions to this rule. ;

[0087] In some embodiments, the reporter moiety can be a FRET pair, so that multiple classifications can be performed in a single excitation and imaging step. As used herein, FRET can include excitation exchange (Forster) transfer or electron exchange (Dexter) transfer.

[0088] Many pH buffers are known to those skilled in the art. The full names of the pH buffers are listed herein. The term "Tris" refers to the pH buffer tris(hydroxymethyl)-aminomethane. The term "Tris-HCl" refers to the pH buffer tris(hydroxymethyl)-aminomethane hydrochloride. The term "triacetate" refers to a pH buffer comprising the acetate salt of tris(hydroxymethyl)-aminomethane. The term "Tricine" refers to the pH buffer N-[tris(hydroxymethyl)methyl]glycine. The term "Bicine" refers to the pH buffer N,N-bis(2-hydroxyethyl)glycine. The term "bis-tris-propane" refers to the pH buffer 1,3-bis[tris(hydroxymethyl)methylamino]propane. The term "HEPES" refers to the pH buffer 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid. The term "MES" refers to the pH buffer 2-(N-morpholino)ethanesulfonic acid. The term "MOPS" refers to the pH buffer 3-(N-morpholino)propanesulfonic acid. The term "MOPSO" refers to the pH buffer 3-(N-morpholino)-2-hydroxypropanesulfonic acid. The term "BES" refers to the pH buffer N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid. The term "TES" refers to the pH buffer 2-[(2-hydroxy-1,1-bis(hydroxymethyl)ethyl)amino]ethanesulfonic acid. The term "CAPS" refers to the pH buffer 3-(cyclohexylamino)-1-propanesulfonic acid. The term "TAPS" refers to the pH buffer N-[tris(hydroxymethyl)methyl]-3-aminopropanesulfonic acid. The term "TAPSO" refers to the pH buffer N-[tris(hydroxymethyl)methyl]-3-amino-2-hydroxypropanesulfonic acid. The term "ACES" refers to the pH buffer N-(2-acetamido)-2-aminoethanesulfonic acid. The term "PIPES" refers to the pH buffer piperazine-1,4-bis(2-ethanesulfonic acid). The term "ethanolamine" refers to the pH buffer also known as 2-aminoethanol.

[0089] The terms "linked," "joined," "attached," and variations thereof include any type of fusion, binding, adhesion, or association between any combination of compounds or molecules that is sufficiently stable to withstand use in a particular procedure. The procedure may include, but is not limited to: transient binding of nucleotides; nucleotide incorporation; deblocking; washing; removal; flow; detection; imaging and / or identification. Such bonds may include, for example, covalent bonding, ionic bonding, hydrogen bonding, dipole-dipole bonding, hydrophilic bonding, hydrophobic bonding, or affinity bonding, bonds or associations involving van der Waals forces, mechanical bonding, and the like. In some embodiments, such bonds occur within a molecule, such as joining the ends of a single-stranded or double-stranded linear nucleic acid molecule together to form a circular molecule. In some embodiments, such bonds may occur between a combination of different molecules or between a molecule and a non-molecule, including, but not limited to, bonds between a nucleic acid molecule and a solid surface; bonds between a protein and a detectable reporter gene portion; bonds between a nucleotide and a detectable reporter gene portion; and the like. Some examples of linkages can be found, for example, in Hermanson, G., Bioconjugation Technology, 2nd ed. (2008); Aslam, M., Dent, A., Bioconjugation: Protein Coupling Techniques for the Biomedical Sciences, London: Macmillan (1998); Aslam, M., Dent, A., Bioconjugation: Protein Coupling Techniques for the Biomedical Sciences, London: Macmillan (1998).

[0090] As used herein, the terms "operably linked" and "operably joined" or related terms refer to the juxtaposition of components. The juxtaposed components can be covalently linked together. For example, two nucleic acid components can be enzymatically linked together, wherein the bond joining the two components together comprises a phosphodiester bond. A first nucleic acid component and a second nucleic acid component can be linked together, wherein the first nucleic acid component can confer a function to the second nucleic acid component. For example, the bond between a primer binding sequence and a sequence of interest forms a nucleic acid library molecule having a portion that can bind to a primer. In another example, a transgene (e.g., a nucleic acid encoding a polypeptide or a nucleic acid sequence of interest) can be linked to a vector, wherein the bond allows the transgene sequence contained in the vector to be expressed or function. In some embodiments, the transgene is operably linked to a host cell regulatory sequence (e.g., a promoter sequence) that affects transgene expression. In some embodiments, the vector comprises at least one host cell regulatory sequence comprising a promoter sequence, an enhancer, a transcription and / or translation initiation sequence, a transcription and / or translation termination sequence, a polypeptide secretion signal sequence, and the like. In some embodiments, the host cell regulatory sequence controls the expression of the level, timing, and / or position of the transgene.

[0091] In some embodiments, the carrier is solid, semi-solid, or a combination thereof. In some embodiments, the carrier is porous, semi-porous, non-porous, or any combination of porosity. In some embodiments, the carrier can be substantially planar, concave, convex, or any combination thereof. In some embodiments, the carrier can be cylindrical, for example, including a capillary or the inner surface of a capillary.

[0092] In some embodiments, the surface of the carrier can be substantially smooth. In some embodiments, the carrier can have a regular or irregular texture comprising bumps, etchings, holes, three-dimensional scaffolds, or any combination thereof.

[0093] In some embodiments, the support comprises beads having any shape including spheres, hemispheres, cylinders, barrels, rings, disks, rods, cones, triangles, cubes, polygons, tubes, or wires.

[0094] The carrier can be made of any material, including but not limited to glass, fused silica, silicon, polymers (e.g., polystyrene (PS), macroporous polystyrene (MPPS), polymethyl methacrylate (PMMA), polycarbonate (PC), polypropylene (PP), polyethylene (PE), high-density polyethylene (HDPE), cyclic olefin polymer (COP), cyclic olefin copolymer (COC), polyethylene terephthalate (PET)), or any combination thereof. Various compositions of both glass and plastic substrates are contemplated.

[0095] In some embodiments, the surface of the carrier is coated with one or more compounds to produce a passivation layer on the carrier. In some embodiments, the carrier includes a low non-specific binding surface that can improve nucleic acid hybridization and amplification performance on the carrier. Generally speaking, the carrier can include one or more layers (e.g., silane layers, polymer films) of covalently or non-covalently attached low binding chemical modification layers, and one or more covalently or non-covalently attached oligonucleotides that can be used to fix multiple nucleic acid template molecules to the carrier.

[0096] In some embodiments, the degree of hydrophilicity (or "wettability" to aqueous solutions) of a surface coating can be assessed, for example, by measuring a water contact angle, wherein a small water droplet is placed on a surface and its contact angle with the surface is measured using, for example, an optical tensiometer. In some embodiments, a static contact angle can be determined. In some embodiments, an advancing or receding contact angle can be determined. In some embodiments, the water contact angle for a hydrophilic low binding carrier surface disclosed herein can be in the range of about 0 degrees to about 30 degrees. In some embodiments, the water contact angle for a hydrophilic low binding carrier surface disclosed herein can be no more than 50 degrees, 40 degrees, 30 degrees, 25 degrees, 20 degrees, 18 degrees, 16 degrees, 14 degrees, 12 degrees, 10 degrees, 8 degrees, 6 degrees, 4 degrees, 2 degrees, or 1 degree. In many cases, the contact angle is no more than 40 degrees. Those skilled in the art will recognize that a given hydrophilic low binding carrier surface disclosed herein can exhibit a water contact angle with a value anywhere within this range.

[0097] The present disclosure provides a plurality of (e.g., two or more) nucleic acid templates fixed to a carrier. In some embodiments, the fixed plurality of nucleic acid templates have the same sequence or have different sequences. In some embodiments, separate nucleic acid template molecules in a plurality of nucleic acid templates are fixed to different sites on the carrier. In some embodiments, two or more separate nucleic acid template molecules in a plurality of nucleic acid templates are fixed to sites on the carrier. In some embodiments, the carrier comprises a plurality of sites arranged in an array. The term "array" refers to a carrier comprising a plurality of sites at predetermined positions on a carrier to form an array of sites. The sites may be discrete and separated by gaps. In some embodiments, the predetermined sites on the carrier may be arranged in rows or columns in one dimension, or in rows and columns in two dimensions. In some embodiments, a plurality of predetermined sites are arranged in an organized manner on the carrier. In some embodiments, a plurality of predetermined sites are arranged in any organized pattern, including straight lines, hexagonal patterns, grid patterns, patterns with reflection symmetry, patterns with rotational symmetry, and the like. The spacing between different pairs of sites may be the same or may be different. In some embodiments, the carrier may have a spacing of about 10 2- 10 15 Sites / mm 2or higher surface density to immobilize nucleic acid template molecules at multiple sites to form a nucleic acid template array. In some embodiments, the carrier comprises at least 10 2 sites, at least 10 3 sites, at least 10 4 sites, at least 10 5 sites, at least 10 6 sites, at least 10 7 sites, at least 10 8 sites, at least 10 9 sites, at least 10 10 sites, at least 10 11 sites, at least 10 12 sites, at least 10 13 sites, at least 10 14 sites, at least 10 15 In some embodiments, the plurality of predetermined sites (e.g., 10 2 -10 15 In some embodiments, the nucleic acid template is immobilized at a plurality of predetermined sites, for example, at 10 or more sites, with a nucleic acid template immobilized thereon to form a nucleic acid template array. In some embodiments, the nucleic acid template is immobilized at a plurality of predetermined sites by hybridization with an immobilized surface capture primer, or the nucleic acid template is covalently attached to a surface capture primer. In some embodiments, the nucleic acid template is immobilized at a plurality of predetermined sites, for example, at 10 or more sites. 2 -10 15 In certain embodiments, the nucleic acid template fixed at a plurality of sites on the carrier comprises a linear nucleic acid template molecule or a circular nucleic acid template molecule or a mixture of a linear molecule and a circular molecule. In certain embodiments, the fixed nucleic acid template is amplified in a clonal manner to produce fixed nucleic acid polymerase colonies (nucleic acid polonies) at the plurality of predetermined sites. In certain embodiments, the independent fixed nucleic acid template molecule comprises a copy of the target sequence being paid attention to, or comprises a concatemer with two or more tandem copies of the target sequence being paid attention to.

[0098] In some embodiments, a vector comprising a plurality of sites located at random positions on the vector is referred to herein as a vector having randomly positioned sites thereon. The positions of the randomly positioned sites on the vector are not predetermined. The plurality of randomly positioned sites are arranged in a disordered and / or unpredictable manner on the vector. In some embodiments, the vector comprises at least 10 2 sites, at least 10 3 sites, at least 10 4 sites, at least 10 5sites, at least 10 6 sites, at least 10 7 sites, at least 10 8 sites, at least 10 9 sites, at least 10 10 sites, at least 10 11 sites, at least 10 12 sites, at least 10 13 sites, at least 10 14 sites, at least 10 15 sites or more, wherein the sites are randomly positioned on the carrier. In some embodiments, the plurality of randomly positioned sites (e.g., 10 2 -10 15 In some embodiments, the nucleic acid template is immobilized at a plurality of randomly located sites by hybridization with an immobilized surface capture primer, or the nucleic acid template is covalently attached to the surface capture primer. In some embodiments, the nucleic acid template is immobilized at a plurality of randomly located sites, for example, at 10 2 -10 15 In certain embodiments, the nucleic acid template fixed at a plurality of sites on the carrier comprises a linear nucleic acid template molecule or a circular nucleic acid template molecule or a mixture of a linear molecule and a circular molecule. In certain embodiments, the fixed nucleic acid template is amplified in a clonal manner to produce a fixed nucleic acid polymerase colony at the plurality of randomly located sites. In certain embodiments, the independent fixed nucleic acid template molecule comprises a copy of the target sequence being paid close attention to, or comprises a concatemer with two or more tandem copies of the target sequence being paid close attention to.

[0099] In some embodiments, for nucleic acid template molecules fixed to predetermined or random sites on a carrier, multiple fixed nucleic acid template molecules on the carrier are fluidically connected to each other to allow a reagent solution (e.g., an enzyme comprising a polymerase, a multivalent molecule, nucleotides, a divalent cation and / or a buffer, etc.) to flow onto the carrier, so that multiple fixed nucleic acid template molecules on the carrier can react with the reagent in a large-scale parallel manner. In some embodiments, the fluid connection of multiple fixed nucleic acid template molecules can be used to perform nucleotide binding assays and / or perform nucleotide polymerization reactions (e.g., primer extension or sequencing) on ​​multiple fixed nucleic acid template molecules, and for detection and imaging for large-scale parallel sequencing. In some embodiments, the term "fixed" and related terms refer to a nucleic acid molecule or enzyme (e.g., a polymerase) attached to a carrier at a predetermined or random position, wherein the nucleic acid molecule or enzyme is directly attached to the carrier by a covalent bond or non-covalent interaction, or the nucleic acid molecule or enzyme is attached to a coating on the carrier.

[0100] As used herein, the term "clonal amplification" and variants thereof refer to that a nucleic acid template molecule has been subjected to one or more amplification reactions in solution or on a carrier. In the case of amplifying the template molecule in solution, the resulting amplicon is distributed on the carrier. Before amplification, the template molecule includes a sequence of interest and at least one universal adapter sequence. In certain embodiments, clonal amplification includes the use of polymerase chain reaction (PCR), multiple displacement amplification (MDA), transcription-mediated amplification (TMA), nucleic acid sequence-based amplification (NASBA), strand displacement amplification (SDA), real-time SDA, bridge amplification, isothermal bridge amplification, rolling circle amplification (RCA), circle-to-circle amplification, helicase-dependent amplification, recombinase-dependent amplification, single-stranded binding (SSB) protein-dependent amplification, or any combination thereof.

[0101] As used herein, the term "sequencing" and its variants include obtaining sequence information from a nucleic acid chain by determining the identity of at least some nucleotides (comprising its core base components) within a nucleic acid template molecule. Although in some embodiments, "sequencing" of a given district of a nucleic acid molecule includes identifying each or each nucleotide in the sequenced district, in some embodiments, "sequencing" includes only determining the identity of some nucleotides in the nucleotides in the district, and still not determining or mistakenly determining the method for the identity of some nucleotides. Any suitable sequencing method can be used. In an exemplary embodiment, sequencing can include labeling or ion-based sequencing methods. In certain embodiments, sequencing methods can include labeled or dye-containing nucleotides or fluorescence-based nucleotide sequencing methods. In certain embodiments, sequencing can include sequencing based on polymerase colonies or bridge sequencing methods. In certain embodiments, sequencing includes adopting synthetic sequencing, hybridization sequencing or a large-scale parallel sequencing platform in conjunction with sequencing programs. Examples of massively parallel sequencing-by-synthesis procedures include polymerase colony sequencing, pyrosequencing (e.g., from 454 Life Sciences; U.S. Pat. Nos. 7,211,390, 7,244,559, and 7,264,929), chain terminator sequencing (e.g., from Illumina; U.S. Pat. No. 7,566,537; Bentley 2006 Current Opinion Genetics and Development 16:545-552; and Bentley et al. 2008 Nature 456:53-59), ion-sensitive sequencing (e.g., from Ion Torrent), probe-anchor ligation sequencing (e.g., from Complete Genomics), and sequencing by PCR. Genomics), DNA nanoball sequencing, nanopore DNA sequencing. Examples of single-molecule sequencing include Heliscope single-molecule sequencing and single-molecule real-time (SMRT) sequencing. Examples of hybridization sequencing include SOLiD sequencing (e.g., from Life Technologies; WO 2006 / 084132). Examples of combined sequencing include Omniome sequencing (e.g., U.S. Patent No. 10,246,744).

[0102] Engineered polymerases exhibiting increased thermal stability

[0103] The present disclosure provides compositions comprising mutant polymerases having amino acid substitutions and / or truncated amino acid sequences, nucleic acids encoding mutant polymerases, and systems and kits comprising mutant polymerases. Further provided herein are methods for using mutant polymerases, comprising methods for binding to nucleic acid duplexes, binding to complementary nucleotides or binding to multivalent molecules having complementary nucleotide units, incorporating complementary nucleotides, extending primers, and nucleic acid sequencing, wherein the methods employ any of the mutant polymerases described herein. The mutant polymerases are engineered to exhibit desired properties, including exonuclease activity. - Activity (exonuclease-minus activity) and increased stability, improved uracil tolerance and / or reduced sequence-specific errors. Additionally, mutant polymerases can be engineered to express higher fractions of soluble expressed enzyme.

[0104] The present disclosure provides mutant polymerase, described mutant polymerase is engineered to reduce post-translational modification, thereby improves protein activity and stability.Recombinant protein is carried out post-translational modification can reduce protein activity, cause misfolding, increase heterogeneity, reduce thermal stability and increase protein aggregation.These modifications have been challenged to realizing stable manufacturability and keeping a long shelf life.Many recombinant protein experiences post-translational modification, comprise phosphorylation, acetylation, ubiquitination, succinylation, methylation, glycosylation (for example, N-connects, O-connects and C-connects), hydroxylation, oxidation, deamidation, nitrosylation, sulfation, SUMOylation, disulfide bonding, proteolysis and / or lipidation (for example, palmitoylation).

[0105] Examples of undesirable effects of post-translational modifications include oxidative damage to sulfur-containing amino acids such as cysteine ​​and methionine. Histidine, tryptophan, lysine, and serine are also known to be susceptible to oxidative damage. Deamidation of asparagine and glutamine may lead to protein degradation, thereby shortening shelf life. Acetylation may involve cleavage of the N-terminal methionine and replacement of the acetyl group. In some proteins, the N-terminal methionine plays an important role in enzyme activity, and removal of this methionine residue can alter enzyme activity. Acetylation may also involve serine and lysine. Methylation may result in changes in hydrophobicity and side chain charge. Ubiquitination may lead to protein degradation.

[0106] Analytical techniques such as mass spectrometry and tandem mass spectrometry can be used to detect post-translational modifications, specifically oxidation of methionine and lysine residues, which enables rational mutagenesis to improve protein yield and stability.

[0107] The present disclosure provides mutant polymerases that are engineered to exhibit a reduced editing mode conformation. Many DNA polymerases have 5' to 3' nucleotide polymerization activity and 3' to 5' exonuclease proofreading activity. Generally speaking, the polymerization site in the polymerase is located at different sites from the proofreading site. During the polymerization mode, the growing end of the template and primer chain is located in the polymerization site. In many family B and C polymerases, the amino acid residues in the finger, thumb, and palm domains constitute at least a part of the polymerization site. In the proofreading mode, the growing end of the primer is physically transferred from the polymerization site to the exonuclease site, and the nucleotides of the error-incorporated end of the growing primer chain are excised by the 3' to 5' proofreading activity of the polymerase. The transfer of the primer end from the polymerization site to the exonuclease site may involve the local unpairing of several base pairs at the primer / template junction, which prevents other chain extension (e.g., polymerization) until the primer end is transferred back to the polymerization site and the primer / template pairing is restored. The amino acid residues that play a role in the transfer of the primer end are unrelated to the amino acid residues involved in the exonuclease activity. The polymerization site and the exonuclease site are located in two different domains. When the correct nucleotide is incorporated, the polymerase sometimes undergoes this polymerization to exonuclease conformational switch unnecessarily, thereby hindering primer extension. In Escherichia coli DNA polymerase III, it has been previously determined that the time scale for primer end transfer and primer / template unpairing is about ten milliseconds, an order of magnitude longer than the time for nucleotide incorporation (Dodd et al., 2020 "Nature Communication (Nature Communication)" 11: 5379, "Polymerization and editing modes of high-fidelity DNA polymerase are connected by a clear path"). Therefore, when the correct nucleotide is incorporated, or when the correct nucleotide unit from the multivalent molecule is bound at the polymerization site, it is expected to design a sequencing polymerase that exhibits a reduced editing mode conformation. It is speculated that the editing mode mutant polymerase will retain 3' to 5' exonuclease activity.

[0108] The key amino acid residues on the path of the primer end that is transferred to the exonuclease site from the polymerization site can be identified by protein modeling and mass spectrometry, without affecting exonuclease activity.At least some of the sudden changes in these key amino acid residues can reduce unnecessary polymerization to the exonuclease conformation switch.For example, the sudden change of the amino acid residue that plays a role in the guiding primer end to the exonuclease site or the sudden change of the amino acid residue of the primer end that is stably transferred at the exonuclease site can reduce the transformation to the editing mode conformation.In another example, the sudden change of the amino acid residue that interacts with the template molecule and makes its stable during polymerization can keep the template molecule at the polymerization site.

[0109] The present disclosure provides mutant polymerases that can be used to perform a two-stage nucleic acid sequencing method. In some embodiments, the first stage generally includes combining a detectably labeled multivalent molecule with a composite polymerase to form a multivalent composite polymerase under conditions suitable for inhibiting nucleotide unit incorporation, and detecting the multivalent composite polymerase. The first stage can be performed using a capture polymerase. In some embodiments, the second stage generally includes polymerase-catalyzed nucleotide incorporation using a stepper polymerase.

[0110] The present disclosure provides mutant polymerases that can be used for either capture or walking events of nucleic acid sequencing. Some of the mutant polymerases can be used for both capture and walking events.

[0111] The present disclosure provides mutant polymerases that can be used to capture multivalent molecules, including a composite mutant polymerase bound to a multivalent molecule having complementary nucleotide units (e.g., exemplary multivalent molecules Figure 2-5 In some embodiments, the multivalent molecule includes a central core attached to a plurality of polymer arms, each polymer arm having a nucleotide unit at the arm end. The multivalent molecule can be labeled with a detectable reporter gene portion. The composite mutant polymerase comprises a mutant polymerase bound to a template / primer duplex. The mutant polymerase is engineered to exhibit reduced sequence-specific errors that occur after certain motif sequences in the primer strand and / or template strand. The sequence-specific errors that capture the polymerase can be characterized by a substantial loss of signal intensity (which can result in base miscalls (e.g., base substitutions) or not called in a specific sequencing cycle). The signal is often restored in the next cycle. The motif sequence that causes the miscall is specific to a given polymerase and can occur on either template strand in the forward or reverse sequencing direction.

[0112] The present disclosure provides mutant polymerases that can be used to bind complementary nucleotides (e.g., unconjugated nucleotides) and incorporate nucleotides into the 3' end of a primer (referred to as stepping events). The mutant polymerases are engineered to exhibit reduced sequence-specific errors characterized by substantial losses of nucleotide incorporation occurring after certain motif sequences in the primer strand and / or template strand. The sequence-specific errors of stepping enzymes can be characterized by large-scale phasing after the sequence motifs. The motif sequences that cause phasing are specific to a given polymerase and can occur on either template strand in the forward or reverse sequencing direction.

[0113] Without wishing to be bound by theory, it is hypothesized that mutant polymerases that exhibit capture or stepping sequence-specific errors at certain sequence motifs during sequencing switch from a nucleotide incorporating conformation to an editing conformation. During the capture event, the editing conformation blocks the binding of complementary nucleotide units from the multivalent molecule, which results in a decrease in signal intensity. During the stepping event, the editing conformation blocks the binding and incorporation of complementary nucleotides or nucleotide analogs, which results in a decrease in signal intensity. Designing capture and stepping polymerases that carry one or more mutation sites that reduce the switch from nucleotide polymerization to editing conformation can reduce capture sequence-specific errors.

[0114] In some embodiments, the mutant polymerases include polypeptides derived from the directed evolution of recently identified novel B-family and A-family polymerases, or fragments thereof, wherein the mutant polymerases exhibit improvements in their specificity while maintaining a high degree of discrimination for correct Watson-Crick base pairing.

[0115] The present disclosure provides polymerases that have been engineered to contain substitution mutations, including polymerases having an amino acid sequence backbone of RLF 89458.1 (e.g., from the Thermococci archaeon, isolate B13_G1) (SEQ ID NO: 1), RLF 78286.1 (e.g., from the Thermococci archaeon, isolate B89_G9) (SEQ ID NO: 2), NOZ 58130.1 (e.g., from the Euryarchaeota archaeon, isolate M_BaxBin.100) (SEQ ID NO: 1714), RMF 90817.1 (e.g., from the Euryarchaeota archaeon, isolate J060 (SEQ ID NO: 2789), MBC 7218772.1 (e.g., from the Hadesarchaea archaeon, isolate MAG-18) (SEQ ID NO: 3). NO: 2790), WP 175059460.1 (e.g., from Thermococcus 2319x1) (SEQ ID NO: 2791), KUO 42443.1 (e.g., from Candidatus Hadarchaem, yellowstonense, isolate YNP_45) (SEQ ID NO: 2792), and NOZ 77387.1 (e.g., from the Euryarchaeota archaea, isolate M_MaxBin.027) (SEQ ID NO: 2793).

[0116] Polypeptides described herein include, but are not limited to, polypeptides with enzymatic activity (such as polymerase activity), and are generally described in terms of families. Typically, a polymerase is an enzyme that binds to a DNA polymerase, RNA polymerase, template-independent polymerase, reverse transcriptase, or other enzyme capable of nucleotide binding and nucleotide incorporation (e.g., primer extension). Many DNA polymerases are known in the art, and in some cases, such enzymes are mutated to produce compositions described herein. Members of the DNA polymerase family are generally defined based on polymerase activity, active site structure, domain homology / function, or sequence homology with other known DNA polymerase family members. For example, DNA polymerases include, but are not limited to, E. coli DNA polymerase I, E. coli DNA polymerase II, or other members of the DNA polymerase family. Known thermostable DNA polymerases include Taq polymerase, Pfu polymerase, and 9°N polymerase or other members of the DNA polymerase family. Wild-type DNA polymerases are or can be obtained from any number of sources, such as eukaryotic, prokaryotic, or viral sources, and in some embodiments, for purposes of this disclosure, are obtained from archaebacterial sources. In some embodiments, the polymerase comprising the amino acid sequence of any one of SEQ ID NOs: 1, 2, 1714, 2789-2793, and 2803 is a member of the DNA polymerase family.

[0117] The polymerase described herein may comprise one or more amino acid substitutions and / or truncations, which increase the thermostability of the polymerase. In certain embodiments, the thermostability of the polymerase can be determined by measuring the temperature at which the polymerase unfolds and / or aggregates. For example, a thermal shift assay using differential scanning fluorimetry can be used to determine the thermostability of the polymerase. Differential scanning fluorimetry can be used to determine the protein unfolding transition (T(m)), during which approximately 50% of the protein is in its native conformation and approximately 50% of the protein is denatured. The protein unfolding transition (T(m)) can be obtained from the melting peak, where the temperature rises along the x-axis and the first derivative curve of fluorescence versus temperature (e.g., dF / dT or dRFU / dT) is drawn along the y-axis. Some proteins exhibit two protein transitions, T(m1) and T(m2). For example, the T(m1) temperature is the temperature at which at least 50% of the protein transitions from folding to unfolding. In another example, the T(m2) temperature is a temperature higher than the T(m1) temperature, where more protein transitions from folding to unfolding at the T(m2) temperature. Completely denatured protein and protein aggregation (T(agg)) can be obtained from a melting curve, where the temperature increase is plotted along the x-axis and the fluorescence (e.g., RFU) is plotted along the y-axis. For example, Table 1-2 (e.g., Figure 31-3 2) The Tm1 and Tm2 of the engineered polymerases are listed.

[0118] In some embodiments, compared to wild-type polymerase or compared to engineered polymerases with mutations that do not confer increased thermal stability, engineered polymerases exhibit protein folding transitions (e.g., thermal stability) at elevated temperatures. In some embodiments, engineered polymerases exhibit increased thermal stability at elevated temperatures of about 72-75°C, or about 75-80°C, or about 80-85°C, or about 85-90°C or higher. Many engineered polymerases in the engineered polymerases described herein exhibit nucleotide binding and incorporation activity within a temperature range of about 25-50°C, or about 45-75°C, or about 65-80°C, or about 80-90°C. Therefore, these engineered polymerases are thermostable (e.g., mesophilic polymerases) within a medium high temperature range. Engineered polymerases described herein are suitable for nucleotide binding, nucleotide unit binding, nucleotide incorporation, and / or nucleic acid sequencing reactions within a temperature range of about 25-50°C, or about 45-75°C, or about 65-80°C, or about 80-90°C or higher. In some embodiments, the mutant polymerase exhibits an increase in thermostability of about 2-4°C, or about 4-6°C, or about 6-8°C, or about 8-10°C.

[0119] In contrast, DNA polymerases that exhibit significantly higher thermostability exceeding 95°C include 9°N, THERMINATOR, VENT, DEEP VENT, Pfu, and Pyrococcus aquatica. Thermostable polymerases, such as 9°N, VENT, DEEP VENT, Pfu, and Pyrococcus aquatica polymerases, are suitable for use in PCR reactions in which typical cycling steps are performed at temperatures exceeding 90-95°C or higher, and may not be suitable for use in nucleotide binding, nucleotide incorporation, and / or nucleic acid sequencing reactions performed at lower temperature ranges. DNA polymerases from Geobacillus stearothermophilus (e.g., Bst polymerase) are generally stable up to 65°C.

[0120] Polymerases variously include DNA polymerases, RNA polymerases, template-independent polymerases, reverse transcriptases, or other enzymes capable of catalyzing nucleotide incorporation. Archaeal polymerases are generally derived from thermophilic organisms and, therefore, may represent a class of thermostable or thermotolerant enzymes. Thus, polypeptide backbones derived from archaeal polymerases provide desirable targets for protein engineering to further enhance the incorporation of reversible terminator nucleotides, which can be improved by employing enzymes with enhanced thermostability or otherwise enhanced resistance to degradation (e.g., by repeated exposure to elevated temperatures, altered buffer conditions, etc.).

[0121] The present disclosure provides compositions and methods including mutant polymerases that exhibit improved shelf life compared to wild-type polymerases or to engineered polymerases having mutations that do not impart improved shelf life. For example, the mutant polymerase can maintain enzymatic activity after being stored for several months or years at a given temperature. In some embodiments, the mutant polymerase can maintain approximately 90-100% activity when stored for 2-12 months at a temperature of approximately 0-positive 27°C, or a temperature of approximately negative 20°C-negative 45°C, or a temperature of approximately negative 45°C-negative 80°C. In some embodiments, the mutant polymerase can maintain approximately 80-90% activity when stored for 2-12 months at a temperature of approximately 0-positive 27°C, or a temperature of approximately negative 20°C-negative 45°C, or a temperature of approximately negative 45°C-negative 80°C. In some embodiments, the mutant polymerase can retain about 70-80% activity when stored for 2-12 months at a temperature of about 0-plus 27° C., or about minus 20° C. to minus 45° C., or about minus 45° C. to minus 80° C. In some embodiments, the mutant polymerase can retain about 70-100% activity when stored for about 12-36 months at a temperature of about 0-plus 27° C., or about minus 20° C. to minus 45° C., or about minus 45° C. to minus 80° C.

[0122] In some embodiments, the wild-type or engineered polymerase can be stored in a storage buffer comprising a solvent, at least one pH buffer, at least one salt, at least one chelating agent, at least one viscosity agent, and at least one detergent. In some embodiments, the solvent comprises water.

[0123] In some embodiments, the buffer comprises MES, HEPES, ACES, Tris and / or Tris-HCl, or any other pH buffer known to those skilled in the art. In some embodiments, the pH of the pH buffer is about 6-6.5, or the pH of the pH buffer is about 6.5-7, or the pH of the pH buffer is about 7-7.5, or the pH of the pH buffer is about 7.5-8, or the pH of the pH buffer is about 8-8.5. In some embodiments, the storage buffer comprises at least one pH buffer at a concentration of about 10-20 mM, or about 20-30 mM, or about 30-40 mM, or about 40-50 mM.

[0124] In some embodiments, the salt comprises a monovalent salt comprising NaCl, KCl, NH2SO4 and / or potassium glutamate. In some embodiments, the storage buffer comprises at least one monovalent salt at a concentration of about 50-100 mM, or about 100-200 mM, or about 200-300 mM, or about 300-400 mM, or about 400-500 mM, or about 500-600 mM, or about 600-800 mM.

[0125] In some embodiments, the chelating agent comprises EDTA (ethylenediaminetetraacetic acid), EGTA (ethylene glycol tetraacetic acid), HEDTA (hydroxyethylethylenediaminetriacetic acid), DPTA (diethylenetriaminepentaacetic acid), NTA (N,N-bis(carboxymethyl)glycine), anhydrous citrate, sodium citrate, calcium citrate, ammonium citrate, diammonium citrate, citric acid, potassium citrate, and / or magnesium citrate. In some embodiments, the storage buffer comprises at least one chelating agent at a concentration of about 0.0125-0.025 mM, or about 0.025-0.05 mM, or about 0.05-0.1 mM, or about 0.1-0.2 mM.

[0126] In some embodiments, the viscosity agent comprises a sugar such as trehalose, sucrose, cellulose, xylitol, mannitol, sorbitol, and / or inositol. In some embodiments, the viscosity agent comprises glycerol or a glycol compound such as ethylene glycol and / or propylene glycol. In some embodiments, the storage buffer comprises about 20-30%, or about 30-40%, or about 40-50%, or about 50-60%, or about 60-70%, or about 70-80% of at least one viscosity agent.

[0127] In some embodiments, the detergent includes an ionic detergent such as SDS (sodium dodecyl sulfate). In some embodiments, the detergent includes a non-ionic detergent such as Triton X-100, Tween 20, Tween 80, or Nonidet P-40. In some embodiments, the detergent includes a zwitterionic detergent such as CHAPS (3-[(3-cholamidopropyl)dimethylammonium]-1-propanesulfonate) or N-dodecyl-N,N-dimethyl-3-ammonium-1-propanesulfonate (DetX). In some embodiments, the detergent includes LDS (lithium dodecyl sulfate), sodium taurodeoxycholate, sodium taurocholate, sodium glycocholate, sodium deoxycholate, or sodium cholate. In some embodiments, the storage buffer comprises about 0.025-0.5%, or about 0.5-0.1%, or about 0.1-0.2%, or about 0.2-0.4%, or about 0.4-0.8%, or about 0.8-1.6% of at least one detergent.

[0128] The present disclosure provides compositions and methods comprising mutant polymerases that exhibit improved ability to bind to complementary nucleotide units of multivalent molecules compared to wild-type polymerases or to engineered polymerases having mutations that do not confer improved binding to complementary nucleotide units of multivalent molecules. Multivalent molecules generally include a central portion (e.g., a core) attached to a plurality of arms, wherein each arm is attached to a nucleotide unit. Multivalent molecules include star-shaped, comb-shaped, cross-linked, bottlebrush-shaped, or dendritic macromolecular configurations (e.g., see Figure 2 ).

[0129] Surprisingly, it was discovered that many of the engineered polymerases described herein exhibited enhanced incorporation rates of nucleotide analogs compared to wild-type polymerases or engineered polymerases with mutations that do not confer enhanced incorporation rates of nucleotide analogs. Some of the engineered polymerases also exhibited one or more desirable properties compared to wild-type polymerases, including increased binding affinity for nucleotide analogs with 3' chain-terminating groups, improved ability to incorporate dATP nucleotides relative to uracil-containing template molecules (e.g., uracil-tolerant mutant polymerases), improved ability to bind to complementary nucleotide units of multivalent molecules, increased thermal stability up to about 90°C, increased shelf life, and reduced sequence-specific errors.

[0130] The present disclosure provides compositions and methods including mutant polypeptides associated with polymerases that exhibit increased ability to bind and discriminate nucleotide analogs, as well as improved incorporation of nucleotide analogs, compared to corresponding wild-type polymerases. Nucleotide analogs include, for example, nucleotides that include a chain-terminating group attached to the 2' or 3' position of the sugar. The chain-terminating group includes an azide, an azido or azidomethyl group, or another type of chain-terminating group. The engineered DNA polymerases exhibit increased incorporation rates of nucleotide analogs compared to the corresponding wild-type polymerases having an amino acid sequence backbone of any one of RLF 89458.1 (SEQ ID NO: 1), RLF 78286.1 (SEQ ID NO: 2), NOZ 58130.1 (SEQ ID NO: 1714), RMF 90817.1 (SEQ ID NO: 2789), MBC 7218772.1 (SEQ ID NO: 2790), WP175059460.1 (SEQ ID NO: 2791), KUO 42443.1 (SEQ ID NO: 2792), and NOZ 77387.1 (SEQ ID NO: 2793).

