Methods and compositions for isothermal DNA amplification

Abstract

The present invention relates to a method for amplifying a DNA template, the method comprising incubating a DNA template with a DNA-dependent RNA polymerase in the presence of ribonucleotides, and amplifying the DNA template with a strand-displacing DNA polymerase. The invention further relates to the use of an RNA polymerase to generate ribonucleotide primers on a DNA template and subsequently amplifying the DNA template with the strand-displacing DNA polymerase, a kit of parts comprising an RNA polymerase and a strand-displacing DNA-dependent DNA polymerase, and the use of the kit of parts for amplifying a DNA template.

Description

[Technical Field]

[0001] Areas: The present invention provides methods, compositions, and kits for replicating or amplifying native or denatured DNA templates under isothermal conditions. More specifically, the present invention is directed to the use of RNA polymerases in methods for amplifying DNA templates without or with exogenously added oligonucleotide primers in reduced form. [Background technology]

[0002] 1. Introduction Several techniques exist for amplifying DNA molecules. These include the polymerase chain reaction (PCR), which involves repeated rounds of reaction at different temperatures, and isothermal reactions, almost all of which involve strand-displacing DNA polymerases. Examples of isothermal reactions include rolling circle amplification (RCA), multiple displacement amplification (MDA), loop-mediated isothermal amplification (LAMP), which involves specific primers that generate pseudo-circular DNA molecules, helicase-dependent amplification (HDA), and nicking enzyme amplification reaction (NEAR), which involves the creation of nicks in the DNA molecule that are used to prime replication.

[0003] Replication of DNA molecules requires a free hydroxyl group to serve as a starting point for the addition of deoxyribonucleotides by DNA polymerase. Typically, a short single-stranded oligonucleotide or primer complementary to a template DNA molecule is added externally to prime the DNA polymerase for replication of the DNA molecule. The single-stranded primer may contain a specific nucleotide sequence to amplify a specific DNA template, or may contain one or more generic and / or degenerate nucleotide sequences to semi-randomly amplify a DNA template. The generic nucleotide sequence includes, for example, random oligoribonucleotides, random hexamer and random nonamer DNA primers.

[0004] Alternatively, DNA replication can be initiated by deoxyribonucleoside monophosphates covalently linked to proteins. These proteins are used in nature by certain bacterial and animal viruses for genome replication in host cells (Salas, 1991, Annu. Rev. Biochem. 60: 39-71), but currently lack widespread application for other purposes. Furthermore, another class of enzymes, called primase polymerases (PrimPols), contain both DNA polymerase and DNA primase activities and can be used for both primer synthesis and DNA molecule replication. Unlike conventional primases, PrimPols generate DNA primers (Lipps et al., 2003, EMBO J 22: 2516-2525). PrimPol, isolated from Thermus thermophiles, was recently found to perform better in amplifying whole genomes from single cells when compared to random priming (Picher et al., 2016. Nature Comm 7: 13296).

[0005] The current state of the art in vitro replication of DNA templates, especially large DNA molecules such as whole genomes, is largely based on multiple displacement amplification (MDA), an isothermal amplification method that uses oligonucleotide primers and strand-displacing DNA polymerases such as Phi29 DNA polymerase to exponentially amplify template DNA molecules (Dean et al., 2001. Genome Res. 11(6): 1095-1099; Dean et al., 2002. Proc. Nath Acad. Sci. 99(8): 5261-5266). Phi29 DNA polymerase combines high processivity, strand displacement activity, and 3' to 5' proofreading exonuclease activity to synthesize DNA fragments >70 kb with a very low error rate (Blanco et al., 1989. J Biol Chem 264: 8935-8940; Garmendia et al., 1992. J Biol Chem 267: 2594-2599; Esteban et al., 1993. J Biol Chem 268: 2719-2726).

