DNA polymerases and related methods

Abstract

Provided herein is an engineered DNA polymerases containing an amino acid sequence selected from: an amino acid sequence that is at least 80% identical to an amino acid sequence selected from: SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:24; and an amino acid sequence that is at least 92% identical to an amino acid sequence selected from: SEQ ID NO:2 and SEQ ID NO:3. Also provided are methods employing the described DNA polymerases.

Description

[0001]DNA POLYMERASES AND RELATED METHODS CROSS-REFERENCING This applicaƟon claims the benefit of U.S. provisional applicaƟon serial no.63 / 610,498, filed on December 15, 2023, which applicaƟon is incorporated by reference herein. SEQUENCE LISTING This applicaƟon isfiled with a Sequence LisƟng in electronic form as a Sequence LisƟng XML, “NEB-474.xml.” created on November 27, 2024, and having a size of 27,332 bytes. The contents of the Sequence LisƟng XML are incorporated by reference herein in their enƟrety. BACKGROUND Sequence-specific DNA amplificaƟon is widely used in research and medical diagnosƟcs. The most common approaches for amplifying DNA strands are polymerase chain reacƟon (PCR) and isothermal amplificaƟon. PCR relies upon exposure of the samples to periods of high temperature in a thermal cycling instrument to separate the strands of DNA duplexes that are formed when the polymerase copies the template strand. Strand separaƟon is necessary to free the newly generated DNA strands so that they can be copied to achieve exponenƟal amplificaƟon. Bst DNA polymerases are enzymes that can copy DNA or RNA strands and have a robust ability to separate the resulƟng duplexes and displace the upstream strand during synthesis. This strand displacing capability removes the need for high temperature strand separaƟon and enables constant temperature (isothermal) amplificaƟon. Tests employing isothermal amplificaƟon, such as LAMP tests, are low-cost compared to PCR because they do not require a thermal cycler or other expensive equipment. Isothermal amplificaƟon techniques conƟnue to gain popularity following their widespread adopƟon for COVID detecƟon, and there is a need for conƟnued broadening of constant temperature test capabiliƟes. Thus, it is desirable to have engineered Bst-like DNA polymerases with enhanced funcƟonality, such as fast reacƟon Ɵmes, low background acƟvity, and thermal stability. SUMMARY Provided herein are engineered DNA polymerases containing an amino acid sequence selected from: an amino acid sequence that is at least 80% idenƟcal to an amino acid sequence selected from: SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:24; and an amino acid sequence that is at least 92% idenƟcal to an amino acid sequence selected from: SEQ ID NO:2 and SEQ ID NO:3. Also provided are methods employing the described DNA polymerases. The methods involve incubaƟng a reacƟon mixture containing (i) a DNA polymerase; (ii) a target nucleic acid; (iii) dNTPs; and (iv) one or more primers, under condiƟons suitable for polynucleoƟde extension of the target nucleic acid to produce copied DNA product. Further provided are composiƟons containing such a DNA polymerase, as well as kits containing such a DNA polymerase and one or more components for carrying out a polynucleoƟde extension reacƟon. The DNA polymerases described herein have enhanced funcƟonality relaƟve to commercially available strand-displacing DNA polymerases, such as fast reacƟon Ɵmes, low background acƟvity, and thermal stability. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows acƟviƟes of exemplary embodiments of DNA polymerases SDpol-1, SDpol-2, SDpol-3, SDpol-4, SDpol-5 and SDpol-6 compared to commercially available enzyme in a LAMP assay, where amplified lambda DNA target is depicted byfilled circles and no template control (NTC) is depicted by open circles. Figure 2 shows thermostability of exemplary embodiments of DNA polymerases using a LAMP assay as a read-out, where acƟvity was maintained aŌer heat challenges of up to 85 ° C. Figure 3 shows LAMP assay performance of exemplary embodiments of DNA polymerases including an embodiment of SDpol-1 and fusion proteins with DNA binding domains Sso7d, BD007, BD023, BD009, BD062, BD093, BD109, BD006, and BD012, where amplified lambda DNA target is depicted byfilled circles and no template control is depicted by open circles. Figure 4 shows a comparison of exemplary embodiments of DNA polymerases BD009- SD-pol1 and Sso7d-SDpol-1 with commercially available enzymes Bst 2.0 and Bst 3.0, in a fluorescence LAMP assay, indicaƟng Ɵme to detecƟon of amplified human genomic DNA. Figures 5A and 5B show a comparison of exemplary embodiments of DNA polymerases BD009-SD-pol1 and Sso7d-SDpol-1 with commercially available enzyme Bst 2.0, in a colorimetric LAMP assay, indicaƟng amplificaƟon of target, where a grey scale hue below 100 arbitrary units (red sample color) indicates no amplificaƟon, and a grey scale hue above 100 arbitrary units indicates amplificaƟon (yellow sample color). Figure 5A shows hue values prior to amplificaƟon; Figure 5B shows hue values aŌer 60 minutes of amplificaƟon. DESCRIPTION This disclosure provides, among other things, DNA polymerases that have improvements in one or more properƟes. For example, the present DNA polymerases are believed to be more heat tolerant relaƟve to commercially available Bst enzymes. Although embodiments of the disclosure are explained in detail, it is to be understood that other embodiments are contemplated. Accordingly, it is not intended that the invenƟon is limited in its scope to the details of construcƟon and arrangement of components set forth in the following descripƟon or illustrated in the drawings. The invenƟon is capable of other embodiments and of being pracƟced or carried out in various ways. Also, in describing the embodiments, specific terminology will be resorted to for the sake of clarity. It must also be noted that, as used in the specificaƟon and the appended claims, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise. For example, reference to a sheet or porƟon is intended also to include the manufacturing of a plurality of sheets or porƟons. References to a sheet containing "a" consƟtuent is intended to include other consƟtuents in addiƟon to the one named. All cited publicaƟons are incorporated by reference herein. Also, in describing the embodiments, terminology will be resorted to for the sake of clarity. It is intended that each term contemplates its broadest meaning as understood by those skilled in the art and includes all technical equivalents which operate in a similar manner to accomplish a similar purpose. Ranges can be expressed herein as from "about" or "approximately" one parƟcular value and / or to "about" or "approximately" another parƟcular value. When such a range is expressed, another embodiment includes from the one parƟcular value and / or to the other parƟcular value. "Comprising" or "containing" or "including" mean that at least the named compound, element, parƟcle, or method step is present in the composiƟon or arƟcle or method, but does not exclude the presence of other compounds, materials, parƟcles, method steps, even if the other such compounds, material, parƟcles, method steps have the same funcƟon as what is named. The term “non-naturally occurring” used in reference to a polypepƟde or composiƟon described herein means that the polypepƟde or composiƟon does not exist in nature. A “non- naturally occurring” composiƟon can differ from naturally occurring composiƟons in one or more of the following respects: (a) having components that are not combined in nature; (b) having components in concentraƟons not found in nature; (c) omiƫng one or components otherwise found in naturally occurring composiƟons; (d) having a form not found in nature, e.g., dried, freeze dried, crystalline, micellular, aqueous; and (e) having one or more addiƟonal components beyond those found in nature (e.g., buffering agents, a detergent, a dye, a solvent or a preservaƟve). The DNA polymerases described herein are examples of non-naturally occurring polypepƟdes, and the