[0131] The data shown in Table 1 and Table 2 provide many exemplary mutant polymerases, compared with its corresponding wild-type polymerase or compared with the engineered polymerase with identical backbone sequence and the sudden change that does not give the thermostability of increase, described exemplary mutant polymerase shows the thermostability of increase.Many mutant polymerases in these mutant polymerases are included in the sudden change of LYP motif and / or other position.In certain embodiments, with respect to the wild-type enzyme of corresponding currently known in the art or enzyme variants, mutant polymerase shows that the incorporation rate of nucleotide analog has increased by about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 500% or 1000%.The exemplary mutant polymerase that shows the thermostability of increase is listed in Table 1 and Table 2.

[0132] The present disclosure provides compositions and methods including mutant polymerases having at least one amino acid residue inserted before or after a LYP motif. In some embodiments, the at least one amino acid residue inserted before or after the LYP motif can move the LYP motif as a unit in a folded polypeptide, which can increase or decrease the activity of the LYP motif in the mutant enzyme. In some embodiments, the insertion of at least one amino acid residue before or after the LYP motif may more effectively modulate the activity of the LYP motif than traditional amino acid substitutions, which are designed to change the amino acid side chains in and around the LYP motif. In some embodiments, the at least one amino acid residue inserted before or after the LYP motif can modulate (e.g., increase or decrease) the incorporation rate of nucleotide analogs compared to a mutant polymerase lacking at least one inserted amino acid residue.

[0133] In some embodiments, a polymerase that binds to a nucleotide (or nucleotide analog) or a multivalent molecule under conditions suitable for inhibiting a polymerase-catalyzed nucleotide incorporation reaction comprises at least one amino acid residue inserted before or after a LYP motif. In some embodiments, a polymerase that binds to a nucleotide (or nucleotide analog) or a multivalent molecule under conditions suitable for initiating a polymerase-catalyzed nucleotide incorporation reaction comprises at least one amino acid residue inserted before or after a LYP motif.

[0134] In some embodiments, at least one amino acid residue inserted before or after the LYP motif comprises any of the 20 amino acids (e.g., W, I, M, P, F, G, A, V, L, H, R, K, D, E, N, Y, C, S, T, or Q). In some embodiments, at least one amino acid residue inserted before or after the LYP motif comprises proline or glycine. Exemplary mutant polymerases include the amino acid sequence of any one of SEQ ID NOs: 1076, 1094, 1197, or 1351.

[0135] In some embodiments, the mutant polypeptide having at least one amino acid residue inserted before or after the LYP motif includes the backbone sequence of any one of RLF 89458.1 (SEQ ID NO: 1), RLF 78286.1 (SEQ ID NO: 2), NOZ 58130.1 (SEQ ID: 1714), RMF 90817.1 (SEQ ID NO: 2789), MBC 7218772.1 (SEQ ID NO: 2790), WP175059460.1 (SEQ ID NO: 2791), KUO 42443.1 (SEQ ID NO: 2792), and NOZ 77387.1 (SEQ ID NO: 2793).

[0136] In some embodiments, at least one amino acid residue can be inserted before or after a LYP motif, wherein the LYP motifs in RLF89458.1 (SEQ ID NO: 1) and RLF 78286 (SEQ ID NO: 2) are located at positions L409, Y410, and P411.

[0137] In some embodiments, at least one amino acid residue may be inserted before or after a LYP motif, wherein the LYP motifs in NOZ58130 (SEQ ID NO: 1714) are located at positions L440, Y441, and P442.

[0138] In some embodiments, at least one amino acid residue can be inserted before or after a LYP motif, wherein the LYP motifs in RMF90817 (SEQ ID NO: 2789) are located at positions L421, Y422, and P423.

[0139] In some embodiments, at least one amino acid residue may be inserted before or after a LYP motif, wherein the LYP motifs in MBC7218772 (SEQ ID NO: 2790) are located at positions L451, Y452, and P453.

[0140] In some embodiments, at least one amino acid residue may be inserted before or after a LYP motif, wherein the LYP motifs in WP175059460 (SEQ ID NO: 2791) are located at positions L411, Y412, and P413.

[0141] In some embodiments, at least one amino acid residue may be inserted before or after a LYP motif, wherein the LYP motifs in KUO42443 (SEQ ID NO: 2792) are located at positions L448, Y449, and P450.

[0142] In some embodiments, at least one amino acid residue may be inserted before or after a LYP motif, wherein the LYP motifs in NOZ77387 (SEQ ID NO: 2793) are located at positions L432, Y433, and P434.

[0143] In some embodiments, mutant polypeptides having at least one amino acid residue inserted before or after the LYP motif include sequences having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity to any one of SEQ ID NOs: 1-1713, 1714-2787, 2789, 2790, 2791, 2792, 2793, and 2803.

[0144] Sites that confer certain activities on a polypeptide may be conserved and can be located by aligning the amino acid sequences of various polymerases. For example, certain residues associated with polymerase activity (e.g., nucleotide incorporation) may be found at residues D405, D539, and / or D541 of a polymerase having a backbone sequence of RLF 89458.1 (SEQ ID NO: 1); or at residues D405, D539, and / or D541 of a polymerase having a backbone sequence of RLF 78286.1 (SEQ ID NO: 2); or at residues D436, D570, and / or D572 of a polymerase having a backbone sequence of NOZ 58130 (SEQ ID NO: 1714); or at residues D417, D551, and / or D553 of a polymerase having a backbone sequence of RMF 90817 (SEQ ID NO: 2789); or at residues D418, D552, and / or D554 of a polymerase having a backbone sequence of MBC 7218772 (SEQ ID NO: 3366). or at residues D444, D582 and / or D584 of a polymerase having a backbone sequence of KUO 42443 (SEQ ID NO: 2792); or at residues D428, D562 and / or D564 of a polymerase having a backbone sequence of NOZ 77387 (SEQ ID NO: 2793).

[0145] The skilled artisan can locate these sites and other functionally equivalent sites in other polymerases by reviewing the sequence alignment shown in Figure 33. Such sites are often found at similar positions in other regions and domains, and polypeptides comprising such domains are consistent with the methods and compositions described herein.

[0146] The mutation in the polymerase described herein comprises one or more changes of the amino acid residue present in the polypeptide. Addition, substitution, deletion and / or truncation are examples of mutations for producing mutant polypeptides. In certain embodiments, substitution comprises exchanging an amino acid for an alternative amino acid, and such alternative amino acid is different from the original amino acid in size, shape, conformation and / or chemical structure. In certain embodiments, mutation is conservative or non-conservative. Conservative mutation comprises replacing an amino acid with an amino acid with a similar chemical property. Addition generally comprises inserting one or more amino acids at the N-terminus, C-terminus or internal position of the polypeptide. In some cases, addition comprises fusion polypeptide, wherein one or more other polypeptides are connected to the polypeptide. In certain embodiments, such other polypeptides comprise a domain with other activity, or a sequence with other functions (e.g., improving expression, assisting purification, improving solubility, attaching to a solid support or other functions). The polypeptide described herein generally comprises one or more non-amino acid groups. Fusion polypeptide optionally comprises amino acids or other chemical linkers connecting one or more proteins. Any number of mutations can be introduced into a polypeptide or portion of a polypeptide described herein, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50 or more mutations.

[0147] In some embodiments, the entire domain (part of a polypeptide with a defined function) is added, deleted, or replaced with a domain from another polypeptide. Exemplary domains include DNA / RNA binding domains, nucleotide binding domains, nuclease domains, subcellular localization domains, such as nuclear localization domains or other domains. In some embodiments, the methods and compositions of the present disclosure include the attachment of a domain that acts as a spacer or tag, and / or provides the attachment of a linker, such as a SNAP tag, an avidin portion, a streptavidin portion, an epitope tag, a fluorescent protein, an affinity tag, a metal binding (i.e., His6 (SEQ ID NO: 2850) or a polyhistidine tag), etc. In some embodiments, one or more mutations are present at any position, such as in an exonuclease domain, a nucleic acid binding domain, a nucleotide binding domain, and / or a catalytic site. The polypeptide includes at least one mutation and can be based on the wild-type backbone sequence of any one of SEQ ID NOs: 1, 2, 1714, 2789, 2790, 2791, 2792, 2793, or 2803.

[0148] As used herein, the term "surrounding" an amino acid residue or sequence position has its ordinary meaning in the art, encompassing and incorporating modifications such as substitutions, deletions, insertions, or post-translational modifications at residues that are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 or more residues away from the named residue, i.e., the N-terminus or C-terminus of the named residue. In some cases, as will be understood by one of ordinary skill in the art, residues that are greater than 12 residues or sequence positions that are N- or C-terminal to a named residue may be considered to "surround" the named residue, based on sequence or structural (i.e., 3-dimensional) context.

[0149] It should be understood that the substitution or modification of the residues described herein may also incorporate or may include non-standard amino acids known in the art, including but not limited to hydroxyproline, N-formylmethionine, selenomethionine, selenocysteine, phosphotyrosine, phosphothistidine, and the like. The mutations, modifications, truncations, substitutions, and the like described herein may be performed by any method known in the art (particularly molecular biology and / or protein engineering). Such methods may include site-directed mutagenesis using mutagenic and / or partially degenerate primers, in vitro gene assembly, gene editing (such as by CRISPR or related methods), and the like. The mutants or engineered proteins described herein may additionally be expressed, isolated, and / or purified by any method known in the art. Related methods are described in: Green, M. and Sambrook, Molecular Cloning: A Laboratory Manual (4th Edition), which is hereby incorporated by reference in its entirety, and in particular discloses methods for modifying, transferring, and expressing recombinant, modified, and engineered gene sequences, as well as extracting, isolating, and / or purifying engineered proteins.

[0150] The polypeptides disclosed herein have been shown to function as nucleotide polymerases that exhibit greater thermostability and greater incorporation rates of 3'-O-azidomethyl-derivatized nucleosides, increased uracil tolerance, and / or improved binding to complementary nucleotide units of multivalent molecules compared to their corresponding wild-type enzymes. The polypeptides disclosed herein can be used for elongation of nucleic acids during replication or synthesis, or can be used to capture / bind nucleotides at nucleotide addition sites, for example, by using non-incorporable or blocked nucleotides, or can be used in the absence of required salts or cofactors. The polypeptides disclosed herein can be used, for example, in polynucleotide sequencing applications, such as sequencing by synthesis and sequencing by binding. The mutant polymerases disclosed herein comprise at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater than 99% sequence identity to any of SEQ ID NOs: 1, 2, 1714, 2789, 2790, 2791, 2792, 2793 or 2803.

[0151] The present disclosure provides engineered DNA polymerases comprising an amino acid sequence backbone of a family B or family A polymerase (which typically comprises replicative polymerases exhibiting high fidelity). Examples of family B-type polymerases include family B archaeal DNA polymerases and Phi29 polymerases. In some embodiments, the engineered DNA polymerase includes a family B archaeal DNA polymerase that can be selected from the following: Pyrococcus, Thermoplasmata, Pyrococcus, Methanococcus, Hadesarchaea, Euryarchaeota, or Candidatus. In some embodiments, the engineered DNA polymerase as a family B polymerase includes an amino acid sequence backbone from 9°N polymerase (comprising THERMINATOR polymerase), VENT polymerase, DEEP VENT polymerase, Pfu polymerase, or Pyrococcus polymerase. In some embodiments, an engineered DNA polymerase belonging to Family A polymerase comprises an amino acid sequence backbone of Geobacillus stearothermophilus (eg, Bst DNA polymerase).

[0152] Can be by one or more mutations are introduced into the amino acid sequence of the DNA polymerase of interest to design and prepare engineered DNA polymerase, and the resulting phenotype of the engineered polymerase can be determined. Any one mutation site or any combination of two or more mutation sites can be transferred from one type of polymerase to a positionally equivalent site (or a functionally equivalent site) in a second type of polymerase. For example, any one or any combination of two or more mutation sites from a DNA polymerase comprising SEQ ID NOs: 1, 2, 1714, 2789, 2790, 2791, 2792, 2793, and 2803, or any of SEQ ID NOs: 3-1713 or 1715-2787, can be introduced into Geobacillus stearothermophilus (e.g., Bst DNA polymerase) (SEQ ID NO: 2794), 9°N polymerase (SEQ ID NO: 2795 or 2796) (including THERMINATOR polymerase; SEQ ID NO: 2797), VENT polymerase (SEQ ID NO: 2798), DEEPVENT polymerase (SEQ ID NO: 2799), Pfu polymerase (SEQ ID NO: 2800), and / or Pyrococcus pyogenes polymerase (SEQ ID NO: 2801), RB69 polymerase (SEQ ID NO: 2802), and Phi29 polymerase (SEQ ID NO: 2803). NO: 2803).

[0153] Exemplary sequence alignments are provided in Figures 33 to 40. The mutation comprises any one or any combination of two or more amino acid substitutions, insertions, deletions and / or truncations.

[0154] The functional equivalent of residue comprises one or more amino acid residues, and described one or more amino acid residues occupy the similar position in the sequence (for example, sequence alignment) and / or three-dimensional structure of enzyme (for example, DNA polymerase) and perform the function substantially the same as the known amino acid residue in known enzyme.Amino acid replacement that is equivalent in function is included in one or more amino acid residues that have identical functional effect in another polypeptide at the specific position in basic polypeptide.Amino acid replacement that is equivalent in function comprises any one or any combination in conservative and / or non-conservative amino acid replacement. Exemplary sequence alignments are provided in Figures 33-40, which list sites in DNA polymerases having a backbone sequence of any one of SEQ ID NOs: 1, 2, 1714, 2789, 2790, 2791, 2792, 2793, and 2803, as well as Geobacillus stearothermophilus (e.g., Bst DNA polymerase) (SEQ ID NO: 2794), 9°N DNA polymerase (relative to SEQ ID NOs: 2795 or 2796), THERMINATOR polymerase (relative to SEQ ID NO: 2797), VENT polymerase (relative to SEQ ID NO: 2798), DEEP VENT polymerase (relative to SEQ ID NO: 2799), Pfu DNA polymerase (relative to SEQ ID NO: 2800), Pyrococcus prostratus DNA polymerase (relative to SEQ ID NO: 2801), RB69 polymerase (relative to SEQ ID NO: 2802), or Phi29 polymerase (relative to SEQ ID NO: 2803). NO: 2803) are examples of amino acid residues at functionally equivalent amino acid positions.

[0155] The wild-type polypeptide sequence is typically the starting point for protein or enzyme engineering to produce a mutant polypeptide. In certain embodiments, the mutant polypeptide differs from the wild-type polypeptide by at least one amino acid residue. The mutant polypeptide typically differs from the nearest wild-type polypeptide by at least one amino acid residue. In certain embodiments, the mutant polypeptide differs from the wild-type polypeptide by at least two amino acid residues. In certain embodiments, the mutant polypeptide differs from the wild-type polypeptide by at least three, four, five, six or more amino acid residues. Typically, the wild-type sequence is the closest wild-type sequence, identified by comparing a polypeptide comprising at least one mutation within the wild-type sequence. In certain embodiments, the wild-type polypeptide sequence comprises the sequence of a naturally occurring polypeptide.

[0156] Amino acid substitution refers to replacing the amino acid residue at the selected position in the polypeptide with a different amino acid having similar or different biochemical properties (such as similar size, shape, conformation, chemical structure, charge and / or hydrophobicity). Amino acid substitution can be conservative or non-conservative amino acid substitution. In some embodiments, the amino acid residue at the selected position in the polypeptide can be replaced with an amino acid with a polar side chain. Examples of amino acids with polar side chains include arginine, asparagine, aspartic acid, glutamine, glutamic acid, histidine, lysine, serine and threonine. In some embodiments, the amino acid residue at the selected position in the polypeptide can be replaced with an amino acid with a non-polar side chain. Examples of amino acids with non-polar side chains include alanine, cysteine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, tyrosine and valine. In some embodiments, the amino acid residue at the selected position in the polypeptide can be replaced with an amino acid with a hydrophobic side chain. Examples of amino acids with hydrophobic side chains include glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tyrosine, and tryptophan. In some embodiments, the amino acid residues at the selected positions in the polypeptide can be replaced with amino acids with uncharged side chains. Examples of amino acids with uncharged side chains include glycine, serine, cysteine, asparagine, glutamine, tyrosine, and threonine. In some embodiments, the amino acid residues at the selected positions in the polypeptide can be replaced with amino acids with positively charged side chains. Examples of amino acids with positively charged side chains include arginine, histidine, and lysine. In some embodiments, the amino acid residues at the selected positions in the polypeptide can be replaced with amino acids with negatively charged side chains. Examples of amino acids with negatively charged side chains include aspartic acid and glutamic acid.

[0157] Exemplary polypeptide mutants described herein are listed in Tables 1-2 ( Figure 31-3 2) are listed.

[0158] In some embodiments, the polypeptide comprises the backbone sequence of RLF 89458.1 and has a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity to any of SEQ ID NOs: 1 or 2, and the polypeptide comprises the sequence of Table 1 Figure 31 At least one mutation among the mutations listed in (e.g., SEQ ID NO: 3-1713).

[0159] In some embodiments, the polypeptide comprises the backbone sequence of NOZ 58130.1 and has a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity to any one of SEQ ID NOs: 1714, and the polypeptide comprises at least one of the mutations listed in Table 2, Figure 32 (e.g., SEQ ID NOs: 1715-2787).

[0160] In some embodiments, the polypeptide comprises the backbone sequence of RMF 90817 and has a sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater than 99% identical to SEQ ID NO: 2787, and the polypeptide comprises at least one mutation at a position equivalent to that listed in Tables 1 and / or 2 (respectively). Figure 31 and 32) (e.g., SEQ ID NOs: 3-1713) (e.g., SEQ ID NOs: 1715-2787). In some embodiments, positionally equivalent amino acid positions are shown in Figures 33 and 36.

[0161] In some embodiments, the polypeptide comprises the backbone sequence of MBC 7218772.1 and has a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity to SEQ ID NO: 2790, and the polypeptide comprises at least one of the mutations and the polypeptide comprises at least one mutation at a position equivalent to a mutation listed in Tables 1 and / or 2, respectively. Figure 31 and 32) (e.g., SEQ ID NOs: 3-1713) (e.g., SEQ ID NOs: 1715-2787). In some embodiments, positionally equivalent amino acid positions are shown in Figures 33 and 37.

[0162] In some embodiments, the polypeptide comprises the backbone sequence of WP 175059460.1 and has a sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater than 99% identical to SEQ ID NO: 2791, and the polypeptide comprises at least one mutation at a position equivalent to that listed in Tables 1 and / or 2 (respectively). Figure 31and 32) (e.g., SEQ ID NOs: 3-1713) (e.g., SEQ ID NOs: 1715-2787). In some embodiments, positionally equivalent amino acid positions are shown in Figures 33 and 38.

[0163] In some embodiments, the polypeptide comprises the backbone sequence of KUO 42443.1 and has a sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater than 99% identical to SEQ ID NO: 2792, and the polypeptide comprises at least one mutation at a position equivalent to that listed in Tables 1 and / or 2 (respectively). Figure 31 and 32) (e.g., SEQ ID NOs: 3-1713) (e.g., SEQ ID NOs: 1715-2787). In some embodiments, positionally equivalent amino acid positions are shown in Figures 33 and 39.

[0164] In some embodiments, the polypeptide comprises the backbone sequence of NOZ 77387.1 and has a sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater than 99% identical to SEQ ID NO: 2793, and the polypeptide comprises at least one mutation at a position equivalent to that listed in Tables 1 and / or 2, respectively. Figure 31 and 32) (e.g., SEQ ID NOs: 3-1713) (e.g., SEQ ID NOs: 1715-2787). In some embodiments, positionally equivalent amino acid positions are shown in Figures 33 and 39.

[0165] In some embodiments, the polypeptide comprises the backbone sequence of Phi29 and has a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity to SEQ ID NO: 2803, and the polypeptide comprises at least one mutation at a position equivalent to that listed in Tables 1 and / or 2 (respectively). Figure 31 and 32) (e.g., SEQ ID NOs: 3-1713) (e.g., SEQ ID NOs: 1715-2787) mutations.

[0166] Segments or portions of larger polypeptides are further described herein. Optionally, the segment has catalytic activity (such as nucleotide incorporation and nucleic acid extension activity), particularly in the context of a nucleotide polymerization or exonuclease domain as described herein. The polypeptides described herein include any full length or segment derived from any one of SEQ ID NOs: 1-1713, 1714-2787, 2789, 2790, 2791, 2792, 2793, and 2803, and at least one additional residue (e.g., +1 residue) at the N-terminus or C-terminus. In some embodiments, both the N- and C-termini have at least two, three, four, five, six, seven, eight, nine, ten, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100 or more additional residues.

[0167] For example, the polypeptides described herein include any of SEQ ID NOs: 1-1713, 1714-2787, 2789, 2790, 2791, 2792, 2793, and 2803 (+1 residue), such as an adjacent N-terminal aspartic acid, an adjacent C-terminal arginine, or a combination thereof, or additional residues, such as residues identified by alignment of any of SEQ ID NOs: 1-1713, 1714-2787, 2789, 2790, 2791, 2792, 2793, and 2803. The polypeptides described herein include any of SEQ ID NOs: 1-1713, 1714-2787, 2789, 2790, 2791, 2792, 2793, and 2803 (+1 residue), such as an adjacent N-terminal glutamine, an adjacent C-terminal histidine, or a combination thereof, or additional residues, such as residues identified by alignment of any of SEQ ID NOs: 1-1713, 1714-2787, 2789, 2790, 2791, 2792, 2793, and 2803. The polypeptides described herein include any of SEQ ID NOs: 1-1713, 1714-2787, 2789, 2790, 2791, 2792, 2793 and 2803 (+1 residue), such as an adjacent N-terminal valine, an adjacent C-terminal cysteine, or a combination thereof, or additional residues, such as residues identified by alignment of any of SEQ ID NOs: 1-1713, 1714-2787, 2789, 2790, 2791, 2792, 2793 and 2803. The polypeptides described herein include any of SEQ ID NOs: 1-1713, 1714-2787, 2789, 2790, 2791, 2792, 2793 and 2803 (+1 residue), such as an adjacent N-terminal threonine, an adjacent C-terminal cysteine, or a combination thereof, or additional residues, such as residues identified by alignment of any of SEQ ID NOs: 1-1713, 1714-2787, 2789, 2790, 2791, 2792, 2793 and 2803. The polypeptides described herein include any of SEQ ID NOs: 1-1713, 1714-2787, 2789, 2790, 2791, 2792, 2793 and 2803 (+1 residue), such as an adjacent N-terminal threonine, an adjacent C-terminal cysteine, or a combination thereof, or additional residues, such as residues identified by alignment of any of SEQ ID NOs: 1-1713, 1714-2787, 2789, 2790, 2791, 2792, 2793 and 2803.The polypeptides described herein include any of SEQ ID NOs: 1-1713, 1714-2787, 2789, 2790, 2791, 2792, 2793, and 2803 (+1 residue), such as an adjacent N-terminal aspartic acid, an adjacent C-terminal leucine, or a combination thereof, or additional residues, such as residues identified by alignment of any of SEQ ID NOs: 1-1713, 1714-2787, 2789, 2790, 2791, 2792, 2793, and 2803. The polypeptides described herein include any of SEQ ID NOs: 1-1713, 1714-2787, 2789, 2790, 2791, 2792, 2793 and 2803 (+1 residue), such as an adjacent N-terminal aspartic acid, an adjacent C-terminal arginine, or a combination thereof, or additional residues, such as residues identified by alignment of any of SEQ ID NOs: 1-1713, 1714-2787, 2789, 2790, 2791, 2792, 2793 and 2803. The polypeptides described herein include any of SEQ ID NOs: 1-1713, 1714-2787, 2789, 2790, 2791, 2792, 2793, and 2803 (+1 residue), such as an adjacent N-terminal threonine, an adjacent C-terminal threonine, or a combination thereof, or additional residues, such as residues identified by alignment of any of SEQ ID NOs: 1-1713, 1714-2787, 2789, 2790, 2791, 2792, 2793, and 2803. The polypeptides described herein include any of SEQ ID NOs: 1-1713, 1714-2787, 2789, 2790, 2791, 2792, 2793 and 2803 (+1 residue), such as an adjacent N-terminal threonine, an adjacent C-terminal asparagine, or a combination thereof, or additional residues, such as residues identified by alignment of any of SEQ ID NOs: 1-1713, 1714-2787, 2789, 2790, 2791, 2792, 2793 and 2803. The polypeptides described herein include any of SEQ ID NOs: 1-1713, 1714-2787, 2789, 2790, 2791, 2792, 2793 and 2803 (+1 residue), such as an adjacent N-terminal threonine, an adjacent C-terminal asparagine, or a combination thereof, or additional residues, such as residues identified by alignment of any of SEQ ID NOs: 1-1713, 1714-2787, 2789, 2790, 2791, 2792, 2793 and 2803.The polypeptides described herein include any of SEQ ID NOs: 1-1713, 1714-2787, 2789, 2790, 2791, 2792, 2793 and 2803 (+1 residue), such as an adjacent N-terminal threonine, an adjacent C-terminal serine, or a combination thereof, or additional residues, such as residues identified by alignment of any of SEQ ID NOs: 1-1713, 1714-2787, 2789, 2790, 2791, 2792, 2793 and 2803.

[0168] Engineered polymerases comprising RLF 89458.1 or RLF 78286.1 backbone sequences

[0169] The present disclosure provides one or more mutant polymerases, wherein the one or more mutant polymerases include the backbone sequence of RLF89458.1 or RLF 78286.1 and are identical to SEQ ID NOs: 1-1713 (Tables 1 and Figure 11-12 ), having 100%, at least 99%, at least 98%, at least 97%, at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, or at least 50% sequence identity to any of RLF 89458.1 and RLF78286.1. The amino acid sequences of RLF 89458.1 and RLF78286.1 differ by an amino acid substitution at position 235, wherein RLF 78286.1 comprises D235E.

[0170] In some embodiments, a mutant polymerase having a backbone sequence of RLF 89458.1 (eg, SEQ ID NO: 1) or RLF 78286.1 (SEQ ID NO: 2) includes various domains.

[0171] In some embodiments, the N-terminal domain includes amino acid residues 1-134 (SEQ ID NO: 2804) (e.g., Figure 29 ).

[0172] In some embodiments, the exonuclease domain (e.g., the 3' to 5' exonuclease domain) includes amino acid residues 135-356 (SEQ ID NO: 2805) (e.g., Figure 29 ).

[0173] In some embodiments, the first palm domain includes amino acid residues 357-454 (SEQ ID NO: 2806) (e.g., Figure 29 ).

[0174] In some embodiments, the finger domain includes amino acid residues 455-504 (SEQ ID NO: 2807) (e.g., Figure 29 ).

[0175] In some embodiments, the second palm domain includes amino acid residues 505-615 (SEQ ID NO: 2808) (e.g., Figure 29 ).

[0176] In some embodiments, the thumb domain includes amino acid residues 616-765 (SEQ ID NO: 2809) (e.g., Figure 29 ).

[0177] The present disclosure provides compositions and methods including mutant polymerases having a backbone sequence of RLF89458.1 (e.g., SEQ ID NO: 1) or RLF 78286.1 (SEQ ID NO: 2) and lacking an exonuclease. - In some embodiments, the mutant polymerase comprises at least one amino acid substitution mutation that reduces 3' to 5' exonuclease activity compared to a polymerase that has an exo-minus mutation. For example, the mutant polymerase comprises at least one amino acid substitution at position D141 and / or E143. In some embodiments, the mutant polymerase comprises mutations D141A, D141V, D141L, D141I, D141F, D141Y, D141N, D141T, D141S, D141R, D141K, D141Q, D141W, D141E, D141M, D141P, D141G, D141H, or D141C. In some embodiments, the mutant polymerase comprises mutations E143A, E143V, E143L, E143I, E143F, E143Y, E143N, E143T, E143S, E143W, E143M, E143P, E143F, E143G, E143H, E143R, E143K, E143D, E143C or E143Q. In some embodiments, position E143 is not mutated. In some embodiments, the mutant polymerase comprises any combination of mutations at sites D141 and E143.

[0178] In some embodiments, the mutant polymerase comprises non-mutant E143E and mutations D141A, D141V, D141L, D141I, D141F, D141Y, D141N, D141T, D141S, D141R, D141K, D141Q, D141W, D141E, D141M, D141P, D141G, D141H, or D141C.

[0179] The present disclosure provides compositions and methods including mutant polymerases that can be used to sequence nucleic acid template molecules containing uracil. The mutant polymerase can exhibit uracil tolerance with an increased ability to incorporate dATP into the 3' end of a nucleic acid primer at a position relative to the uracil base in the nucleic acid template molecule. The mutant polymerase can also bind to adenine-bearing nucleotide units of a multivalent molecule at a position relative to the uracil base in the nucleic acid template molecule. Compared to wild-type polymerases or compared to engineered polymerases with mutations that do not confer uracil tolerance, the mutant polymerase can exhibit increased uracil tolerance. Mutant polymerases having a backbone sequence of uracil-resistant RLF 89458 or RLF 78286 (e.g., SEQ ID NO: 1 or 2, respectively) can include mutations at positions Y7, T9, H89, Q91, V93, and / or R97. In some embodiments, the mutant polymerase comprises the backbone sequence of RLF 89458 or RLF 78286 (e.g., SEQ ID NO: 1 or 2, respectively), and has an amino acid substitution at position Y7, wherein the mutation comprises any of Y7A, Y7F, Y7N, Y7D, Y7R, Y7W, Y7H, or Y7Q. In some embodiments, the mutant polymerase comprises the backbone sequence of RLF 89458 or RLF 78286 (e.g., SEQ ID NO: 1 or 2, respectively), and has an amino acid substitution at position T9, wherein the mutation comprises any of T9N, T9E, T9S, T9L, T9I, T9D, T9A, or T9R. In some embodiments, the mutant polymerase comprises the backbone sequence of RLF 89458 or RLF 78286 (e.g., SEQ ID NO: 1 or 2, respectively) and has an amino acid substitution at position H89, wherein the mutation comprises any of H89D, H89A, H89Y, H89R, H89N, H89Q, H89K, H89F, H89L, or H89V. In some embodiments, the mutant polymerase comprises the backbone sequence of RLF 89458 or RLF 78286 (e.g., SEQ ID NO: 1 or 2, respectively) and has an amino acid substitution at position Q91, wherein the mutation comprises any of Q91L, Q91H, Q91R, Q91W, Q91A, Q91K, Q91N, Q91P, Q91V, or Q91Y. In some embodiments, the mutant polymerase comprises the backbone sequence of RLF 89458 or RLF 78286 (e.g., SEQ ID NO: 1 or 2, respectively), and has an amino acid substitution at position V93, wherein the mutation comprises any of V93A, V93M, V93E, V93F, V93Y, V93G, V93S, V93K, V93T, or V93I.In some embodiments, the mutant polymerase comprises the backbone sequence of RLF 89458 or RLF 78286 (e.g., SEQ ID NO: 1 or 2, respectively), and has an amino acid substitution at position R97, wherein the mutation comprises any of R97C, R97H, R97S, R97P, R97L, R97A, R97N, R97Q, R97E, R97I, R97K, R97M, or R97T.

[0180] Other uracil-tolerant mutant polymerases having backbone sequences of NOZ 58130 (SEQ ID NO: 1714), RMF 90817 (SEQ ID NO: 2789), MBC 7218772 (SEQ ID NO: 2790), WP 175059460 (SEQ ID NO: 2791), KUO 42443 (SEQ ID NO: 2792), or NOZ 77387 (SEQ ID NO: 2793) may contain mutations at positions equivalent to Y7, T9, H89, Q91, V93, and / or R97 in RLF 89458 (SEQ ID NO: 1). Figure 33 shows a sequence alignment of these different polymerases and their positionally equivalent amino acid residues.

[0181] The present disclosure provides compositions and methods comprising mutant polymerases having a backbone sequence of RLF89458.1 (e.g., SEQ ID NO: 1) or RLF 78286.1 (SEQ ID NO: 2) and comprising at least one amino acid substitution mutation in a LYP motif (e.g., at positions L409, Y410, and P411). In some embodiments, at least one mutation in the LYP motif can increase the incorporation rate of a nucleotide analog. In some embodiments, any one or any combination of the first, second, and / or third positions of the LYP motif can be mutated. For example, mutations in the LYP motif include AAG, AAP, AAV, AAI, AGA, AGG, AGI, AGP, AGV, FAA, FAG, FAI, FAP, FAV, FGA, FGG, FGP, F GV, LAG, LAI, LAP, LGG, LGI, LGV, SAA, SAG, SAI, SAV, SGA, SGG, SGI, YAA, YAG, YAI, YAP, YGA, YGG, YGI , YGP, LAA, LAV, LGP, LGA, FGI, SGV, YAV, YGV, SYP, SAP, AAA, SGP, LFP, IFP, VFP, LMP, VMP, IMP, LLP, V LP, ILP, LDP, VDP, IDP, LTP, VTP, ITP, LIP, TIP, NNP, NDP, NAP, SYG, SSG, SSS, CAG, ASG, SSG, MFG and FTA.

[0182] In some embodiments, the polymerase comprises the backbone sequence of RLF 89458 or RLF 78286 (e.g., SEQ ID NO: 1 or 2, respectively) and has a substitution mutation at position L409 comprising a nonpolar amino acid or a polar uncharged amino acid. In some embodiments, the amino acid substitution mutation at position L409 comprises valine, glycine, threonine, alanine, serine, isoleucine, leucine, phenylalanine, tyrosine, or methionine. SEQ ID NOs: 1-1713 include exemplary amino acid substitution mutations at position L409.

[0183] In some embodiments, the polymerase comprises the backbone sequence of RLF 89458 or RLF 78286 (e.g., SEQ ID NO: 1 or 2, respectively) and has a substitution mutation at position Y410 comprising a nonpolar amino acid or a polar uncharged amino acid. In some embodiments, the amino acid substitution mutation at position Y410 comprises threonine, serine, glycine, alanine, valine, isoleucine, or tyrosine. SEQ ID NOs: 1-1713 include exemplary amino acid substitution mutations at position Y410.

[0184] In some embodiments, the polymerase comprises the backbone sequence of RLF 89458 or RLF 78286 (e.g., SEQ ID NO: 1 or 2, respectively) and has a substitution mutation at position P411 comprising a polar uncharged amino acid, a nonpolar amino acid, or a positively charged amino acid. In some embodiments, the amino acid substitution mutation at position P411 comprises serine, glycine, alanine, valine, cysteine, lysine, isoleucine, threonine, or proline. SEQ ID NOs: 1-1713 include exemplary amino acid substitution mutations at position P411.

[0185] The present disclosure provides compositions and methods comprising mutant polymerases having a backbone sequence of RLF89458 or RLF78286 (e.g., SEQ ID NO: 1 or 2, respectively) and having at least one mutation that reduces post-translational modification. In some embodiments, the polymerase comprises at least one mutation or any combination of mutations of methionine, lysine, histidine, tryptophan, and / or cysteine ​​residues. In some embodiments, the polymerase comprises an amino acid substitution at position M1, M129, M159, M313, M329, M467, and / or M759. In some embodiments, the mutation at position M1 comprises M1F, M1I, M1L, M1S, M1N, M1A, M1V, M1Y, M1Q, M1K, M1V, or M1A. In some embodiments, the mutation at position M129 includes M129I, M129V, M129K, M129L, M129E, M129F, M129N, M129S, M129R or M129Y. In some embodiments, the mutation at position M159 includes M159W, M159F or M159Y. In some embodiments, the mutation at position M313 includes M313I, M313K, M313L, M313V, M313D, M313R, M313E, M313A, M313L or M313N. In some embodiments, the mutation at position M329 includes M329L, M329S, M329W, M329A, M329R, M329I, M329Q, M329N or M329E. In some embodiments, the mutation at position M467 comprises M467V, M467K, M467D, M467T, M467R, M467E, M467Q or M467L. In some embodiments, the mutation at position M759 comprises M759T, M759S, M759N, M759R, M759E, M759D or M759A.