[0006] Although natural DNA generally exists as a double-helical structure, the use of specific or degenerate random oligonucleotides, such as hexamer primers, requires denatured or single-stranded DNA for efficient priming. Denaturation of DNA by thermal or chemical methods can result in strand breaks and other harmful damage to the initial template DNA, which is highly undesirable and potentially detrimental to downstream analysis. Furthermore, the use of random primers often results in biased amplification of the template DNA, sequencing errors due to mispriming, and / or nonspecific amplification due to self-priming of the random oligonucleotides (see, e.g., Hansen et al., 2010. Nucleic Acids Res 38: el31; van Gurp et al., 2013. PLoS ONE 8(12): e85583; Sabina and Leamon 2015. Methods Mol Biol. 1347:15-41). Some of these problems have been partially resolved by the use of PrimPol, isolated from Thermus thermophilus (Picher et al., 2016. Nature Comm 7: 13296), which possesses endogenous primase activity without the need for exogenously added oligonucleotides. However, both random oligonucleotides and PrimPol require or prefer single-stranded or denatured DNA as templates for efficient priming. Denaturation can be achieved by increasing the temperature or by adding a highly alkaline solution followed by neutralization of the solution prior to replication. This additional denaturation step tends to increase the risk of contamination due to artifacts such as damage or cleavage of the template DNA. Therefore, this additional denaturation step is preferably avoided, especially in high-throughput DNA replication methods and for delicate or rare DNA templates. Therefore, there is a need for a DNA replication method that does not require an additional step of denaturing the oligonucleotide primer and / or DNA template molecules. [Prior art documents] [Patent documents]

[0007] [License 1] US 5,744,312 [Non-licensed literature]

[0008] [Non-licensed Document 1] Salas, 1991. Annu. Rev. Biochem. 60: pp. 39~71 [Non-licensed Document 2] Lipps, 2003. EMBO J 22: pp. 2516-2525 [Non-licensed Document 3] Picherら, 2016. Nature Comm 7: 13296 pages [Non-licensed Document 4] Dean, 2001. Genome Res. 11(6): pages 1095~1099 [Non-licensed Document 5] Dean, 2002. Proc. Nath Acad. Sci. 99(8): pages 5261~5266 [Non-licensed Document 6] Blanco, 1989. J Biol Chem 264: pages 8935~8940 [Non-licensed Document 7] Garmendia, 1992. J Biol Chem 267: 2594~2599 pages [Non-licensed Document 8] Esteban, 1993. J Biol Chem 268: 2719~26 pages [Non-licensed Document 9] Hansen, 2010. Nucleic Acids Res 38: el31 [Non-licensed Document 10] van Gurpら, 2013. PLoS ONE 8(12): e85583 [Non-licensed Document 11] Sabina and Leamon 2015. Methods Mol Biol. 1347:15~41 pages [Non-licensed Document 12] McAllister and Raskin, 1993. Mol Microbiol. 10: 1~6 pages [Non-licensed Document 13] Kostyuk, 1995. FEBS Lett. 369: Pages 165~168 [Non-licensed Document 14] Sousa, 1995. EMBO J. 14(18): pages 4609~4621 [Non-licensed Document 15] Gudima, 1998. FEBS Lett. 439: Pages 302~306 [Non-licensed Document 16] Padilla, 2002. Nucl. Acids Res. 30(24): el38 [Non-licensed Document 17] Boulain, 2013. Protein Eng Des Sel. 26(11): pages 725~734 [Non-licensed Document 18] Ikeda, 1993. Biochemistry 32(35): pages 9115~9124 [Non-licensed Document 19] Fujimura and Roop, 1976. J Biol Chem 251: 2168~2174. [Non-licensed Document 20] Kaboord and Benkovic, 1995. Curr Biol 5: 149-157 [Non-licensed Document 21] Matsumoto, 1989. Gene 84: 247 pages [Non-licensed Document 22] Jungら, 1987. Proc Natl Aced Sci USA 84: 8287 pages [Non-licensed Document 23] Zhu and Ito, 1994. Biochim Biophys Acta 1219: 267-276. [Non-licensed Document 24] de Vega, 2010. PNAS 107: pages 16506~16511 [Non-licensed Document 25] Suzuki et al., 2005. Nucleic Acids Symp Series 49: 97-98 [Non-Patent Document 26] Golomb et al., 1977. J Virol 21: pp. 743-752 [Non-Patent Document 27] Stump and Hall, 1993. Nucleic Acids Res 21: pp. 5480-5484. [Non-patent document 28] Maslak and Martin, 1993. Biochemistry 32: 4281-4285 [Non-Patent Document 29] Li et al., 1996. Biochemistry 35: 3722-3727 [Non-Patent Document 30] Revyakin et al., 2006. Science 314: 1139-1143 [Non-Patent Document 31] Cheng et al., 1994. Proc Natl Acad Sci 91: 5695-5699 [Non-Patent Document 32] Schneider et al., 2012. Nature Methods 9: 671-675 Summary of the Invention

[0009] 2. Brief Description of the Invention Provided herein are methods, compositions, and kits for amplifying template or target DNA in the absence or reduced amount of oligonucleotide primers. The target DNA may be a circular molecule, such as a plasmid, cosmid, or circularized DNA padlock probe, or a linear molecule, such as a genome, one or more genome fragments, or synthetically or enzymatically generated nucleic acid fragments. The template DNA molecule may be single-stranded or double-stranded.