composiƟons comprising the DNA polymerases, including those created when performing the related methods, are examples of non-naturally occurring composiƟons. This disclosure relates to strand-displacing DNA polymerases designed using algorithmic strategies. As background, strand displacing polymerases, exemplified by the well-known Bst polymerase, belong to the “A” family of DNA polymerases, which in their naƟve state contain an N-terminal 5’-3’ exonuclease domains upstream of a 3’-5’ exonuclease domain and a polymerase domain. The 5’-3’ exonuclease acƟvity can be eliminated by mutaƟons or truncaƟons while maintaining the DNA polymerase acƟvity (see, e.g., Riggs et al., Biochim Biophys Acta 1996;1307:178-86). The crystal structure of a truncated Bst polymerase from Bacillus stearothermophilus has been solved (see, e.g., Kiefer JR et al, Structure 1997;5:95-108) and many mutaƟons in Bst polymerases are known (see, e.g., Oscorbin et al. Comput Struct Biotechnol J.2023 Sep 12;21:4519-4535). Variants of a DNA polymerase described herein can thus be designed using sequence alignments and published structural informaƟon. In various embodiments, provided herein are DNA polymerases comprising an amino acid sequence selected from: an amino acid sequence that is at least 80% idenƟcal to an amino acid sequence selected from: SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:24; and an amino acid sequence that is at least 92% idenƟcal to an amino acid sequence selected from: SEQ ID NO:2 and SEQ ID NO:3. In an embodiment, the DNA polymerase includes an amino acid sequence selected from amino acid sequences at least 80% idenƟcal to an amino acid sequence selected from: SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:24. In an embodiment, the DNA polymerase can comprise an amino acid sequence at least 85% idenƟcal, at least 88% idenƟcal, at least 90% idenƟcal, at least 92% idenƟcal, at least 93% idenƟcal, at least 95% idenƟcal, at least 96% idenƟcal, at least 97% idenƟcal, at least 98% idenƟcal or at least 99% idenƟcal to any of SEQ ID NOS: 4-6. In an embodiment, the DNA polymerase comprises an amino acid sequence that is idenƟcal to SEQ ID NO: 4, SEQ ID NO:5, SEQ ID NO:6 or SEQ ID NO:24. In an embodiment, the DNA polymerase includes an amino acid sequence selected from amino acid sequences at least 92% idenƟcal to SEQ ID NO:2, at least 93% idenƟcal, at least 95% idenƟcal, at least 96% idenƟcal, at least 97% idenƟcal, at least 98% idenƟcal or at least 99% idenƟcal to SEQ ID NO:2. In an embodiment, the DNA polymerase comprises an amino acid sequence that containsfive or fewer amino acid subsƟtuƟons, four or fewer amino acid subsƟtuƟons, three or fewer amino acid subsƟtuƟons, two or fewer amino acid subsƟtuƟons, or one amino acid subsƟtuƟon relaƟve to SEQ ID NO:2. In an embodiment, the DNA polymerase comprises an amino acid sequence idenƟcal to SEQ ID NO:2. The DNA polymerase can contain one or more of the following amino acids, which are present in SEQ ID NO:2 and different from the corresponding stretch of amino acids of a truncated version of the wild type Bst NCBI Reference Sequence: WP_108438052.1 (provided herein as SEQ ID NO:1): M at posiƟon 1, E at posiƟon 4, I at posiƟon 11, E at posiƟon 17, T at posiƟon 18, E at posiƟon 27, S at posiƟon 28, Q at posiƟon 41, L at posiƟon 45, F at posiƟon 47, H at posiƟon 55, I at posiƟon 58, P at posiƟon 59, T at posiƟon 60, V at posiƟon 62, S at posiƟon 66, A at posiƟon 68, R at posiƟon 70, E at posiƟon 74, V at posiƟon 82, I at posiƟon 89, H at posiƟon 95, K at posiƟon 100, I at posiƟon 102, D at posiƟon 103, S at posiƟon 116, E at posiƟon 117, S at posiƟon 118, N at posiƟon 119, T at posiƟon 132, Q at posiƟon 135, E at posiƟon 139, A at posiƟon 148, V at posiƟon 149, N at posiƟon 153, V at posiƟon 154, K at posiƟon 164, Y at posiƟon 167, K at posiƟon 170, E at posiƟon 171, T at posiƟon 172,M174I at posiƟon 174, E at posiƟon 176, K at posiƟon 178, E at posiƟon 179, Y at posiƟon 183, E at posiƟon 184, M at posiƟon 191, S at posiƟon 194, H at posiƟon 195, K at posiƟon 206, E at posiƟon 210, Q at posiƟon 213, E at posiƟon 217, E at posiƟon 225, E at posiƟon 236, T at posiƟon 237, I at posiƟon 259, I at posiƟon 283, Q at posiƟon 309, T at posiƟon 331, E at posiƟon 357, V at posiƟon 360, N at posiƟon 379, Q at posiƟon 389, D at posiƟon 407, S at posiƟon 434, G at posiƟon 438, Q at posiƟon 453, D at posiƟon 464, E at posiƟon 525, R at posiƟon 531, K at posiƟon 558, N at posiƟon 566. In an embodiment, a DNA polymerase includes an amino acid sequences that is at least 92% idenƟcal, at least 93% idenƟcal, at least 95% idenƟcal, at least 96% idenƟcal, at least 97% idenƟcal, at least 98% idenƟcal or at least 99% idenƟcal to SEQ ID NO:3. In an embodiment, the DNA polymerase comprises an amino acid sequence idenƟcal to SEQ ID NO:3. In an embodiment, the DNA polymerase comprises an amino acid sequence that containsfive or fewer amino acid subsƟtuƟons, four or fewer amino acid subsƟtuƟons, three or fewer amino acid subsƟtuƟons, two or fewer amino acid subsƟtuƟons, or one amino acid subsƟtuƟon relaƟve to SEQ ID NO:3. In an embodiment, the DNA polymerase can contain one or more of the following amino acids, which are present in SEQ ID NO:3 and different from the corresponding stretch of amino acids of a wild type Bst NCBI Reference Sequence: GCD81562.1 (provided herein as SEQ ID NO:7): M at posiƟon 292, E at posiƟon 293,P at posiƟon 294,T at posiƟon 296, E at posiƟon 300, S at posiƟon 302, K at posiƟon 304, E at posiƟon 307, I at posiƟon 309, E at posiƟon 312, T at posiƟon 315, E at posiƟon 327, N at posiƟon 343,V at posiƟon 358, K at posiƟon 360, E at posiƟon 361,T at posiƟon 367,A at posiƟon 381,I at posiƟon 387 , K at posiƟon 390,D at posiƟon 393, T at posiƟon 422,D at posiƟon 423,K at posiƟon 460, Q at posiƟon 465, E at posiƟon 466, K at posiƟon 468, I at posiƟon 516, K at posiƟon 519, Q at posiƟon 522, E at posiƟon 575, K at posiƟon 599K, V at posiƟon 643, E at posiƟon 647, A at posiƟon 653,A at posiƟon 675, D at posiƟon 680, E at posiƟon 696, G at posiƟon 728, E at posiƟon 739, E at posiƟon 753, E at posiƟon 754, D at posiƟon 758, A at posiƟon 814, E at posiƟon 818,Q at posiƟon 823, E at posiƟon 841, K at posiƟon 848. With reference to an amino acid, “posiƟon” refers to the place such amino acid occupies in the primary sequence of a polypepƟde numbered from its amino terminus to its carboxy terminus. A posiƟon in one primary sequence can correspond to a posiƟon in a second primary sequence, for example, where the two posiƟons are opposite one another when the two primary sequences are aligned using an alignment algorithm (e.g., BLAST (Journal of Molecular Biology.215 (3): 403–410) using default parameters (e.g., expect threshold 0.05, word size 3, max matches in a query range 0, matrix BLOSUM62, Gap existence 11 extension 1, and condiƟonal composiƟonal score matrix adjustment) or custom parameters). An amino acid posiƟon in one sequence can correspond to a posiƟon within a funcƟonally equivalent moƟf or structural moƟf that can be idenƟfied within one or more other sequence(s) in a database by alignment of the moƟfs. Analogously, with reference to a nucleoƟde, “posiƟon” refers to the place such nucleoƟde occupies in the nucleoƟde sequence of an oligonucleoƟde or polynucleoƟde numbered from its 5’ end to its 3’ end. In an embodiment, a DNA polymerase described herein can be a fusion protein. As used herein, the term “fusion protein” means a non-naturally occurring polypepƟde containing two or more amino acid segments that are not joined in their naturally occurring states. A fusion protein can be constructed for a variety of purposes, such as for ease of purificaƟon (e.g., poly- His, chiƟn binding domain, maltose binding protein, glutathione S-transferase (GST), alpha maƟng factor or SNAP-Tag® (New England Biolabs, Ipswich, MA)); for detecƟon (e.g., a fluorescent protein for direct detecƟon, an enzyme for indirect detecƟon such as horse radish peroxidase); for protein translocaƟon within a cell, Ɵssue or organism; for protein interacƟon with other targets (e.g., DNA binding domain, which can be non-specific or specific); for