[0186] In some embodiments, the polymerase comprises the backbone sequence of RLF 89458 or RLF 78286 (e.g., SEQ ID NO: 1 or 2, respectively), and has amino acid substitutions to reduce post-translational modifications, including mutations at positions K240, K306, K371, K429, K468, K476, and / or K592. In some embodiments, the mutation at position K240 comprises K240S, K240E, K240R, K240D, K240N, K240Q, or K240A. In some embodiments, the mutation at position K306 comprises K306R, K306N, K306Q, K306A, K306V, K306I, or K306F. In some embodiments, the mutation at position K371 includes K371R, K371D, K371N, K371Q, K371Y, K371T, K371V or K371L. In some embodiments, the mutation at position K429 includes K429R, K429S, K429M, K429A, K429N, K429D, K429Q, K429H, K429Y, K429V, K429L or K429E. In some embodiments, the mutation at position K468 includes K468R, K468E, K468Y, K468T or K468L. In some embodiments, the mutation at position K476 includes K476R, K476D, K476A, K476F or K476R. In some embodiments, the mutation at position K592 comprises K592Q, K592R, K592W, K592Y, K592A, K592F, K592I, K592T, K592N, or K592S.

[0187] In some embodiments, the polymerase comprises the backbone sequence of RLF 89458 or RLF 78286 (e.g., SEQ ID NO: 1 or 2, respectively), and has amino acid substitutions to reduce post-translational modifications, including mutations at positions W299 and / or W767. In some embodiments, the mutation at position W299 comprises W299F, W299E, W299N, W299Q, W299Y, W299A, or W299F. In some embodiments, the mutation at position W767 comprises W767H, W767Y, W767F, W767S, W767R, W767D, W767A, or W767N.

[0188] In some embodiments, the polymerase comprises the backbone sequence of RLF 89458 or RLF 78286 (e.g., SEQ ID NO: 1 or 2, respectively), and has amino acid substitutions to reduce post-translational modifications, including a mutation at position H601. In some embodiments, the mutation at position H601 comprises H601R, H601I, H601A, H601T, H601V, H601L, H601N, or H601K.

[0189] In some embodiments, the polymerase comprises the backbone sequence of RLF 89458 or RLF 78286 (e.g., SEQ ID NO: 1 or 2, respectively), and has amino acid substitutions to reduce post-translational modifications, including a mutation at position C428. In some embodiments, the mutation at position C428 comprises C428Y.

[0190] The present disclosure provides compositions and methods comprising mutant polymerases having a backbone sequence of RLF89458 or RLF 78286 (e.g., SEQ ID NO: 1 or 2, respectively) and having at least one mutation that removes an unpaired cysteine, which can reduce oxidative damage to the polymerase. In some embodiments, removing at least one unpaired cysteine ​​increases the thermal stability of the mutant polymerase and / or reduces protein aggregation. In some embodiments, removing at least one unpaired cysteine ​​increases the amount of protein in lysate preparation. In some embodiments, the polymerase comprises at least one mutation or any combination of mutations of the cysteine ​​at positions 223 and / or 509. In some embodiments, the mutation at position C223 comprises the amino acid substitution C223L, C223M, C223A, C223S, C223P, C223K, C223N, C223D, or C223V. In some embodiments, the mutation at position C509 comprises the amino acid substitution C509V, C509Y, C509S, C509M, C509A, C509N, C509D, C509H, or C509Q.

[0191] In some embodiments, the polymerase comprises the backbone sequence of RLF 89458 or RLF 78286 (e.g., SEQ ID NO: 1 or 2, respectively) and has amino acid substitutions that remove unpaired cysteines, including amino acid substitutions at positions C509 and V419. In some embodiments, the mutation at C509 comprises any of C509V, C509Y, C509S, C509M, C509A, C509N, C509D, C509H, or C509Q. In some embodiments, the mutation at V419 comprises any of V419I, V419L, or V419R.

[0192] In some embodiments, the polymerase comprises the backbone sequence of RLF 89458 or RLF 78286 (e.g., SEQ ID NO: 1 or 2, respectively) and has amino acid substitutions that remove unpaired cysteines, including amino acid substitutions at positions C509 and Q497. In some embodiments, the mutation at C509 comprises any of C509V, C509Y, C509S, C509M, C509A, C509N, C509D, C509H, or C509Q. In some embodiments, the mutation at Q497 comprises Q497H, Q497G, Q497M, Q497N, Q497F, Q497L, Q497R, Q497K, Q497T, Q497E, Q497D, or Q497Y.

[0193] In some embodiments, the polymerase comprises the backbone sequence of RLF 89458 or RLF 78286 (e.g., SEQ ID NO: 1 or 2, respectively) and has amino acid substitutions that remove unpaired cysteines, including amino acid substitutions at positions C509 and S512. In some embodiments, the mutation at C509 comprises any of C509V, C509Y, C509S, C509M, C509A, C509N, C509D, C509H, or C509Q. In some embodiments, the mutation at S512 comprises S512R, S512D, S512E, S512H, S512F, S512K, S512W, or S512D.

[0194] The present disclosure provides compositions and methods comprising mutant polymerases having a backbone sequence of RLF89458 or RLF78286 (e.g., SEQ ID NO: 1 or 2, respectively) and having at least one mutation that increases thermostability compared to a wild-type polymerase or an engineered polymerase having a mutation that does not confer increased thermostability. In some embodiments, the polymerase exhibits thermostability at elevated temperatures of about 72-75°C, or about 75-80°C, or about 80-85°C, or about 85-90°C, or higher. In some embodiments, a thermal shift assay using differential scanning fluorimetry can be used to determine the thermostability of the polymerase.

[0195] In some embodiments, the polymerase comprises the backbone sequence of RLF 89458 or RLF 78286 (e.g., SEQ ID NO: 1 or 2, respectively) and has amino acid substitutions that increase thermal stability, including amino acid substitutions at one or more positions comprising D141 and / or E143 that also confer exonuclease activity. In some embodiments, the polymerase engineered to exhibit increased thermal stability comprises an amino acid substitution at position M329 alone or in combination with D141 and / or E143. In some embodiments, the polymerase engineered to exhibit increased thermal stability comprises an amino acid substitution at position D315 alone or in combination with D141 and / or E143. In some embodiments, the polymerase (e.g., polymerase enzyme) engineered to exhibit increased protein production in lysate preparation comprises the amino acid substitution D141N alone or in combination with non-mutated E143 or any amino acid substitution of E143. The increased protein production in lysate preparation can increase the yield of active polymerase during manufacturing.

[0196] In some embodiments, the mutation at D141 comprises D141A, D141V, D141L, D141I, D141F, D141Y, D141N, D141T, D141S, D141R, D141K, D141Q, D141W, D141E, D141M, D141P, D141G, D141H, or D141C.

[0197] In some embodiments, the mutation at E143 comprises E143A, E143V, E143L, E143I, E143F, E143Y, E143N, E143T, E143S, E143W, E143M, E143P, E143G, E143H, E143R, E143K, E143D, E143C, or E143Q.

[0198] In some embodiments, the mutation at M329 comprises M329L, M329S, M329W, M329A, M329R, M329I, M329Q, M329N, or M329E.

[0199] In some embodiments, mutations at D315 include D315A, D315E, D315R, D315W, D315L, D315W, D315F.

[0200] The present disclosure provides compositions and methods comprising mutant polymerases having a backbone sequence of RLF89458 or RLF 78286 (e.g., SEQ ID NO: 1 or 2, respectively) and a C-terminal truncation that increases the thermostability of the polymerase compared to a non-truncated polymerase having the same wild-type or engineered backbone sequence. The C-terminal region of the polymerase may be disordered and may cause protein aggregation. Thus, truncation at the C-terminal region can reduce protein aggregation and improve stability. In some embodiments, the polymerase exhibits thermostability at elevated temperatures of about 72-75°C, or about 75-80°C, or about 80-85°C, or about 85-90°C or higher. In some embodiments, a thermal shift assay performed using differential scanning fluorimetry can be used to determine the thermostability of the polymerase.

[0201] In some embodiments, the polymerase comprises the backbone sequence of RLF 89458 or RLF 78286 (e.g., SEQ ID NO: 1 or 2, respectively), and is engineered to exhibit increased thermostability, wherein the polymerase comprises a truncation at an amino acid position comprising K464 (truncated), R465 (truncated), E475 (truncated), Y481 (truncated), E616 (truncated), E620 (truncated), E755 (truncated), Y756 (truncated), Q757 (truncated), R758 (truncated), M759 (truncated), T762 (truncated), W767 (truncated), or M770 (truncated). In Tables 1 and 2, truncations are designated with a "^."

[0202] The present disclosure provides compositions and methods comprising mutant polymerases having a backbone sequence of RLF89458 or RLF78286 (e.g., SEQ ID NO: 1 or 2, respectively) and an N-terminal truncation that increases the thermostability of the polymerase compared to a non-truncated polymerase having the same wild-type or engineered backbone sequence. In some embodiments, the engineered polymerase comprises a deleted methionine at position 1. Table 1 lists various engineered polymerases designated "M1X" having a deleted methionine at position 1 and their Tm1 and Tm2 data.

[0203] The present disclosure provides compositions and methods comprising mutant polymerases having a backbone sequence of RLF89458 or RLF78286 (e.g., SEQ ID NO: 1 or 2, respectively) and having at least one mutation that reduces polymerization to exonuclease conformational switching compared to the wild-type polymerase or compared to an engineered polymerase having a mutation that does not reduce conformational switching. In some embodiments, protein modeling can identify key amino acid residues that interact with a primer or primer end, wherein these amino acid residues may play a role in interacting with the primer or transferring the primer end from the polymerization site to the exonuclease site. In some embodiments, the polymerase comprises an amino acid substitution at any one or any combination of positions V610, D613, Q664, E668, P677, and / or D671.

[0204] In some embodiments, the mutation at V610 comprises V610D, V610A, V610K, V610S, V610T, V610N, V610R, or V610Q.

[0205] In some embodiments, the mutation at D613 comprises D613S, D613E, D613R, D613K, D613N, D613Q, D613A, D613V, D613Y, or D613F.

[0206] In some embodiments, the mutation at Q664 comprises Q664A, Q664L, Q664V, Q664F, Q664I, Q664R, Q664K, Q664T, Q664N, or Q664M.

[0207] In some embodiments, the mutation at E668 comprises E668G, E668K, E668M, E668A, E668P, E668S, E668R, E688N, E688D, E668Y, or E668Q.

[0208] In some embodiments, the mutation at P677 comprises P677L, P677R, P677K, or P677A.

[0209] In some embodiments, the mutation at D671 comprises D671G, D671R, D671Y, D671S, D671A, D671K, or D671N.

[0210] The present disclosure provides compositions and methods comprising mutant polymerases having a backbone sequence of RLF89458 or RLF78286 (e.g., SEQ ID NO: 1 or 2, respectively) and having at least one mutation that reduces protein aggregation by reducing intermolecular interactions compared to the wild-type polymerase or compared to an engineered polymerase having a mutation that does not confer reduced aggregation. In some embodiments, the mutant polymerase aggregates at elevated temperatures of about 72-75°C, or about 75-80°C, or about 80-85°C, or about 85-90°C, or higher, compared to the wild-type polymerase or compared to an engineered polymerase having a mutation that does not confer reduced aggregation. In some embodiments, a thermal shift assay using differential scanning fluorimetry can be used to determine the aggregation temperature of a protein.

[0211] In some embodiments, the polymerase comprises the backbone sequence of RLF 89458 or RLF 78286 (e.g., SEQ ID NO: 1 or 2, respectively) and has aggregation-reducing amino acid substitutions, including amino acid substitutions at one or more positions N11, K507, K511, and / or K637.

[0212] In some embodiments, the mutation at position N11 comprises N11 S, N11A, N11R, N11Q, N11E, N11K, N11T, or N11D.

[0213] In some embodiments, the mutation at position K507 comprises K507L, K507E, K507S, K507A, K507N, K507Q, K507E, K507T, or K507D.

[0214] In some embodiments, the mutation at position K511 comprises E511K, E511S, E511A, E511R, E511N, E511T, E511Q, E511L, E511D, or E511A.

[0215] In some embodiments, the mutation at position K637 comprises K637M, K637A, K637N, K637Q, K637E, K637S, or K637T.

[0216] The present disclosure provides compositions and methods including mutant polymerases having RLF89458.1 (eg, SEQ ID NO: 1) or RLF 78286.1 (SEQ ID NO: 2). 39, A40, L41, L42, E43, D44, D45, E46, S47, I48, E49, D50, N51, L52, E53, D54, D55, E56, S57, I58, E59, D60, D61, E62, K63, G64, E65, F66, K67, I68, E69, K70, K71, E72, K72, G73, E74, E75, F76, K77, I78, E79, K80, K81, K82, K83, G84, E85, F86, K87, I89, E90, I51, K52, K53, I54, T55, R58, G56, E57, R58, H59, G60, K61, K62, V63, I65, I66, R67, V68, E69, K70, V71, K72, K73, K74, F75, L76, G77, E78, P79 , I80, E81, V82, W83, K84, L85, V86, F87, E88, H89, P90, Q91, D92, V93, P94, A95, I96, R97, D98, A99, I100, R101, S102, H103, P104, A105, V106 , R107, E108, I109, F110, E111, Y112, D113, I114, P115, F116, A117, K118, R119, Y120, L121, I122, D123, K124, L126, V127, P128, M129, E130 , G131, G132, E133, L135, K136, L137, L138, A139, F140, D141, I142, E143, T144, F145, Y146, H147, E148, D150, E151, E156, M159, S166, W173 , K174, I176, Y180, A190, I191, K192, L195, L198, R199, Q196, P203, V205, L207, Y209, G211, N213, F214, D215, F216, A217, Y218, I219, K220 , C223, E224, K225, G227, L228, K229, F230, T231, I232, G233, R234, S2 37. E238, P239, K240, I241, Q242, R243, M244, G245, D246, R247, A249,E251、L258、Y261、P262、V264、R265、H266、T267、I268、R269、L270、P271、T272、Y273、T274、L275、E276、A277、V278、V282、F283、K285、K286、K287、E288、 K289、V290、Y291、A292、I295、E297、A298、W299、K300、S301、E302、L305、K306、R307、V308、A309、Q310、Y311、M313、D315、R317、A318、Y320、E321、P328、 V331、M329、E332、L333、A334、I337、G338、Q339、V341、D343、S345、S347、S348、T349、G350、N351、L352、V353、W355、Y356、L357、R359、V360、Y362、N365、 E366、L367、K371、P372、G373、G374、E375、E376、Y377、Q378、M381、S383、S384、Y385、I386、G388、Y389、E394、K395、G396、E399、S400、A402、Y403、L404、 F406、R407、S408、L409、Y410、P411、S412、I413、V415、H417、V419、P421、D4 22、T423、L424、E425、E427、C428、K429、N430、Y431、V433、A434、I436、Y439、 R440、K443、K446、G447、F448、I449、P450、S451、I452、L453、E454、D455、I4 57、T459、K462、V463、K464、R465、M467、K468、T470、I471、D472、I474、E475、 K476、M478、Y481、R484、A485、L486、K487、I488、N491、S492、Y493、Y494、G4 95、Q497、G498、Y499、P500、K501、S506、K507、E508、C509、E511、S512、V513、 T514、G517、R518、H519、I521、T523、A528、E529、K534、V535、Y537、A538、D539、T540、D541、G542、F543、F544、I547、N549、E550、K551、P552、I555、S557、K558, A559, K560, K561, L563, K564, H565, E568, K569, G572, M573, E575, E577, L583, G585, F586, V588, T589, K592, Y593, L595, I596 , D599, H601, T604, R605, G606, L607, V609, V610, R611, R612, D613, E616, I617, K619, E620, T621, Q622, A623, K624, V625, L626, E62 7. V628, I629, L630, R631, E632, G633, S634, I635, E636, K637, A638, A639, G640, I641, V642, K644, V645, V646, E647, D648, L649, A6 50. N651, Y652, R653, V654, V656, E657, K658, I660, H662, E663, Q664, I665, T666, R667, E668, K670, D671, Y672, K673, A674, T675, G 676, P677, H678, V679, A680, I681, A682, K683, R684, L685, Q686, A687, R688, G689, I690, K691, V692, K693, P694, T696, I698, S699, Y700, V701, V702, L703, K704, G705, S706, K707, K708, I709, D711, R712, V713, I714, L715, F716, D717, E718, Y719, D720, S721, S722 768, M770, K771, Q772, L773, L774, E775, A776, F777, G778, Y779, K780, E781, K782, D783, L784, E785, Y786, Q787, R788, M789, K790, Q791, T792, G793, L794, G795, A796, W797, L798, and / or M799.

[0217] The present disclosure provides compositions and methods including mutant polymerases having RLF89458.1 (eg, SEQ ID NO: 1) or RLF 78286.1 (SEQ ID NO: 2). NO: 2) backbone sequence, and including amino acid substitution mutations at any one or any combination of positions comprising the following: M1F, M1I, M1L, M1S, M1N, M1A, M1V, M1Y, M1Q, M1K, M1V, M1A, L3I, D4R, D4A, D6S, D6R, Y7A, Y7F, Y7N, Y7D, Y7R, Y7W, Y7H, Y7Q, I8S, T9N, T9E, T9S, T9L, T9I, T9D, T9A, T9R, E10V, E10D, E10K, E10R, E10A, E10N, N11S, N11A, N11R, N11Q , N11E, N11K, N11T, N11D, G12S, G12D, G12E, K13E, P14Q, P14N, P14S, V15I, I16T, I16N, I16F, R17H, R17C, I18V, I18L, F19Y, F19S, F19I, K20M , K20E, K21E, E22G, E22V, E22K, K23E, K23M, K23R, K23A, K23N, G24S, E25K, F26L, K27M, I28F, I28N, I28T, I28V, I28F, E29V, E29D, Y30F, Y30N , Y30D, Y30K, D31V, R32C, R32S, N33S, F34S, F34I, E35K, E35G, E35D, P36L, P36A, P36G, P36V, P36M, P36T, P36K, Y37N, Y37F, I38T, I38N, Y39F , A40G, A40V, A40T, L41P, L41F, L41Y, L41D, L41E, L42P, L42Q, E43V, E43K, E43D, D44N, D44G, D45V, E46V, E46S, S47N, S47G, S47R, S47A, I48V , E49G, E49K, D50V, D50G, D50N, D50E, I51K, I51F, I51V, K52I, K52R, K53E, I54T, I54N, I54F, I54K, I54V, T55I, T55S, T55A, G56D, G56S, G56V , G56A, E57G, E57K, R58C, R58L, R58H, H59L, H59Y, G60S, G60D, K61M, K6 1T, K62N, K62E, K62R, K62T, K62V, V63A, V63I, V63D, I65T, I65V, I65F,I65N、I66V、I66T、I66N、I66K、R67C、V68M、V68A、E69K、K70I、V71I、K72H、K72R、K72V、K72Q、K73E、K74E、K74R、K74N、K74Q、F75C、L76Q、G77D、G77S、E78K、E78G、E78N、E78S、E78R、P79S、I80F、I80N、I80K、I80S、I80R、E81D、E81V、V82A、W83R、K84R、L85V、L85Q、L85A、V86D、V86I、V86A、V86Y、F87I、F87L、F87C、E88K、E88D、E88N、E88T、H89D、H89A、H89Y、H89R、H89N、H89Q、H89K、H89F、H89L、H89V、P90L、P90S、P90D、P90R、P90A、P90G、P90V、P90M、P90T、P90K、Q91L、Q91H、Q91R、Q91W、Q91A、Q91K、Q91N、Q91P、Q91V、Q91Y、D92N、D92V、D92E、D92R、V93A、V93M、V93E、V93F、V93Y、V93G、V93S、V93K、V93T、V93I、V93L、P94L、P94W、P94Y、P94Q、P94F、P94S、A95V、I96T、I96K、I96S、R97C、R97H、R97S、R97P、R97L、R97A、R97N、R97Q、R97E、R97I、R97K、R97M、R97T、D98E、D98N、D98V、A99T、A99K、I100T、R101C、R101H、S102N、S102G、S102E、H103R、H103L、H103Q、H103Y、P104T、P104L、A105S、V106A、V106T、R107C、R107S、R107V、E108V、I109K、I109N、I109F、F110L、F110S、F110Y、E111V、E111G、Y112C、D113G、D113Y、1114T、1114A、I114G、I114V、I114M、I114T、I114K、P115C、P115L、P115S、P115R、P115F、F116L、F116S、F116A、A117T、A117V、A117K、K118M、K118R、K118A、K118Q、K118Y、K118N、R119H、R119S、R119C、R119A、R119G、R119V、R119M、R119T、R119K、R119Y、Y120C、Y120N、L121M、I122V、I122F、I122N、I122D、D123G、D123E、D123N、D123V、K124N、K124E、K124R、L126F、L126P、L126Q、V127M、V127I、P128L、P128M、M129I、M129V、M129K、M129L、M129E、M129F、M129N、M129S、M129R、M129Y、E130D、E130G、E130V、E130K、E130T、G131S、G132S、G132D、E133K、L135M、L135P、L135Q、K136E、K136R、K136L、L137F、L137M、L138P、A139E、F140Y、F140L、F140S、D141A、D141V、D141L、D141I、D141F、D141Y、D141N、D141T、D141S、D141R、D141K、D141Q、D141W、D141E、D141M、D141P、D141G、D141H、D141C、I142V、I142F、I142A、E143A、E143V、E143L、E143I、E143F、E143Y、E143N、E143T、E143S、E143W、E143M、E143P、E143G、E143H、E143R、E143K、E143D、E143C、E143Q、T144F、F145L、Y146C、Y146A、Y146E、Y146S、Y146E、Y146K、Y146T、Y146R、H147E、H147R、H147E、H147D、H147N、H147Q、H147K、H147A、E148S、E148D、E148R、E148A、D150R、D150E、E151R、E156P、M159W、M159F、M159Y、S166E、W173R、W173A、W173Q、W173N、W173H、W173F、K174N、K174D、K174E、K174Q、I176V、Y180F、A190V、A190M、1191L、K192L、L195A、R199H、Q196R、L198I、L198V、P203S、V205A、L207I、L207V、Y209A、Y209E、Y209W、G211S、N213E、N213W、N213Y、F214A、F214E、F214W、F214V、D215A、D215N、D215Q、D215E、D215R、D215K、D215P、F216L、A217N、A217T、A217Q、A217S、Y218H、I219V、I219L、K220R、K220N、K220Q、K220H、K220I、K220L、K220M、K220Y、C223V、C223E、C223S、C223L、C223M、C223A、C223P、C223K、C223N、C223D、E224V、E224R、E224K、K225E、G227S、L228P、L228I、L228V、K229R、K229N、K229Q、K229H、F230L、T231I、T231P、I232F、G233D、R234C、S237G、S237C、E238S、E238R、P239S、K240S、K240E、K240R、K240D、K240N、K240Q、K240A、I241T、I241Q、I241E、I241N、I241A、I241S、I241D、I241P、Q242N、Q242S、Q242R、Q242D、Q242E、Q242V、R243E、M244T、M244K、G245D、G245S、G245R、G245A、G245N、G245K、D246R、D246L、D246E、D246V、D246Q、R247E、R247D、R247S、R247H、A249G、A249V、E251S、E251R、E251A、L258I、L258Q、L258F、Y261A、Y261P、Y261T、P262S、P262R、P262L、P262D、P262E、P262Q、V264I、V264A、R265D、R265I、H266F、H266W、H266Y、H266R、H266I、H266L、H266A、H266K、T267A、T267F、T267M、T267V、T267W、T267Y、T267I、T267S、I268A、I268F、I268M、I268V、I268W、I268Y、R269L、R269K、R269S、R269T、R269V、R269N、R269H、R269Y、L270R、P271S、P271F、P271E、P271L、P271Q、P271N、T272A、T272Y、T272V、T272S、T272L、T272E、T272C、T272R、T272W、T272N、T272F、T272H、T272K、Y273A、Y273W、T274E、T274W、T274S、T274D、T274V、T274A、T274R、T274N、L275P、L275M、E276K、A277V、V278M、V282L、V282T、V282G、V282I、F283L、K285I、K285Q、K286E、K286 P、K287R、E288G、E288K、K289E、K289Q、K289N、K289I、K289R、V290E、Y291F、 Y291N、Y291D、Y291K、Y291R、Y291Q、Y291A、A292N、A292T、A292I、I295N、E29 7G、A298G、W299F、W299E、W299N、W299Q、W299Y、W299A、W299F、K300S、K300E、S301N、S301T、E302G、L305P、K306R、K306N、K306Q、K306A、K306V、K306I、K 306F、R307C、V308I、V308A、A309S、Q310R、Y311A、Y311E、Y311W、Y311F、M313I、M313K、M313L、M313V、M313D、M313R、M313E、M313A、M313L、M313N、D315A、 D315E、D315R、D315W、D315L、D315W、D315F、R317C、R317K、A318V、Y320F、E321L、P328A、M329L、M329S、M329W、M329A、M329R、M329I、M329Q、M329N、M329 E、V331A、E332K、E332G、E332Q、L333A、L333V、L333I、L333F、A334S、I337V、G338D、Q339N、V341L、D343E、D343N、D343R、D343A、S345C、S345R、S347N、S34 7T、S347R、S347A、S348C、T349A、T349E、T349F、T349I、T349L、T349N、T349S、T349Y、G350S、N351S、N351Q、N351R、N351I、N351Y、N351K、N351A、N351E、L 352M、V353Q、V353E、W355R、W355F、Y356N、Y356C、Y356L、Y356F、L357P、R359H、V360A、V360D、V360I、V360K、Y362I、Y362E、Y362F、Y362N、Y362D、Y362K、Y362R、Y362T、N365S、E366A、E366D、E366N、E366Q、E366R、L367P、K371R、K371D、K371N、K371Q、K371Y、K371T、K371V、K371L、P372S、P372M、G373S、G373D、G374E、E375R、E375K、E376K、Y377L、Q378R、Q378A、Q378E、Q378K、M381I、M381R、M381V、M381D、M381L、S383G、S383Q、S383E、S383T、S384A、S384R、S384D、S384Q、S384E、S384T、Y385R、Y385S、Y385F、Y385K、Y385H、Y385W、Y385A、Y385M、Y385F、Y385D、I386A、I386N、I386D、I386E、I386K、I386T、I386L、G388S、G388R、Y389R、Y389S、Y389F、Y389N、Y389D、Y389K、Y389A、Y389Q、Y389E、Y389I、E394G、K395R、G396S、E399D、S400N、S400D、A402T、A402V、Y403H、Y403L、Y403D、Y403Q、Y403E、Y403H、Y403K、Y403F、Y403W、Y403R、L404Q、F406Y、F406R、F406I、R407N、R407K、R407A、R407L、R407V、R407I、S408A、S408G、L409S、L409F、L409A、L409Y、L409I、L409V、L409T、L409N、L409C、L409M、Y410A、Y410G、Y410F、Y410M、Y410L、Y410D、Y410T、Y410I、Y410N、Y410V、Y410E、Y410S、Y410L、P411G、P411A、P411I、P411V、P411S、P411T、P411L、S412N、S412A、S412G、I413F、I413V、V415M、V415K、V415R、V415N、V415T、V415I、H417I、H417R、H417F、H417Y、H417V、V419I、V419L、V419R、P421S、D422V、T423I、T423L、L424Q、E425N、E427G、E427R、C428Y、K429R、K429S、K429M、K429A、K429N、K429D、K429Q、K429H、K429Y、K429V、K429L、K429E、N430E、Y431A、Y431D、V433A、A434V、A434D、A434P、I436T、I436F、I436A、I436R、I436N、I436D、I436Q、I436E、I436H、I436K、I436S、Y439H、R440H、K443R、K446P、G447D、F448I、F448L、I449N、I449F、P450L、S451N、S451T、S451A、S451D、I452L、L453Q、E454D、E454N、E454T、E454G、D455N、I457L、T459E、K462D、V463M、V463I、K464C、R465C、R465T、M467V、M467K、M467D、M467T、M467R、M467E、M467Q、M467L、K468R、K468E、K468Y、K468T、K468L、T470S、T470A、I471K、I471Q、I471S、I471V、I471K、D472V、D472E、D472N、I474C、I474F、I474V、I474L、E475C、K476R、K476D、K476A、K476F、K476R、M478L、Y481C、Y481A、Y481F Y481T、Y481V、Y481W、R484D、R484N、R484K、R484S、R484T、R484V、R484L、A485S、A485T、A485L、A485V、A485G、A485R、L486I、K487M、K487R、K487N、K487A、K487Q、K487Y、I488A、I488V、I488S、I488T、I488M、I488R、I488N、I488Q、I488E、I488K、I488L、N491T、N491S、N491A、N491I、S492G、S492Y、S492D、S492K、S492T、S492N、S492E、Y493T、Y493S、Y493I、Y493F、Y493W、Y494A、Y494N、Y494G、Y494F、Y494W、G495S、Q497H、Q497G、Q497M、Q497N、Q497F、Q497L、Q497R、Q497K、Q497T、Q497E、Q497D、Q497Y、G498R、G498D、G498E、G498F、G498I、G498S、Y499F、P500A、K501R、K501A、K501Q、K501Y、K501N、S506C、S506R、S506A、S506L、S506T、S506N、S506D、S506H、S506V、K507L、K507E、K507S、K507A、K507N、K507Q、K507E、K507T、K507D、E508Q、E508C、C509V、C509Y、C509S、C509M、C509A、C509N、C509D、C509H、C509Q、E511K、E511S、E511A、E511R、E511N、E511T、E511Q、E511L、E511D、E511A、S512R、S512D、S512E、S512H、S512F、S512K、S512W、S512D、V513T、V5131、V513L、V513M、V513F、V513A、V513S、T514A、T514G、T514S、T514V、T514I、T514S、G517A、G517S、G517V、G517T、R518C、H519N、H519Y、H519E、H519Q、I521N、I521T、I521E、I521H、T523I、T523A、A528L、A528I、E529N、K534N、K534S、K534R、V535N、V535K、V535S、V535R、Y537H、A538V、A538G、D539A、D539G、D539E、D539V、D539L、D539S、D539N、D539I、T540I、D541A、D541G、D541E、G542S、G542E、G542D、G542N、G542R、G542T、F543L、F544H、F544Y、I547F、I547T、I547P、N549G、E550A、K551D、P552L、I555V、S557C、S557K、K558A、K558V、K558Q、K558R、A559K、K561N、K561E、L653M、K564S、K564Q、H565Y、H565S、H565N、E568K、K569E、G572S、M573I、M573V、M573K、M573D、M573A、M573R、M573L、E575K、E577D、L583P、G585D、G585A、G585I、G585V、G585Y、G585F、G585T、F586I、V588E、V588T、T589K、K592Q、K592R、K592W、K592Y、K592A、K592F、K592I、K592T、K592N、K592S、Y593R、Y593N、Y593E、Y593H、Y593K、Y593F、Y593A、L595V、I596T、D599E、H601R、H601I、H601A、H601T、H601V、H601L、H601N、H601K、T604S、R605K、R605N、R605Q、R605H、R605D、R605E、R605S、R605T、R605Y、R605A、G606S、G606R、G606Y、G606Q、G606N、G606E、G606D、L607F、V609I、V609L、V610D、V610A、V610K、V610S、V610T、V610N、V610R、V610Q、R611M、R611E、R612E、R612H、R612F、R612W、R612M、R612S、R612N、R612G、R612L、R612I、D613S、D613E、D613R、D613K、D613N、D613Q、D613A、D613V、D613Y、D613F、E616C、E616G、I617V、K619R、K619A、K619S、K619T、K619V、E620D、E620K、E620C、E620V、T621I、T621S、Q622L、A623T、A623C、A623K、K624I、K624R、V625F、L626I、E627K、V628L、V628I、V628A、I629F、I629C、L630Q、L630M、R631H、R631C、R631D、R631K、E632G、E632C、E632D、E632H、G633S、G633D、S634C、S634D、I635V、I635N、I635T、E636G、E636K、E636D、K637M、K637A、K637N、K637Q、K637E、K637S、K637T、A638E、A638V、A638T、A639T、A639V、G640D、G640R、I641F、I641V、I641A、V642I、V642A、K644E、V645E、V645I、V645M、V646A、V646D、E647G、E647D、E647K、D648V、D648C、D648L、D648G、L649Q、A650E、A650V、A650T、A650N、A650S、N651S、N651K、Y652H、Y652C、Y652M、Y652L、Y652F、R653C、R653H、R653Y、R653E、V654M、V656I、V656I、E657V、K658R、K658E、K658I、K658L、I660V、H662V、E663K、E663R、E663S、E663M、E663Q、E663V、Q664A、Q664L、Q664V、Q664F、Q664I、Q664R、Q664K、Q664T、Q664N、Q664M、I665V、I665F、I665P、I665M、I665F、T666A、R667E、E668G、E668K、E668M、E668A、E668P、E668S、E668R、E688N、E688D、E668Y、E668Q、K670E、K670I、K670R、K670S、D671G、D671R、D671Y、D671S、D671A、D671K、D671N、Y672F、K673I、K673Y、K673R、K673S、K673E、A674T、A674V、A674S、T675S、T675I、T675A、G676S、G676F、G676E、G676Y、G676R、G676L、G676Q、P677L、P677R、P677K、P677A、H678R、H678K、H678Q、H678F、H678W、V679S、V679M、A680V、A680I、A680D、I681T、I681L、I681F、I681V、A682T、A682S、K683R、R684H、L685E、Q686R、Q686C、Q686L、Q686A、A687C、A687T、A687S、R688S、G689S、G689D、I690V、I690F、I690R、I690N、I690Q、I690K、I690T、I690Y、K691R、K691V、K691T、K691D、K691E、K691Q、K691N、V692I、K693M、K693V、K693Q、K693R、K693A、K693T、K693N、K693Y、K693S、K693E、K693D、P694R、T696S、T696I、T696L、T696M、I698K、I698M、I698F、I698Q、S699I、S699G、Y700W、Y700S、V701I、V702A、V702I、L703P、K704E、K704I、K704N、G705D、S706N、S706C、S706G、K707I、K707G, K707N, K708M, K708R, I709F, I709V, I709L, D711G, R712C, V713I, V713A, I714F, L715P, L715Q, F716L, D717N, E718 K, E718V, Y719F, D720V, D720Y, D720E, S721N, S721C, S721G, S721P, S722G, R723H, K725E, K725L, K725R, Y726F, P728S, P72 8L, P728A, Y730H, Y731H, I732T, I732F, I732N, H733R, H733E, N734Y, N734R, N734P, N734D, N734K, N734T, Q735H, Q735R, V7 36A, P738L, A739V, V740I, L741A, L741Q, L741E, R742K, R742L, R742C, I743V, I743E, L744A, E745V, E745F, E745R, A746V, A 746G, F747L, F747Y, G748V, G748K, Y749F, Y749E, K750N, K750R, E751K, E751D, E751M, K752E, K752L, D753V, D753E, D753G , L754Y, L754S, E755G, E755Q, E755D, E755K, E755Y, E755R, Y756C, Y756F, Y756I, Y756R, Y756Q, Y756K, Q757L, Q757H, Q757 S, Q757M, R758H, R758A, R758K, M759T, M759S, M759N, M759R, M759E, M759D, M759A, Q761L, T762N, G765S, G765R, G765L, G76 5F, G765Y, G765I, G765T, G765D, G765E, W767H, W767Y, W767F, W767S, W767R, W767D, W767A, W767N, M770S, M770T and / or M770N. ,

[0218] In some embodiments, the mutant polymerase has a backbone sequence of RLF 89458.1 (e.g., SEQ ID NO: 1) or RLF78286.1 (SEQ ID NO: 2) and includes amino acid deletions at any one or any combination of positions comprising M1 (deleted), R58 (deleted), V93 (deleted) and / or E755 (deleted).

[0219] In some embodiments, the mutant polymerase has a backbone sequence of RLF 89458.1 (e.g., SEQ ID NO: 1) or RLF78286.1 (SEQ ID NO: 2) and a substitution mutation at position M1. In some embodiments, the amino acid substitution at position M1 comprises any of the 20 natural amino acids (i.e., W, I, M, P, F, G, A, V, L, H, R, K, D, E, N, C, S, T, or Q) or an unnatural amino acid known to those skilled in the art.