[0010] The present invention provides a method for amplifying a template DNA molecule, comprising the steps of: a) providing a template DNA molecule; b) providing an RNA polymerase, a DNA polymerase, and a combination of ribonucleotides and deoxyribonucleotides; and c) incubating the materials in a suitable buffer for a suitable period of time to allow replication and amplification of the template DNA molecule.

[0011] The template DNA molecule, RNA polymerase, DNA polymerase, and nucleotides are preferably incubated in the appropriate buffer at a constant temperature within the range of 3°C, 6°C, or 12°C for an appropriate period of time.

[0012] The RNA polymerase is preferably a member of the family of single-subunit RNA polymerases. Preferred RNA polymerases are selected from the group of T7 RNA polymerase, T3 RNA polymerase, SP6 RNA polymerase, or mutants thereof.

[0013] The DNA polymerase is preferably a DNA-dependent DNA polymerase with strand displacement activity. Preferred DNA polymerases are selected from Phi29, Bst, Bent DNA polymerase, or combinations or variants thereof.

[0014] In a preferred method of the invention, said ribonucleotides preferably comprise at least one ribonucleotide having a purine nucleobase.

[0015] Replication and amplification of a template DNA molecule by the methods of the present invention may further be carried out in the presence of at least one oligonucleotide primer complementary to the template DNA molecule, a mixture of random oligonucleotide primers, or a combination thereof.

[0016] In the methods of the present invention, one of the nucleotides or ribonucleotides, preferably at least one of the nucleotides or ribonucleotides, is preferably modified or labeled with a detectable label.

[0017] The amplification products replicated and amplified by the methods of the present invention may be detected directly or indirectly by various methods known in the art, such as by DNA binding agents or DNA intercalating agents, or by biotin- or fluorophore-labeled probes, such as oligonucleotides or molecular beacons that are complementary to the template DNA molecule.

[0018] The present invention further provides the use of an RNA polymerase to provide a primer on a DNA template and, optionally, to amplify the DNA template from the primer by a strand-displacing DNA polymerase in the presence of deoxyribonucleotides, said providing a primer preferably comprising at least one ribonucleotide having a purine nucleobase, more preferably two or three ribonucleotides selected from GTP, ATP, and CTP.

[0019] The RNA polymerases of the present invention may be used to amplify single-stranded or double-stranded template DNA molecules, including genomic DNA, in linear or circular form. The RNA polymerases of the present invention may be used for biomedical purposes, including but not limited to, therapeutic purposes, including gene therapy and vaccines, or forensic or diagnostic purposes, such as in microbiological or genetic diagnostics, liquid-phase immunoassays and / or immunohistochemistry, including genotyping, DNA sequencing, detection of viral or bacterial DNA, or detection of nucleic acid mutations.

[0020] The invention further provides a kit of parts comprising parts carrying an RNA polymerase, a strand-displacing DNA polymerase, or a mixture thereof, and, optionally, a suitable buffer.

[0021] Said kit of parts may further comprise ribonucleotides, deoxyribonucleotides, and / or mixtures thereof.

[0022] The present invention further provides the use of a kit of parts according to the invention for the amplification of a template DNA molecule.

[0023] 3. Description of the drawings [Brief explanation of the drawings]