chemical modificaƟon (e.g., to introduce a modificaƟon site). Other kinds of funcƟonal domains can also be joined to a DNA polymerase amino acid sequence described herein. A DNA polymerase described herein can be joined with such domains at its N-terminus, C-terminus, and or the middle porƟon, or at more than one locaƟon. Segments of a fusion protein can opƟonally be separated by a linker. PolypepƟde components of a fusion protein can be joined by one or more pepƟde bonds, disulfide linkages, and / or other covalent bonds. Therefore, provided herein are DNA polymerases that are fusion proteins, comprising a DNA polymerase described herein joined to an exogenous amino acid sequence. In an embodiment, the exogenous amino acid sequence comprises a purificaƟon tag. Fusion proteins of the subject DNA polymerases with poly-His purificaƟon tags are described, for example, in Example 1. In an embodiment, the exogenous amino acid sequence comprises a DNA binding protein domain. In parƟcular embodiments, the DNA binding protein domain can be a DNA binding protein domain listed in Table 1 (see, e.g., US 2016 / 0160193). Example 3 describes a variety of subject DNA polymerases that are N-terminal fusions with DNA binding protein domains, including Sso7d, BD007, BD023, BD009, BD062, BD093, BD109, BD006, and BD012. In an embodiment, the exogenous amino acid sequence comprises another type of funcƟonal domain, such as those described herein above. Table 1: DNA binding domains Name AbbreviaƟon Accession DNA-binding protein Tfx BD-51gi|499321160 The DNA polymerases described herein, including the fusion proteins, can have one or more acƟviƟes selected from strand-displacing DNA polymerase acƟvity, and strand-displacing DNA polymerase acƟvity with reverse transcriptase acƟvity. In an embodiment, the polypepƟde has strand-displacing DNA polymerase acƟvity. In an embodiment, the DNA polymerase has strand-displacing polymerase acƟvity and reverse transcriptase acƟvity. The DNA polymerases described herein are believed to not have substanƟal 5’-3’ exonuclease acƟvity. The DNA polymerases described herein can be thermostable relaƟve to known Bst polymerases. As such, a DNA polymerase described herein can retain its desired acƟvity at temperatures of over 65°C, over 70°C, over 75°C, over 80°C, and over 85°C. As non-limiƟng examples, such DNA polymerases can be present in a reacƟon mixture that is exposed to heat, e.g., when input target nucleic acid is heat denatured for binding to primers, when sample is treated to inacƟvate nucleases, cell lysis, and other steps facilitated by heat treatment. The DNA polymerases described herein can be used for any purpose in which their acƟvity is necessary or desired (generally, their DNA polymerase acƟvity, and for some applicaƟons, their strand-displacing DNA polymerase acƟvity, and opƟonally for some applicaƟons, reverse transcriptase acƟvity). Accordingly, provided herein are methods, which involve incubaƟng a reacƟon mixture containing (i) a DNA polymerase;(ii) a target nucleic acid;(iii) dNTPs; and (iv) one or more primers, under condiƟons suitable for polynucleoƟde extension of the target nucleic acid to produce copied DNA product (e.g., presence of divalent caƟons). In an embodiment, the condiƟons can be isothermal. In an embodiment, the DNA polymerase comprises an amino acid sequence selected from: an amino acid sequence that is at least 80% idenƟcal to an amino acid sequence selected from: SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:24; and an amino acid sequence that is at least 95% idenƟcal to an amino acid sequence selected from: SEQ ID NO:2 and SEQ ID NO:3. As used herein, the term “target nucleic acid” means the substrate for a DNA polymerase described herein. As used herein, the term "nucleic acid" means a polymeric form of nucleoƟdes of any length, such as deoxyribonucleoƟdes or ribonucleoƟdes, or analogs thereof. For example, a nucleic acid can be DNA, RNA or the DNA product of RNA subjected to reverse transcripƟon (cDNA). Non-limiƟng examples of nucleic acids include coding or non-coding regions of a gene or gene fragment, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant nucleic acids, branched nucleic acids, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. Other examples of nucleic acids include, without limitaƟon, cDNA, aptamers, and pepƟde nucleic acids. A nucleic acid can contain modified nucleoƟdes, such as methylated nucleoƟdes and nucleoƟde analogs ("analogous" forms of purines and pyrimidines are well known in the art). If present, modificaƟons to the nucleoƟde structure can be imparted before or aŌer assembly of the polymer. A nucleic acid can be a single-stranded, double-stranded, parƟally single-stranded, or parƟally double-stranded DNA or RNA, depending on the applicaƟon. As used herein, the term “DNA polymerase” means an enzyme capable of catalyzing polynucleoƟde extension. A DNA polymerase can also have reverse transcriptase acƟvity. As used herein, the term "polynucleoƟde extension" means the synthesis of DNA catalyzed by a DNA polymerase resulƟng in polymerizaƟon of individual nucleoside triphosphates using a primer as a point of iniƟaƟon. Generally, a primer is hybridized to a target nucleic acid to form a primer-template complex. The primer-template complex is contacted with the DNA polymerase and nucleoside triphosphates (dNTPs) in a suitable environment to permit the addiƟon of nucleoƟdes to the 3ʹ end of the primer, thereby producing a copied DNA product complementary to at least a porƟon of the target nucleic acid. In strand-displacing polynucleoƟde extension the resulƟng duplexes can be separated by the DNA polymerase, as the upstream strand is displaced during DNA synthesis. This strand-displacing polynucleoƟde extension permits isothermal amplificaƟon of nucleic acid targets, as newly synthesized strands are liberated from duplexes and become available for copying by the polymerase. CondiƟons suitable for polynucleoƟde extension, including strand-displacing polynucleoƟde extension, are known in the art (see, e.g., Sambrook et al., supra. See also Ausubel et al., Short Protocols in Molecular Biology (4th ed., John Wiley & Sons 1999)). In general, a reacƟon mixture for carrying out polynucleoƟde extension using a DNA polymeraseincludes dNTPs, a divalent caƟon (e.g., Mg2+, Mn2+, Co2+, Cd2+), one or more primers, andopƟonally can include an anƟbody, anƟbody-like molecule, an aptamer, or other enƟty to inhibit the DNA polymerase or another reacƟon component under selected condiƟons (such as temperature or salt concentraƟon). Exemplary condiƟons for polynucleoƟde extension using Bst enzymes are widely published (e.g., US9127258B2, US9963687B2, US11492673B2). The design of aptamers is well known (see, e.g., Byun J. Life (Basel).2021 Feb 28;11(3):193). A reacƟon mixture can have a pH range of about 6.5 -10, such as about 7.5 – 9.0, and polynucleoƟde extension can be carried out at a temperature range of about 37 – 80 ° C, including about 50-70 ° C, and about 70 ° C. Higher reacƟon temperatures can have benefits for high GC content or structured DNA / RNA template, speed, and / or reducƟon of nonspecific amplificaƟon. The DNA polymerases described herein are inacƟvated at higher temperatures than some commercially available Bst polymerases (for example, see Example 2). As used herein, the term "reverse transcriptase" means a DNA polymerase that can producefirst-strand cDNA from an RNA template. Such enzymes are commonly referred to as RNA-directed DNA polymerases and have IUBMB acƟvity EC 2.7.7.49. In some cases, a reverse transcriptase can generate a complementary DNA strand using either single-stranded RNA or DNA as a template, although as used herein the term “reverse transcriptase acƟvity” refers to the ability of the DNA polymerase to generate a complementary DNA strand using an RNA template. Thus, in the context of reverse transcripƟon, “template” means the substrate RNA for the reverse transcriptase to make a copied DNA product. An RNA template can be complex (e.g., total RNA, polyA+ RNA, mRNA, etc.) or not complex (e.g., an enriched RNA or an in vitro transcribed