[0220] In some embodiments, the mutant polymerase has a backbone sequence of RLF 89458.1 (e.g., SEQ ID NO: 1) or RLF78286.1 (SEQ ID NO: 2) and a substitution mutation at position Y7. In some embodiments, the amino acid substitution at position Y7 comprises any of the 20 natural amino acids (i.e., W, I, M, P, F, G, A, V, L, H, R, K, D, E, N, C, S, T, or Q) or an unnatural amino acid known to those skilled in the art.

[0221] In some embodiments, the mutant polymerase has a backbone sequence of RLF 89458.1 (e.g., SEQ ID NO: 1) or RLF78286.1 (SEQ ID NO: 2) and a substitution mutation at position K74. In some embodiments, the amino acid substitution at position K74 comprises any of the 20 natural amino acids (i.e., W, I, M, P, F, G, A, V, L, H, R, K, D, E, N, C, S, T, or Q) or an unnatural amino acid known to those skilled in the art.

[0222] In some embodiments, the mutant polymerase has a backbone sequence of RLF 89458.1 (e.g., SEQ ID NO: 1) or RLF78286.1 (SEQ ID NO: 2) and a substitution mutation at position E88. In some embodiments, the amino acid substitution at position E88 comprises any of the 20 natural amino acids (i.e., W, I, M, P, F, G, A, V, L, H, R, K, D, E, N, C, S, T, or Q) or an unnatural amino acid known to those skilled in the art.

[0223] In some embodiments, the mutant polymerase has a backbone sequence of RLF 89458.1 (e.g., SEQ ID NO: 1) or RLF78286.1 (SEQ ID NO: 2) and a substitution mutation at position V93. In some embodiments, the amino acid substitution at position V93 comprises any of the 20 natural amino acids (i.e., W, I, M, P, F, G, A, L, H, R, K, D, E, N, Y, C, S, T, or Q) or an unnatural amino acid known to those skilled in the art.

[0224] In some embodiments, the mutant polymerase has a backbone sequence of RLF 89458.1 (e.g., SEQ ID NO: 1) or RLF78286.1 (SEQ ID NO: 2) and a substitution mutation at position A217. In some embodiments, the amino acid substitution at position A217 comprises any of the 20 natural amino acids (i.e., W, I, M, P, F, G, A, L, H, R, K, D, E, N, Y, C, S, T, or Q) or an unnatural amino acid known to those skilled in the art.

[0225] In some embodiments, the mutant polymerase has a backbone sequence of RLF 89458.1 (e.g., SEQ ID NO: 1) or RLF78286.1 (SEQ ID NO: 2) and a substitution mutation at position Y261. In some embodiments, the amino acid substitution at position Y261 comprises any of the 20 natural amino acids (i.e., W, I, M, P, F, G, A, V, L, H, R, K, D, E, N, C, S, T, or Q) or an unnatural amino acid known to those skilled in the art.

[0226] In some embodiments, the mutant polymerase has a backbone sequence of RLF 89458.1 (e.g., SEQ ID NO: 1) or RLF78286.1 (SEQ ID NO: 2) and a substitution mutation at position T267. In some embodiments, the amino acid substitution at position T267 comprises any of the 20 natural amino acids (i.e., W, I, M, P, F, G, A, V, L, H, R, K, D, E, N, Y, C, S, or Q) or an unnatural amino acid known to those skilled in the art.

[0227] In some embodiments, the mutant polymerase has a backbone sequence of RLF 89458.1 (e.g., SEQ ID NO: 1) or RLF78286.1 (SEQ ID NO: 2) and a substitution mutation at position 1268. In some embodiments, the amino acid substitution at position 1268 comprises any of the 20 natural amino acids (i.e., W, M, P, F, G, A, V, L, H, R, K, D, E, N, Y, C, S, T, or Q) or is an unnatural amino acid known to those skilled in the art.

[0228] In some embodiments, the mutant polymerase has a backbone sequence of RLF 89458.1 (e.g., SEQ ID NO: 1) or RLF78286.1 (SEQ ID NO: 2) and a substitution mutation at position E366. In some embodiments, the amino acid substitution at position E366 comprises any of the 20 natural amino acids (i.e., W, M, P, F, G, A, V, L, H, R, K, D, E, N, Y, C, S, T, or Q) or an unnatural amino acid known to those skilled in the art.

[0229] In some embodiments, the mutant polymerase has a backbone sequence of RLF 89458.1 (e.g., SEQ ID NO: 1) or RLF78286.1 (SEQ ID NO: 2) and a substitution mutation at position 1436. In some embodiments, the amino acid substitution at position 1436 comprises any of the 20 natural amino acids (i.e., W, M, P, F, G, A, V, L, H, R, K, D, E, N, Y, C, S, T, or Q) or is with an unnatural amino acid known to those skilled in the art.

[0230] In some embodiments, the mutant polymerase has a backbone sequence of RLF 89458.1 (e.g., SEQ ID NO: 1) or RLF78286.1 (SEQ ID NO: 2) and a substitution mutation at position A485. In some embodiments, the amino acid substitution at position A485 comprises any of the 20 natural amino acids (i.e., W, I, M, P, F, G, V, L, H, R, K, D, E, N, Y, C, S, T, or Q) or an unnatural amino acid known to those skilled in the art.

[0231] In some embodiments, the mutant polymerase has a backbone sequence of RLF 89458.1 (e.g., SEQ ID NO: 1) or RLF78286.1 (SEQ ID NO: 2) and a substitution mutation at position T514. In some embodiments, the amino acid substitution at position T514 comprises any of the 20 natural amino acids (i.e., W, I, M, P, F, G, A, V, L, H, R, K, D, E, N, Y, C, S, or Q) or an unnatural amino acid known to those skilled in the art.

[0232] In some embodiments, the mutant polymerase has a backbone sequence of RLF 89458.1 (e.g., SEQ ID NO: 1) or RLF78286.1 (SEQ ID NO: 2) and a substitution mutation at position D671. In some embodiments, the amino acid substitution at position D671 comprises any of the 20 natural amino acids (i.e., W, I, M, P, F, G, A, V, L, H, R, K, E, N, Y, C, S, T, or Q) or an unnatural amino acid known to those skilled in the art.

[0233] Engineered polymerase comprising the NOZ 58130.1 backbone sequence

[0234] The present disclosure provides compositions and methods comprising mutant polymerases having a backbone sequence of NOZ58130.1 and 100%, at least 99%, at least 98%, at least 97%, at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, or at least 50% sequence identity to any one of SEQ ID NOs: 1714-2787 (Tables 2 and 32 at FIG. 32 ). Figure 13 ).

[0235] In some embodiments, a mutant polymerase having a backbone sequence of NOZ 58130.1 (eg, SEQ ID NO: 1714) includes various domains.

[0236] In some embodiments, the N-terminal domain includes amino acid residues 1-162 (SEQ ID NO: 2810) (e.g., Figure 30 ).

[0237] In some embodiments, the exonuclease domain (e.g., 3' to 5' exonuclease domain) includes amino acid residues 163-405 (SEQ ID NO: 2811) (e.g., Figure 30 ).

[0238] In some embodiments, the palm domain includes amino acid residues 406-640 (SEQ ID NO: 2812) (e.g., Figure 30 ).

[0239] In some embodiments, the finger subdomain within the palm domain includes amino acid residues 480-527 (SEQ ID NO: 2813) (e.g., Figure 30 ).

[0240] In some embodiments, the thumb domain includes amino acid residues 641-792 (SEQ ID NO: 2814) (e.g., Figure 30 ).

[0241] The present disclosure provides compositions and methods comprising a mutant polymerase having a backbone sequence of NOZ58130.1 (SEQ ID NO: 1714) and a mutant polymerase lacking an exonuclease. - In some embodiments, the mutant polymerase comprises at least one amino acid substitution mutation that reduces 3' to 5' exonuclease activity compared to a mutant polymerase. For example, the mutant polymerase comprises at least one amino acid substitution at position D168 and / or E170. In some embodiments, the mutant polymerase comprises mutations D168A, D168V, D168L, D168I, D168F, D168Y, D168N, D168K, D168T, D168S, D168W, D168M, D168P, D168G, D168H, D168R, D168E, D168C, or D168Q. In some embodiments, the mutant polymerase comprises mutations E170A, E170V, E170L, E170I, E170F, E170Y, E170N, E170K, E170T, E170S, E170W, E170M, E170P, E170G, E170H, E170R, E170D, E170C, or E170Q. In some embodiments, the mutant polymerase comprises any combination of mutations at positions D168 and E170. SEQ ID NOs: 1714-2787 include exemplary amino acid substitution mutations at positions D168 and E170.

[0242] The present disclosure provides compositions and methods comprising mutant polymerases having a backbone sequence of NOZ58130.1 (SEQ ID NO: 1714) and comprising at least one amino acid substitution mutation in a LYP motif (e.g., at positions L440, Y441, and P442). In some embodiments, at least one mutation in the LYP motif can increase the incorporation rate of a nucleotide analog. In some embodiments, any one or any combination of the first, second, and / or third positions of the LYP motif can be mutated. For example, mutations in the LYP motif include YAG, FAG, YGP, YAP, FGP, SAP, AAA, YGA, YAA, FGA, FTA, AAG, AAP, AAV, AAI, AGA, AGG, AGI, AGP, AGV, FAA, FAI, FAP, FAV, FGG, FGV, LAG, LAI, LAP, LGG, LGI, LGV, SAA, SAG, SAI, S AV, SGA, SGG, SGI, YAI, YGG, YGI, LAA, LAV, LGP, LGA, FGI, SGV, YAV, YGV, SYP, SGP, LFP, IFP, VF P, LMP, VMP, IMP, LLP, VLP, ILP, LDP, VDP, IDP, LTP, VTP, ITP, LIP, TIP, NNP, NDP, NAP, MFG and SYG.

[0243] In some embodiments, the polymerase comprises the backbone sequence of NOZ 58130.1 (SEQ ID NO: 1714) and has a substitution mutation at position L440 comprising a nonpolar amino acid or a polar uncharged amino acid. In some embodiments, the amino acid substitution mutation at position L440 comprises valine, glycine, threonine, alanine, serine, isoleucine, leucine, phenylalanine, tyrosine, or methionine. SEQ ID NOs: 1714-2787 include exemplary amino acid substitution mutations at position L440.

[0244] In some embodiments, the polymerase comprises the backbone sequence of NOZ 58130.1 (SEQ ID NO: 1714) and has a substitution mutation at position Y441 comprising a nonpolar amino acid or a polar uncharged amino acid. In some embodiments, the amino acid substitution mutation at position Y441 comprises threonine, serine, glycine, alanine, valine, isoleucine, or tyrosine. SEQ ID NOs: 1715-2787 include exemplary amino acid substitution mutations at position Y441.

[0245] In some embodiments, the polymerase comprises the backbone sequence of NOZ 58130.1 (SEQ ID NO: 1714) and has a substitution mutation at position P442 comprising a polar uncharged amino acid, a nonpolar amino acid, or a positively charged amino acid. In some embodiments, the amino acid substitution mutation at position P442 comprises serine, glycine, alanine, valine, cysteine, lysine, isoleucine, threonine, or proline. SEQ ID NOs: 1715-2787 include exemplary amino acid substitution mutations at position P442.

[0246] The present disclosure provides compositions and methods comprising mutant polymerases having a backbone sequence of NOZ58130.1 (SEQ ID NO: 1714) and having at least one mutation that reduces post-translational modification compared to a wild-type polymerase or an engineered polymerase lacking a mutation that reduces post-translational modification. In some embodiments, the polymerase comprises at least one mutation or any combination of mutations in methionine, lysine, tryptophan, and / or serine residues. In some embodiments, the polymerase is engineered to comprise any one or any combination of amino acid substitution mutations at W135, M187, W329, K335, M389, S473, M527, K552, M629, W641, K650, K711, M723, and / or W791.

[0247] In some embodiments, the polymerase comprises the backbone sequence of NOZ 58130.1 (SEQ ID NO: 1714) and has a mutation at position W135, including W135S, W135L, W135R, W135Y, W135F, W135D, W135A, W135V, or W135G.

[0248] In some embodiments, the polymerase comprises the backbone sequence of NOZ 58130.1 (SEQ ID NO: 1714) and has a mutation at position M187, including M187S, M187L, M187R, M187Y, M187I, M187T, M187A, or M187V.

[0249] In some embodiments, the polymerase comprises the backbone sequence of NOZ 58130.1 (SEQ ID NO: 1714) and has a mutation at position W329, including W329Y, W329F, W329L, W329D, W329A, or W329V.

[0250] In some embodiments, the polymerase comprises the backbone sequence of NOZ 58130.1 (SEQ ID NO: 1714) and has a mutation at position K335, including K335R, K335L, K335S, or K335A.

[0251] In some embodiments, the polymerase comprises the backbone sequence of NOZ 58130.1 (SEQ ID NO: 1714) and has a mutation at position M389, including M389D, M389E, M389L, M389Y, M389S, M389A, or M389V.

[0252] In some embodiments, the polymerase comprises the backbone sequence of NOZ 58130.1 (SEQ ID NO: 1714) and has a mutation at position S473, including S473K, S473R, S473T, S473Q, or S473A.

[0253] In some embodiments, the polymerase comprises the backbone sequence of NOZ 58130.1 (SEQ ID NO: 1714) and has a mutation at position M527, including M527H, M527G, M527Q, M527L, M527D, M527A, or M527V.

[0254] In some embodiments, the polymerase comprises the backbone sequence of NOZ 58130.1 (SEQ ID NO: 1714) and has a mutation at position M549, including M549N, M549Y, M549H, M549T, M549D, M549R, M549A, or M549V.

[0255] In some embodiments, the polymerase comprises the backbone sequence of NOZ 58130.1 (SEQ ID NO: 1714) and has a mutation at position K552, including K552R, K552T, K552N, K552Q, or K552A.

[0256] In some embodiments, the polymerase comprises the backbone sequence of NOZ 58130.1 (SEQ ID NO: 1714) and has a mutation at position M629, including M629L, M629A, M629D, M629R, or M629V.

[0257] In some embodiments, the polymerase comprises the backbone sequence of NOZ 58130.1 (SEQ ID NO: 1714) and has a mutation at position W641, including W641R, W641A, W641L, W641F, W641Y, or W641V.

[0258] In some embodiments, the polymerase comprises the backbone sequence of NOZ 58130.1 (SEQ ID NO: 1714) and has a mutation at position K650, including K650T, K650C, K650A, K650R, or K650S.

[0259] In some embodiments, the polymerase comprises the backbone sequence of NOZ 58130.1 (SEQ ID NO: 1714) and has a mutation at position K711, including K711R, K711L, K711T, or K711D.

[0260] In some embodiments, the polymerase comprises the backbone sequence of NOZ 58130.1 (SEQ ID NO: 1714) and has a mutation at position M723, including M723S, M723I, M723T, M723N, M723R, M723L, M723A, or M723C.

[0261] In some embodiments, the polymerase comprises the backbone sequence of NOZ 58130.1 (SEQ ID NO: 1714) and has a mutation at position W791, including W791R, W791Y, W791D, W791S, W791L, W791A, or W791V.

[0262] The present disclosure provides compositions and methods comprising mutant polymerases having a backbone sequence of NOZ58130.1 (SEQ ID NO: 1714) and having at least one mutation that removes an unpaired cysteine, which can reduce oxidative damage to the polymerase. In some embodiments, removing at least one unpaired cysteine ​​increases the thermal stability of the mutant polymerase and / or reduces protein aggregation. In some embodiments, removing at least one unpaired cysteine ​​increases the amount of protein in lysate preparation. In some embodiments, the polymerase comprises at least one mutation or any combination of mutations of the cysteine ​​at positions 362 and / or 539. In some embodiments, the mutation at position C362 comprises the amino acid substitution C362A, C362L, C362I, C362S, C362F, C362Y, C362V, C362P, C362K, C362N, or C362D. In some embodiments, the mutation at position C539 comprises the amino acid substitution C539A, C539V, C539L, C539S, C539Y, C539D, C539K, C539N, or C539P.

[0263] In some embodiments, the polymerase comprises the backbone sequence of NOZ 58130.1 (SEQ ID NO: 1714) with a non-mutated or mutated C362 and an amino acid substitution of R536C.

[0264] In some embodiments, the polymerase comprises the backbone sequence of NOZ 58130.1 (SEQ ID NO: 1714) and has a non-mutated or mutated C539 and an amino acid substitution R536C.

[0265] In some embodiments, the polymerase comprises the backbone sequence of NOZ 58130.1 (SEQ ID NO: 1714) with a non-mutated or mutated C362 and an amino acid substitution of D451C.

[0266] The present disclosure provides compositions and methods comprising mutant polymerases having a backbone sequence of NOZ58130.1 (SEQ ID NO: 1714) and a C-terminal truncation that increases the thermostability of the polymerase compared to a non-truncated polymerase having the same wild-type or engineered backbone sequence. The C-terminal region of the polymerase may be disordered and may cause protein aggregation. Therefore, truncation at the C-terminal region can reduce protein aggregation and improve stability. In some embodiments, the polymerase exhibits thermostability at elevated temperatures of about 72-75°C, or about 75-80°C, or about 80-85°C, or about 85-90°C or higher. In some embodiments, a thermal shift assay performed using differential scanning fluorimetry can be used to determine the thermostability of the polymerase.

[0267] In some embodiments, the polymerase comprises the backbone sequence of NOZ 58130.1 (SEQ ID NO: 1714) and has a C-terminal truncation for increased thermal stability, comprising M723 (truncated), G773 (truncated), D777 (truncated), E781 (truncated), T784 (truncated), Q785 (truncated), R790 (truncated), W791 (truncated), or F792 (truncated). In Table 2, truncations are designated with a "^".

[0268] The present disclosure provides compositions and methods comprising mutant polymerases having a backbone sequence of NOZ58130.1 (SEQ ID NO: 1714) and at least one mutation that reduces polymerization to exonuclease conformational switching compared to wild-type polymerase or compared to engineered polymerases having mutations that do not reduce conformational switching. In some embodiments, protein modeling can identify key amino acid residues that interact with a primer or primer end, wherein these amino acid residues may play a role in interacting with the primer or in shifting the primer end from the polymerization site to the exonuclease site. In some embodiments, the polymerase comprises an amino acid substitution at any one or any combination of positions Q95, I186, V304, L313, and / or E318.

[0269] In some embodiments, the polymerase comprises the backbone sequence of NOZ 58130.1 (SEQ ID NO: 1714) and has a mutation at Q95, including Q95L, Q95H, Q95R, Q95W, Q95A, Q95K, Q95N, or Q95P.

[0270] In some embodiments, the polymerase comprises the backbone sequence of NOZ 58130.1 (SEQ ID NO: 1714) and has a mutation at 1186, including 1186R or 1186N.

[0271] In some embodiments, the polymerase comprises the backbone sequence of NOZ 58130.1 (SEQ ID NO: 1714) and has a mutation at V304, including V304D.

[0272] In some embodiments, the polymerase comprises the backbone sequence of NOZ 58130.1 (SEQ ID NO: 1714) and has a mutation at L313, including L313M, L313D, L313F, L313K, L313R, L313A, or L313E.

[0273] In some embodiments, the polymerase comprises the backbone sequence of NOZ 58130.1 (SEQ ID NO: 1714) and has a mutation at E318, including E318V.

[0274] The present disclosure provides compositions and methods comprising mutant polymerases having a backbone sequence of NOZ58130 (SEQ ID NO: 1714) and having at least one mutation that reduces protein aggregation by reducing intermolecular interactions compared to wild-type polymerase or compared to engineered polymerases having mutations that do not confer reduced aggregation. In some embodiments, the mutant polymerase aggregates at elevated temperatures of about 72-75°C, or about 75-80°C, or about 80-85°C, or about 85-90°C, or higher, compared to wild-type polymerase or compared to engineered polymerases having mutations that do not confer reduced aggregation. In some embodiments, a thermal shift assay using differential scanning fluorimetry can be used to determine the aggregation temperature of a protein. In some embodiments, the mutant polymerase comprises a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity to any one of SEQ ID NOs: 1714-2787 and comprises at least one mutation that reduces protein aggregation by reducing intermolecular interactions.

[0275] In some embodiments, the polymerase comprises the backbone sequence of NOZ 58130 (SEQ ID NO: 1714) and has aggregation-reducing amino acid substitutions, including amino acid substitutions at one or more positions E76, K254, R537, E541, and / or K664.

[0276] In some embodiments, the mutation at position E76 comprises E76N, E76A, E76R, E76K, E76T, E76V, E76S, E76Q, E76D, or E76I.

[0277] In some embodiments, the mutation at position K254 comprises K254E, K254D, K254A, K254N, K254T, K254V, K254S, K254Q, K254G, K254I, or K254R.

[0278] In some embodiments, the mutation at position R537 comprises R537S, R537T, R537N, R537Q, R537A, R537V, R537D, R537I, R537E, R537G, R537K, or R537L.

[0279] In some embodiments, the mutation at position E541 comprises E541A, E541R, E541N, E541K, E541T, E541V, E541S, E541Q, E541D, or E541I.

[0280] In some embodiments, the mutation at position K664 comprises K664R, K664A, K664N, K664Q, K664E, K664S, K664T, K664D, K664G, K664V, or K664I.

[0281] In some embodiments, the mutant polymerase comprises the backbone sequence of NOZ 58130.1 (SEQ ID NO: 1714) and a deleted portion of any of the domains, including the N-terminal domain (SEQ ID NO: 2810), the exonuclease domain (SEQ ID NO: 2811), the first palm domain (SEQ ID NO: 2812), the finger domain (SEQ ID NO: 2813), and the thumb domain (SEQ ID NO: 2814).

[0282] In some embodiments, the mutant deletion polymerase comprises a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater than 99% sequence identity to any one of SEQ ID NOs: 1714-2787 and comprises a deleted portion of any of the domains.

[0283] In some embodiments, the mutant deletion polymerase comprises the backbone sequence of NOZ 58130.1 (SEQ ID NO: 1714), and the deleted portion comprises I130-P160 or any subregion thereof. In some embodiments, the deleted portion comprises the N-terminus of any one of I130, T134, L136, L138, R143, G151, or E156. In some embodiments, the deleted portion comprises the C-terminus of any one of P160, E156, V152, A147, G145, or L136.

[0284] In some embodiments, the deleted portion includes any of I130-P160, L136-P160, L138-P160, R143-P160, I130-V152, L136-V152, L138-V152, R143-V152, I130-A147, L136-A147, L138-A147, R143-A147, I130-G145, L136-G145, L138-G145, R143-G145, I130-L136, G151-P160, G151-V152, E156-P160, or T134-E156.

[0285] In some embodiments, the portion deleted in the mutant polymerase (e.g., having a backbone sequence of NOZ 58130.1, SEQ ID NO: 1714) is a sequence or region not present in other archaeal Family B polymerases, including RLF89458 (SEQ ID NO: 1), RLF 78286 (SEQ ID NO: 2), WP 175059460 (SEQ ID NO: 2791), 9°N (SEQ ID NOs: 2795 and 2796), THERMINATOR (SEQ ID NO: 2797), VENT (SEQ ID NO: 2798), DEEP VENT (SEQ ID NO: 2799), Pfu (SEQ ID NO: 2800), and Pyrococcus aquatica (SEQ ID NO: 2801). For example, see the sequence alignments in Figures 33 and 35, which illustrate regions present in NOZ 58130 but not in other archaeal Family B polymerases. In some embodiments, the deleted portion of the mutant polymerase comprises the 23-amino acid sequence TWLRLEVEERDGRALLRGVEQLE (SEQ ID NO: 2788). In some embodiments, the deleted portion is larger or smaller than SEQ ID NO: 2788.

[0286] The present disclosure provides compositions and methods including mutant polymerases having NOZ58130.1 (e.g., SEQ ID NO: 1714) backbone sequence and includes amino acid substitution mutations at any one or any combination of positions including: Y14, E15, V17, E18, R20, F26, L25, L27, G29, F34, V35, V36, F41, S42, P43, F45, L48, P49, G50, R55, E58, L61, A62, S63, A65, E67, A68, I69, K71, V72, I74, E76, K77, L79, F80, T82, P83, R84, V85, A86, L87, T90, V91, S92, H93, P94, Q95, D 96, V97, P98, R99, I100, R101, E102, R103, R105, E108, D111, L112, 1113 , N114, E115, H116, D117, I118, V121, R122, R123, Y124, I126, R128, I13 0. K131, P132, L133, T134, W135, L136, E139, G145, R150, E153, E156, E1 57. E158, R163, V164, A165, V167, D168, I169, E170, V171, Y172, N173T, P 174, E184, I186, M187, V190, T192, S193, E197, L200, V205, G207, E209, Q210, Q215, D216, M220, L222, E226, K229, G231, Y233, I236, V237, G238 , N240, T241, S243, F244, Y248, R252, L253, K254, L260, L265, D266, L26 9. S272, G275, A276, L277, I282, A286, V288, L290, Y291, P292, I293, V29 4. R295, H297, V298, K299, N301, S302, Y303, V304, S307, V309, L312, L3 13. G314, E318, K319, L320, D321, G322, R324, L325, F326, T327, W329, D 330, E331, K335, R336, L338, L339, A343, Y342, L344, D346, A352, A354, K356, L360, C362, I367, A376, M378, T379, V384, L387, M389, R390, T393,L398, I399, P400, E407, Y408, A409, R413, Y416, R422, V429, V434, F435, D436, F437, S439, L440, Y441, P442, S443, I444, I445, V446, T454, A465, S473, F479, I480, R496, D504, F511, A515, S522, F523, Y524, Y526, M527, R536, R537, E538, C539, E541, V543, A544, F546, A547, M549, I551, K552, M555, A558, E559, F562, L564, E565, V566, D570, D572, V576, V577, I578, P580, L585, A586, Q587, K588, K592, V593, E595, M597, I601, F608, L613, V 615, T616, R619, L622, L623, K628, M629, V631, F636, V637, R638, R639, D640, W641, A642, K650, I655, L656, A 660, K664, A665, L668, I673, E674, R675, R677, S682, D685, T687, Y689, T690, Q691, R695, S698, S701, E703, V 707, A708, K711, E718, V719, M723, I724, I729, T730, K734, G735, S737, Q738, T741, D752, D758, N759, I761, I765, R767, I772, Y774, L779, K780, E781, G782, I783, T784, Q785, T786, S787, L788, S789, R790, W791, and / or F792.

[0287] The present disclosure provides compositions and methods comprising mutant polymerases having a backbone sequence of NOZ58130.1 (e.g., SEQ ID NO: 1714) and comprising amino acid substitution mutations at any one or any combination of positions comprising: Y14F, Y14D, Y14I, Y14N, E15I, V17E, E18S, E18N, E18D, R20K, L25F, F26Y, F26S, F26I, L27Q, L27K, G29E, G29K, F34K, V35M, V35K, V36F, V36N, V36T, V36I, V36E, F41S, F41I, F41S, Y, S42K, S42G, S42D, S42E, P43L, P43A, P43G, P43V, P43M, P43T, P43K, F45T, F45N, F45I, L48D, P49V, P49K, P49D, P4 9E, P49L, G50K, R55G, R55K, R55E, R55I, E58V, L61I, L61S, L61A, L61T, A62D, A62S, A62V, A62G, S63G, S63K, S63E, A 65L, A65Y, A65H, E67M, E67K, E67V, A68R, I69A, I69D, I69V, K71T, K71V, K71F, K71N, K71I, V72T, V72N, V72I, I74K, E76Q, E76N, E76A, E76R, E76K, E76T, E76V, E76S, E76D, E76I, K77E, L79F, F80L, T82K, T82G, T82N, T82S, T82E, P83R , R84N, R84K, R84S, V85R, V85E, A86V, L87W, T90D, T90I, T90A, T90V, V91I, V91L, V91C, V91F, H93D, H93A, H93Y, P94 L, P94S, P94D, P94R, P94A, P94G, P94V, P94M, P94T, P94K, Q95L, Q95H, Q95R, Q95W, Q95A, Q95K, Q95N, Q95P, D96N, D9 6V, V97S, V97A, V97F, V97Y, V97Q, P98L, P98W, P98Y, P98Q, P98F, P98S, R99V, R99A, I100T, I100K, I100S, R101C, R1 01H, R101S, R101P, R101L, E102N, E102V, E102D, R103T, R103A, R105C, R105H, E108N, D111C, D111S, D111R, L112N,L112Q、L112T、I113K、I113N、I113F、I113A、I113D、I113N、I113T、L114N、L114T、E115V、E115G、H116C、H116Y、H116L、D117G、D117Y、I118T、I118A、I118G、I118V、I118M、I118T、I118K、V121T、V121K、V121A、R122S、R122M、R122K、R123H、R123S、R123C、R123A、R123G、R123V、R123M、R123T、R123K、R123Y、Y124R、Y124C、I126V、I126F、I126N、I126D、R128K、I130L、K131I、P132L、P132M、L133I、L133V、L133K、L133L、L133E、L133M、T134E、W135S、W135L、W135R、W135Y、W135F、W135D、W135A、W135V、W135G、L136D、E139V、G145S、R150A、R150V、R150L、R150K、R150F、E153A、E153V、E153L、E153K、E153R、E153F、E156N、E156R、E157A、E157V、E157L、E157K、E157R、E157F、E157D、E157G、E157T、E158S、E158G、R163E、R163L、R163K、R163H、V164F、V164L、V164M、V164I、A165P、A165L、V167I、V167F、D168A、D168V、D168L、D168I、D168F、D168Y、D168N、D168T、D168S、D168W、D168M、D168P、D168G、D168H、D168R、D168E、D168C、D168K、D168Q、I169V、I169F、I169A、I169R、I169W、E170A、E170V、E170L、E170I、E170F、E170Y、E170N、E170T、E170S、V171F、V171T、V171R、Y172R、Y172T、N173T、P174R、E184N、I186R、I186L、I186N、M187S、M187L、M187R、M187Y、M187I、M187T、M187A、M187V、V190Y、T192D、S193E、E197A、L200I、V205I、G207D、E209P、Q210Y、Q215S、D216T、M220I、L222K、E226R、K229E、G231K、Y233P、I2 36L、V237I、G238T、N240G、N240T、N240S、T241G、S243N、F244S、Y248D、Y248 R、R252A、R252S、L253V、L253E、L253C、K254E、K254R、K254A、K254N、K254Q、 K254S、K254T、K254D、K254G、K254V、K254I、L260F、L265D、D266G、L269P、S27 2Q、G275N、G275K、G275S、G275R、A276M、A276N、A276Q、A276D、L277R、L277M 、L277D、I282V、A286I、V288F、L290I、Y291A、Y291P、Y291G、Y291D、Y291N、P 292R、I293V、V294M、V294I、R295A、H297F、H297Y、V298I、K299N、N301R、N301P、S302N、S302T、Y303G、Y303D、Y303A、V304D、V304A、V304E、V304H、V304I、 V304L、V304M、V304P、V304R、V304T、V304W、V304Y、V304F、V304G、V304K、V3 04N、V304Q、V304S、S307A、V309Y、L312V、L312I、L313M、L313D、L313F、L313 K、L313R、L313A、L313E、G314S、G314D、G314K、G314R、G314E、E318V、K319V、 K319R、L320V、D321F、G322D、G322S、R324E、L325R、F326N、F326T、F326A、T32 7Q、W329Y、W329F、W329L、W329D、W329A、W329V、D330N、D330E、E331N、E331R、K335R、K335L、K335S、K335A、R336L、L338E、L339V、Y342N、Y342A、Y342R、A 343S、L344M、D346G、D346A、D346R、A352L、A352E、A352D、A352Q、A354G、K356R、K356D、K356E、L3601、L360Q、L360V、L360M、C362A、C362L、C362I、C362S、C362F、C362Y、C362V、C362P、C362K、C362N、C362D、I367L、A376C、A376R、A376S、M378R、M378T、M378A、T379D、T379K、T379N、T379S、V384Q、V384E、L387 N、L387C、L387Y、L387F、M389D、M389E、M389L、M389Y、M389S、M389A、M389V、R390M、L398D、I399A、I399N、I399R、I399F、P400H、P400N、P400S、E407R、Y40 8R、A409R、A409Q、R413Q、R413T、Y416H、R422V、R422T、R422D、V429W、V434H 、V434L、V434Y、F435L、D436T、F437Y、F437R、F437I、S439A、S439G、S439R、L4 40、L440Y、L440F、L440S、L440A、Y441、Y441A、Y441G、Y441T、P442、P442G、P 442A、S443R、S443N、S443A、S443G、I444F、I445L、I445F、V446M、V446K、V446 R、V446N、V446T、D451C、T454I、T454L、T454R、A465V、A465D、A465P、S473K、 S473R、S473T、S473Q、S473A、F479I、F479L、I480F、I480Y、R496T、R496A、R49 6G、R496C、D504E、F511Y、F511L、F511V、A515L、A515S、A515T、A515V、A515G 、A515R、S522D、S522K、S522T、S522N、S522E、S522G、S522Y、F523A、F523S、F5 23T、F523V、F523I、F523Y、Y524A、Y524N、Y524G、Y524F、Y524L、Y526C、M527H、M527G、M527Q、M527L、M527D、M527A、M527V、R536C、R537K、R537E、R537G、R 537S、R537L、R537A、R537N、R537Q、R537T、R537D、R537V、R537I、E538A、E538C、C539A、C539V、C539L、C539S、C539Y、C539D、C539K、C539N、C539P、E541K、E541S、E541A、E541R、E541N、E541T、E541V、E541Q、E541D、E541I、V543T、V543I、V543A、V543S、V543G、A544G、A544S、A544T、F546W、A547G、M549N、M549Y、M549H、M549T、M549D、M549R、M549A、M549V、I551N、I551T、I551E、I551H、I551L、I551V、I551A、K552R、K552T、K552N、K552Q、K552A、M555Y、M555I、A558I、E559N、E559K、E559D、F562E、F562N、F562Q、F562R、L564F、E565N、E565K、E565S、E565R、V566N、V566K、V566S、V566R、D570A、D570G、D570E、D570V、D570L、D570S、D572A、D572G、D572E、V576A、V577T、I578F、I578T、I578P、I578R、I578T、I578E、I578N、P580G、L585K、L585S、L585E、L585Q、L585R、L585T、L585V、A586K、K588N、K588Q、K588R、K588S、K592A、K592G、K592R、K592T、K592N、K592S、V593I、V593A、E595K、M597L、I601L、F608Y、L613F、V615E、V615T、T616K、R619E、L622V、L623I、K628R、K628I、K628H、M629L、M629A、M629D、M629R、M629V、M629I、V631T、F636I、V637D、V637N、V637R、R638D、R639D、R639N、D640R、D640K、W641R、W641A、W641L、W641F、W641Y、W641V、A642S、K650T、K650C、K650A、K650R、K650S、I655L、I655V、I655A、L656I、A660G、A665E、A665V、A665T、K664R、K664A、K664N、K664Q、K664E、K664S、K664T、K664D、K664G、K664V、K664I、L668I、I673T、E674G、E674D、E674K、R675V、R675C、R675L、R675D, R677E, R677V, R677T, R677N, R677A, S682P, D685R, D685E, D685I, D685L, D685K, T687V, Y689D, Y689N, T690K, T6 90R, T690S, T690M, T690Q, T690V, T690E, T690N, Q691S, Q691T, R695K, S698D, S698K, S698R, S698G, S698Y, S698D, S701 T, S701V, S701A, S701R, S701E, E703R, E703S, E703K, V7071, V707D, V707A, A708V, K711R, K711L, K711T, K711D, E718R, E718V, E718K, V719I, M723S, M723I, M723T, M723N, M723R, M723L, M723A, M723C, I724D, I724V, I729V, T730L, K734I, K73 4G, K734N, G735M, G735R, G735K, G735S, G735P, G735T, G735E, S737R, S737E, Q738D, Q738S, Q738E, Q738, Q738R, T741I, D752Q, D752T, D758N, N759P, N759D, N759K, N759T, N759Y, N759R, I761V, I765V, R767E, I772L, I772Y, I772F, Y774F, Y77 4E, L779G, L779Q, L779D, L779K, L779Y, L779E, K780C, K780F, K780I, K780R, K780Q, K780Y, E781L, E781H, E781S, E781M, E781Q, G782H, G782A, G782K, G782R, Q785L, T786N, S789G, W791R, W791Y, W791D, W791S, W791L, W791A, W791V, and / or F792R.

[0288] In some embodiments, the mutant polymerase has a backbone sequence of NOZ 58130.1 (SEQ ID NO: 1714) and a substitution mutation at position Y14. In some embodiments, the amino acid substitution at position Y14 comprises any of the 20 natural amino acids (i.e., W, I, M, P, F, G, A, V, L, H, R, K, D, E, N, C, S, T, or Q) or an unnatural amino acid known to those skilled in the art.

[0289] In some embodiments, the mutant polymerase has a backbone sequence of NOZ 58130.1 (SEQ ID NO: 1714) and a substitution mutation at position L48. In some embodiments, the amino acid substitution at position L48 comprises any of the 20 natural amino acids (i.e., W, I, M, P, F, G, A, V, L, H, R, K, D, E, N, C, S, T, or Q) or an unnatural amino acid known to those skilled in the art.

[0290] In some embodiments, the mutant polymerase has a backbone sequence of NOZ 58130.1 (SEQ ID NO: 1714) and a substitution mutation at position E76. In some embodiments, the amino acid substitution at position E76 comprises any of the 20 natural amino acids (i.e., W, I, M, P, F, G, A, V, L, H, R, K, D, E, N, C, S, T, or Q) or an unnatural amino acid known to those skilled in the art.

[0291] In some embodiments, the mutant polymerase has a backbone sequence of NOZ 58130.1 (SEQ ID NO: 1714) and a substitution mutation at position V97. In some embodiments, the amino acid substitution at position V97 comprises any of the 20 natural amino acids (i.e., W, I, M, P, F, G, A, L, H, R, K, D, E, N, Y, C, S, T, or Q) or an unnatural amino acid known to those skilled in the art.

[0292] In some embodiments, the mutant polymerase has a backbone sequence of NOZ 58130.1 (SEQ ID NO: 1714) and a substitution mutation at position R 122. In some embodiments, the amino acid substitution at position R 122 comprises any of the 20 natural amino acids (i.e., W, I, M, P, F, G, A, V, L, H, K, D, E, N, Y, C, S, T, or Q) or an unnatural amino acid known to those skilled in the art.

[0293] In some embodiments, the mutant polymerase has a backbone sequence of NOZ 58130.1 (SEQ ID NO: 1714) and a substitution mutation at position Y 124. In some embodiments, the amino acid substitution at position Y 124 comprises any of the 20 natural amino acids (i.e., W, I, M, P, F, G, A, V, L, H, K, D, E, N, Y, C, S, T, or Q) or an unnatural amino acid known to those skilled in the art.

[0294] In some embodiments, the mutant polymerase has a backbone sequence of NOZ 58130.1 (SEQ ID NO: 1714) and a substitution mutation at position R 150. In some embodiments, the amino acid substitution at position R 150 comprises any of the 20 natural amino acids (i.e., W, I, M, P, F, G, A, V, L, H, K, D, E, N, Y, C, S, T, or Q) or an unnatural amino acid known to those skilled in the art.

[0295] In some embodiments, the mutant polymerase has a backbone sequence of NOZ 58130.1 (SEQ ID NO: 1714) and a substitution mutation at position K 254. In some embodiments, the amino acid substitution at position K 254 comprises any of the 20 natural amino acids (i.e., W, I, M, P, F, G, A, V, L, H, K, D, E, N, Y, C, S, T, or Q) or an unnatural amino acid known to those skilled in the art.

[0296] In some embodiments, the mutant polymerase has a backbone sequence of NOZ 58130.1 (SEQ ID NO: 1714) and a substitution mutation at position V 304. In some embodiments, the amino acid substitution at position V 304 comprises any of the 20 natural amino acids (i.e., W, I, M, P, F, G, A, V, L, H, K, D, E, N, Y, C, S, T, or Q) or an unnatural amino acid known to those skilled in the art.

[0297] In some embodiments, the mutant polymerase has a backbone sequence of NOZ 58130.1 (SEQ ID NO: 1714) and a substitution mutation at position C 362. In some embodiments, the amino acid substitution at position C 362 comprises any of the 20 natural amino acids (i.e., W, I, M, P, F, G, A, V, L, H, R, K, D, E, N, Y, S, T, or Q) or an unnatural amino acid known to those skilled in the art.

[0298] In some embodiments, the mutant polymerase has a backbone sequence of NOZ 58130.1 (SEQ ID NO: 1714) and a substitution mutation at position R496. In some embodiments, the amino acid substitution at position R496 comprises any of the 20 natural amino acids (i.e., W, I, M, P, F, G, A, V, L, H, K, D, E, N, Y, C, S, T, or Q) or an unnatural amino acid known to those skilled in the art.

[0299] In some embodiments, the mutant polymerase has a backbone sequence of NOZ 58130.1 (SEQ ID NO: 1714) and a substitution mutation at position A515. In some embodiments, the amino acid substitution at position A515 comprises any of the 20 natural amino acids (i.e., W, I, M, P, F, G, V, L, H, R, K, D, E, N, Y, C, S, T, or Q) or an unnatural amino acid known to those skilled in the art.

[0300] In some embodiments, the mutant polymerase has a backbone sequence of NOZ 58130.1 (SEQ ID NO: 1714) and a substitution mutation at position R537. In some embodiments, the amino acid substitution at position R537 comprises any of the 20 natural amino acids (i.e., W, I, M, P, F, G, A, V, L, H, K, D, E, N, Y, C, S, T, or Q) or an unnatural amino acid known to those skilled in the art.

[0301] In some embodiments, the mutant polymerase has a backbone sequence of NOZ 58130.1 (SEQ ID NO: 1714) and a substitution mutation at position E559. In some embodiments, the amino acid substitution at position E559 comprises any of the 20 natural amino acids (i.e., W, I, M, P, F, G, A, V, L, H, R, K, D, N, Y, C, S, T, or Q) or an unnatural amino acid known to those skilled in the art.

[0302] In some embodiments, the mutant polymerase has a backbone sequence of NOZ 58130.1 (SEQ ID NO: 1714) and has a substitution mutation at position K664. In some embodiments, the amino acid substitution at position K664 includes any of the 20 natural amino acids (i.e., W, I, M, P, F, G, A, V, L, H, R, K, D, N, Y, C, S, T, or Q) or is with an unnatural amino acid known to those skilled in the art.

[0303] In some embodiments, the mutant polymerase has a backbone sequence of NOZ 58130.1 (SEQ ID NO: 1714) and includes an amino acid deletion at any position including D117 (deleted).

[0304] In some embodiments, the mutant polymerase has a backbone sequence of NOZ 58130.1 (SEQ ID NO: 1714) and includes truncations at amino acid positions comprising: Q587 (truncated), M723 (truncated), G773 (truncated), Y774 (truncated), D777 (truncated), E781 (truncated), G782 (truncated), T784 (truncated), Q785 (truncated), R790 (truncated), W791 (truncated), or F792 (truncated). The truncated polymerase can exhibit increased thermostability compared to a non-truncated polymerase having the same backbone sequence. In Table 2, truncations are designated with the symbol "^."

[0305] In some embodiments, the mutant polymerase has a backbone sequence of NOZ 58130.1 (SEQ ID NO: 1714) and includes a truncated portion at the C-terminus. In some embodiments, the truncated mutant polymerase includes a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater than 99% sequence identity to any one of SEQ ID NOs: 1714-2787 and a truncated portion at the C-terminus. In some embodiments, the truncated portion can be 2-5, 5-10, 10-15, or 15-20 amino acids in length. In some embodiments, the truncated portion includes G782-F792 and is 11 amino acids in length. The truncated polymerase can exhibit increased thermostability compared to a non-truncated polymerase with the same backbone sequence. In Table 2, the truncated portion is designated with the letter "X."

[0306] Compositions comprising engineered polymerases

[0307] The present disclosure provides polymerases that are mutated at two or more positions to increase the thermostability of the enzyme, exhibit improved binding of a nucleotide reagent and / or improved binding and incorporation of a nucleotide reagent, increased incorporation rate of a nucleotide analog, improved uracil tolerance, and / or reduced sequence-specific sequencing errors, compared to a wild-type polymerase comprising the amino acid sequence of any one of SEQ ID NOs: 1, 2, 1714, 2789-2793, and 2803. For example, the mutant polymerase exhibits increased thermostability over a temperature range of about 25-50° C., or about 45-75° C., or about 65-90° C. In another example, the mutant polymerase exhibits an increased incorporation rate of a nucleotide analog that includes a chain terminating moiety (e.g., a blocking moiety) at the 2′ sugar position and / or at the 3′ sugar position. The mutant polymerase can exhibit increased uracil tolerance. The mutant polymerase can exhibit improved binding to a complementary nucleotide unit of a multivalent molecule. In some embodiments, the mutant polymerase comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 99% identical or higher to any one of SEQ ID NOs: 1, 2, 1714, 2789-2793, and 2803.

[0308] In some embodiments, the mutant polymerase comprises the backbone sequence of RLF 89458.1 or RLF 78286.1 and comprises the amino acid sequence of any one of SEQ ID NOs: 1-1713, and comprises an exonuclease-conferring - Amino acid substitutions that confer activity: D141A, D141V, D141L, D141I, D141F, D141Y, D141N, D141T, D141S, D141R, D141K, D141Q, D141W, D141E, D141M, D141P, D141G, D141H, or D141C. In some embodiments, the mutant polymerase comprises the backbone sequence of RLF 89458.1 or RLF 78286.1 and comprises the amino acid sequence of any one of SEQ ID NOs: 1-1713, and comprises the following amino acid substitutions that confer exonuclease activity: - Active amino acid substitutions: non-mutated E143 or mutated E143A, E143V, E143L, E143I, E143F, E143Y, E143N, E143T, E143S, E143W, E143M, E143P, E143F, E143G, E143H, E143R, E143K, E143D, E143C or E143Q.

[0309] In some embodiments, the mutant polymerase comprises the backbone sequence of NOZ 58130.1 and comprises the amino acid sequence of any one of SEQ ID NOs: 1714-2787 and 2249-2479, and comprises an amino acid substitution that confers exonuclease activity comprising any one of the following: D168A, D168V, D168L, D168I, D168F, D168Y, D168N, D168K, D168T, D168S, D168W, D168M, D168P, D168G, D168H, D168R, D168E, D168C, or D168Q. In some embodiments, the mutant polymerase comprises the backbone sequence of NOZ 58130 and comprises the amino acid sequence of any one of SEQ ID NOs: 1714-2787, and comprises an amino acid substitution that confers exonuclease activity comprising any one of the following: - Active amino acid substitutions: E170A, E170V, E170L, E170I, E170F, E170Y, E170N, E170K, E170T, E170S, E170W, E170M, E170P, E170G, E170H, E170R, E170D, E170C, or E170Q.

[0310] The present disclosure provides engineered Archaeal Family B DNA or Family A polymerases comprising a Geobacillus stearothermophilus (e.g., Bst DNA polymerase) (SEQ ID NO: 2794), 9°N polymerase (SEQ ID NO: 2795 or 2796) (including THERMINATOR polymerase; SEQ ID NO: 2797), VENT polymerase (SEQ ID NO: 2798), DEEP VENT polymerase (SEQ ID NO: 2799), Pfu polymerase (SEQ ID NO: 2800), and / or Pyrococcus prostratus polymerase (SEQ ID NO: 2801), and RB69 polymerase (SEQ ID NO: 2802) mutated in one or more positions, said polymerases being positionally equivalent (or functionally equivalent sites) to RLF 89458.1 (SEQ ID NO: 1 or 3-1713) or RLF 78286.1 (SEQ ID NO: 2803). Amino acid substitution at any one or any combination of positions in the polymerase backbone sequence of NO: 2): M1, L3, D4, D6, Y7, I8, T9, E10, N11, G12, K13, P14, V15, I16, R17, I18, F19, K20, K21, E22, K23, G24, E25, F26, K27, I28, E29, Y30, D31, R32, N33, F34, E3 5. P36, Y37, I38, Y39, A40, L41, L42, E43, D44, D45, E46, S47, I48, E49, D50, I51, K52, K53, I54, T5 5. R58, G56, E57, R58, H59, G60, K61, K62, V63, I65, I66, R67, V68, E69, K70, V71, K72, K73, K74, F75 , L76, G77, E78, P79, I80, E81, V82, W83, K84, L85, V86, F87, E88, H89, P90, Q91, D92, V93, P94, A95 , I96, R97, D98, A99, I100, R101, S102, H103, P104, A105, V106, R107, E108, I109, F110, E111, Y112 , D113, I114, P115, F116, A117, K118, R119, Y120, L121, I122, D123, K124, L126, V127, P128, M129 , E130, G131, G132, E133, L135, K136, L137, L138, A139, F140, D141, I142, E143, T144, F145, Y146,H147、D150、E151、M159、W173、K174、I176、Y180、A190、I191、K192、L195、L1 98、R199、Q196、P203、V205、L207、Y209、G211、N213、F214、F216、A217、Y218、 I219、C223、E224、G227、L228、F230、T231、I232、G233、R234、S237、E238、P2 39、K240、I241、Q242、R243、M244、G245、D246、R247、A249、E251、L258、Y261、 P262、V264、R265、H266、T267、I268、R269、L270、P271、T272、Y273、T274、L275、E276、A277、V278、V282、F283、K285、K286、K287、E288、K289、V290、Y291、 A292、I295、E297、A298、W299、K300、S301、L305、K306、R307、V308、A309、Y311、M313、D315、R317、A318、Y320、E321、P328、M329、V331、E332、L333、A334、 I337、G338、Q339、V341、D343、S345、S347、S348、T349、G350、N351、L352、V353、W355、Y356、L357、R359、V360、Y362、N365、E366、L367、K371、P372、G373、 E376、Q378、M381、S384、Y385、I386、G388、Y389、E394、G396、A402、Y403、L4 04、F406、R407、S408、L409、Y410、P411、S412、I413、V415、H417、V419、P421、 D422、T423、L424、E427、C428、K429、A434、I436、R440、K443、G447、F448、I4 49、P450、S451、I452、L453、E454、D455、I457、V463、K464、R465、M467、K468、 D472、I474、E475、K476、Y481、R484、A485、L486、K487、I488、N491、S492、Y4 93、Y494、G495、Q497、G498、Y499、P500、K501、S506、K507、E508、C509、E511、S512、V513、T514、G517、R518、H519、I521、T523、A528、E529、K534、V535、A538、E539、D541、G542、I547、I555、P552、S557、K558、A559、K560、K 561、L563、K564、H565、E568、K569、G572、M573、E575、E577、L583、G585、F586、V588、T589、K592、L595、I596、H601、T604、G606、V609、V610、R6 11、R612、D613、E616、I617、K619、E620、T621、Q622、A623、K624、V625、L626、E627、V628、I629、L630、R631、E632、G633、S634、I635、E636、K63 7、A638、A639、G640、I641、V642、V645、V646、E647、D648、L649、A650、N 651、Y652、R653、V654、V656、E657、K658、I660、H662、E663、Q664、I665、 T666、R667、E668、K670、D671、Y672、K673、A674、T675、G676、P677、H67 8、V679、A680、I681、A682、K683、R684、L685、Q686、A687、R688、G689、I 690, K691, V692, K693, P694, T696, I698, S699, Y700, V701, V702, L703, K704, G705, S706, K707, K708, I709, D711, R712, V713, I714, L715, F7 16、D717、E718、D720、S721、S722、R723、K725、Y726、P728、Y730、Y731、I732、H733、N734、Q735、V736、P738、A739、V740、L741、R742、I743、L74 4、E745、A746、F747、G748、Y749、K750、E751、K752、D753、L754、E755、Y756、Q757、R758、M759、K760、Q761、T762、G763、L764、G765、A766、W767、L768 and / or M770. Based on the sequence alignment shown in Figure 34, those skilled in the art can determine the positionally equivalent positions (or functionally equivalent sites) in Geobacillus stearothermophilus (e.g., Bst DNA polymerase) (SEQ ID NO: 2794), 9°N polymerase (SEQ ID NO: 2795 or 2796) (including THERMINATOR polymerase; SEQ ID NO: 2797), VENT polymerase (SEQ ID NO: 2798), DEEP VENT polymerase (SEQ ID NO: 2799), Pfu polymerase (SEQ ID NO: 2800) and / or Pyrococcus aquatica polymerase (SEQ ID NO: 2801) and RB69 polymerase (SEQ ID NO: 2802).

[0311] The present disclosure provides engineered Archaeal Family B DNA or Family A polymerases comprising a Geobacillus stearothermophilus (e.g., Bst DNA polymerase) (SEQ ID NO: 2794), a 9°N polymerase (SEQ ID NO: 2795 or 2796) (including a THERMINATOR polymerase; SEQ ID NO: 2797), a VENT polymerase (SEQ ID NO: 2798), a DEEP VENT polymerase (SEQ ID NO: 2799), a Pfu polymerase (SEQ ID NO: 2800), and / or a Pyrococcus prostratus polymerase (SEQ ID NO: 2801), and a RB69 polymerase (SEQ ID NO: 2802) mutated in one or more positions, said polymerases being positionally equivalent (or functionally equivalent sites) to a polymerase having a NOZ 58130.1 (SEQ ID NO: 2802) comprising: NOS: 1714-2787) of the backbone sequence of any one of the following positions: Y14, E18, F26, L27, G29, F34, V35, V36, F41, S42, P43, F45, L48, P49, R55, L61, A62, S63, A65, E67, I69, K71, V72, E76, K77, T82, P83, R84, V85, T90 , V91, S92, H93, P94, Q95, D96, V97, P98, R99, I100, R101, E102, R103, R105, E108, D111, L112, I113, N1 14. E115, H116, D117, I118, V121, R122, R123, Y124, I126, I130, P132, L133, W135, G145, R150, E153, E1 56. E157, E158, R163, V164, A165, V167, D168, I169, E170, V171, Y172, N173T, P174, E184, I186, M187, V190, L200, V205, M220, K229, Y233, I236, V237, G238, N240, F244, Y248, R252, L253, K254, L260, L269, G275, A276, L277, I282, A286, V288, L290, Y291, P292, I293, V294, R295, H297, V298, N301, S302, Y303, V304, S307, V309, L312, L313, G314, E318, K319, L320, D321, G322, L325, F326, T327, W329, D330, E331,K335, L338, L339, A343, Y342, L344, D346, A352, A354, K356, L360, C362, I367, A376, M378, T379, V384, L387, M389, R390, T39 3. L398, I399, P400, E407, Y408, A409, R413, Y416, R422, V429, V434, F435, D436, F437, S439, L440, Y441, P442, S443, I444, I 445, V446, T454, A465, S473, F479, I480, R496, D504, F511, A515, S522, F523, Y524, Y526, M527, R536, R537, E538, C539, E541 , V543, A544, F546, A547, M549, I551, K552, M555, A558, E559, F562, L564, E565, V566, D570, D572, V576, V577, I578, P580, L58 5. A586, Q587, K588, K592, V593, E595, M597, I601, F608, L613, V615, T616, R619, L622, L623, K628, M629, V631, F636, V637, R 638, R639, D640, W641, A642, K650, I655, L656, A660, K664, A665, L668, I673, E674, R675, R677, S682, D685, T687, Y689, T690, 782, I783, T784, Q785, T786, S787, L788, S789, R790, W791, and / or F792. Based on the sequence alignment shown in FIG35 , one skilled in the art can determine that the enzymes of Geobacillus stearothermophilus (e.g., Bst DNA polymerase) (SEQ ID NO: 2794), 9°N polymerase (SEQ ID NO: 2795 or 2796) (including THERMINATOR polymerase; SEQ ID NO: 2797), VENT polymerase (SEQ ID NO: 2798),Positionally equivalent positions (or functionally equivalent sites) in DEEPVENT polymerase (SEQ ID NO: 2799), Pfu polymerase (SEQ ID NO: 2800), and / or Pyrococcus aquatica polymerase (SEQ ID NO: 2801) and RB69 polymerase (SEQ ID NO: 2802).

[0312] The present disclosure provides engineered Archaeal Family B DNA or Family A polymerases comprising a Geobacillus stearothermophilus (e.g., Bst DNA polymerase) (SEQ ID NO: 2794), a 9°N polymerase (SEQ ID NO: 2795 or 2796) (including a THERMINATOR polymerase; SEQ ID NO: 2797), a VENT polymerase (SEQ ID NO: 2798), a DEEP VENT polymerase (SEQ ID NO: 2799), a Pfu polymerase (SEQ ID NO: 2800), and / or a Pyrococcus prostratus polymerase (SEQ ID NO: 2801), and a RB69 polymerase (SEQ ID NO: 2802) mutated in one or more positions, said polymerases being positionally equivalent (or functionally equivalent sites) to an RMF 90817.1 (SEQ ID NO: 2802) having a sequence comprising: NO: 2789) backbone sequence) at any one or any combination of positions in the polymerase: Y11, D15, F23, K25, I28, L29, F34, Q35, P36, F38, H43, E49, G55, A56, V57, R62, R67, I75, L76, S77, H78, P79, S80, E81, V82, P83, K84, I85, R86, E87, E88 8. R90, E96, I98, E100, H101, D102, I103, A106, R108, I111, P117, L118, E138, G139, R144, V145, M1 46. ​​D149, I150, E151, T152, A234, Y272, C307, R312, E333, A357, V365, L368, F374, L390, V415, D41 7. F418, S420, L421, Y422, P423, I425, V427, T435, P445, F459, A496, S503, F504, Y505, M508, K518 , E519, C520, S523, V524, T525, M530, T532, D551, D553, V559, R566, A567, M568, R576, I596, T597, 767, S768, L769, Q770, R771, W772, and / or F773.Based on the sequence alignment shown in Figure 36, one skilled in the art can determine positionally equivalent positions (or functionally equivalent sites) in Bacillus stearothermophilus (e.g., Bst DNA polymerase) (SEQ ID NO: 2794), 9°N polymerase (SEQ ID NO: 2795 or 2796) (including THERMINATOR polymerase; SEQ ID NO: 2797), VENT polymerase (SEQ ID NO: 2798), DEEP VENT polymerase (SEQ ID NO: 2799), Pfu polymerase (SEQ ID NO: 2800) and / or Pyrococcus pyocyaneus polymerase (SEQ ID NO: 2801) and RB69 polymerase (SEQ ID NO: 2802).

[0313] The present disclosure provides engineered Archaeal Family B DNA or Family A polymerases comprising amino acid substitutions at any one or any combination of positions of Geobacillus stearothermophilus (e.g., Bst DNA polymerase) (SEQ ID NO: 2794), 9°N polymerase (SEQ ID NO: 2795 or 2796) (including THERMINATOR polymerase; SEQ ID NO: 2797), VENT polymerase (SEQ ID NO: 2798), DEEP VENT polymerase (SEQ ID NO: 2799), Pfu polymerase (SEQ ID NO: 2800), and / or Pyrococcus aquatica polymerase (SEQ ID NO: 2801), and RB69 polymerase (SEQ ID NO: 2802) mutated in one or more positions that are positionally equivalent (or functionally equivalent sites) to a polymerase having a backbone sequence of MBC 7218772.1 (SEQ ID NO: 2790) comprising I10, C468, and / or T560. Based on the sequence alignment shown in Figure 37, one skilled in the art can determine positionally equivalent positions (or functionally equivalent sites) in Bacillus stearothermophilus (e.g., Bst DNA polymerase) (SEQ ID NO: 2794), 9°N polymerase (SEQ ID NO: 2795 or 2796) (including THERMINATOR polymerase; SEQ ID NO: 2797), VENT polymerase (SEQ ID NO: 2798), DEEP VENT polymerase (SEQ ID NO: 2799), Pfu polymerase (SEQ ID NO: 2800) and / or Pyrococcus pyocyaneus polymerase (SEQ ID NO: 2801) and RB69 polymerase (SEQ ID NO: 2802).

[0314] The present disclosure provides engineered Archaeal Family B DNA or Family A polymerases comprising a Geobacillus stearothermophilus (e.g., Bst DNA polymerase) (SEQ ID NO: 2794), a 9°N polymerase (SEQ ID NO: 2795 or 2796) (including a THERMINATOR polymerase; SEQ ID NO: 2797), a VENT polymerase (SEQ ID NO: 2798), a DEEP VENT polymerase (SEQ ID NO: 2799), a Pfu polymerase (SEQ ID NO: 2800), and / or a Pyrococcus prostratus polymerase (SEQ ID NO: 2801), and a RB69 polymerase (SEQ ID NO: 2802) mutated in one or more positions, said polymerases being positionally equivalent (or functionally equivalent sites) to a WP 175059460.1 (SEQ ID NO: 2802) having a sequence comprising: NO: 2791) backbone sequence amino acid substitution at any one or any combination of positions in the polymerase: Y7, D11, I51, K61, V93, A117, M129, D141, I142, E143, T144, A223, E302, E323, D407, F408, S410, L411, Y412, P413 , R487, A488, S495, Y496, K510, T517, I524, K562, A563, R564, S572, T593, R605, K652, D675, K695, T700, R712, R759, Y760, Q761, S762, S763, K764, Q765, and / or T766. Based on the sequence alignment shown in Figure 38, one skilled in the art can determine positionally equivalent positions (or functionally equivalent sites) in Bacillus stearothermophilus (e.g., Bst DNA polymerase) (SEQ ID NO: 2794), 9°N polymerase (SEQ ID NO: 2795 or 2796) (including THERMINATOR polymerase; SEQ ID NO: 2797), VENT polymerase (SEQ ID NO: 2798), DEEP VENT polymerase (SEQ ID NO: 2799), Pfu polymerase (SEQ ID NO: 2800) and / or Pyrococcus pyocyaneus polymerase (SEQ ID NO: 2801) and RB69 polymerase (SEQ ID NO: 2802).

[0315] The present disclosure provides engineered Archaeal Family B DNA or Family A polymerases comprising a Geobacillus stearothermophilus (e.g., Bst DNA polymerase) (SEQ ID NO: 2794), a 9°N polymerase (SEQ ID NO: 2795 or 2796) (including a THERMINATOR polymerase; SEQ ID NO: 2797), a VENT polymerase (SEQ ID NO: 2798), a DEEP VENT polymerase (SEQ ID NO: 2799), a Pfu polymerase (SEQ ID NO: 2800), and / or a Pyrococcus prostratus polymerase (SEQ ID NO: 2801), and a RB69 polymerase (SEQ ID NO: 2802) mutated in one or more positions, said polymerases being positionally equivalent (or functionally equivalent sites) to a KUO 42443.1 (SEQ ID NO: 2802) having a sequence comprising: : SEQ ID NO: 2792). The amino acid substitutions may be made at any one or any combination of positions in the backbone sequence of a polymerase comprising the following: Y7, D170, E172, T557, and / or S558. Based on the sequence alignment shown in FIG39, one skilled in the art can determine positionally equivalent positions (or functionally equivalent sites) in Geobacillus stearothermophilus (e.g., Bst DNA polymerase) (SEQ ID NO: 2794), 9°N polymerase (SEQ ID NO: 2795 or 2796) (including THERMINATOR polymerase; SEQ ID NO: 2797), VENT polymerase (SEQ ID NO: 2798), DEEP VENT polymerase (SEQ ID NO: 2799), Pfu polymerase (SEQ ID NO: 2800), and / or Pyrococcus prostratus polymerase (SEQ ID NO: 2801), and RB69 polymerase (SEQ ID NO: 2802).

[0316] The present disclosure provides engineered Archaeal Family B DNA or Family A polymerases comprising a Geobacillus stearothermophilus (e.g., Bst DNA polymerase) (SEQ ID NO: 2794), a 9°N polymerase (SEQ ID NO: 2795 or 2796) (including a THERMINATOR polymerase; SEQ ID NO: 2797), a VENT polymerase (SEQ ID NO: 2798), a DEEP VENT polymerase (SEQ ID NO: 2799), a Pfu polymerase (SEQ ID NO: 2800), and / or a Pyrococcus prostratus polymerase (SEQ ID NO: 2801), and a RB69 polymerase (SEQ ID NO: 2802) mutated in one or more positions, said polymerases being positionally equivalent (or functionally equivalent sites) to an Archaeal Family B DNA or Family A polymerase having a sequence comprising NOZ 77387.1 (SEQ ID NO: 2794). 40 ), those skilled in the art can determine the positional equivalents (or functionally equivalent sites) in Geobacillus stearothermophilus (e.g., BstDNA polymerase) (SEQ ID NO: 2794), 9°N polymerase (SEQ ID NO: 2795 or 2796) (including THERMINATOR polymerase; SEQ ID NO: 2797), VENT polymerase (SEQ ID NO: 2798), DEEP VENT polymerase (SEQ ID NO: 2799), Pfu polymerase (SEQ ID NO: 2800), and / or Pyrococcus aquaticus polymerase (SEQ ID NO: 2801), and RB69 polymerase (SEQ ID NO: 2802).

[0317] The present disclosure provides polymerases operably linked to a detectable reporter moiety. Any of the polymerases described herein can be labeled with a detectable reporter moiety, comprising a polymerase having a mutant amino acid sequence backbone of any polymerase described herein, comprising any of SEQ ID NOs: 1-2787, 2789-2793, Geobacillus stearothermophilus (e.g., Bst DNA polymerase) (SEQ ID NO: 2794), 9°N polymerase (SEQ ID NO: 2795 or 2796) (including THERMINATOR polymerase; SEQ ID NO: 2797), VENT polymerase (SEQ ID NO: 2798), DEEP VENT polymerase (SEQ ID NO: 2799), Pfu polymerase (SEQ ID NO: 2800), and / or Pyrococcus aquatica polymerase (SEQ ID NO: 2801), RB69 polymerase (SEQ ID NO: 2802), and Phi29 (SEQ ID NO: 2803).

[0318] In some embodiments, the detectable reporter gene portion produces a detectable signal caused by a chemical or physical change (e.g., heat, light, electricity, pH, salt concentration, enzymatic activity, or adjacent events such as FRET). In some embodiments, the detectable reporter gene portion includes a luminescent moiety, a fluorescent moiety, or a quencher. In some embodiments, the detectable portion includes a fluorescent moiety that behaves as a FRET donor or acceptor. The detectable reporter gene portion can be attached to a polymerase at the N-terminus, the C-terminus, or any internal position. The detectable reporter gene portion is attached to a polymerase in a manner that does not interfere with the ability of the polymerase to bind to a nucleic acid template molecule, a nucleic acid primer, or a nucleotide. The detectable reporter gene portion is attached to a polymerase in a manner that does not interfere with the catalytic activity of the polymerase (comprising nucleotide incorporation).

[0319] The present disclosure provides recombinant fusion polypeptides comprising any of the DNA polymerases described herein operably linked to any one or any combination of two or more exogenous amino acid sequences for affinity purification, cleavage, and / or solubilization. In some embodiments, the recombinant fusion polypeptide includes a polymerase having a mutant amino acid sequence backbone of any polymerase described herein, including any of SEQ ID NOs: 1-2787, 2789-2793, Geobacillus stearothermophilus (e.g., Bst DNA polymerase) (SEQ ID NO: 2794), 9°N polymerase (SEQ ID NO: 2795 or 2796) (including THERMINATOR polymerase; SEQ ID NO: 2797), VENT polymerase (SEQ ID NO: 2798), DEEP VENT polymerase (SEQ ID NO: 2799), Pfu polymerase (SEQ ID NO: 2800), and / or Pyrococcus prostrata polymerase (SEQ ID NO: 2801), and RB69 polymerase (SEQ ID NO: 2802), and Phi29 (SEQ ID NO: 2803), operably linked to an affinity purification tag, a cleavage tag, and / or a solubility tag.

[0320] In some embodiments, the recombinant fusion polypeptide comprises any of a wild-type and mutant polymerase, wherein the wild-type and mutant polymerase is operably linked to at least one affinity purification tag sequence at its N-terminus and / or C-terminus, wherein the affinity purification tag sequence comprises a histidine tag (e.g., a His-tag), a FLAG tag, a T7 tag, a StrepII tag, an S tag (e.g., from pancreatic RNase A), an HA tag (e.g., from human influenza hemagglutinin protein), and / or a c-Myc tag. In some embodiments, the affinity purification tag sequence comprises a plurality of histidine residues, e.g., 3-10 histidine residues (SEQ ID NO: 2851). In some embodiments, the affinity purification tag sequence comprises a plurality of consecutive histidine residues, e.g., 3-10 consecutive histidine residues (SEQ ID NO: 2851).

[0321] In some embodiments, the wild-type polymerase and the engineered polymerase are recombinant proteins that can be expressed by host cells containing cloned expression vectors. It has been previously demonstrated that recombinant proteins carrying affinity purification His tags can be post-translationally modified by E. coli expression host cells. Examples of post-translational modifications include N-phosphogluconylation, in which a gluconic acid derivative is attached to a His tag at the N-terminus of the recombinant protein. N-phosphogluconylation is catalyzed by the E. coli 6-phosphoglucono-1,5-lactone pathway (Geoghegan 1999 Analytical Biochemistry 267: 169-184; Aon 2008 Applied and Environmental Microbiology 74(4): 950-958). Another example of a post-translational modification includes N-glucosylation, in which the N-phosphogluconylated recombinant protein further modifies the phosphatase of the E. coli host cell. N-glucosaminidation of recombinant proteins originates from non-enzymatic acylation modifications and is known to reduce protein activity, increase susceptibility to oxidation, and reduce protein crystallization. Therefore, N-phosphoglucosaminidation and / or N-glucosaminidation are undesirable post-translational modifications that may adversely affect the production and shelf life of recombinant proteins containing engineered polymerases.

[0322] It has been previously demonstrated that N-glucosylation can be reduced by changing host cell culture conditions, adapting modified purification methods, or site-directed mutagenesis of amino acid residues near the N-terminus of recombinant proteins. For example, recombinant proteins carrying an N-terminal His tag, in which the amino acid sequence of the His tag is modified, exhibit reduced N-glucosylation (Martos-Maldonado et al., 2018, Nature Communications, 9:3307 (doi: 10.1038 / s41467-018-05695-3)).

[0323] In some embodiments, any engineered polymerase comprising any of SEQ ID NOs: 1-2787 can be linked to a conventional His tag at the N-terminus or C-terminus. In some embodiments, the conventional His tag comprises the sequence MGSSHHHHHH (SEQ ID NO: 2815), MGSSHHHHHHGS (SEQ ID NO: 2816), or MGSSHHHHHHSSG (SEQ ID NO: 2817).

[0324] In some embodiments, the engineered polymerase can be linked to a modified His tag at the N-terminus or C-terminus to produce a tagged polymerase that exhibits reduced N-phosphogluconylation and / or N-gluconylation when expressed by an expression host cell containing the cloned expression vector, wherein the modified His tag comprises the sequence MGSDKIHHHHHH (SEQ ID NO: 2818), MAHHHHHH (SEQ ID NO: 2819), MHHHHHH (SEQ ID NO: 2820), MRGSPHHHHHH (SEQ ID NO: 2821), MRGSHHHHHH (SEQ ID NO: 2822), MHHHHHHSSG (SEQ ID NO: 2823), or GGHHHHHH (SEQ ID NO: 2824).

[0325] In some embodiments, the engineered polymerase can be linked to a His tag at the N-terminus or C-terminus that is modified by replacing one or more amino acid residues susceptible to N-phosphoglucosaminidation and / or N-glucosaminidation. In some embodiments, the engineered polymerase can be linked to a modified N-terminal His tag at the N-terminus or C-terminus to generate a tagged polymerase that exhibits reduced N-phosphoglucosaminidation and / or N-glucosaminidation when expressed by an expression host cell containing a cloned expression vector, wherein the modified His tag comprises a glycine substituted at position 2 with a phenylalanine (F) or a proline (P). In some embodiments, the modified His tag comprises the sequence MFGSDKIHHHHHH (SEQ ID NO: 2825), MPGSDKIHHHHHH (SEQ ID NO: 2826), MFGSSHHHHHHGS (SEQ ID NO: 2827), MPGSSHHHHHHGS (SEQ ID NO: 2828), MFRGSPHHHHHH (SEQ ID NO: 2829), MPRGSPHHHHHH (SEQ ID NO: 2830), MFRGSHHHHHH (SEQ ID NO: 2831), MPRGSHHHHHH (SEQ ID NO: 2832), MFGSSHHHHHHSSGLVPRGSH (SEQ ID NO: 2846), MPGSSHHHHHHSSGLVPRGSH (SEQ ID NO: 2847), MPSSHHHHHHSSGLVPRGS (SEQ ID NO: 2848), or MPGSSHHHHHHSSGLVPRGS (SEQ ID NO: 2849). NO: 2849).