product). As used herein, the term “primer” means an oligonucleoƟde that is capable, upon forming a duplex with a nucleic acid template, of acƟng as a point of iniƟaƟon of polynucleoƟde extension and being extended from its 3’ end along the template so that copied DNA product is formed. The sequence of nucleoƟdes added during the extension process is determined by the sequence of the template polynucleoƟde. Primers are of a length compaƟble with their use in synthesis of copied DNA products, and can be in the range of between 6 to 100 nucleoƟdes in length, such as 10 to 75, 15 to 60, 15 to 40, 18 to 30, 20 to 40, 21 to 50, 22 to 45, 25 to 40, and so on, more typically in the range of between 15 to 60 nucleoƟdes long, and any length between the stated ranges. Primers are selected for a parƟcular applicaƟon (e.g., applicaƟons such WGA and MDA can use primers as small as 6-mers; SDA can use primers in the range of 40-mers; LAMP can use primers of about 17 to 50) and are usually single-stranded and contain a 3’ hydroxyl group. A primer can be a DNA oligonucleoƟde, RNA oligonucleoƟde, or hybrid. A primer used for polynucleoƟde extension using a DNA polymerase can contain a feature (e.g., chemical moiety, sequence, modified nucleoƟde, etc.) for the detecƟon or immobilizaƟon of the primer so long as such feature(s) do not destroy the ability of the primer to act as a point of iniƟaƟon of DNA synthesis. For example, primers can contain an addiƟonal nucleic acid sequence at the 5' end that does not hybridize to the target nucleic acid, but that facilitates cloning or sequencing of the amplified product or introduces a site to facilitate exponenƟal amplificaƟon (e.g., T7 RNA polymerase promoter or nicking enzyme recogniƟon site). A primer can include convenƟonal nucleoƟdes, unconvenƟonal nucleoƟdes (e.g., ribonucleoƟdes or labeled nucleoƟdes), nucleoƟde analogs, and mixtures thereof, as suitable for a parƟcular applicaƟon. Among the uses of DNA polymerases for polynucleoƟde extension are isothermal DNA amplificaƟon approaches that rely on the strand displacement acƟvity of the DNA polymerase. The term “isothermal” as used herein means a constant temperature, as opposed to cycling between temperatures. Such isothermal amplificaƟon methods include strand displacement amplificaƟon (SDA) (see, e.g., Milla et al, Biotechniques 1998; 24:392-6), linear target isothermal mulƟmerizaƟon and amplificaƟon (LIMA) (see, e.g., Hafner et al., Biotechniques 2001;30: 852-6), loop-mediated isothermal amplificaƟon (LAMP) (see, e.g., Notomi, et al., E63 Nucleic Acids Res 2000; 28), nicking enzyme amplificaƟon reacƟon (NEAR)(see, e.g., US20090081670A1); recombinase polymerase amplificaƟon (RPA) (see, e.g., Piepenburg et al., PLoS Biol. 2006;4(7):e204); recombinase-assisted amplificaƟon (RAA)(see, e.g., Chen et al., Analyst 2020;145:440-4); whole genome amplificaƟon, MulƟple-strand Displacement AmplificaƟon (MDA) (e.g., extending DNA isolated from Ɵssue (fresh, frozen, or preserved); see, e.g., Aviel- Ronen S, et al. BMC Genomics.2006 Dec 12;7:312), and HDA (helicase dependent amplificaƟon) (see, e.g., Vincent et al., EMBO J 2004; 5(8):795-800) and library prep, including whole genome amplificaƟon. Thus, in some embodiments, a DNA polymerase described herein is used in an isothermal reacƟon. In some embodiments, the isothermal reacƟon is a LAMP reacƟon. LAMP reacƟons use several primers (generally, from four to six primers) that bind to locaƟons on the target nucleic acid (“LAMP primers”). Thus, a method, composiƟon or kit described herein can involve or include one or more, two or more, three or more, four or more,five or more, six or more, or a greater number of primers, e.g., for performing a one or more polynucleoƟde extension reacƟons using a described DNA polymerase (as LAMP primers and in other reacƟon contexts). Guidance for selecƟng LAMP primers, including use of online soŌware such as NEB LAMP primer design tool, PrimerExplorer, LAMP Designer OpƟgene, and Premier BiosoŌ, is well known in the art (see, for example, Parida et al., Rev. Med. Virol, 2008, 18:407-421 and Nagamine et al. Mol. Cell Probes 2002, 16, 223-229). VariaƟons of LAMP reacƟons include reverse transcripƟon loop- mediated isothermal amplificaƟon (RT-LAMP), mulƟplex loop-mediated amplificaƟon (M-LAMP). RT-LAMP reacƟons use reverse transcriptase acƟvity combined with DNA polymerase acƟvity. In an embodiment, a DNA polymerase described herein can possess reverse transcriptase acƟvity. Thus, such DNA polymerases can also be useful in RT-LAMP reacƟons. DNA polymerases described herein can also be used together with other reverse transcriptase enzymes for RT- LAMP reacƟons to detect specific RNA sequences in a sample. PolynucleoƟde extension employing a DNA polymerase can be detected and / or analyzed using various detecƟon methods. Examples include detecƟon of labels such as dyes and dye combinaƟons (e.g., detecƟng dyes associated with copied DNA products); real-Ɵme fluorescence; gel electrophoresis; AC susceptometry (see, for example, Tian et al, Biosens. Bioelectron.2016, 86, 420-425), and turbidimetric analysis. Many dyes can be observed by eye in colorimetric detecƟon or visual observaƟon offluorescence, in addiƟon to or instead of observaƟon by instrumentaƟon. A variety dyes are useful for detecƟng products of polynucleoƟde extension, e.g., molecular beacons, FRET-like dyes, metallochromic indicators, such as 4-(2-pyridylazo) resorcinol (PAR), hydroxynaphthol blue, calcein, malachite green, leuco crystal violet, DNA intercalaƟng dyes such as SYBR Green dyes, EvaGreen, Bromo-PAPS, Goldview dye, Miami Yellow, GelRed dye, SYTO dyes, and berberine. Colorimetric dyes that are pH sensiƟve are useful for colorimetric LAMP reacƟons; examples include phenol red, cresol red, m- cresol purple, bromocresol purple, neutral red, phenolphthalein, naphtholphthalein, and thymol blue; andfluorescent dyes such as 2',7’-Bis-(2-Carboxyethyl)-5-(and-6)-carboxyfluorescein or a carboxyl seminaphthorhodafluor (e.g. SNARF-1). Generally, dyes are selected based on factors such as signal to noise, threshold Ɵme, opƟcal set-up. LAMP sensiƟvity has been improved by reducing background and enhancing signal and these improvements can be used in the methods described herein. See for example: US 9,121,046, US 9,546,358, US 9,074,249, US 9,074,243, US 9,157,073, and US 9,127,258 in addiƟon to US 9,580,748, US 9,034,606, and US 10,597,647 all incorporated in enƟrety by reference. Examples 1-3 describe use offluorescent LAMP reacƟons; Example 4 describes use of colorimetric LAMP reacƟons, and Example 5 describes use of a cas / LAMP reacƟon with fluorescence detecƟon. A DNA polymerase described herein can be used in methods that employ its reverse transcriptase acƟvity. Therefore, in some embodiments, a DNA polymerase is used in a reverse transcripƟon reacƟon. In some embodiments, the reverse transcripƟon reacƟon is carried out in a reacƟon mixture containing an RNA template, one or more primer(s), and a DNA polymerase described herein. The reacƟon mixture typically contains all four standard deoxyribonucleoside triphosphates (dNTPs), a buffer, and a divalent caƟon, and opƟonally can include another reverse transcriptase. The ability of the DNA polymerases described herein to extend DNA and RNA templates is broadly useful in a wide variety of applicaƟons. Among these uses is detecƟng target nucleic acids in samples for research and medical diagnosƟcs. For instance, the DNA polymerases can be used in detecƟng DNA or RNA in diverse samples such as samples obtained from humans, animals, plants, environments (e.g., soils, waters, vehicles, homes, hospitals, airports), and food products, to detect targets of interest such as pathogens (e.g., viruses, bacteria, fungi, parasites) and DNA in forensic and archaeological samples. Thus, as used herein, the term “sample” means a natural or man-made substance suspected of containing a target nucleic acid, such as a biologicalfluid, cell, Ɵssue, or fracƟon thereof, food or environmental substance that can contain or be contaminated by a target nucleic acid. A sample can be derived from a prokaryote or eukaryote