[0326] In some embodiments, the tagged polymerase that exhibits reduced N-phosphoglucosamylation and / or N-glucosamylation also exhibits increased enzymatic activity and / or reduced oxidation compared to a tagged polymerase carrying an unmodified His tag. In some embodiments, the tagged polymerase that exhibits reduced N-phosphoglucosamylation and / or N-glucosamylation also exhibits an increased fraction of active polymerase in a preparation batch (e.g., a manufacturing batch) and / or an increased shelf life compared to a tagged polymerase carrying an unmodified His tag.

[0327] In some embodiments, the engineered polymerase comprises any of SEQ ID NOs: 1-2787 and 2789-2793 operably linked to at least one polypeptide cleavage sequence at its N-terminus and / or C-terminus, or the polypeptide cleavage sequence can be positioned between the affinity tag sequence and the N-terminus or C-terminus of the polymerase sequence. In some embodiments, the polypeptide cleavage sequence can be recognized and cleaved by a protease or by reducing conditions. In some embodiments, the polypeptide cleavage sequence comprises a thrombin cleavage sequence, a TEV cleavage sequence (e.g., from tobacco etch virus, including AcTEV and ProTEV), a factor Xa cleavage sequence, an enterokinase cleavage sequence, and a SUMO cleavage sequence (e.g., small ubiquitin-like modifier including Ulp1, Senp2, and SUMOstar).

[0328] In some embodiments, the engineered polymerase can be linked to a tag comprising a thrombin cleavage sequence at the N-terminus or C-terminus. For example, a conventional thrombin cleavage sequence comprises the sequence LVPRGS (SEQ ID NO: 2833), wherein cleavage occurs after arginine (R).

[0329] In some embodiments, the engineered polymerase can be linked to a tag at the N-terminus or C-terminus that includes a modified thrombin cleavage sequence to generate a tagged polymerase that exhibits reduced N-phosphogluconylation and / or N-gluconylation when expressed by an expression host cell containing the cloned expression vector, wherein the modified thrombin cleavage sequence includes an additional amino acid residue inserted after the arginine at position 4. In some embodiments, the modified thrombin cleavage sequence includes the sequence LVPRAGSH (SEQ ID NO: 2834), LVPRGGSH (SEQ ID NO: 2835), or LVPRSGSH (SEQ ID NO: 2836).

[0330] In some embodiments, the engineered polymerase can be linked to a His tag comprising a thrombin cleavage sequence at the N-terminus or the C-terminus. In some embodiments, the His tag comprising a conventional thrombin cleavage sequence comprises the following sequence:

[0331] MGSSHHHHHHSSGLVPRGSH (SEQ ID NO: 2837) or MGSSHHHHHHSSGLVPRGS (SEQ ID NO: 2838). In some embodiments, the His tag comprising a thrombin cleavage sequence comprises the following sequence: MGSSHHHHHHSSGLHHRSGLVPRGSH (SEQ ID NO: 2839), MGSSHHHHHHSSGLVPRQS (SEQ ID NO: 2840), or MGSSHHHHHHSSGLVPGGSH (SEQ ID NO: 2841).

[0332] In some embodiments, the engineered polymerase can be linked at the N-terminus or C-terminus to a His tag comprising a modified thrombin cleavage sequence to generate a tagged polymerase that exhibits reduced N-phosphogluconylation and / or N-gluconylation when expressed by an expression host cell containing the cloned expression vector, wherein the His tag comprising a modified thrombin cleavage sequence comprises the sequence MGSSHHHHHHSSGLVPRAGSH (SEQ ID NO: 2842), MGSSHHHHHHSSGLVPRGGSH (SEQ ID NO: 2843), or MGSSHHHHHHSSGLVPRSGSH (SEQ ID NO: 2844).

[0333] In some embodiments, the recombinant fusion polypeptide comprises any of the wild-type and mutant polymerases described herein operably linked at its N-terminus and / or C-terminus to at least one exogenous amino acid sequence comprising maltose binding protein (MBP), small ubiquitin-like modifier (SUMO), and glutathione S-transferase (GST) to improve solubility.

[0334] The present disclosure provides a composition comprising: one or more mutant polymerases and at least one nucleic acid template molecule and at least one nucleic acid primer. In some embodiments, the one or more mutant polymerases may or may not bind to at least one nucleic acid template molecule and at least one nucleic acid primer. In some embodiments, the primer provides a starting site for nucleotide polymerization. In some embodiments, the primer includes a 3' extendable end for a polymerase-catalyzed nucleotide incorporation reaction, or the primer includes a 3' non-extendable end. In some embodiments, the nucleic acid template molecule comprises at least one uridine nucleotide or lacks a uridine nucleotide. In some embodiments, the mutant polymerase comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99% identical or a higher level of sequence identity to any one of SEQ ID NOs: 1-2787 and 2789-2793. In some embodiments, the mutant polymerase comprises a sequence that confers exonuclease- In some embodiments, the polymerase comprises at least one mutation that increases the thermostability of the enzyme, improves nucleotide reagent binding and / or improves nucleotide reagent binding and incorporation, increases the incorporation rate of nucleotide analogs, improves uracil tolerance, and / or reduces sequence-specific sequencing errors, compared to its corresponding wild-type polymerase.

[0335] The present disclosure provides a composition comprising: one or more mutant polymerases and at least one nucleic acid template molecule having a self-priming 3' end. In some embodiments, the one or more mutant polymerases may or may not bind to at least one nucleic acid template molecule having a self-priming 3' end. In some embodiments, the self-priming 3' end of the template molecule provides a starting site for nucleotide polymerization. In some embodiments, the nucleic acid template molecule comprises at least one uridine nucleotide or lacks a uridine nucleotide. In some embodiments, the mutant polymerase comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99% identical or a higher level of sequence identity to any one of SEQ ID NOs: 1-2787 and 2789-2793. In some embodiments, the mutant polymerase comprises an amino acid sequence that confers a nuclease exonuclease - Active amino acid substitutions. In some embodiments, the polymerase comprises at least one mutation that increases the thermostability of the enzyme, increases the rate of incorporation of nucleotide analogs, and / or increases uracil tolerance compared to its corresponding wild-type polymerase.

[0336] In some embodiments, the composition comprises: one or more mutant polymerases that bind to a nucleic acid duplex, each nucleic acid duplex comprising a nucleic acid template hybridized to a nucleic acid primer, thereby forming a composite polymerase. In some embodiments, the primer provides an initiation site for nucleotide polymerization. In some embodiments, the mutant polymerase binds to a nucleic acid template molecule having a self-priming 3′ end to form a composite polymerase that lacks a separate primer molecule. In some embodiments, the nucleic acid template molecule comprises at least one uridine nucleotide or lacks a uridine nucleotide. In some embodiments, the mutant polymerase comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99% identical, or more identical to any one of SEQ ID NOs: 1-2787 and 2789-2793. In some embodiments, the mutant polymerase is a recombinant polymerase.

[0337] In some embodiments, the composition includes one or more mutant polymerases, at least one nucleic acid template molecule, and a start site for nucleotide polymerization, wherein the mutant polymerase is in solution, the nucleic acid template molecule is in solution, and the start site (e.g., a primer) is in solution. In some embodiments, the composition includes one or more mutant polymerases, at least one nucleic acid template molecule, and a start site for nucleotide polymerization, wherein the composition includes any combination of the mutant polymerase in solution, the nucleic acid template molecule in solution or fixed to a support, and the start site (e.g., a primer) in solution or fixed to a support. In some embodiments, the composition includes one or more mutant polymerases, at least one nucleic acid template molecule, and a start site for nucleotide polymerization, wherein the composition includes any combination of the mutant polymerase in solution or fixed to a support, the nucleic acid template molecule in solution or fixed to a support, and the start site (e.g., a primer) in solution or fixed to a support.

[0338] In some embodiments, the mutant polymerase exhibits increased thermostability compared to a wild-type polymerase or compared to an engineered polymerase having the same backbone sequence (comprising a polymerase having an amino acid sequence of any one of SEQ ID NOs: 1-2787 and 2789-2793). For example, the mutant polymerase exhibits increased thermostability over a temperature range of about 25-50°C or about 45-90°C.

[0339] In some embodiments, the mutant polymerase exhibits an increased rate of incorporation of a nucleotide analog, wherein the nucleotide analog includes a chain terminating moiety (e.g., a blocking moiety) at the sugar 2' position and / or at the sugar 3' position, compared to a wild-type polymerase or compared to an engineered polymerase with the same backbone sequence (comprising a polymerase having an amino acid sequence of any one of SEQ ID NOs: 1-2787 and 2789-2793).

[0340] In some embodiments, the mutant polymerase exhibits increased uracil tolerance compared to the wild-type polymerase, compared to the wild-type polymerase, or compared to an engineered polymerase with the same backbone sequence (comprising a polymerase having the amino acid sequence of any one of SEQ ID NOs: 1-2787 and 2789-2793).

[0341] In some embodiments, the mutant polymerase exhibits an increased ability to bind to complementary nucleotide units of a multivalent molecule compared to a wild-type polymerase or compared to an engineered polymerase with the same backbone sequence (comprising a polymerase having an amino acid sequence of any one of SEQ ID NOs: 1-2787 and 2789-2793).

[0342] In some embodiments, the mutant polymerase exhibits reduced switching from a polymerizing conformation to an exonuclease conformation (e.g., reduced switching to an editing mode) compared to a wild-type polymerase or compared to an engineered polymerase with the same backbone sequence (comprising a polymerase having an amino acid sequence of any one of SEQ ID NOs: 1-2787 and 2789-2793).

[0343] In some embodiments, a composition comprises: one or more mutant polymerases and a plurality of nucleic acid duplexes, each nucleic acid duplex comprising a nucleic acid template hybridized to a nucleic acid primer. In some embodiments, the one or more polymerases and the nucleic acid duplexes further comprise a nucleotide reagent. The one or more mutant polymerases may or may not bind to the nucleic acid duplexes. The one or more mutant polymerases may or may not bind to the nucleotide reagent. In some embodiments, the one or more mutant polymerases bind to the nucleic acid duplexes comprising the nucleic acid template hybridized to the nucleic acid primers, thereby forming a composite polymerase. In some embodiments, the composite polymerase further comprises a nucleotide reagent. In some embodiments, the mutant polymerase comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99% identical, or more identical to any one of SEQ ID NOs: 1-2787 and 2789-2793. In some embodiments, the mutant polymerase is a recombinant polymerase.

[0344] In certain embodiments, nucleotide reagents include any one or any combination of nucleotides and / or multivalent molecules. In certain embodiments, nucleotides include typical nucleotides. In certain embodiments, nucleotides include unlabeled nucleotides. In certain embodiments, nucleotides include detectably labeled nucleotides, and each detectably labeled nucleotide includes a detectable reporter gene portion engaged with a phosphate moiety in the phosphate moiety of a core base or phosphate chain. In certain embodiments, nucleotides include nucleotides carrying removable or non-removable chain termination moieties. In certain embodiments, reversible chain termination nucleotides can be detectably labeled or unlabeled. In certain embodiments, independent multivalent molecules include a central core attached to a plurality of polymer arms, and each polymer arm has a nucleotide unit at the end of the arm.

[0345] In some embodiments, the composite polymerase further comprises a nucleotide reagent comprising a nucleotide. In some embodiments, the nucleotide can be bound to the composite polymerase without being incorporated. In some embodiments, the complementary nucleotide can be bound to the composite polymerase without undergoing polymerase-catalyzed incorporation to form a ternary complex, wherein the complementary nucleotide binds to the 3' end of the primer at a position opposite to the complementary nucleotide in the template strand.

[0346] In certain embodiments, at least one nucleotide in a plurality of nucleotides includes a base, a sugar and at least one phosphate group. In certain embodiments, at least one nucleotide in a plurality of nucleotides includes an aromatic base, a five-carbon sugar (e.g., ribose or deoxyribose) and one or more phosphate groups (e.g., 1-10 phosphate groups). A plurality of nucleotides may include at least one type of nucleotide selected from the group consisting of: dATP, dGTP, dCTP, dTTP and dUTP. A plurality of nucleotides may include a mixture of any combination of two or more types of nucleotides selected from the group consisting of: dATP, dGTP, dCTP, dTTP and / or dUTP.

[0347] In some embodiments, at least one of the plurality of nucleotides comprises a chain of one, two, or three phosphorus atoms, wherein the chain is typically attached to the 5' carbon of the sugar moiety via an ester or phosphoramide bond. In some embodiments, at least one of the plurality of nucleotides is an analog with a phosphorus chain in which the phosphorus atoms are linked together with an intervening O, S, NH, methylene, or ethylene. In some embodiments, the phosphorus atoms in the chain comprise substituted side groups (comprising O, S, or BH3). In some embodiments, the chain comprises a phosphate group substituted with an analog comprising a phosphoramide, phosphorothioate, phosphorodithioate, and O-methylphosphoramidite group.

[0348] In some embodiments, at least one nucleotide in a plurality of nucleotides includes a nucleotide analog having a chain termination portion (e.g., a blocking portion) at the sugar 2' position, at the sugar 3' position, or at the sugar 2' and 3' positions. In some embodiments, the chain termination portion can inhibit the polymerase-catalyzed incorporation of subsequent nucleotide units or free nucleotides in the nascent chain during the primer extension reaction. In some embodiments, the chain termination portion is attached to the 3' sugar hydroxyl position, wherein the sugar includes a ribose or deoxyribose moiety. In some embodiments, the chain termination portion can be removed / cut from the 3' sugar hydroxyl position to produce a nucleotide with a 3'OH sugar group, which can be extended with subsequent nucleotides in a polymerase-catalyzed nucleotide incorporation reaction. In some embodiments, the chain termination portion includes an alkyl, alkenyl, alkynyl, allyl, aryl, benzyl, an azide group, an amine group, an amide group, a ketone group, an isocyanate group, a phosphate group, a thio group, a disulfide group, a carbonate group, a urea group, a silyl group, or an acetal group. In some embodiments, the chain termination portion can be cut / removed from the nucleotide, for example, by reacting the chain termination portion with a chemical agent, a pH change, light, or heat. In some embodiments, the chain-terminating moieties alkyl, alkenyl, alkynyl, and allyl groups can be cleaved with tetrakis(triphenylphosphine)palladium(0) (Pd(PPh3)4), with piperidine, or with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ). In some embodiments, the chain-terminating moieties aryl and benzyl groups can be cleaved with H2Pd / C. In some embodiments, the chain-terminating moieties amine, amide, ketone, isocyanate, phosphate, thio, disulfide can be cleaved with a phosphine or thiol group (including β-mercaptoethanol or dithiothreitol (DTT)). In some embodiments, the chain-terminating moiety carbonate can be cleaved with potassium carbonate in MeOH (K2CQ3), with triethylamine in pyridine, or with Zn in acetic acid (AcOH). In some embodiments, the chain-terminating moieties urea and silyl can be cleaved with tetrabutylammonium fluoride, pyridine-HF, with ammonium fluoride, or with triethylamine trihydrofluoride. In some embodiments, the chain-terminating moiety can be cleaved / removed with nitrous acid. In some embodiments, the chain-terminating moiety can be cleaved / removed using a solution comprising nitrite (e.g., a combination of nitrite and an acid such as acetic acid, sulfuric acid, or nitric acid). In some further embodiments, the solution can include an organic acid.

[0349] In some embodiments, at least one nucleotide in the plurality of nucleotides comprises a terminator nucleotide analog having a chain-terminating moiety (e.g., a blocking moiety) at the sugar 2' position, at the sugar 3' position, or at both the sugar 2' and 3' positions. In some embodiments, the chain-terminating moiety comprises an azide, an azido, or an azidomethyl group. In some embodiments, the chain-terminating moiety comprises a 3'-O-azido or a 3'-O-azidomethyl group. In some embodiments, the chain-terminating moieties azide, azido, and azidomethyl groups can be cleaved / removed with a phosphine compound. In some embodiments, the phosphine compound comprises a derivatized trialkylphosphine moiety or a derivatized triarylphosphine moiety. In some embodiments, the phosphine compound comprises tris(2-carboxyethyl)phosphine (TCEP), or bissulfotriphenylphosphine (BS-TPP), or tris(hydroxypropyl)phosphine (THPP). In some embodiments, the cleaving agent comprises 4-dimethylaminopyridine (4-DMAP). In some embodiments, the chain-terminating portion comprising one or more of 3′-O-amino, 3′-Q-aminomethyl, 3′-Q-methylamino, or derivatives thereof can be cleaved with nitrous acid by a mechanism utilizing nitrous acid or using a solution comprising nitrous acid. In some embodiments, the chain-terminating portion comprising one or more of 3′-Q-amino, 3′-O-aminomethyl, 3′-Q-methylamino, or derivatives thereof can be cleaved using a solution comprising nitrite. In some embodiments, for example, nitrite can be combined or contacted with an acid such as acetic acid, sulfuric acid, or nitric acid. In some further embodiments, for example, nitrite can be combined or contacted with an organic acid (e.g., formic acid, acetic acid, propionic acid, butyric acid, isobutyric acid, etc.). In some embodiments, the chain-terminating portion comprises a 3′-acetal portion that can be cleaved with a palladium deblocking reagent (e.g., Pd(0)).

[0350] In some embodiments, the nucleotide analog comprises a chain terminating moiety selected from the group consisting of: 3′-deoxynucleotide, 2′,3′-dideoxynucleotide, 3′-methyl, 3′-azido, 3′-azidomethyl, 3′-O-azidoalkyl, 3′-Q-ethynyl, 3′-Q-aminoalkyl, 3′-Q-fluoroalkyl, 3′-fluoromethyl, 3′-difluoromethyl, 3′-trifluoromethyl, 3′-sulfonyl, 3′-malonyl, 3′-amino, 3′-O-amino, 3′-thiol, 3′-aminomethyl, 3′-ethyl, 3′butyl, 3′-tert-butyl, 3′-fluorenylmethyloxycarbonyl, 3′tert-butoxycarbonyl, 3′-O-alkylhydroxyamino, 3′-phosphorothioate, and 3-O-benzyl, or a derivative thereof.

[0351] In some embodiments, a plurality of nucleotides include a plurality of nucleotides lacking a detectable reporter moiety (e.g., a fluorophore). In some embodiments, a plurality of nucleotides include a plurality of nucleotides labeled with a detectable reporter moiety. The detectable reporter moiety includes a fluorophore. In some embodiments, the fluorophore is attached to a nucleotide base. In some embodiments, the fluorophore is attached to a nucleotide base with a linker that can be cut / removed from the base.

[0352] In some embodiments, the cleavable linker on the base includes a cleavable portion comprising an alkyl, alkenyl, alkynyl, allyl, aryl, benzyl, an azide group, an amine group, an amide group, a ketone group, an isocyanate group, a phosphate group, a thio group, a disulfide group, a carbonate group, a urea group, or a silyl group. In some embodiments, the cleavable linker on the base can be cleaved / removed from the base by reacting the cleavable portion with a chemical agent, a pH change, light, or heat. In some embodiments, the cleavable portion of the alkyl, alkenyl, alkynyl, and allyl groups can be cleaved with tetrakis(triphenylphosphine)palladium(0) (Pd(PPh3)4), with piperidine, or with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ). In some embodiments, the cleavable portion of the aryl and benzyl groups can be cleaved with H2Pd / C. In some embodiments, cleavable moieties such as amines, amides, ketones, isocyanates, phosphates, thios, and disulfides can be cleaved with phosphine or thiol groups, including β-mercaptoethanol or dithiothreitol (DTT). In some embodiments, cleavable moieties such as carbonates can be cleaved with potassium carbonate in MeOH (K2CQ3), with triethylamine in pyridine, or with Zn in acetic acid (AcOH). In some embodiments, cleavable moieties such as ureas and silyls can be cleaved with tetrabutylammonium fluoride, pyridine-HF, with ammonium fluoride, or with triethylamine trihydrofluoride.

[0353] In some embodiments, the cleavable linker on the base comprises a cleavable moiety comprising an azide, an azido, or an azidomethyl group. In some embodiments, the cleavable moieties azide, an azido, and an azidomethyl group can be cleaved / removed with a phosphine compound. In some embodiments, the phosphine compound comprises a derivatized trialkylphosphine moiety or a derivatized triarylphosphine moiety. In some embodiments, the phosphine compound comprises tris(2-carboxyethyl)phosphine (TCEP), or bissulfotriphenylphosphine (BS-TPP), or tris(hydroxypropyl)phosphine (THPP). In some embodiments, the cleaving agent comprises 4-dimethylaminopyridine (4-DMAP).

[0354] In some embodiments, the chain-terminating moiety (e.g., at the sugar 2' and / or sugar 3' position) and the cleavable linker on the base have the same or different cleavable moieties. In some embodiments, the chain-terminating moiety attached to the base (e.g., at the sugar 2' and / or sugar 3' position) and the detectable reporter moiety can be chemically cleaved / removed with the same chemical agent. In some embodiments, the chain-terminating moiety attached to the base (e.g., at the sugar 2' and / or sugar 3' position) and the detectable reporter moiety can be chemically cleaved / removed with different chemical agents.

[0355] In some embodiments, the composition comprises: one or more mutant polymerases and a nucleic acid duplex, each nucleic acid duplex comprising a nucleic acid template hybridized to a nucleic acid primer. In some embodiments, the one or more polymerases and the nucleic acid duplex further comprise a plurality of nucleotide reagents. In some embodiments, the one or more polymerases and the nucleic acid duplex further comprise a plurality of multivalent molecules. The one or more mutant polymerases may or may not bind to the nucleic acid duplex. The one or more mutant polymerases may or may not bind to one or more of the multivalent molecules. In some embodiments, the one or more mutant polymerases bind to a nucleic acid duplex comprising a nucleic acid template hybridized to a nucleic acid primer, thereby forming a composite polymerase. In some embodiments, the composite polymerase further comprises at least one nucleotide reagent (e.g., a plurality of multivalent molecules). In some embodiments, the mutant polymerase comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99% identical, or a higher level of sequence identity to any one of SEQ ID NOs: 1-2787 and 2789-2793. In some embodiments, the mutant polymerase is a recombinant polymerase.

[0356] In certain embodiments, the nucleotide reagent comprises any one or any combination of nucleotides and / or multivalent molecules. In certain embodiments, the nucleotide comprises a typical nucleotide. In certain embodiments, the nucleotide comprises an unlabeled nucleotide. In certain embodiments, the nucleotide comprises a nucleotide analog, comprising a detectably labeled nucleotide and / or a nucleotide carrying a removable or non-removable chain termination portion. In certain embodiments, an independent multivalent molecule comprises a central core attached to a plurality of polymer arms, each polymer arm having a nucleotide unit at the end of the arm.

[0357] In some embodiments, multivalent molecules generally include a central portion (e.g., a core) attached to a plurality of arms, wherein each arm is attached to a nucleotide unit. Multivalent molecules include star-shaped, comb-shaped, cross-linked, bottle-brush-shaped, or dendritic macromolecular configurations. In some embodiments, multivalent molecules may include 2-4, 4-10, 10-20, or up to 64 arms. In some embodiments, the arms may radiate from the central portion.

[0358] In some embodiments, at least one multivalent molecule in a plurality of multivalent molecules comprises: (a) a core; and (b) a plurality of nucleotide arms, the plurality of nucleotide arms comprising (i) a core attachment portion, (ii) a spacer (e.g., comprising a PEG portion), (iii) a linker, and (iv) a nucleotide unit, wherein the core is attached to the plurality of nucleotide arms, wherein the spacer is attached to the linker, wherein the linker is attached to the nucleotide unit. In some embodiments, the nucleotide unit comprises a base, a sugar, and at least one phosphate group, and the linker is attached to the nucleotide unit via the base. In some embodiments, the linker comprises an aliphatic chain or an oligoethylene glycol chain, wherein both linker chains have 2-6 subunits. In some embodiments, the linker further comprises an aromatic portion. Exemplary multivalent molecules are Figure 2-5 An exemplary nucleotide arm is shown in Figure 6 Exemplary spacers are shown in Figure 7 (top). Various exemplary linkers are shown in Figure 7 (bottom) and Figure 8 Examples of various linkers that can be joined / attached to nucleotide units are shown in Figure 9A -D, where the 5-position of the pyrimidine base or the 7-position of the purine base is attached to the linker via propargylamine attachment (see also Figure 10 ).

[0359] In certain embodiments, the nucleotide arms are designed so that the nucleotide units of the nucleotide arms can interact with polymerase in a manner similar to free nucleotides. The nucleotide units of the nucleotide arms can be combined with the polymerase compounded with nucleic acid template and nucleic acid primer (for example, nucleotide association). The nucleotide units can also be dissociated from the composite polymerase, or recombined with the same composite polymerase, or combined with the different composite polymerases near the multivalent molecule. Because the multivalent molecule includes a plurality of nucleotide arms, the nucleotide units of a single multivalent molecule can be combined with a plurality of composite polymerases simultaneously. The multivalent molecule effectively increases the local concentration of nucleotide, and this can enhance the signal in the nucleotide binding reaction.

[0360] In some embodiments, the nucleotide units of the multivalent molecule can be bound to the composite polymerase without being incorporated. In some embodiments, the complementary nucleotide units of the multivalent molecule can be bound to the composite polymerase without being subjected to polymerase-catalyzed incorporation, wherein the complementary nucleotide units are bound to the 3' end of the primer at a position opposite to the complementary nucleotide in the template strand.

[0361] In some embodiments, the nucleotide unit of the multivalent molecule can be combined with a composite polymerase and undergo primer extension by being incorporated into the 3' end of the extendable primer (e.g., compounded with a polymerase), thereby causing primer extension. When the nucleotide unit comprises a sugar 3'OH, then subsequent nucleotides can be incorporated into the nascent extended primer. When the nucleotide unit comprises a sugar 3'OH substituted with a blocking group, then subsequent nucleotides are blocked and cannot be incorporated into the nascent extended primer chain. The nucleotide unit (of the multivalent molecule) can be combined with the 3' end of the primer at a position relative to the complementary nucleotide in the template chain. The nucleotide unit can undergo nucleotide incorporation in a polymerase-catalyzed reaction, thereby extending the primer by one nucleotide.

[0362] In certain embodiments, the core of multivalent molecule, joint and / or nucleotide unit can be marked with a detectable reporter gene portion (e.g., fluorophore) in a manner that allows to distinguish the different multivalent molecules carrying different types of nucleotide units. For example, the core unit of the first multivalent molecule is marked with a first fluorophore, wherein the first multivalent molecule includes multiple nucleotide arms with dGTP nucleotide units. The core unit of the second multivalent molecule is marked with a second fluorophore (different from the first fluorophore), wherein the second multivalent molecule includes multiple nucleotide arms with dATP nucleotide units. The combination and incorporation events of nucleotide units can be detected, and the specific base (as a part of multivalent molecule) of the nucleotide unit can be identified based on the detection and identification of the detectable reporter gene portion on the core. In another example, the joint and / or nucleotide unit of the first multivalent molecule is marked with a first fluorophore, wherein the first multivalent molecule includes multiple nucleotide arms with dGTP nucleotide units. The joint and / or nucleotide unit of the second multivalent molecule is marked with a second fluorophore (different from the first fluorophore), wherein the second multivalent molecule includes multiple nucleotide arms with dATP nucleotide units. Binding and incorporation events of nucleotide units can be detected, and the specific base of the nucleotide unit (as part of a multivalent molecule) can be identified based on detection and identification of a detectable reporter moiety on the core. In some embodiments, the core, linker, and nucleotide unit are not labeled with a detectable reporter moiety.

[0363] In some embodiments, at least one nucleotide unit attached to the nucleotide arm of a multivalent molecule can be labeled with a detectable reporter moiety (e.g., a fluorophore) in a manner that allows for distinguishing between different multivalent molecules carrying different types of nucleotide units. For example, the nucleotide unit of a first multivalent molecule is labeled with a first fluorophore, wherein the first multivalent molecule includes a plurality of nucleotide arms with dGTP nucleotide units. The nucleotide unit of a second multivalent molecule is labeled with a second fluorophore (different from the first fluorophore), wherein the second multivalent molecule includes a plurality of nucleotide arms with dATP nucleotide units. The binding and incorporation events of nucleotide units can be detected, and the specific bases of nucleotide units (as part of a multivalent molecule) can be identified based on the detection and identification of the detectable reporter moiety on the nucleotide unit.

[0364] In some embodiments, an individual multivalent molecule in the plurality of multivalent molecules comprises a core attached to a plurality of nucleotide arms, and wherein the plurality of nucleotide arms have the same type of nucleotide units selected from the group consisting of: dATP, dGTP, dCTP, dTTP, and dUTP.

[0365] In some embodiments, the nucleotide unit of at least one multivalent molecule comprises an aromatic base, a five-carbon sugar (e.g., ribose or deoxyribose) and one or more phosphate groups (e.g., 1-10 phosphate groups). A plurality of multivalent molecules may comprise a type of multivalent molecule having a type of nucleotide unit selected from the group consisting of: dATP, dGTP, dCTP, dTTP, and dUTP. A plurality of nucleotides may comprise a mixture of any combination of two or more types of multivalent molecules, wherein the individual multivalent molecules in the mixture comprise a nucleotide unit selected from the group consisting of: dATP, dGTP, dCTP, dTTP, and / or dUTP.

[0366] In some embodiments, the plurality of composite mutant DNA polymerases further comprises a first binding complex and a second binding complex, and a multivalent molecule that forms an avidity complex, wherein (i) the first binding complex comprises a first nucleic acid primer, a first DNA polymerase, and a first multivalent molecule, and the first nucleic acid primer, the first DNA polymerase, and the first multivalent molecule bind to a first portion of a concatemer template molecule, thereby forming a first binding complex (e.g., Figures 44-46 ), wherein the first nucleotide unit of the multivalent molecule is bound to the first DNA polymerase, and (ii) the second binding complex comprises a second nucleic acid primer, a second DNA polymerase, and the first multivalent molecule, the second nucleic acid primer, the second DNA polymerase, and the first multivalent molecule being bound to a second portion of the same concatemer template molecule, thereby forming a second binding complex (e.g., Figures 44-46), wherein the second nucleotide unit of the multivalent molecule binds to the second DNA polymerase, wherein the first binding complex and the second binding complex comprising the same multivalent molecule form an affinity complex (e.g., Figure 47 In some embodiments, the first polymerase comprises any mutant polymerase described herein. In some embodiments, the second polymerase comprises any mutant polymerase described herein. The concatemer template molecule comprises tandem repeats of a sequence of interest and at least one universal sequencing primer binding site. The first nucleic acid primer and the second nucleic acid primer can bind to the sequencing primer binding site along the concatemer template molecule.

[0367] In some embodiments, in the system, the plurality of complex DNA polymerases further comprises a first binding complex and a second binding complex and a multivalent molecule, which form an affinity complex, wherein (i) the first binding complex comprises a first nucleic acid primer, a first DNA polymerase, and a first multivalent molecule, the first nucleic acid primer, the first DNA polymerase, and the first multivalent molecule bind to a first template molecule, thereby forming a first binding complex, wherein the first nucleotide unit of the multivalent molecule binds to the first DNA polymerase, and (ii) the second binding complex comprises a second nucleic acid primer, a second DNA polymerase, and a first multivalent molecule, the second nucleic acid primer, the second DNA polymerase, and the first multivalent molecule bind to a second template molecule, thereby forming a second binding complex, wherein the second nucleotide unit of the multivalent molecule binds to the second DNA polymerase, wherein the first binding complex and the second binding complex comprising the same multivalent molecule form an affinity complex. In some embodiments, the first polymerase comprises any mutant polymerase described herein. In some embodiments, the second polymerase comprises any mutant polymerase described herein. In some embodiments, the first template molecule and the second template molecule are template molecules amplified in a clonally manner. In some embodiments, the first template molecule and the second template molecule are positioned very close to each other. For example, the first template molecule and the second template molecule amplified in a clonally amplified manner include linear template molecules generated by bridge amplification and are fixed to the same position or feature on the support. The first template molecule and the second template molecule include a sequence of interest and at least one universal sequencing primer binding site. The first nucleic acid primer and the second nucleic acid primer can bind to the sequencing primer binding sites on the first template molecule and the second template molecule, respectively.

[0368] In some embodiments, at least one of the plurality of multivalent molecules comprises a nucleotide unit having a chain of one, two, or three phosphorus atoms, wherein the chain is typically attached to the 5' carbon of the sugar moiety via an ester or phosphoramide bond. In some embodiments, at least one nucleotide unit is a nucleotide analog having a phosphorus chain, wherein the phosphorus atoms are linked together with an intervening O, S, NH, methylene, or ethylene. In some embodiments, the phosphorus atom in the chain comprises a substituted side group (comprising O, S, or BH3). In some embodiments, the chain comprises a phosphate group (e.g., 1-10 phosphate groups) substituted with an analog comprising a phosphoramide, a phosphorothioate, a phosphorodithioate, and an O-methylphosphoramidite group.

[0369] In some embodiments, an individual multivalent molecule in the plurality of multivalent molecules comprises a core attached to a plurality of nucleotide arms, and wherein the individual nucleotide arms comprise nucleotide units having a chain terminating moiety (e.g., a blocking moiety) at the sugar 2' position, at the sugar 3' position, or at both the sugar 2' and 3' positions.

[0370] In some embodiments, at least one multivalent molecule in a plurality of multivalent molecules includes a nucleotide unit comprising a nucleotide analog having a chain termination portion (e.g., a blocking portion) at the sugar 2' position, at the sugar 3' position, or at the sugar 2' and 3' positions. In some embodiments, the chain termination portion can inhibit the polymerase-catalyzed incorporation of subsequent nucleotide units or free nucleotides in the nascent chain during the primer extension reaction. In some embodiments, the chain termination portion is attached to the 3' sugar hydroxyl position, wherein the sugar includes a ribose or deoxyribose portion. In some embodiments, the chain termination portion can be removed / cut from the 3' sugar hydroxyl position to produce a nucleotide with a 3'OH sugar group, which can be extended with subsequent nucleotides in a polymerase-catalyzed nucleotide incorporation reaction. In some embodiments, the chain termination portion includes an alkyl, alkenyl, alkynyl, allyl, aryl, benzyl, an azide group, an amine group, an amide group, a ketone group, an isocyanate group, a phosphate group, a thio group, a disulfide group, a carbonate group, a urea group, or a silyl group. In some embodiments, the chain-terminating moiety can be cleaved / removed from the nucleotide, for example, by reacting the chain-terminating moiety with a chemical agent, a change in pH, light, or heat. In some embodiments, the chain-terminating moiety alkyl, alkenyl, alkynyl, and allyl groups can be cleaved with tetrakis(triphenylphosphine)palladium(0) (Pd(PPh3)4), with piperidine, or with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ). In some embodiments, the chain-terminating moiety aryl and benzyl groups can be cleaved with H2Pd / C. In some embodiments, the chain-terminating moiety amine, amide, ketone, isocyanate, phosphate, thio, disulfide can be cleaved with a phosphine or thiol group (including β-mercaptoethanol or dithiothreitol (DTT)). In some embodiments, the chain-terminating moiety carbonate can be cleaved with potassium carbonate in MeOH (K2CQ3), with triethylamine in pyridine, or with Zn in acetic acid (AcOH). In some embodiments, the chain terminating moieties urea and silyl can be cleaved with tetrabutylammonium fluoride, pyridine-HF, with ammonium fluoride, or with triethylamine trihydrofluoride.