and therefore can include cells from, for example, animals, plants, or fungi as well as viruses. Accordingly, a sample includes a specimen obtained from one or more individuals or can be derived from such a specimen. For example, a sample can be a Ɵssue secƟon obtained by biopsy, or cells that are placed in or adapted to Ɵssue culture. Exemplary samples include biological specimens such a cheek swab, nasopharyngeal swab, throat swab, nasopharynxflush through, amnioƟcfluid, skin biopsy, organ biopsy, tumor biopsy, blood, urine, saliva, semen, sputum, cerebral spinalfluid, tears, mucus, and the like. A sample can be further fracƟonated, if desired, to a fracƟon containing parƟcular cell types. For example, a blood sample can be fracƟonated into serum or into fracƟons containing parƟcular types of blood cells. If desired, a sample can be a combinaƟon of samples from an individual such as a combinaƟon of a Ɵssue andfluid, or a combinaƟon of samples from more than one individual (e.g., pooled samples, maternal sample containing fetal nucleic acid). Prior to analysis, a sample can be processed to preserve the integrity of nucleic acid targets. Such methods include the use of appropriate buffers and / or inhibitors, including nuclease, protease, and phosphatase inhibitors, that preserve or minimize changes in the molecules in the sample, including ƟssuefixaƟves (e.g., in the case of FFPE preserved Ɵssues). Also provided by the present disclosure are composiƟons including a DNA polymerase described herein. Such a composiƟon can include one or more DNA polymerases and one or more substances selected for purposes such as storage stability (including a substance such as a solid support, gel, or soluƟon), detecƟon of presence, concentraƟon, or acƟvity of the polymerase, and for performing a method using the polymerase (e.g., providing the DNA polymerase with other components for isothermal amplificaƟon, referenced in some instances as a reacƟon mixture). A composiƟon can contain components for polynucleoƟde extension (e.g., isothermal amplificaƟon of a nucleic acid target), such as dNTPs. ComposiƟons containing dNTPs can include one, two, three of all four of dATP, dTTP, dGTP and dCTP, and can include one or more modified dNTPs, such as forms that are resistant to, or suscepƟble, to a parƟcular enzymaƟc or chemical conversion, or that are detectable. Examples of modified dNTPs include alpha-phosphorothioate dNTPs, dUTP, dITP, labeled dNTPs such as, e.g.,fluorescein- or cyanine-dye family dNTPs. A DNA polymerase composiƟon can include any of (including one or more of) a buffer such as an enzyme storage bugger (e.g., containing a buffering agent such as Tris, MOPS, CAPS, HEPES, Bis- Tris), an excipient, a salt (e.g., NaCl, MgSO4, KCL, (NH4)2SO4), MgCl2, CaCl2), a protein (e.g., albumin, an enzyme, such as a UDG, a reverse transcriptase or another polymerase), a dye (e.g., for detecƟng the presence, concentraƟon or acƟvity of the DNA polymerase), a stabilizer, a detergent (for example, ionic, non-ionic, and / or zwiƩerionic detergents, a poloxamer), a polynucleoƟde such as one or more primers and / or control polynucleoƟdes (e.g., a plasmid, linear RNA or DNA), a cell (e.g., intact, digested, or any cell-free extract), a biological sample, an anƟbody or aptamer (e.g., for inhibiƟng or otherwise affecƟng the acƟvity of a DNA polymerase), a crowding agent, a reacƟon mixture (generally, containing some or all components for carrying out a polynucleoƟde extension using the DNA polymerase), a sugar (e.g., a mono, di, tri, tetra, or higher saccharide), a starch, cellulose, a glass-forming agent (e.g., for lyophilizaƟon), a lipid, an oil, aqueous soluƟon, a support (e.g., a matrix such as a bead,filter paper, slide) and / or (non- naturally occurring) combinaƟons thereof. CombinaƟons can include, for example, two or more of the listed components (e.g., a salt and a buffer) or a plurality of a single listed component (e.g., two different salts or two different sugars). In an embodiment, the DNA polymerase is provided in a soluƟon. The soluƟon can contain glycerol or be glycerol-free. In another embodiment, the DNA polymerase composiƟon includes a solid support. OpƟonally, the DNA polymerase can be aƩached non-covalently (e.g., dried or lyophilized on, or associated by hybridizaƟon or other non-covalent aƩachment) or covalently to the solid support. In an embodiment, the soluƟon or solid support can also include a component such as a buffering agent; a salt; a primer; an aptamer; another component for polynucleoƟde extension. Also provided by the present disclosure are kits for using a DNA polymerase herein. A kit can include one or more DNA polymerases together with one or more other components useful for carrying out a method involving polynucleoƟde extension, such as an isothermal amplificaƟon reacƟon including those described herein above. A kit can therefore contain components for polynucleoƟde extension of a nucleic acid target, such as dNTPs. A kit containing dNTPs can include one, two, three of all four of dATP, dTTP, dGTP and dCTP, and can include one or more modified dNTPs, such as forms that are resistant to, or suscepƟble, to a parƟcular enzymaƟc or chemical conversion, or that are detectable. Examples of modified dNTPs include alpha-phosphorothioate dNTPs, dUTP, dITP, labeled dNTPs such as, e.g.,fluorescein- or cyanin-dye family dNTPs. Examples herein describe inclusion of dUTP in LAMP reacƟons to reduce carryover contaminaƟon. IncorporaƟon of dUTP by a DNA polymerase is commonly used during amplicon generaƟon, and excision of incorporated uracil in copied DNA product and can be catalyzed by a uracil DNA glycosidase (UDG). A kit can include a composiƟon such as a buffer and / or reacƟon mixture in any convenient form, such as in soluƟon, concentrated form, dried form, disposed in, on, or within a solid support (e.g., a tube, plate, pellet, membrane, bead). Such a composiƟon can contain components useful for enabling use of the DNA polymerase in a parƟcular assay format, e.g., to promote a parƟcular aspect of the DNA polymerase enzymaƟc acƟvity, a molecular interacƟon, a stability profile, and other desirable properƟes. Accordingly, a buffer and / or reacƟon mixture can contain one or more salts (e.g., NaCl, MgSO4, KCl, (NH4)2SO4), detergents (ionic, non-ionic, zwiƩerionic), poloxamers, preservaƟves, inhibitors of unwanted acƟviƟes, crowding agents, reducing agents (e.g., DTT, TCEP), catalysts, dyes, (e.g., dyes described herein such as DNA intercalaƟng dyes and colorimetric dyes (e.g., Bromo-PAPS, phenol red)), and other substances. In an embodiment, the reacƟon mixture is suitable for receiving and extending a target nucleic acid in the presence of the DNA polymerase and one or more primers. A reacƟon mixture can include components useful for carrying out a parƟcular protocol. For example, as described in US20210285065A1, which is incorporated herein by reference, a LAMP reacƟon can be carried out in the presence of one or more of guanidine hydrochloride, guanidine thiocyanate, guanidine chloride, guanidine sulfate, or arginine; as such a reacƟon mixture can contain one or more of these components. In an embodiment, the DNA polymerase is in a form selected from: dried form, lyophilized form, and soluƟon form, wherein the soluƟon is opƟonally glycerol-free. In some embodiments, a kit includes one or more oligonucleoƟdes that bind to a predetermined nucleic acid template, e.g., one or more primers for isothermal amplificaƟon of a target nucleic acid. Primers, if included, can be, for example, one or more isothermal amplificaƟon primers, exonuclease-resistant primers, chemically modified primers, e.g., forfluorescence or lateralflow detecƟon, sequencing primers, or combinaƟons thereof. In some embodiments, a kit does not include primers or includes a limited number of primers, in instances where the kit user provides primers appropriate for their selected target nucleic acid. In some embodiments, a kit includes a control, such as a control polynucleoƟde (e.g., a plasmid, linear RNA or DNA, control primer (e.g., rAcƟn control). In some embodiments, a kit