[0371] In some embodiments, at least one of the plurality of multivalent molecules comprises a nucleotide unit comprising a terminator nucleotide analog having a chain terminating moiety (e.g., a blocking moiety) at the sugar 2′ position, at the sugar 3′ position, or at both the sugar 2′ and 3′ positions. In some embodiments, the chain terminating moiety comprises an azide, an azido, or an azidomethyl group. In some embodiments, the chain terminating moiety comprises a 3′-O-azido or a 3′-O-azidomethyl group. In some embodiments, the chain terminating moieties azide, azido, and azidomethyl can be cleaved / removed with a phosphine compound. In some embodiments, the phosphine compound comprises a derivatized trialkylphosphine moiety or a derivatized triarylphosphine moiety. In some embodiments, the phosphine compound comprises tris(2-carboxyethyl)phosphine (TCEP), or bissulfotriphenylphosphine (BS-TPP), or tris(hydroxypropyl)phosphine (THPP). In some embodiments, the cleaving agent comprises 4-dimethylaminopyridine (4-DMAP) 。 .

[0372] In some embodiments, at least one multivalent molecule in the plurality of multivalent molecules comprises a nucleotide unit comprising a chain terminating moiety selected from the group consisting of: a 3′-deoxynucleotide, a 2′,3′-dideoxynucleotide, a 3′-methyl, a 3′-azido, a 3′-azidomethyl, a 3′-Q-azidoalkyl, a 3′-O-ethynyl, a 3′-Q-aminoalkyl, a 3′-O-fluoroalkyl, a 3′-fluoromethyl, a 3′-difluoromethyl, a 3′-trifluoromethyl, a 3′-sulfonyl, a 3′-malonyl, a 3′-amino, a 3′-Q-amino, a 3′-thiol, a 3′-aminomethyl, a 3′-ethyl, a 3′butyl, a 3′-tert-butyl, a 3′-fluorenylmethyloxycarbonyl, a 3′-tert-butoxycarbonyl, a ′-Q-alkylhydroxylamino, a 3′-phosphorothioate, and a 3-O-benzyl group or a derivative thereof.

[0373] In some embodiments, at least one multivalent molecule in the plurality of multivalent molecules comprises a core attached to a plurality of nucleotide arms, wherein the core is labeled with a detectable reporter moiety. In some embodiments, the detectable reporter moiety comprises a fluorophore.

[0374] In some embodiments, at least one multivalent molecule in the plurality of multivalent molecules comprises a nucleotide unit attached to a plurality of nucleotide arms, wherein the nucleotide unit is labeled with a detectable reporter moiety. In some embodiments, the detectable reporter moiety comprises a fluorophore.

[0375] In some embodiments, at least one multivalent molecule in the plurality of multivalent molecules comprises at least one linker that is part of a nucleotide arm, wherein the linker is labeled with a detectable reporter moiety. In some embodiments, the detectable reporter moiety comprises a fluorophore.

[0376] In some embodiments, the core comprises a streptavidin-type or avidin-type portion, and the core attachment portion comprises biotin. In some embodiments, the core comprises a streptavidin-type or avidin-type portion (comprising avidin protein), and any derivatives, analogs, and other non-natural forms of avidin that can be bound to at least one biotin portion. Other forms of avidin portions include natural and recombinant avidin and streptavidin and derived molecules, such as non-glycosylated avidin and truncated streptavidin. For example, the avidin portion includes deglycosylated forms of avidin, bacterial streptavidin produced by Streptomyces (e.g., Streptomyces avidinii), and derived forms, such as N-acyl avidins, such as N-acetyl, N-phthaloyl, and N-succinyl avidin, and the commercially available product ExtrAvidin. TM 、Captavidin TM , Neutravidin TM and NeutraliteAvidin TM . Exemplary multivalent molecules in Figure 2-3 and 5, where a universal core is conjugated to multiple nucleotide arms. Exemplary multivalent molecules are shown in Figure 4 , where a universal dendrimer core is conjugated to multiple nucleotide arms. An exemplary design of a multivalent molecule is shown in Figure 5 , which shows a core (e.g., a streptavidin core) attached / bound to a plurality of nucleotide arms, wherein the nucleotide arms include a core attachment moiety (e.g., biotin), a spacer, a linker, and a nucleotide unit. An exemplary biotinylated nucleotide arm including biotin, a spacer, a linker, and a nucleotide unit is shown in FIG. Figure 6 Shown in.

[0377] In some embodiments, the composition comprises: one or more mutant polymerases, the one or more mutant polymerases binding to nucleic acid duplexes, each nucleic acid duplex comprising a nucleic acid template hybridized to a nucleic acid primer, thereby forming a composite polymerase, and the composition further comprises at least one cation. In some embodiments, the at least one cation is selected from the group consisting of strontium, barium, sodium, magnesium, potassium, manganese, calcium, lithium, nickel, and cobalt. In some embodiments, the cation comprises a catalytic divalent cation that promotes polymerase-catalyzed nucleotide incorporation, wherein the catalytic divalent cation comprises magnesium or manganese. In some embodiments, the cation comprises a non-catalytic divalent cation that inhibits polymerase-catalyzed nucleotide incorporation, wherein the non-catalytic divalent cation comprises strontium, barium, and / or calcium.

[0378] In some embodiments, the composition includes: one or more mutant polymerases, the one or more mutant polymerases being bound to a nucleic acid duplex, each nucleic acid duplex including a nucleic acid template molecule hybridized with a nucleic acid primer, thereby forming a composite polymerase. In some embodiments, the nucleic acid template molecule includes a linear nucleic acid molecule, or a circular nucleic acid molecule, or a mixture of both a linear nucleic acid molecule and a circular nucleic acid molecule. In some embodiments, the nucleic acid template molecule in a plurality of nucleic acid template molecules includes the same target sequence of interest or different target sequences of interest. In some embodiments, the nucleic acid template molecule includes an amplified nucleic acid molecule. In some embodiments, the nucleic acid template molecule includes a template molecule or a single nucleic acid template molecule amplified in a clonally amplified manner. In some embodiments, the nucleic acid template molecule includes a copy of the target sequence of interest. In some embodiments, the nucleic acid template molecule includes two or more tandem copies (e.g., concatemers) of the target sequence of interest. In some embodiments, the nucleic acid template molecule includes at least one uridine nucleotide or lacks a uridine nucleotide. In some embodiments, the primer provides a starting site for nucleotide polymerization. In some embodiments, the nucleic acid primer includes an extendable 3' end or a non-extendable 3' end. In some embodiments, the mutant polymerase comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 99% identical or higher to any one of SEQ ID NOs: 1-2787 and 2789-2793.

[0379] In some embodiments, the composite polymerase is immobilized to a support, wherein any one of the nucleic acid template, nucleic acid primer, and / or polymerase is immobilized to the support. In some embodiments, the composition comprises a plurality of composite polymerases immobilized to the support. In some embodiments, about 10 2 -10 15 In certain embodiments, a plurality of composite polymerases are fixed to the carrier at different sites on the carrier. In certain embodiments, a plurality of composite polymerases are fixed to predetermined sites (e.g., positions) on the carrier. In certain embodiments, a plurality of composite polymerases are fixed to random sites (e.g., positions) on the carrier. In certain embodiments, a plurality of composite mutant DNA polymerases are connected to each other through fluid communication to allow a reagent solution (e.g., an enzyme comprising a polymerase, a multivalent molecule, nucleotides and / or a divalent cation, etc.) to flow onto the carrier so that the plurality of composite polymerases fixed on the carrier can react with the reagent solution in a large-scale parallel manner.

[0380] In some embodiments, the carrier includes a planar or non-planar carrier. The carrier can be solid or semi-solid. In some embodiments, the carrier can be porous, semi-porous or non-porous. In some embodiments, the surface of the carrier can be coated with one or more compounds to produce a passivation layer on the carrier. In some embodiments, the passivation layer forms a porous or semi-porous layer. In some embodiments, nucleic acid primers or templates or polymerases can be attached to the passivation layer to fix primers, templates and / or polymerases to the carrier. In some embodiments, the carrier includes a low non-specific binding surface that can improve nucleic acid hybridization and amplification performance on the carrier. Generally speaking, the carrier can include one or more layers (e.g., silane layers, polymer films) in a low binding chemical modification layer of covalent or non-covalent attachment, and one or more oligonucleotides that can be used to fix multiple nucleic acid template molecules to the carrier of the covalent or non-covalent attachment. In some embodiments, the carrier can include a functionalized polymer coating that is at least covalently attached to a portion of the carrier by a chemical group on the carrier, a primer grafted to the functionalized polymer coating, and a water-soluble protective coating on the primer and the functionalized polymer coating. In some embodiments, the functionalized polymer coating comprises poly(N-(5-azidoacetamidopentyl)acrylamide-co-acrylamide) (PAZAM). In some embodiments, the support comprises a surface coating comprising at least one hydrophilic polymer coating and at least one layer of a plurality of oligonucleotides. The hydrophilic polymer coating may comprise polyethylene glycol (PEG). The hydrophilic polymer coating may comprise branched PEG having at least 4 branches. In some embodiments, the low nonspecific binding coating has a degree of hydrophilicity that can be measured as a water contact angle, wherein the water contact angle does not exceed 45 degrees.

[0381] In some embodiments, a composition comprises a plurality of composite polymerases comprising at least a first composite polymerase and a second composite polymerase, wherein: (a) the first composite polymerase comprises a first mutant polymerase bound to a first nucleic acid duplex comprising a first nucleic acid template molecule hybridized to a first nucleic acid primer, and (b) the second composite polymerase comprises a second mutant polymerase bound to a second nucleic acid duplex comprising a second nucleic acid template molecule hybridized to a second nucleic acid primer. In some embodiments, the first nucleic acid template molecule and the second nucleic acid template molecule comprise the same or different sequences. In some embodiments, the first nucleic acid template molecule and the second nucleic acid template molecule are clonally amplified. In some embodiments, the first nucleic acid template molecule and / or the second nucleic acid template molecule comprise at least one uridine nucleotide or lack uridine nucleotides. In some embodiments, the first primer and the second primer comprise an extendable 3' end or a non-extendable 3' end. In some embodiments, the first mutant polymerase and the second mutant polymerase comprise an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99% identical, or more, to any of SEQ ID NOs: 1-2787 and 2789-2793. In some embodiments, the first mutant polymerase and the second mutant polymerase are recombinant polymerases.

[0382] In some embodiments, a plurality of composite polymerases (including a first composite polymerase and a second composite polymerase) are immobilized to a support. In some embodiments, the density of the plurality of composite polymerases comprises about 10% of the density of the composite polymerases immobilized to the support. 2 -10 15 Pieces / mm 2 In some embodiments, the first nucleic acid template molecule and the second nucleic acid template molecule are fixed to different sites on the carrier. In some embodiments, the carrier comprises a plurality of sites arranged in an array. In some embodiments, the sites on the carrier are arranged in rows or columns in one dimension, or in rows and columns in two dimensions. In some embodiments, the plurality of sites are arranged on the carrier in a random or organized manner or a combination of the two. In some embodiments, the plurality of sites are arranged in any pattern (including a straight line or a hexagonal pattern). In some embodiments, the carrier comprises about 10 2 -10 15 Sites / mm 2 or more to form a nucleic acid template array. In some embodiments, the nucleic acid templates fixed at multiple sites, for example, are arranged in an array of about 10 2 -10 15 Sites / mm 2In certain embodiments, the present invention provides a kind of nucleic acid template molecule that is fixed on the carrier.In certain embodiments, the nucleic acid template molecule that is fixed on the carrier is connected with each other fluid to allow reagent solution (for example, multiple enzymes (for example, polymerase), multiple nucleotides and / or multiple multivalent molecules) to flow onto the carrier so that the multiple nucleic acid template molecules that are fixed on the carrier can react with multiple reagents in a large-scale parallel manner.In certain embodiments, the fluid communication of multiple nucleic acid polymerase communities that are fixed on the carrier can be used for carrying out nucleotide binding assay and / or carrying out nucleotide incorporation assay (for example, primer extension or sequencing) on ​​multiple nucleic acid polymerase communities substantially simultaneously.In certain embodiments, the fluid communication of multiple nucleic acid polymerase communities that are fixed on the carrier can be used for carrying out detection and imaging to carry out large-scale parallel sequencing.In certain embodiments, term " fixed " and related terms refer to that nucleic acid molecule or enzyme are directly attached to carrier or directly attached to the coating on carrier by covalent bond or non-covalent interaction.In certain embodiments, low non-specific binding coating has a certain degree of hydrophilicity, and described hydrophilicity can be measured as water contact angle, and wherein water contact angle is no more than 45 degree.

[0383] In some embodiments, the binding complex comprises a mutant polymerase, a nucleic acid template molecule duplexed with a primer, and a nucleotide reagent. In some embodiments, the binding complex comprises (i) a mutant polymerase, a nucleic acid template molecule duplexed with a primer, and a nucleotide, or the binding complex comprises (ii) a mutant polymerase, a nucleic acid template molecule duplexed with a primer, and a nucleotide unit of a multivalent molecule. In some embodiments, the mutant polymerase comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99% identical or higher to any one of SEQ ID NOs: 1-2787 and 2789-2793. In some embodiments, the binding complex has a retention time of greater than about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1 second. In some embodiments, the retention time of the binding complex is 1-30 seconds. The residence time of the bound complex is greater than about 0.1-0.25 seconds, or about 0.25-0.5 seconds, or about 0.5-0.75 seconds, or about 0.75-1 second, or about 1-2 seconds, or about 2-3 seconds, or about 3-4 seconds, or about 4-5 seconds, or about 5-10 seconds or about 10-30 seconds, and / or wherein the method is or can be performed at a temperature of at or above 15°C, at or above 20°C, at or above 25°C, at or above 35°C, at or above 37°C, at or above 42°C, at or above 55°C, at or above 60°C, or at or above 72°C, or at or above 80°C, or within the limits of any of the foregoing. In some embodiments, the residence time of the binding complex can be greater than 1 second, greater than 2 seconds, greater than 3 seconds, greater than 5 seconds, greater than 10 seconds, greater than 15 seconds, greater than 20 seconds, greater than 30 seconds, greater than 60 seconds, greater than 120 seconds, greater than 360 seconds, greater than 3600 seconds or longer or continue the time within the range limited by any two or more values ​​in these values. The binding complex (e.g., a ternary complex) remains stable before being subjected to conditions that result in the dissociation of the interaction between any one of a polymerase, a template molecule, a primer and / or a nucleotide unit or a nucleotide. For example, dissociation conditions include: contacting the binding complex with any one of detergent, EDTA and / or water or any combination thereof. In some embodiments, the disclosure provides the method, wherein the binding complex is deposited on a surface, or attached or hybridized to the surface, and the surface shows a contrast-to-noise ratio greater than 20 in the detection step. In some embodiments, the present disclosure provides the method, wherein the contacting is performed under conditions that stabilize the binding complex when the nucleotide or nucleotide unit is complementary to the next base of the template nucleic acid, and destabilize the binding complex when the nucleotide or nucleotide unit is not complementary to the next base of the template nucleic acid.

[0384] The present disclosure provides a composition comprising a reaction mixture comprising: (a) one or more mutant polymerases; (b) a nucleic acid template molecule; (c) a nucleic acid primer having a 3' extendable end or a 3' non-extendable end; and (d) a plurality of nucleotides or a plurality of multivalent molecules. In some embodiments, the one or more mutant polymerases do not bind to the nucleic acid template molecule. In some embodiments, the one or more mutant polymerases do not bind to the nucleic acid primer. In some embodiments, the one or more mutant polymerases bind to a nucleic acid duplex comprising a nucleic acid template hybridized with a nucleic acid primer, thereby forming a composite polymerase. In some embodiments, the nucleic acid template molecule comprises at least one uridine nucleotide or lacks a uridine nucleotide. In some embodiments, the plurality of nucleotides comprises at least one uridine nucleotide or lacks a uridine nucleotide. In some embodiments, the mutant polymerase comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99% identical or higher to any one of SEQ ID NOs: 1-2787 and 2789-2793.

[0385] In some embodiments, the reaction mixture further comprises (e1) at least one non-catalytic divalent cation that allows at least one nucleotide to bind to the complex polymerase, or allows at least one multivalent molecule to bind to the complex polymerase, but inhibits polymerase-catalyzed incorporation. In some embodiments, the non-catalytic divalent cation comprises strontium, barium, and / or calcium.

[0386] In some embodiments, the reaction mixture further comprises (e2) at least one catalytic divalent cation, wherein the at least one catalytic divalent cation allows at least one nucleotide to bind to the composite polymerase, or allows at least one multivalent molecule to bind to the composite polymerase, and the catalytic divalent cation promotes the incorporation of polymerase catalysis. In some embodiments, the catalytic divalent cation comprises magnesium and / or manganese. In some embodiments, the nucleic acid template and the nucleic acid primer are in solution. In some embodiments, the nucleic acid template and / or the nucleic acid primer are fixed to a carrier or a coating on a carrier.

[0387] In some embodiments, the reaction mixture is suitable for use in performing a nucleotide binding reaction (or a multivalent molecule binding reaction). In some embodiments, the reaction mixture is suitable for use in performing a nucleotide incorporation reaction (or an incorporation reaction of nucleotide units of a multivalent molecule). In some embodiments, the reaction mixture is suitable for use in performing a primer extension reaction in which a nucleotide is incorporated into the 3′ end of an extendable primer (or a nucleotide unit of a multivalent molecule is incorporated into the 3′ end of an extendable primer).

[0388] Reagent test kit

[0389] The present disclosure provides a kit comprising at least one mutant polymerase comprising an amino acid sequence at least 80%, 85%, 90%, 95%, 99% identical or higher to any one of SEQ ID NOs: 1-2787 and 2789-2793.

[0390] In some embodiments, the kit further comprises at least one cation. In some embodiments, the at least one cation is selected from the group consisting of strontium, barium, sodium, magnesium, potassium, manganese, calcium, lithium, nickel, and cobalt.

[0391] In some embodiments, the kit further comprises a plurality of nucleic acid primers having an extendable 3' end or a non-extendable 3' end. In some embodiments, at least one of the primers can be fixed to a support. In some embodiments, the fixed primer (e.g., a capture primer) can be used to hybridize with a nucleic acid template. In some embodiments, at least one of the primers comprises a sequencing primer that can hybridize with an adapter sequence (e.g., a universal adapter sequence) attached to a template molecule.

[0392] In certain embodiments, test kit further includes a plurality of nucleotides. In certain embodiments, at least one nucleotide in a plurality of nucleotides includes base, sugar and at least one phosphate group. In certain embodiments, at least one nucleotide in a plurality of nucleotides includes aromatic base, five-carbon sugar (for example, ribose or deoxyribose) and one or more phosphate groups (for example, 1-10 phosphate groups). A plurality of nucleotides can include at least one type of nucleotide selected from the group consisting of: dATP, dGTP, dCTP, dTTP and dUTP. A plurality of nucleotides can include a mixture of any combination of two or more types of nucleotides selected from the group consisting of: dATP, dGTP, dCTP, dTTP and / or dUTP.

[0393] In some embodiments, in the kit, at least one of the plurality of nucleotides comprises a chain of one, two, or three phosphorus atoms, wherein the chain is typically attached to the 5' carbon of the sugar moiety via an ester or phosphoramide bond. In some embodiments, at least one of the plurality of nucleotides is an analog with a phosphorus chain in which the phosphorus atoms are linked together with an intervening O, S, NH, methylene, or ethylene. In some embodiments, the phosphorus atoms in the chain comprise substituted side groups (comprising O, S, or BH3). In some embodiments, the chain comprises a phosphate group substituted with an analog comprising a phosphoramide, phosphorothioate, phosphorodithioate, and O-methylphosphoramidite group.

[0394] In certain embodiments, in the test kit, at least one nucleotide in a plurality of nucleotides includes a terminator nucleotide analog with a chain termination portion (e.g., a blocking portion) at a sugar 2' position, at a sugar 3' position, or at a sugar 2' and 3' position. In certain embodiments, the chain termination portion can inhibit the polymerase-catalyzed incorporation of subsequent nucleotide units or free nucleotides in the nascent chain during the primer extension reaction. In certain embodiments, the chain termination portion is attached to the 3' sugar hydroxyl position, wherein the sugar includes a ribose or deoxyribose moiety. In certain embodiments, the chain termination portion can be removed / cut from the 3' sugar hydroxyl position to produce a nucleotide with a 3'OH sugar group, which can be extended with subsequent nucleotides in a polymerase-catalyzed nucleotide incorporation reaction. In certain embodiments, the chain termination portion includes an alkyl, alkenyl, alkynyl, allyl, aryl, benzyl, an azide group, an amine group, an amide group, a ketone group, an isocyanate group, a phosphate group, a thio group, a disulfide group, a carbonate group, a urea group, or a silyl group. In certain embodiments, the test kit may further include a chemical agent for cutting the chain termination portion. For example, the kit includes tetrakis(triphenylphosphine)-palladium(0) (Pd(PPh3)4) with piperidine, or with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ), H2Pd / C, or phosphine, or with any one or any combination of thiol groups (including β-mercaptoethanol or dithiothreitol (DTT)). In some embodiments, the kit includes chemicals including potassium carbonate (K2CO3) in MeOH, triethylamine in pyridine, or Zn in acetic acid (AcOH). In some embodiments, the kit includes chemicals including tetrabutylammonium fluoride, pyridine-HF, ammonium fluoride, or triethylamine trihydrofluoride. In some embodiments, the kit includes chemicals including nitrous acid. In some embodiments, the kit includes a solution including a nitrite, such as a combination of a nitrite and an acid such as acetic acid, sulfuric acid, or nitric acid. In some further embodiments, the solution may include an organic acid (e.g., formic acid, acetic acid, propionic acid, butyric acid, isobutyric acid, etc.).

[0395] In some embodiments, in the kit, at least one of the multiple nucleotides includes a terminator nucleotide analog having a chain termination portion (e.g., a blocking portion) at the sugar 2' position, at the sugar 3' position, or at the sugar 2' and 3' positions. In some embodiments, the chain termination portion includes an azide, an azido group, or an azidomethyl group. In some embodiments, the chain termination portion includes a 3'-O-azido group or a 3'-O-azidomethyl group. In some embodiments, the kit may include a chemical agent for cutting the chain termination portion. For example, the kit includes any one or any combination of phosphine compounds, the phosphine compound including a derivatized trialkylphosphine portion or a derivatized triarylphosphine portion. In some embodiments, the phosphine compound includes tris(2-carboxyethyl)phosphine (TCEP), or bissulfotriphenylphosphine (BS-TPP), or tris(hydroxypropyl)phosphine (THPP). In some embodiments, the cutting agent includes 4-dimethylaminopyridine (4-DMAP).

[0396] In some embodiments, in the kit, the nucleotide analog comprises a chain terminating moiety selected from the group consisting of: 3′-deoxynucleotide, 2′,3′-dideoxynucleotide, 3′-methyl, 3′-azido, 3′-azidomethyl, 3′-O-azidoalkyl, 3′-O-ethynyl, 3′-O-aminoalkyl, 3′-O-fluoroalkyl, 3′-fluoromethyl, 3′-difluoromethyl, 3′-trifluoromethyl, 3′-sulfonyl, 3′-malonyl, 3′-amino, 3′-O-amino, 3′-thiol, 3′-aminomethyl, 3′-ethyl, 3′butyl, 3′-tert-butyl, 3′-fluorenylmethyloxycarbonyl, 3′-tert-butoxycarbonyl, 3′-O-alkylhydroxyamino, 3′-phosphorothioate, and 3-O-benzyl, or a derivative thereof.

[0397] In some embodiments, in the kit, the plurality of nucleotides comprises a plurality of nucleotides labeled with a detectable reporter moiety. The detectable reporter moiety comprises a fluorophore. In some embodiments, the fluorophore is attached to a nucleotide base. In some embodiments, the fluorophore is attached to a nucleotide base using a linker that can be cleaved / removed from the base.

[0398] In some embodiments, in the kit, the cleavable linker on the base includes a cleavable portion comprising the following: an alkyl, alkenyl, alkynyl, allyl, aryl, benzyl, an azide group, an amine group, an amide group, a ketone group, an isocyanate group, a phosphate group, a thio group, a disulfide group, a carbonate group, a urea group or a silyl group. In some embodiments, the kit may further include a chemical agent for cleaving the cleavable linker on the base. For example, the kit includes tetrakis(triphenylphosphine)-palladium(0) (Pd(PPh3)4) with piperidine, or with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ), H2Pd / C, or phosphine or with any one or any combination of a thiol group (comprising β-mercaptoethanol or dithiothreitol (DTT)). In some embodiments, the kit includes a chemical agent comprising potassium carbonate (K2CO3) in MeOH, triethylamine in pyridine or Zn in acetic acid (AcOH). In some embodiments, the kit comprises chemicals including tetrabutylammonium fluoride, pyridine-HF, ammonium fluoride, or triethylamine trihydrofluoride.

[0399] In certain embodiments, in the kit, the cleavable linker on the base includes a cleavable portion comprising an azide, an azido or an azidomethyl group. In certain embodiments, the kit may include a chemical agent for cleaving the cleavable linker on the base. For example, the kit includes any one or any combination of phosphine compounds, the phosphine compound including a derived trialkylphosphine moiety or a derived triarylphosphine moiety. In certain embodiments, the phosphine compound includes tris (2-carboxyethyl) phosphine (TCEP), or bissulfotriphenylphosphine (BS-TPP) or tris (hydroxypropyl) phosphine (THPP). In certain embodiments, the cutting agent includes 4-dimethylaminopyridine (4-DMAP).

[0400] In some embodiments, in the kit, the chain-terminating portion (e.g., at the sugar 2' and / or sugar 3' position) and the cleavable linker on the base have the same or different cleavable portions. In some embodiments, the chain-terminating portion (e.g., at the sugar 2' and / or sugar 3' position) and the detectable reporter portion attached to the base can be chemically cleaved / removed using the same chemical agent. In some embodiments, the chain-terminating portion (e.g., at the sugar 2' and / or sugar 3' position) and the detectable reporter portion attached to the base can be chemically cleaved / removed using different chemical agents.

[0401] The present disclosure provides a kit comprising at least one mutant polymerase comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99% identical or higher to any one of SEQ ID NOs: 1-2787 and 2789-2793, and the kit further comprising a plurality of multivalent molecules. In some embodiments, at least one multivalent molecule in the plurality of multivalent molecules comprises: (a) a core; and (b) a plurality of nucleotide arms comprising (i) a core attachment portion, (ii) a spacer (e.g., comprising a PEG portion), (iii) a linker, and (iv) a nucleotide unit, wherein the core is attached to the plurality of nucleotide arms, wherein the spacer is attached to the linker, wherein the linker is attached to the nucleotide unit. Exemplary multivalent molecules are Figure 2-5 An exemplary nucleotide arm is shown in Figure 6 Exemplary spacers are shown in Figure 7 (top). Various exemplary linkers are shown in Figure 7 (bottom) and Figure 8 Examples of various linkers that can be joined / attached to nucleotide units are shown in Figure 9A -D, where the 5-position of the pyrimidine base or the 7-position of the purine base is attached to the linker via propargylamine attachment (see also Figure 10 In some embodiments, the nucleotide unit comprises a base, a sugar, and at least one phosphate group, and the linker is attached to the nucleotide unit via the base. In some embodiments, the linker comprises an aliphatic chain or an oligoethylene glycol chain, wherein both linker chains have 2-6 subunits. In some embodiments, the linker further comprises an aromatic moiety.

[0402] In some embodiments, in the kit, an individual multivalent molecule of the plurality of multivalent molecules comprises a core attached to a plurality of nucleotide arms, and wherein the plurality of nucleotide arms have the same type of nucleotide units selected from the group consisting of: dATP, dGTP, dCTP, dTTP, and dUTP.

[0403] In some embodiments, in the kit, the nucleotide unit of at least one multivalent molecule includes an aromatic base, a five-carbon sugar (e.g., ribose or deoxyribose) and one or more phosphate groups (e.g., 1-10 phosphate groups). The plurality of multivalent molecules can include a type of multivalent molecule having a type of nucleotide unit selected from the group consisting of: dATP, dGTP, dCTP, dTTP, and dUTP. The plurality of nucleotides can include a mixture of any combination of two or more types of multivalent molecules, wherein the individual multivalent molecules in the mixture include a nucleotide unit selected from the group consisting of: dATP, dGTP, dCTP, dTTP, and / or dUTP.

[0404] In some embodiments, in the test kit, at least one of the plurality of multivalent molecules comprises a nucleotide unit having a chain of one, two or three phosphorus atoms, wherein the chain is typically attached to the 5' carbon of the sugar moiety via an ester or phosphoramide bond. In some embodiments, at least one nucleotide unit is a nucleotide analog with a phosphorus chain, wherein the phosphorus atoms are linked together with an intervening O, S, NH, methylene or ethylene. In some embodiments, the phosphorus atom in the chain comprises a substituted side group (comprising O, S or BH3). In some embodiments, the chain comprises a phosphate group substituted with an analog comprising a phosphoramide, a thiophosphate, a dithiophosphate and an O-methylphosphoramidite group.

[0405] In some embodiments, in the kit, an individual multivalent molecule of the plurality of multivalent molecules comprises a core attached to a plurality of nucleotide arms, and wherein the individual nucleotide arms comprise nucleotide units having a chain terminating moiety (e.g., a blocking moiety) at the sugar 2' position, at the sugar 3' position, or at both the sugar 2' and 3' positions.

[0406] In some embodiments, in the test kit, at least one multivalent molecule in a plurality of multivalent molecules includes a nucleotide unit comprising a terminator nucleotide analogue having a chain termination portion (e.g., a blocking portion) at a sugar 2' position, at a sugar 3' position, or at a sugar 2' and 3' position. In some embodiments, the chain termination portion can inhibit the polymerase-catalyzed incorporation of subsequent nucleotide units or free nucleotides in the nascent chain during the primer extension reaction. In some embodiments, the chain termination portion is attached to the 3' sugar hydroxyl position, wherein the sugar includes a ribose or deoxyribose portion. In some embodiments, the chain termination portion can be removed / cut from the 3' sugar hydroxyl position to produce a nucleotide with a 3'OH sugar group, which can be extended with subsequent nucleotides in a polymerase-catalyzed nucleotide incorporation reaction. In some embodiments, the chain termination portion includes an alkyl, alkenyl, alkynyl, allyl, aryl, benzyl, an azide group, an amine group, an amide group, a ketone group, an isocyanate group, a phosphate group, a thio group, a disulfide group, a carbonate group, a urea group, or a silyl group. In some embodiments, the test kit may also include a chemical agent for cutting the chain termination portion of the nucleotide unit of the multivalent molecule. For example, the kit includes tetrakis(triphenylphosphine)-palladium(0) (Pd(PPh3)4) with piperidine, or with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ), H2Pd / C, or phosphine, or with any one or any combination of thiol groups (including β-mercaptoethanol or dithiothreitol (DTT)). In some embodiments, the kit includes chemicals including potassium carbonate (K2CO3) in MeOH, triethylamine in pyridine, or Zn in acetic acid (AcOH). In some embodiments, the kit includes chemicals including tetrabutylammonium fluoride, pyridine-HF, ammonium fluoride, or triethylamine trihydrofluoride.

[0407] In some embodiments, in the kit, at least one of the plurality of multivalent molecules comprises a nucleotide unit comprising a terminator nucleotide analog having a chain-terminating portion (e.g., a blocking portion) at the sugar 2' position, at the sugar 3' position, or at the sugar 2' and 3' positions. In some embodiments, the chain-terminating portion comprises an azide, an azido group, or an azidomethyl group. In some embodiments, the chain-terminating portion comprises a 3'-O-azido group or a 3'-O-azidomethyl group. In some embodiments, the kit may comprise a chemical agent for cleaving the chain-terminating portion of the nucleotide unit of the multivalent molecule. For example, the kit comprises any one or any combination of phosphine compounds, the phosphine compound comprising a derivatized trialkylphosphine portion or a derivatized triarylphosphine portion. In some embodiments, the phosphine compound comprises tris(2-carboxyethyl)phosphine (TCEP), or bissulfotriphenylphosphine (BS-TPP), or tris(hydroxypropyl)phosphine (THPP). In some embodiments, the cleaving agent comprises 4-dimethylaminopyridine (4-DMAP).

[0408] In some embodiments, in the kit, at least one multivalent molecule in the plurality of multivalent molecules comprises a nucleotide unit comprising a chain terminating moiety selected from the group consisting of: a 3′-deoxynucleotide, a 2′,3′-dideoxynucleotide, a 3′-methyl, a 3′-azido, a 3′-azidomethyl, a 3′-O-azidoalkyl, a 3′-O-ethynyl, a 3′-O-aminoalkyl, a 3′-O-fluoroalkyl, a 3′-fluoromethyl, a 3′-difluoromethyl, a 3′-trifluoromethyl, a 3′-sulfonyl, a 3′-malonyl, a 3′-amino, a 3′-O-amino, a 3′-thiol, a 3′-aminomethyl, a 3′-ethyl, a 3′-butyl, a 3′-tert-butyl, a 3′-fluorenylmethyloxycarbonyl, a 3′-tert-butoxycarbonyl, a 3′-O-alkylhydroxyamino, a 3′-phosphorothioate, and a 3-O-benzyl group or a derivative thereof.

[0409] In some embodiments, in the kit, at least one of the plurality of multivalent molecules comprises a core attached to a plurality of nucleotide arms. In some embodiments, the core, at least one linker, and / or at least one nucleotide unit is labeled with a detectable reporter moiety. In some embodiments, the detectable reporter moiety comprises a fluorophore.

[0410] In some embodiments, in the kit, a separate multivalent molecule comprises a core having an avidin-like portion, and the core attachment portion comprises biotin. In some embodiments, the core comprises a streptavidin-type or avidin-type portion (comprising avidin protein), and any derivatives, analogs, and other non-natural forms of avidin that can be bound to at least one biotin portion. Other forms of avidin moieties include natural and recombinant avidin and streptavidin and derived molecules, for example, non-glycosylated avidin and truncated streptavidin. For example, the avidin moiety includes a deglycosylated form of avidin, bacterial streptavidin produced by Streptomyces (e.g., Streptomyces avidinus), and derived forms, for example, N-acyl avidins, such as N-acetyl, N-phthaloyl, and N-succinyl avidin, and the commercially available product ExtrAvidin. TM 、Captavidin TM , Neutravidin TM and Neutralite Avidin TM .

[0411] In certain embodiments, the test kit includes one or more containers containing at least one mutant polymerase, cation, primer, multiple nucleotides and / or multiple multivalent molecules. The mutant polymerase, cation, primer and / or multiple nucleotides can be combined in any combination and can be contained in a single container, or can be contained in a separate container or any combination thereof. The mutant polymerase, cation, primer and / or multiple multivalent molecules can be combined in any combination and can be contained in a single container, or can be contained in a separate container or any combination thereof.

[0412] The kit may include instructions for use of the kit for performing a nucleotide binding reaction, a nucleotide incorporation reaction, and / or a nucleic acid sequencing reaction using a plurality of nucleotides. The kit may include instructions for use of the kit for performing a multivalent molecule binding reaction, a multivalent molecule incorporation reaction, and / or a nucleic acid sequencing reaction using a plurality of multivalent molecules.

[0413] Nucleic acids encoding engineered polymerases, vectors, and host cells

[0414] The present disclosure provides nucleic acids encoding any of the mutant polymerases described herein, comprising an amino acid sequence at least 80%, 85%, 90%, 95%, 99% identical or higher to any of SEQ ID NOs: 1-2787 and 2789-2793.

[0415] The present disclosure provides a vector operably linked to at least one nucleic acid (e.g., a transgene) encoding any one of the mutant polymerases described herein, the mutant polymerase comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99% identical or higher to any one of SEQ ID NOs: 1-2787 and 2789-2793. In some embodiments, the vector comprises at least one host cell regulatory sequence comprising a promoter sequence, an enhancer, a transcription and / or translation initiation sequence, a transcription and / or translation termination sequence, a polypeptide secretion signal sequence, and the like. The promoter sequence can be a constitutive promoter sequence or an inducible promoter sequence. In some embodiments, the promoter sequence in the vector can be operably linked to at least one nucleic acid encoding the mutant polymerase to control expression of the mutant polymerase by the host cell. In some embodiments, the vector comprises an expression vector.

[0416] The present disclosure provides a host cell comprising a vector (e.g., an expression vector) operably linked to at least one nucleic acid (e.g., a transgene) encoding any one of the mutant polymerases described herein, the mutant polymerase comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99% identical, or more, to any one of SEQ ID NOs: 1-2787 and 2789-2793. In some embodiments, the vector comprises a promoter sequence operably linked to the at least one nucleic acid encoding the mutant polymerase, wherein the promoter sequence contr...

Claims

1. An engineered polymerase comprising an amino acid sequence at least 85% identical to SEQ ID NO: 1, and has a first amino acid substitution selected from the group consisting of D141N, D141V, D141A, D141L, D141I, D141F, D141Y, D141T, D141S, D141R, D141K, D141Q, D141W, D141E, D141M, D141P, D141G, D141H, and D141C, and The engineered polymerase has a second amino acid substitution selected from the group consisting of E143A, E143V, E143L, E143I, E143F, E143Y, E143N, E143T, E143S, E143W, E143M, E143P, E143F, E143G, E143L, E143H, E143R, E143K, E143D, E143C and E143Q.

2. An engineered polymerase comprising an amino acid sequence at least 85% identical to SEQ ID NO: 1 and having a non-mutated position E143E and an amino acid substitution selected from the group consisting of: D141N, D141V, D141A, D141L, D141I, D141F, D141Y, D141T, D141S, D141R, D141K, D141Q, D141W, D141E, D141M, D141P, D141G, D141H and D141C.

3. The engineered polymerase of claim 1 or 2, further comprising an amino acid substitution selected from the group consisting of V93A, V93M, V93E, V93F, V93Y, V93G, V93S, V93K, V93T and V93I.

4. The engineered polymerase of claim 1 or 2, further comprising amino acid substitutions L409S, Y410A and P411G.

5. The engineered polymerase of claim 1 or 2, further comprising an amino acid substitution selected from the group consisting of: M1F, M1I, M1L, M1S, M1N, M1A, M1V, M1Y, M1Q, M1K, M1V, and M1A.

6. The engineered polymerase of claim 1 or 2, further comprising an amino acid substitution selected from the group consisting of: M129I, M129V, M129K, M129L, M129E, M129F, M129N, M129S, M129R, and M129Y.

7. The engineered polymerase of claim 1 or 2, further comprising an amino acid substitution selected from the group consisting of: M159W, M159F, and M159Y.

8. The engineered polymerase of claim 1 or 2, further comprising an amino acid substitution selected from the group consisting of: M313I, M313K, M313L, M313V, M313D, M313R, M313E, M313A, M313L, and M313N.

9. The engineered polymerase of claim 1 or 2, further comprising an amino acid substitution selected from the group consisting of: M329L, M329S, M329W, M329A, M329R, M329I, M329Q, M329N and M329E.

10. The engineered polymerase of claim 1 or 2, further comprising an amino acid substitution selected from the group consisting of: M467V, M467K, M467D, M467T, M467R, M467E, M467Q, and M467L.

11. The engineered polymerase of claim 1 or 2, further comprising an amino acid substitution selected from the group consisting of: M759T, M759S, M759N, M759R, M759E, M759D and M759A.

12. The engineered polymerase of claim 1 or 2, further comprising an amino acid substitution selected from the group consisting of: K240R, K240D, K240N, K240Q, K240A.

13. The engineered polymerase of claim 1 or 2, further comprising an amino acid substitution selected from the group consisting of K306R, K306N, K306Q, K306A, K306V, K306I and K306F.

14. The engineered polymerase of claim 1 or 2, further comprising an amino acid substitution selected from the group consisting of K371R, K371D, K371N, K371Q, K371Y, K371T, K371V, and K371L.

15. The engineered polymerase of claim 1 or 2, further comprising an amino acid substitution selected from the group consisting of K429R, K429S, K429M, K429A, K429N, K429D, K429Q, K429H, K429Y, K429V and K429L.

16. The engineered polymerase of claim 1 or 2, further comprising an amino acid substitution selected from the group consisting of K468R, K468E, K468Y, K468T and K468L.

17. The engineered polymerase of claim 1 or 2, further comprising an amino acid substitution selected from the group consisting of: K476R, K476D, K476A, and K476F.

18. The engineered polymerase of claim 1 or 2, further comprising an amino acid substitution selected from the group consisting of K592Q, K592R, K592W, K592Y, K592A, K592F, K592I, K592T, K592N, and K592S.

19. The engineered polymerase of claim 1 or 2, further comprising an amino acid substitution selected from the group consisting of W299F, W299E, W299N, W299Q, W299Y, W299A and W299F.

20. The engineered polymerase of claim 1 or 2, further comprising an amino acid substitution selected from the group consisting of: H601R, H601I, H601A, H601T, H601V, H601L, and H601N.

21. The engineered polymerase of claim 1 or 2, further comprising an amino acid substitution selected from the group consisting of C428Y.

22. The engineered polymerase of claim 1 or 2, further comprising a deleted methionine at position 1.

23. The engineered polymerase of claim 1 or 2, further comprising an amino acid substitution selected from the group consisting of C223V, C223E, C223S, C223L, C223M, C223A, C223P, C223K, C223N, and C223D.

24. The engineered polymerase of claim 1 or 2, further comprising an amino acid substitution selected from the group consisting of: C509V, C509Y, C509S, C509M, C509A, C509N, C509D, C509H, and C509Q.

25. The engineered polymerase of claim 1 or 2, further comprising a truncation at the following positions: K464, R465, E475, Y481, E616, E620, E755, Y756, Q757, R758, M759, T762, W767, or M770.

26. The engineered polymerase of claim 1 or 2, further comprising an amino acid substitution selected from the group consisting of: V610D, V610A, V610K, V610S, V610T, V610N, V610R, and V610Q.

27. The engineered polymerase of claim 1 or 2, further comprising an amino acid substitution selected from the group consisting of D613S, D613E, D613R, D613K, D613N, D613Q, D613A, D613V, D613Y, and D613F.

28. The engineered polymerase of claim 1 or 2, further comprising an amino acid substitution selected from the group consisting of Q664A, Q664L, Q664V, Q664F, Q664I, Q664R, Q664K, Q664T, Q664N, and Q664M.

29. The engineered polymerase of claim 1 or 2, further comprising an amino acid substitution selected from the group consisting of E668G, E668K, E668M, E668A, E668P, E668S, E668R, E688N, E688D, E668Y, and E668Q.

30. The engineered polymerase of claim 1 or 2, further comprising an amino acid substitution selected from the group consisting of: P677L, P677R, P677K and P677A.

31. The engineered polymerase of claim 1 or 2, further comprising an amino acid substitution selected from the group consisting of D671G, D671R, D671Y, D671S, D671A, D671K, and D671N.

32. The engineered polymerase of claim 1 or 2, further comprising an amino acid substitution selected from the group consisting of: N11S, N11A, N11R, N11Q, N11E, N11K, and N11T.

33. The engineered polymerase of claim 1 or 2, further comprising an amino acid substitution selected from the group consisting of K507L, K507E, K507S, K507A, K507N, K507Q, K507E, and K507T.

34. The engineered polymerase of claim 1 or 2, further comprising an amino acid substitution selected from the group consisting of: E511K, E511S, E511A, E511R, E511N, and E511T.

35. The engineered polymerase of claim 1 or 2, further comprising an amino acid substitution selected from the group consisting of K637M, K637A, K637N, K637Q, K637E, K637S and K637T.

36. An engineered polymerase comprising an amino acid sequence at least 85% identical to SEQ ID NO: 1714, and has a first amino acid substitution selected from the group consisting of D168A, D168V, D168L, D168I, D168F, D168Y, D168N, D168T, D168S, D168W, D168M, D168P, D168G, D168H, D168R, D168E, D168C, D168K or D168Q, and The engineered polymerase has a second amino acid substitution selected from the group consisting of E143A, E143V, E143L, E143I, E143F, E143Y, E143N, E143T, E143S, E143W, E143M, E143P, E143G, E143H, E143R, E143K, E143D, E143C, or E143Q.

37. The engineered polymerase of claim 36, further comprising amino acid substitutions at positions L440, Y441, and P442 of the LYP motif, wherein the LYP motif mutations comprise: L440F, Y441G and P442P; or ●L440S, Y441G, P442P.

38. The engineered polymerase of claim 36, further comprising an amino acid substitution selected from the group consisting of: Y14F, Y14D, Y14I, and Y14N.

39. The engineered polymerase of claim 36, further comprising an amino acid substitution selected from the group consisting of W135S, W135L, W135R, W135Y, W135F, W135D, W135A, and W135V.

40. The engineered polymerase of claim 36, further comprising an amino acid substitution selected from the group consisting of: M187S, M187L, M187R, M187Y, M187I, M187T, M187A, and M187V.

41. The engineered polymerase of claim 36, further comprising an amino acid substitution selected from the group consisting of W329Y, W329F, W329L, W329D, W329A, and W329V.

42. The engineered polymerase of claim 36, further comprising an amino acid substitution selected from the group consisting of: K335R, K335L, K335S, and K335A.

43. The engineered polymerase of claim 37, further comprising an amino acid substitution selected from the group consisting of: M389D, M389E, M389L, M389Y, M389S, M389A, and M389V.

44. The engineered polymerase of claim 36, further comprising an amino acid substitution selected from the group consisting of S473K, S473R, S473T, S473Q, and S473A.

45. The engineered polymerase of claim 36, further comprising an amino acid substitution selected from the group consisting of: M527H, M527G, M527Q, M527L, M527D, M527A, and M527V.

46. ​​The engineered polymerase of claim 36, further comprising an amino acid substitution selected from the group consisting of: M549N, M549Y, M549H, M549T, M549D, M549R, M549A, and M549V.

47. The engineered polymerase of claim 36, further comprising an amino acid substitution selected from the group consisting of K552R, K552T, K552N, K552Q, and K552A.

48. The engineered polymerase of claim 36, further comprising an amino acid substitution selected from the group consisting of: M629L, M629A, M629D, M629R, and M629V.

49. The engineered polymerase of claim 36, further comprising an amino acid substitution selected from the group consisting of W641R, W641A, W641L, W641F, W641Y, and W641V.

50. The engineered polymerase of claim 36, further comprising an amino acid substitution selected from the group consisting of K650T, K650C, K650A, K650R, and K650S.

51. The engineered polymerase of claim 36, further comprising an amino acid substitution selected from the group consisting of K711R, K711L, K711T, and K711D.

52. The engineered polymerase of claim 36, further comprising an amino acid substitution selected from the group consisting of: M723S, M723I, M723T, M723N, M723R, M723L, M723A, and M723C.

53. The engineered polymerase of claim 36, further comprising an amino acid substitution selected from the group consisting of W791R, W791Y, W791D, W791S, W791L, W791A, and W791V.

54. The engineered polymerase of claim 36, further comprising an amino acid substitution selected from the group consisting of: C362A, C362L, C362I, C362S, C362F, C362Y, C362V, C362P, C362K, C362N, and C362D.

55. The engineered polymerase of claim 36, further comprising an amino acid substitution selected from the group consisting of: C539A, C539V, C539L, C539S, C539Y, C539D, C539K, C539N and C539P.

56. The engineered polymerase of claim 36, further comprising a truncation at the following positions: M723, G773, D777, E781, T784, Q785, R790, W791, or F792.

57. The engineered polymerase of claim 36, further comprising an amino acid substitution selected from the group consisting of: Q95L, Q95H, Q95R, Q95W, Q95A, Q95K, Q95N and Q95P.

58. The engineered polymerase of claim 36, further comprising amino acid substitution 1186R.

59. The engineered polymerase of claim 36, further comprising the amino acid substitution V304D.

60. The engineered polymerase of claim 36, further comprising an amino acid substitution selected from the group consisting of L313M, L313D, L313F, L313K, L313R, L313A, and L313E.

61. The engineered polymerase of claim 37, further comprising the amino acid substitution E318V.

62. The engineered polymerase of any one of claims 1, 2, 36 or 37, further comprising a plurality of engineered polymerases, a plurality of nucleic acid template molecules, and a plurality of nucleotide polymerization initiation sites having 3' extendable ends.

63. The engineered polymerase of any one of claims 1, 2, 36 or 37, wherein the plurality of nucleic acid template molecules comprises linear nucleic acid molecules, circular nucleic acid molecules, or a mixture of linear and circular nucleic acid molecules.

64. The engineered polymerase of any one of claims 1, 2, 36 or 37, wherein the plurality of nucleic acid template molecules comprises clonally amplified template molecules.

65. The engineered polymerase of any one of claims 1, 2, 36 or 37, wherein at least one of the nucleic acid template molecules in the plurality of nucleic acid template molecules comprises one copy of a target sequence of interest, or comprises a concatemer having two or more tandem copies of a target sequence of interest.

66. An engineered polymerase according to any one of claims 1, 2, 36 or 37, wherein a separate nucleotide polymerization start site among the multiple nucleotide polymerization start sites comprises a nucleic acid primer that hybridizes to a portion of at least one of the nucleic acid template molecules, or wherein the separate nucleotide polymerization start site among the multiple nucleotide polymerization start sites comprises a self-priming end portion of at least one of the nucleic acid template molecules.

67. The engineered polymerase of claim 62, wherein the plurality of polymerases, the plurality of nucleic acid template molecules, and the plurality of nucleotide polymerization initiation sites form a plurality of composite polymerases, each composite polymerase comprising a polymerase that binds to a nucleic acid duplex, wherein the duplex comprises a nucleic acid template molecule hybridized to a nucleic acid primer.

68. The engineered polymerase of claim 67, wherein the plurality of nucleic acid template molecules comprise the same target sequence of interest or different target sequences of interest.

69. The engineered polymerase of claim 67, wherein the plurality of complex polymerases further comprises a plurality of multivalent molecules, wherein individual multivalent molecules in the plurality comprise: (a) Nucleus; and (b) a plurality of nucleotide arms, the plurality of nucleotide arms comprising: (i) a core attachment portion, (ii) a spacer, (iii) a linker, and (iv) a nucleotide unit, wherein the core is attached to the plurality of nucleotide arms via its core attachment portion, wherein the spacer is attached to the linker, and wherein the linker is attached to the nucleotide unit.

70. The engineered polymerase of claim 69, wherein the linker comprises an aliphatic chain having 2-6 subunits or an oligoethylene glycol chain having 2-6 subunits.

71. The engineered polymerase of claim 69, wherein the plurality of nucleotide arms attached to a given core have the same type of nucleotide units, and wherein the nucleotide units comprise dATP, dGTP, dCTP, dTTP, or dUTP.

72. The engineered polymerase of claim 69, wherein the plurality of multivalent molecules comprises one type of multivalent molecule, wherein each multivalent molecule in the plurality has the same type of nucleotide unit selected from the group consisting of: dATP, dGTP, dCTP, dTTP, and dUTP.

73. The engineered polymerase of claim 69, wherein the plurality of multivalent molecules comprises a mixture of any combination of two or more types of multivalent molecules, each type having a nucleotide unit selected from the group consisting of: dATP, dGTP, dCTP, dTTP and / or dUTP.

74. The engineered polymerase of claim 69, wherein at least one multivalent molecule of the plurality of multivalent molecules comprises a core labeled with a fluorophore.

75. The engineered polymerase of claim 69, wherein at least one multivalent molecule of the plurality of multivalent molecules comprises a nucleotide unit labeled with a fluorophore.

76. The engineered polymerase of claim 67, wherein the plurality of composite polymerases further comprises a plurality of nucleotides, wherein individual nucleotides in the plurality of nucleotides comprise an aromatic base, a pentose sugar, and 1-10 phosphate groups.

77. The engineered polymerase of claim 76, wherein the plurality of nucleotides comprises a type of nucleotide selected from the group consisting of: dATP, dGTP, dCTP, dTTP, and dUTP.

78. The engineered polymerase of claim 76, wherein the plurality of nucleotides comprises a mixture of any combination of two or more types of nucleotides selected from the group consisting of: dATP, dGTP, dCTP, dTTP, and / or dUTP.

79. The engineered polymerase of claim 76, wherein at least one nucleotide in the plurality of nucleotides is labeled with a fluorophore.

80. The engineered polymerase of claim 76, wherein the plurality of nucleotides lacks a fluorophore label.

81. An engineered polymerase according to claim 76, wherein at least one of the nucleotides in the plurality of nucleotides comprises a removable chain-terminating portion attached to the 3′ carbon position of a sugar group, wherein the removable chain-terminating portion comprises an alkyl group, an alkenyl group, an alkynyl group, an allyl group, an aryl group, a benzyl group, an azide group, an azido group, an O-azidomethyl group, an amine group, an amide group, a ketone group, an isocyanate group, a phosphate group, a thio group, a disulfide group, a carbonate group, a urea group or a silyl group, and wherein the removable chain-terminating portion can be cleaved with a chemical compound to produce an extendable 3′ OH portion on the sugar group.

82. The engineered polymerase of claim 67, wherein the plurality of complex polymerases further comprises a plurality of non-catalytic divalent cations that inhibit polymerase-catalyzed nucleotide incorporation, wherein the non-catalytic divalent cations comprise strontium or barium.

83. The engineered polymerase of claim 67, wherein the plurality of composite polymerases further comprises a plurality of catalytic divalent cations that facilitate polymerase-catalyzed nucleotide incorporation, wherein the catalytic divalent cations comprise magnesium or manganese.

84. The engineered polymerase of claim 67, wherein the plurality of complex polymerases is immobilized to a support or to a coating on the support.

85. The engineered polymerase of claim 84, wherein the density of the plurality of composite polymerases immobilized to the carrier is 10 2 -10 12 / mm 2 .

86. The engineered polymerase of claim 84, wherein the plurality of immobilized composite polymerases are immobilized to predetermined sites on the support or to random sites on the support.

87. An engineered polymerase according to claim 84, wherein the coating comprises at least one hydrophilic polymer coating, and the at least one hydrophilic polymer coating comprises unbranched polyethylene glycol (PEG), or wherein the coating comprises at least one hydrophilic polymer coating, and the at least one hydrophilic polymer coating comprises branched polyethylene glycol (PEG) having at least 4 branches.

88. The engineered polymerase of claim 87, wherein the water contact angle of the hydrophilic polymer coating does not exceed 45 degrees.

89. The engineered polymerase of claim 84, wherein the plurality of immobilized composite polymerases are fluidically connected to each other to allow a reagent solution to flow onto the carrier, such that the plurality of immobilized composite polymerases on the carrier react with the reagent solution in a massively parallel manner.

90. The engineered polymerase of claim 67, wherein the plurality of complex polymerases further comprises a first binding complex and a second binding complex, wherein (i) the first binding complex comprises a first nucleic acid primer, a first polymerase, and a first multivalent molecule, wherein the first nucleic acid primer, the first polymerase, and the first multivalent molecule bind to a first portion of a concatemer template molecule to form a first binding complex, wherein a first nucleotide unit of the multivalent molecule binds to the first polymerase, and (ii) the second binding complex comprises a second nucleic acid primer, a second polymerase, and the first multivalent molecule, wherein the second nucleic acid primer, the second polymerase, and the first multivalent molecule bind to a second portion of the same concatemer template molecule, thereby forming a second binding complex, wherein a second nucleotide unit of the multivalent molecule binds to the second polymerase, The first binding complex and the second binding complex comprising the same multivalent molecule form an affinity complex.

91. A method for forming a plurality of composite polymerases, the method comprising: A plurality of engineered polymerases are contacted with (i) a plurality of nucleic acid template molecules and (ii) a plurality of nucleic acid primers under conditions suitable for forming a plurality of composite polymerases, each composite polymerase comprising a polymerase that binds to a nucleic acid duplex, wherein the nucleic acid duplex comprises a nucleic acid template molecule hybridized to a nucleic acid primer, wherein the plurality of engineered polymerases comprises an amino acid sequence according to any one of claims 1, 2, 36 or 37.

92. The method of claim 91, wherein the plurality of nucleic acid template molecules comprises linear nucleic acid molecules, circular nucleic acid molecules, or a mixture of linear nucleic acid molecules and circular nucleic acid molecules.

93. The method of claim 91, wherein the plurality of nucleic acid template molecules comprises clonally amplified template molecules.

94. The method of claim 91, wherein an individual nucleic acid template molecule in the plurality of nucleic acid molecules comprises one copy of a target sequence of interest, or wherein an individual nucleic acid template molecule in the plurality of nucleic acid molecules comprises a concatemer having two or more tandem copies of a target sequence of interest.

95. The method of claim 91, wherein the plurality of nucleic acid molecules comprise the same target sequence of interest or different target sequences of interest.

96. The method of claim 91, further comprising: The plurality of composite polymerases is contacted with a plurality of multivalent molecules, wherein individual multivalent molecules in the plurality comprise: (a) a core; and (b) a plurality of nucleotide arms, the plurality of nucleotide arms comprising: (i) a core attachment portion, (ii) a spacer, (iii) a linker, and (iv) a nucleotide unit, wherein the core is attached to the plurality of nucleotide arms via its core attachment portion, wherein the spacer is attached to the linker, and wherein the linker is attached to the nucleotide unit.

97. The method of claim 96, wherein the linker comprises an aliphatic chain having 2-6 subunits or an oligoethylene glycol chain having 2-6 subunits.

98. The method of claim 96, wherein the plurality of nucleotide arms attached to a given core have the same type of nucleotide units, and wherein the type of nucleotide units comprises dATP, dGTP, dCTP, dTTP, or dUTP.

99. The method of claim 96, wherein the plurality of multivalent molecules comprises one type of multivalent molecule, wherein each multivalent molecule in the plurality has the same type of nucleotide unit selected from the group consisting of: dATP, dGTP, dCTP, dTTP, and dUTP.

100. The method of claim 96, wherein the plurality of multivalent molecules comprises a mixture of any combination of two or more types of multivalent molecules, each type having a nucleotide unit selected from the group consisting of: dATP, dGTP, dCTP, dTTP and / or dUTP.

101. The method of claim 96, wherein at least one of the plurality of multivalent molecules is labeled with a fluorophore.

102. The method of claim 96, wherein at least one multivalent molecule of the plurality of multivalent molecules comprises a core labeled with a fluorophore.

103. The method of claim 96, wherein at least one multivalent molecule of the plurality of multivalent molecules comprises one or more nucleotide units labeled with a fluorophore.

104. The method of claim 96, wherein the contacting is performed under conditions suitable for binding the complementary nucleotide units of at least one of the multivalent molecules to at least one of the composite polymerases.

105. The method of claim 96, further comprising contacting the plurality of complex polymerases with a plurality of non-catalytic divalent cations that inhibit polymerase-catalyzed nucleotide incorporation, wherein the non-catalytic divalent cations comprise strontium or barium.

106. The method of claim 91, further comprising: The plurality of composite polymerases is contacted with a plurality of nucleotides, wherein individual nucleotides in the plurality of nucleotides include an aromatic base, a pentose sugar, and 1-10 phosphate groups.

107. The method of claim 106, wherein the plurality of nucleotides comprises a type of nucleotide selected from the group consisting of: dATP, dGTP, dCTP, dTTP, and dUTP.

108. The method of claim 106, wherein the plurality of nucleotides comprises a mixture of any combination of two or more types of nucleotides selected from the group consisting of: dATP, dGTP, dCTP, dTTP, and / or dUTP.

109. The method of claim 106, wherein at least one nucleotide in the plurality of nucleotides is labeled with a fluorophore.

110. The method of claim 106, wherein the plurality of nucleotides lacks a fluorophore label.

111. A method according to claim 106, wherein at least one of the nucleotides in the plurality of nucleotides comprises a removable chain-terminating portion attached to the 3′ carbon position of a sugar group, wherein the removable chain-terminating portion comprises an alkyl group, an alkenyl group, an alkynyl group, an allyl group, an aryl group, a benzyl group, an azide group, an azido group, an O-azidomethyl group, an amine group, an amide group, a ketone group, an isocyanate group, a phosphate group, a thio group, a disulfide group, a carbonate group, a urea group, a silyl group or an acetal group, and wherein the removable chain-terminating portion can be cleaved with a chemical compound to produce an extendable 3′OH portion on the sugar group.

112. The method of claim 106, wherein the contacting is performed under conditions suitable for binding at least one complementary nucleotide from the plurality of nucleotides to at least one complex polymerase.

113. The method of claim 106, further comprising contacting the plurality of composite polymerases with a plurality of catalytic divalent cations that promote polymerase-catalyzed nucleotide incorporation, wherein the catalytic divalent cations comprise magnesium or manganese.

114. The method of claim 91, wherein the plurality of complex polymerases are immobilized to a support or to a coating on the support.

115. The method according to claim 114, wherein the density of the plurality of composite polymerases immobilized to the carrier is 10 2 -10 12 / mm 2 .

116. The method according to claim 114, wherein the plurality of immobilized composite polymerases are immobilized to predetermined sites on the carrier, or the plurality of immobilized composite polymerases are immobilized to random sites on the carrier.

117. A method according to claim 114, wherein the coating comprises at least one hydrophilic polymer coating, and the at least one hydrophilic polymer coating comprises unbranched polyethylene glycol (PEG), or wherein the coating comprises at least one hydrophilic polymer coating, and the at least one hydrophilic polymer coating comprises branched polyethylene glycol (PEG) having at least 4 branches.

118. The method of claim 117, wherein the hydrophilic polymer coating has a water contact angle of no more than 45 degrees.

119. The method of claim 91, wherein the plurality of immobilized composite polymerases are fluidically connected to each other to allow a reagent solution to flow onto the carrier, such that the plurality of immobilized composite polymerases on the carrier react with the reagent solution in a massively parallel manner.

120. The method of claim 96, comprising forming a plurality of binding complexes, the method comprising the steps of: a) combining a first nucleic acid primer, a first polymerase, and a first multivalent molecule with a first portion of a concatemer template molecule, thereby forming a first binding complex, wherein a first nucleotide unit of the first multivalent molecule is bound to the first polymerase; as well as b) combining a second nucleic acid primer, a second polymerase, and the first multivalent molecule with a second portion of the same concatemer template molecule, thereby forming a second binding complex, wherein the second nucleotide unit of the first multivalent molecule is bound to the second polymerase, The first binding complex and the second binding complex comprising the same multivalent molecule form an affinity complex.

121. A method for determining the sequence of a nucleic acid template, the method comprising: a) contacting a plurality of first polymerases with (i) a plurality of nucleic acid templates and (ii) a plurality of nucleic acid primers, each nucleic acid template comprising a target sequence of interest, wherein the contacting is performed under conditions suitable for binding the plurality of first polymerases to the plurality of nucleic acid template molecules and the plurality of nucleic acid primers, thereby forming a plurality of first composite polymerases, each first composite polymerase comprising a first polymerase bound to a nucleic acid duplex, wherein the nucleic acid duplex comprises a nucleic acid template molecule hybridized to a nucleic acid primer, wherein the plurality of first polymerases comprises an amino acid sequence according to any one of claims 1, 2 or 37; b) contacting the plurality of first composite polymerases with a plurality of multivalent molecules to form a plurality of multivalent binding complexes, wherein an individual multivalent molecule of the plurality comprises a core attached to a plurality of nucleotide arms, and each nucleotide arm is attached to a nucleotide unit, wherein the contacting is performed under conditions suitable for binding complementary nucleotide units of the multivalent molecules to at least two first composite polymerases of the plurality of first composite polymerases, thereby forming a plurality of multivalent binding complexes, and wherein the conditions are suitable for inhibiting incorporation of the complementary nucleotide units into primers of the plurality of multivalent binding complexes; c) detecting the plurality of multivalent binding complexes; as well as d) identifying the bases of the complementary nucleotide units in the plurality of multivalent binding complexes, thereby determining the sequence of the nucleic acid template molecule.

122. The method of claim 121, further comprising: e) dissociating the plurality of multivalent binding complexes by removing the plurality of first polymerases and the multivalent molecules bound thereto, and retaining a plurality of nucleic acid duplexes; f) contacting the plurality of retained nucleic acid duplexes with the plurality of second polymerases under conditions suitable for binding of the plurality of second polymerases to the plurality of retained nucleic acid duplexes of step (e), thereby forming a plurality of second composite polymerases, each second composite polymerase comprising a second polymerase that binds to the nucleic acid duplexes, wherein the plurality of second polymerases comprises an amino acid sequence according to any one of claims 1, 2 or 37; and g) contacting the plurality of second composite polymerases with a plurality of nucleotides, wherein the contacting is performed under conditions suitable for binding complementary nucleotides from the plurality of nucleotides to at least two of the second composite polymerases, thereby forming a plurality of nucleotide binding complexes, and the conditions are suitable for promoting incorporation of the bound complementary nucleotides into primers of the nucleotide binding complexes.

123. The method of claim 122, wherein the plurality of nucleotides in step (g) comprises a plurality of unlabeled nucleotides.

124. The method of claim 122, wherein the plurality of nucleotides in step (g) comprises a plurality of labeled nucleotides, and the method further comprises: h) detecting the complementary nucleotide incorporated into the primer of the nucleotide composite polymerase; as well as i) identifying the base of the complementary nucleotide incorporated into the primer of the nucleotide composite polymerase.

125. The method of claim 121, wherein step (b) of contacting the plurality of first complex polymerases with the plurality of multivalent molecules is performed in the presence of a non-catalytic divalent cation that inhibits polymerase-catalyzed nucleotide incorporation, wherein the non-catalytic divalent cation comprises strontium or barium.

126. The method of claim 122, wherein step (g) of contacting the plurality of second complex polymerases with the plurality of nucleotides is performed in the presence of a catalytic divalent cation that promotes polymerase-catalyzed nucleotide incorporation, wherein the catalytic divalent cation comprises magnesium or manganese.

127. The method of claim 121, wherein the plurality of nucleic acid template molecules in step (a) comprises clonally amplified template molecules.

128. The method of claim 121, wherein an individual nucleic acid template molecule in the plurality of nucleic acid molecules of step (a) comprises one copy of a target sequence of interest, or comprises a concatemer having two or more tandem copies of a target sequence of interest.

129. The method of claim 121, wherein the nucleic acid template molecules in the plurality of nucleic acid molecules in step (a) comprise the same target sequence of interest or different target sequences of interest.

130. The method of claim 121, wherein an individual multivalent molecule of the plurality of multivalent molecules comprises: (a) Nucleus; and (b) a plurality of nucleotide arms, the plurality of nucleotide arms comprising: (i) a core attachment portion, (ii) a spacer, (iii) a linker, and (iv) a nucleotide unit, wherein the core is attached to the plurality of nucleotide arms via its core attachment portion, wherein the spacer is attached to the linker, and wherein the linker is attached to the nucleotide unit.

131. The method of claim 130, wherein the linker comprises an aliphatic chain having 2-6 subunits or an oligoethylene glycol chain having 2-6 subunits.

132. The method of claim 130, wherein the plurality of nucleotide arms attached to a given core have the same type of nucleotide units, and wherein the type of nucleotide units comprises dATP, dGTP, dCTP, dTTP, or dUTP.

133. The method of claim 130, wherein the plurality of multivalent molecules comprises one type of multivalent molecule, wherein each multivalent molecule in the plurality has the same type of nucleotide unit selected from the group consisting of: dATP, dGTP, dCTP, dTTP, and dUTP.

134. The method of claim 130, wherein the plurality of multivalent molecules comprises a mixture of any combination of two or more types of multivalent molecules, each type having a nucleotide unit selected from the group consisting of: dATP, dGTP, dCTP, dTTP and / or dUTP.

135. The method of claim 130, wherein at least one multivalent molecule of the plurality of multivalent molecules is labeled with a fluorophore.

136. The method of claim 130, wherein at least one multivalent molecule of the plurality of multivalent molecules comprises a core labeled with a fluorophore.

137. The method of claim 130, wherein at least one multivalent molecule in the plurality of multivalent molecules comprises one or more nucleotide units labeled with a fluorophore.

138. The method of claim 122, wherein individual nucleotides of the plurality of nucleotides in step (g) comprise an aromatic base, a pentose sugar, and 1-10 phosphate groups.

139. A method according to claim 138, wherein the plurality of nucleotides of step (g) comprises one type of nucleotide selected from the group consisting of: dATP, dGTP, dCTP, dTTP and dUTP, or a mixture of any combination of two or more types of nucleotides selected from the group consisting of: dATP, dGTP, dCTP, dTTP and / or dUTP.

140. The method of claim 122, wherein at least one of the nucleotides in the plurality of nucleotides in step (g) is labeled with a fluorophore.

141. The method of claim 122, wherein the plurality of nucleotides in step (g) lack a fluorophore label.

142. A method according to claim 122, wherein at least one of the nucleotides in the plurality of nucleotides in step (g) comprises a removable chain-terminating portion attached to the 3′ carbon position of the sugar group, wherein the removable chain-terminating portion comprises an alkyl group, an alkenyl group, an alkynyl group, an allyl group, an aryl group, a benzyl group, an azide group, an azido group, an O-azidomethyl group, an amine group, an amide group, a ketone group, an isocyanate group, a phosphate group, a thio group, a disulfide group, a carbonate group, a urea group or a silyl group, and wherein the removable chain-terminating portion can be cleaved with a chemical compound to produce an extendable 3′OH portion on the sugar group.

143. The method of claim 121, wherein the plurality of first complex polymerases in step (a) are immobilized to a carrier or to a coating on the carrier.

144. The method of claim 143, wherein the density of the plurality of first complex polymerases immobilized to the support is 10 2 -10 12 / mm 2 .

145. The method of claim 143, wherein the plurality of first complex polymerases are immobilized to predetermined sites on the support or to random sites on the support.

146. A method according to claim 143, wherein the coating comprises at least one hydrophilic polymer coating, and the at least one hydrophilic polymer coating comprises unbranched polyethylene glycol (PEG), or wherein the coating comprises at least one hydrophilic polymer coating, and the at least one hydrophilic polymer coating comprises branched polyethylene glycol (PEG) having at least 4 branches.

147. The method of claim 146, wherein the water contact angle of the hydrophilic polymer coating does not exceed 45 degrees.

148. The method of claim 121, wherein a plurality of immobilized first complex polymerases are fluidically connected to each other to allow a reagent solution to flow onto the carrier, so that the plurality of immobilized first complex polymerases on the carrier react with the reagent solution in a large-scale parallel manner.

149. The method of claim 121, comprising forming a plurality of binding complexes, the method comprising the steps of: a) combining a first nucleic acid primer, a first polymerase, and a first multivalent molecule with a first portion of a concatemer template molecule, thereby forming a first binding complex, wherein a first nucleotide unit of the first multivalent molecule is bound to the first polymerase; as well as b) combining a second nucleic acid primer, a second polymerase, and the first multivalent molecule with a second portion of the same concatemer template molecule, thereby forming a second binding complex, wherein the second nucleotide unit of the first multivalent molecule is bound to the second polymerase, The first binding complex and the second binding complex comprising the same multivalent molecule form an affinity complex.

150. The method of claim 121, further comprising: a) contacting the plurality of polymerases and the plurality of nucleic acid primers with different portions of a concatemer nucleic acid template molecule to form at least a first composite polymerase and a second composite polymerase on the same concatemer template molecule; b) contacting the plurality of multivalent molecules with the at least first composite polymerase and the second composite polymerase on the same concatemer template molecule under conditions suitable for binding a single multivalent molecule from the plurality of multivalent molecules to the first composite polymerase and the second composite polymerase, wherein at least a first nucleotide unit of the single multivalent molecule is bound to the first composite polymerase, the first composite polymerase comprising a first primer that hybridizes to a first portion of the concatemer template molecule, thereby forming a first binding complex, and wherein at least a second nucleotide unit of the single multivalent molecule is bound to the second composite polymerase, the second composite polymerase comprising a second primer that hybridizes to a second portion of the concatemer template molecule, thereby forming a second binding complex, and wherein said contacting is performed under conditions suitable for inhibiting polymerase-catalyzed incorporation of the bound first nucleotide unit and second nucleotide unit into said first bound complex and said second bound complex, and ● wherein said first binding complex and said second binding complex bound to the same multivalent molecule form an affinity complex; c) detecting the first binding complex and the second binding complex on the same concatemer template molecule; as well as d) identifying the first nucleotide unit in the first binding complex, thereby determining the sequence of the first portion of the concatemer template molecule, and identifying the second nucleotide unit in the second binding complex, thereby determining the sequence of the second portion of the concatemer template molecule.

Images