includes target-specific primers (e.g., to detect a pathogen). In some embodiments, a kit includes LAMP primers. A kit can contain components for carrying out a Cas / LAMP protocol, such as a cas enzyme and a guide RNA. A kit can contain an oligonucleoƟde probe labeled to facilitate detecƟon, e.g., hybridizaƟon-basedfluorescence or lateralflow detecƟon. A kit can include an aptamer, e.g., for binding to a DNA polymerase to control the condiƟons under which the DNA polymerase has acƟvity (e.g., to reduce off-target amplificaƟon) or for binding to another component in the kit (e.g., another enzyme such as a reverse transcriptase). A kit can also include instrucƟons for pracƟcing a desired method (e.g., extending a target nucleic acid, detecƟng a target nucleic acid, DNA sequencing, DNA labeling) via any communicaƟon means. For example, the instrucƟons can be printed (e.g., on paper or plasƟc), and / or electronic (e.g., provided on a device such as a portable drive, or remotely accessible such as on a web applicaƟon, phone applicaƟon, video, or voice transmission), and / or via demonstraƟon. A kit can include one or more other enzymes, as suitable for a parƟcular purpose. For example, a kit for performing RT-LAMP can opƟonally include a reverse transcriptase in cases where the selected DNA polymerase reverse transcriptase acƟvity is insufficient under the selected reacƟon condiƟons. Components of a kit can be provided in a single container or compartment (e.g., for a single step use) or mulƟple containers or compartments (e.g., for combining, for sequenƟal use, for parallel use, or another desired workflow). A kit can include a sample collecƟon container, which opƟonally can contain a reagent, e.g., for stabilizing the sample (e.g., a poloxamer) or preparing it for assay. In a specific embodiment, a kit includes a DNA polymerase described herein and a component, wherein the component is opƟonally selected from a storage buffer; a reacƟon mixture; a primer, an aptamer. In DNA polymerase is in a form selected from: dried form, lyophilized form, aqueous soluƟon form. In an embodiment, the reacƟon mixture can contain a buffering agent and a salt. The kit can also include dNTPs, as described in more detail above. In an embodiment, the polymerase can be provided in a separate tube from the reacƟon mixture. In an embodiment, a reacƟon mixture is suitable for receiving and extending a target nucleic acid in the presence of the DNA polymerase and one or more primers. Other formats of reacƟon mixtures, which require addiƟon of certain components prior to use are also provided. A kit can contain a DNA polymerase that is thermostable at a parƟcular temperature, as described herein above, e.g., to enable use of reacƟon mixtures that will contain a DNA polymerase during a heaƟng process (e.g., heat lysis of cells, heat denaturaƟon of nucleic acids). EMBODIMENTS Embodiment 1. A DNA polymerase comprising an amino acid sequence selected from: an amino acid sequence that is at least 80% idenƟcal to an amino acid sequence selected from: SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6; and an amino acid sequence that is at least 95% idenƟcal to an amino acid sequence selected from: SEQ ID NO:2 and SEQ ID NO:3. Embodiment 2. The DNA polymerase of embodiment 1, comprising an amino acid sequence that is at least 90% idenƟcal to an amino acid sequence selected from: SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6. Embodiment 3. The DNA polymerase of embodiment 1 or 2, comprising an amino acid sequence that is at least 95% idenƟcal to an amino acid sequence selected from: SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6. Embodiment 4. The DNA polymerase of any of embodiments 1-3, comprising an amino acid sequence that is idenƟcal to an amino acid sequence selected from: SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6. Embodiment 5. A DNA polymerase, wherein the DNA polymerase is a fusion protein comprising an amino acid sequence of any of embodiments 1-4 and an exogenous amino acid sequence. Embodiment 6. The DNA polymerase of embodiment 5, wherein the exogenous amino acid sequence comprises a DNA binding domain. Embodiment 7. The DNA polymerase of embodiment 5 or 6, wherein the exogenous amino acid sequence comprises a purificaƟon tag. Embodiment 8. A method, comprising: incubaƟng a reacƟon mixture comprising (i) a DNA polymerase of any embodiment above; (ii) a target nucleic acid; (iii) dNTPs; and (iv) one or more primers, under condiƟons suitable for polynucleoƟde extension of the target nucleic acid to produce copied DNA product. Embodiment 9. The method of embodiment 8, wherein the condiƟons are isothermal. Embodiment 10. The method of embodiment 9, wherein the one or more primers comprise Loop-Mediated Isothermal AmplificaƟon (LAMP) primers. Embodiment 11. A composiƟon, comprising a DNA polymerase of any of embodiments 1-7. Embodiment 12. The composiƟon of embodiment 11, further comprising a soluƟon, wherein the soluƟon opƟonally comprises glycerol. Embodiment 13. The composiƟon of embodiment 11, further comprising a solid support. Embodiment 14. The composiƟon of any of embodiments 11-13, further comprising a component selected from a buffering agent; a salt; a primer; an aptamer. Embodiment 15. A kit, comprising: (i) a DNA polymerase of any of embodiments 1-7 and (ii) a component, wherein the component is opƟonally selected from a storage buffer; a reacƟon mixture; a primer, an aptamer. Embodiment 16. The kit of embodiment 15, wherein the DNA polymerase is in a form selected from: dried form, lyophilized form, aqueous soluƟon form. Embodiment 17. The kit of embodiment 15 or 16, comprising a reacƟon mixture, wherein the reacƟon mixture comprises a buffering agent and a salt. Embodiment 18. The kit of any of embodiments 15-17, wherein the kit further comprises dNTPs. Embodiment 19. The kit of any of embodiments 15-18, wherein the DNA polymerase is provided in a separate tube from the reacƟon mixture. Embodiment 20. The kit of embodiment 19, wherein the reacƟon mixture is suitable for receiving and extending a target nucleic acid in the presence of the DNA polymerase and one or more primers. Embodiment 21. The kit of any of embodiments 15-20, further comprising one or more addiƟonal enzymes, opƟonally selected from a reverse transcriptase and a uracil DNA glycosidase (UDG). Embodiment 22. The kit of any of embodiments 15-21, further comprising a dye. Embodiment 23. The kit of embodiment 22, wherein the dye is selected from a fluorescent dye and a colorimetric dye. Embodiment 24. The kit of embodiment 22, wherein the dye is a pH sensiƟve dye. Embodiment 25. The kit of any of embodiments 22-24, wherein the dye is selected from Bromo-PAPS and phenol red. The skilled arƟsan will understand that thefigures, described above, and examples, described below, are for illustraƟon purposes only. Neither thefigures nor the examples are intended to limit the scope of the disclosed teachings in any way. EXAMPLES Example 1. Improved performance of syntheƟc DNA polymerases influorescent LAMP assays This example shows acƟviƟes of DNA polymerases in afluorescence LAMP assay using lambda DNA, including lower non template amplificaƟon relaƟve to a commercially available enzyme. DNA polymerases SDpol-1, SDpol-2, SDopol-3, SDpol-4, SDpol-5 and SDpol-6 (SEQ ID NOS: 2, 3, 4, 5, 6, and 24, respecƟvely) were each expressed with an N-terminal His6tag and purified using NEBExpress Ni Spin Columns. Their acƟviƟes were compared to that of BST 2.0 (New England Biolabs, Ipswich, MA) purified in the same manner. LAMP reacƟons contained 20 mM Tris-HCl (pH 8.8), 10 mM (NH4)2SO4, 50 mM KCl, 8 mM MgSO4, 0.1% Tween 20, 1.4 mM dNTPs, 0.5X LAMPfluorescent dye (NEB), and 10 ng of lambda DNA template. Each reacƟon contained six primers at the following concentraƟons: 1.6 µM FIP (CAGCCAGCCGCAGCACGTTCGCTCATAGGAGATATGGTAGAGCCGC (SEQ ID NO:17)), 1.6 µM BIP (GAGAGAATTTGTACCACCTCCCACCGGGCACATAGCAGTCCTAGGGACAGT (SEQ ID NO:18)), 0.2 µM F3 (GGCTTGGCTCTGCTAACACGTT, SEQ ID NO:19), 0.2 µM B3 (GGACGTTTGTAATGTCCGCTCC (SEQ ID NO:20)), 0.4 µM LoopF (ACCATCTATGACTGTACGCC (SEQ ID NO:21)), and 0.4 µM LoopB (CTGCATACGACGTGTCT (SEQ ID NO:22)).25 µL LAMP reacƟons were set up in triplicate with and without lambda DNA template and run at 65 ℃ in a CFX96 Touch Real-Time PCR machine (Bio- Rad), monitoringfluorescence in the SYBR channel every 15 s. Figure 1 shows Cq values (converted to minutes) for each enzyme, with and without lambda DNA template. SDpol-1, SDpol-2, SDpol-3, SDpol-5 and SDpol-6 amplified this target with lower non-template control amplificaƟon relaƟve to BST 2.0, while SDpol-4 was comparable. Example 2. Improved thermostability of DNA polymerases This example shows improved thermostability of DNA polymerases relaƟve to a commercially available Bst enzyme in afluorescence LAMP assay using a lambda DNA target. Thermostability of SDpol-1, SDpol-3, SDpol-4, SDpol-6 was demonstrated by performing one-minute heat challenges ranging from 65℃ to 85℃ before carrying out an isothermal LAMP reaction. LAMP reactions contained the same components as Example 1, except that the dNTP mix included 0.7 mM dUTP. LAMP reactions were carried out at 65 ℃ for 80 minutes. As shown in Figure 2, for SDpol-1, SDpol-3, and SDpol-4, acƟviƟes were unaffected by heat challenge at 65℃, 66℃, 69℃ and 73℃, and SDpol-6 acƟvity was unaffected to 85℃. Example 3. DNA polymerase fusions with DNA binding domains This example shows acƟviƟes of DNA polymerase fusion proteins in afluorescence LAMP assay using a lambda DNA target. Fusion proteins were prepared by adding N-terminal His-tags and N-terminal DNA binding domains Sso7d, BD007, BD023, BD009, BD062, BD093, BD109, BD006, BD012, as described below, to the SDpol-1 polypepƟde. AcƟvity of these fusion proteins was compared to that of SDpol-1 alone, using the same LAMP assay as in Example 1, except the KCl concentraƟon was increased to 100 mM. SEQ ID NO:8, Sso7d: ATVKFKYKGEEKEVDISKIKKVWRVGKMISFTYDEGGGKTGRGAVSEKDAPKELLQMLEKQKK SEQ ID NO:9, BD007: KRRPTINDVAKLAGVSISTVSRYLKDPSQVSEKLGERIREAIKKLGYKPNKIAQGLRTGD SEQ ID NO:10, BD023: HKKLNPKSMKRENKKMVLRYLIESGPHSRVEIARKTGLAQSAIWRIIEELVNEGLVEEKGTATGRRRKAVTYGPT RSFITS SEQ ID NO:11, BD009: KKKYVTIRDIAEKAGVSINTVSRALNNKPDISEETRRKILKIAQELGYVKNATASSLRSK SEQ ID NO:12, BD062: NTGAQGVSEMSRMKIISVQLPQSLIHGLDALVKRGIYPNRSEAIRVAIRELLKKELYKEEIQEEIPEYVVK SEQ ID NO:13, BD093: IINPQARLTPLELEILEIIKQKKSITITEIKEILSERRKSEYPLSLVSEYISRLERKGYVKKIAKGRKKFVEALI SEQ ID NO:14, BD109: GRKVRTQQNEILNLLNEKEKAVLRAILEHGGEIKQEDLPELVGYSRPTISKVIQELENKGLIKREKSGKTFVVKIER KIKLD SEQ ID NO:15, BD006: RGTVKWFDSKKGYGFITMENGEDIFVHWSAIQMDGFKTLRENETVEFEVQKGTKGPQAVNVRPVR SEQ ID NO:16, BD012: RIGEKLRKLRLSRGLTQEELAERTDLSRSFISQLESDKTSPSIDTLERILEALGTDLKHF Figure 3 shows the acƟviƟes of these DNA polymerases with and without lambda DNA template. As is shown, the DNA-binding domain fusion proteins had faster performance than SDpol-1 in this LAMP assay. Example 4. Fast target amplificaƟon of DNA polymerases influorescent LAMP This example shows acƟviƟes of DNA polymerase fusion proteins in afluorescence LAMP assay using a human genomic DNA target compared to commercially available Bst enzymes. Two DNA polymerases (Sso7d-SDpol-1 and BD009-SDpol-1) were compared to Bst 2.0 (NEB) and Bst 3.0 (NEB) in 25 µLfluorescent LAMP reacƟon using a human genomic DNA target in triplicate at 10 ng, 1 ng total DNA inputs including no template control (NTC). The LAMP reacƟon was performed at 65℃ for 1 hour, and LAMPfluorescent dye (NEB) was spiked into each reacƟon to monitor thefluorescence change over Ɵme. The data was collected in a Biorad OPUS Real-Time PCR instrument monitoringfluorescence in the SYBR channel every 15 seconds, and the threshold cycle values were converted to Ɵme to detecƟon (minutes) for reporƟng. Each reacƟon contained six primers at the following concentraƟons: 1.6 µM FIP 1.6 µM BIP, 0.2 µM F3, 0.2 µM B3, 0.4 µM LoopF, and 0.4 µM LoopB. The dUTP and AntarcƟc Thermolabile UDG (New England Biolabs) were added to the reacƟons to prevent carryover contaminaƟon. As shown in Figure 4, Bst 3.0 was faster than Bst 2.0 to detect the DNA target, and Sso7d-SDpol-1 and BD009-SDpol-1 both showed fast amplificaƟon (lower Ɵme to detecƟon values) similar to Bst 3.0. Sso7d-SDpol-1 and BD009-SDpol-1 showed no NTC amplificaƟon however, Bst 3.0 induced NTC amplificaƟon within 30 minutes. Example 5. Fast target amplificaƟon using DNA polymerases in colorimetric LAMP This example shows an acƟvity comparison between Bst 2.0 and two embodiments of DNA polymerases (Sso7d-SDpol-1 and BD009-SDpol-1) in a colorimetric LAMP assay. Sso7d-SDpol-1 and BD009-SDpol-1 were compared to Bst 2.0 in 25 µL LAMP reacƟon using a human genomic DNA target in triplicate at 10 ng, 1 ng, 0.1 ng total DNA inputs including no template control (NTC). The samples were incubated at 65℃ for 1 hour, and 5-Bromo-PAPS dye and MnCl2were spiked into each reacƟon to monitor the color change over Ɵme for visual detecƟon. Bromo-PAPs-MnCl2complex produces red color in the absence of amplificaƟon. Following amplificaƟon, red color change to yellow due to precipitaƟon of MnCl2by inorganic pyrophosphate byproducts. The plates were scanned before and aŌer the reacƟon and, each well color was quanƟfied by taking the mean hue of the subset of pixels associated with each well. Before and aŌer amplificaƟon hue values (grey scale) are presented in Figures 5A and 5B, respecƟvely. In thefigure, hue values larger than 100 represent amplified samples (color change to yellow), and smaller than 100 represents samples with no amplificaƟon (red color). Each reacƟon contained six primers at the following concentraƟons: 1.6 µM FIP 1.6 µM BIP, 0.2 µM F3, 0.2 µM B3, 0.4 µM LoopF, and 0.4 µM LoopB. The dUTP and AT UDG were added to the reacƟons to prevent carryover contaminaƟon. As shown in Figure 5A and 5B, Sso7d-SDpol-1 and BD009-SDpol-1 showed fast amplificaƟon, and even at low template input (0.1 ng) the sample color change to yellow as reflected in the above-100 data points in Figure 5B. The DNA polymerases showed no NTC amplificaƟon aŌer 1 hour. Example 6. Improved LAMP detecƟon by Cas enzyme collateral acƟvity This example shows high performance of a DNA polymerase in a LAMP / Cas assay. This assay is described in Joung J, et al. N Engl J Med.2020 Oct 8;383(15):1492-1494. doi: 10.1056 / NEJMc2026172. RT-LAMP reacƟons were incubated at 45–65 °C. Primers targeƟng SARS-CoV-2 Gene N and condiƟons for the Bst 2.0 control reacƟon were taken from Joung et al, N Engl J Med 2020. ReacƟons were set up in 25 µL volume with 0–10000 copies of SARS-CoV-2 RNA (Control 16, Twist Biosciences) in buffer containing 20 mM Tris pH 8.8, 50 mM KCl, 10 mM (NH4)2SO4, 7 mM (BD009-SDPol-1) or 8 mM (Bst 2.0) MgSO4, 1.4 mM each dNTP, 0.1% v / v Tween-20. For real-Ɵme monitoring of amplificaƟon, reacƟons contained 1 µM SYTO™-82 (ThermoFisher) measured via the HEX channel of a Bio-Rad CFX96 instrument. To independently monitor amplificaƟon with a sequence-specific mechanism, reacƟons also contained 0.5 µM Alicyclobacillus acidiphilus Cas12b (AapCas12b), 0.5 µM guide RNA (from Joung et al; 5ʹ- GUCUAGAGGACAGAAUUUUUCAACGGGUGUGCCAAUGGCCACUUUCCAGGUGGCAAAGCCCGUUG AGCUUCUCAAAUCUGAGAAGUGGCACCGAAGAACGCUGAAGCGCUG (SEQ ID NO:23)), and 0.2 µM reporter DNA (5ʹ-FAM-T10-BHQ1) measured via the FAM channel. Simultaneous measurement in both channels allows for determinaƟon of amplificaƟon specificity as the SYTO-82 signal will arise from spurious nonspecific amplificaƟon while the FAM Cas12b signal is only present when the targeted sequence is amplified, confirming the correct idenƟty of the amplified DNA. In reacƟons with Bst 2.0, RNA was confidently detected to 1000 copies but required >30 minutes of incubaƟon. ReacƟons with BD009-SDpol-1 were significantly faster to detecƟon down to 10 copies. Analysis of the Cas12b FAM signal allowed for specificity determinaƟon, and BD009- SDpol-1 was confirmed to be producing specific amplificaƟon product with 100 copies RNA input. LAMP Ɵme was calculated by an average of 2 replicates using the HEX threshold and Cas RFU from the FAM signal subtracƟng baseline signal from endpointfluorescence. Results are shown in Table 2. Table 2: LAMP / Cas acƟvity Bst 2.0 BD009-SDpol-1 10000 30.0 2200 8.7 5228 1000 38.3 950 11.7 5429

Claims

CLAIMS What is claimed is:

1. A DNA polymerase comprising an amino acid sequence selected from: an amino acid sequence that is at least 80% idenƟcal to an amino acid sequence selected from: SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:24; and an amino acid sequence that is at least 92% idenƟcal to an amino acid sequence selected from: SEQ ID NO:2 and SEQ ID NO:

3.

2. The DNA polymerase of claim 1, comprising an amino acid sequence that is at least 90% idenƟcal to an amino acid sequence selected from: SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:

24.

3. The DNA polymerase of claim 1 or 2, comprising an amino acid sequence that is at least 95% idenƟcal to an amino acid sequence selected from: SEQ ID NO: 2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:

24.

4. The DNA polymerase of any of claims 1-3, comprising an amino acid sequence that is idenƟcal to an amino acid sequence selected from: SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 SEQ ID NO:6, and SEQ ID NO:

24.

5. A DNA polymerase, wherein the DNA polymerase is a fusion protein comprising an amino acid sequence of any of claims 1-4 and an exogenous amino acid sequence.

6. The DNA polymerase of claim 5, wherein the exogenous amino acid sequence comprises a DNA binding domain.

7. The DNA polymerase of claim 5 or 6, wherein the exogenous amino acid sequence comprises a purificaƟon tag.

8. A method, comprising: incubaƟng a reacƟon mixture comprising: (i) a DNA polymerase of any of claims 1-7; (ii) a target nucleic acid; (iii) dNTPs; and (iv) one or more primers, under condiƟons suitable for polynucleoƟde extension of the target nucleic acid to produce a copy of the target nucleic acid.

9. The method of claim 8, wherein the condiƟons are isothermal.

10. The method of claim 9, wherein the one or more primers comprise Loop-Mediated Isothermal AmplificaƟon (LAMP) primers.

11. A composiƟon comprising: (i) an aqueous soluƟon of or (ii) a freeze-dried / lyophilized form of the DNA polymerase of any of claims 1-7.

12. The composiƟon of claim 11, wherein the DNA polymerase is in a soluƟon that comprises glycerol.

13. The composiƟon of claim 11, further comprising a solid support.

14. The composiƟon of any of claims 11-13, further comprising one or more components selected from a buffering agent, a salt, a primer, and an aptamer.

15. A kit comprising: a DNA polymerase of any of claims 1-7; and a reacƟon buffer.

16. The kit of claim 15, further comprising dNTPs.

17. The kit of claim 15 or 16, wherein the DNA polymerase is in a form selected from: dried form, lyophilized form, soluƟon form.

18. The kit of any of claims 15-17, wherein the reacƟon buffer comprises a buffering agent and a salt.

19. The kit of claim 18, wherein the DNA polymerase is provided in a separate tube from reacƟon buffer.

20. The kit of any of claims 15-19, wherein the reacƟon mixture is suitable for receiving and extending a target nucleic acid in the presence of the DNA polymerase and one or more primers.

21. The kit of any of claims 15-20, further comprising one or more addiƟonal enzymes, opƟonally selected from a reverse transcriptase and a uracil DNA glycosidase (UDG).

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