[0024] [Figure 1] Priming of DNA amplification by RNA polymerase. Figure 1A. Amplification products of circular plasmid DNA following priming with random hexamers or with different RNA polymerases. RNA polymerase priming occurs without exogenously added oligonucleotide primers and appears to be independent of its cognate promoter sequence. Templates used: Lane 1, no template control; Lane 2, p53 (no RNA polymerase promoter); Lane 3, plasmid p3 containing a single T7 promoter; Lane 4, plasmid pB327 containing two inverted T7 promoters; Lane 5, plasmid pBSK containing T3 and T7 promoters; Lane 6, pGem-7 plasmid containing T7 and SP6 promoters. Figure 1B. Restriction enzyme analysis (BsaI) illustrates faithful amplification of pB327 plasmid DNA following priming with random hexamers or with different RNA polymerases in the presence or absence of purinergic ribonucleotides. Lane 1, marker; lane 2, hexamer primer (H); lane 3, hexamer primer and GTP / ATP; lane 4, T7 RNA polymerase and GTP / ATP; lane 5, T3 RNA polymerase and GTP / ATP; lane 6, SP6 RNA polymerase and GTP / ATP; lane 7, T7 RNA polymerase without GTP / ATP; lane 8, digestion control, 250 ng pB327 template. [Figure 2A]Priming by RNA polymerase in the presence of different ribonucleotides. Figure 2A. Ribonucleotide dependence of RNA polymerase-mediated priming of p53 DNA amplification. Lane -, no ribonucleotides; Lane G, 0.5 mM GTP; Lane A, 0.5 mM ATP; Lane U, 0.5 mM UTP; Lane C, 0.5 mM CTP; Lane Pu, 0.25 mM GTP and 0.25 mM ATP; Lane Py, 0.25 mM UTP and 0.25 mM CTP; Lane N, 0.125 mM of all four ribonucleotides. Figure 2B. DNA amplification of plasmid pB327 in the presence of RNA polymerase and decreasing ribonucleotide concentrations. The concentrations of GTP and ATP in micromolar are indicated above each lane. (-) indicates a no-template control containing 1 mM GTP and ATP. [Figure 2B] Priming by RNA polymerase in the presence of different ribonucleotides. Figure 2A. Ribonucleotide dependence of RNA polymerase-mediated priming of p53 DNA amplification. Lane -, no ribonucleotides; Lane G, 0.5 mM GTP; Lane A, 0.5 mM ATP; Lane U, 0.5 mM UTP; Lane C, 0.5 mM CTP; Lane Pu, 0.25 mM GTP and 0.25 mM ATP; Lane Py, 0.25 mM UTP and 0.25 mM CTP; Lane N, 0.125 mM of all four ribonucleotides. Figure 2B. DNA amplification of plasmid pB327 in the presence of RNA polymerase and decreasing ribonucleotide concentrations. The concentrations of GTP and ATP in micromolar are indicated above each lane. (-) indicates a no-template control containing 1 mM GTP and ATP. [Figure 3] Amplification of native or denatured pB327 plasmid DNA in the presence of RNA polymerase for different incubation times. Reactions in lanes 1-4 contained 10 ng of pB327 native, undenatured plasmid DNA as the starting template, while lanes 5-8 contained 10 ng of pB327 that had been heat-denatured prior to amplification. All reactions contained 50 μM hexamer in addition to the RNA polymerase indicated on each lane. [Figure 4]Amplification of a single-stranded circular M13mp18 DNA template followed by XbaI digestion. The lower band represents the correct product migrating approximately 7 kb. Lane 1, no priming control; lane 2, 50 μM hexamer primer; lane 3, 0.5 U / μl T7 RNA polymerase; lane 4, 0.5 U / μl T3 RNA polymerase; lane 5, 0.5 U / μl SP6 RNA polymerase; lane 6, 0.5 U / μl T7 RNA polymerase and 50 μM hexamer primer; lane 7, 0.5 U / μl T3 RNA polymerase and 50 μM hexamer primer; lane 8, 0.5 U / μl SP6 RNA polymerase and 50 μM hexamer primer. [Figure 5] Specific amplification of genomic DNA. Figure 5A. Amplification of 1 ng / μl HeLa genomic DNA template. Figure 5B. Nonspecific amplification with hexamer primers in the absence of template. Lane 1, no priming control; Lane 2, 50 μM hexamer primer; Lane 3, 0.5 U / μl T7 RNA polymerase; Lane 4, 0.5 U / μl T3 RNA polymerase; Lane 5, 0.5 U / μl SP6 RNA polymerase; Lane 6, 0.5 U / μl T7 RNA polymerase and 50 μM hexamer primer; Lane 7, 0.5 U / μl T3 RNA polymerase and 50 μM hexamer primer; Lane 8, 0.5 U / μl SP6 RNA polymerase and 50 μM hexamer primer. [Figure 6A] RNA polymerase enhances and increases the yield of hexamer-primed amplification of circular DNA templates. Figure 6A. Enhanced amplification of native p53 plasmid DNA in the presence of RNA polymerase (RNAP). Amplification times in hours are indicated above the lanes. Figure 6B. (Top and bottom) Quantification of DNA amplification over time and relative activity of RNA polymerase. Figure 6C. Enhanced amplification of denatured p53 plasmid DNA mediated by RNA polymerase in the presence of the indicated hexamer concentrations (in micromolar). The template used was 0.5 ng / μl of denatured p53 plasmid DNA, which does not contain T7, T3, or SP6 promoter sequences. [Figure 6B]RNA polymerase enhances and increases the yield of hexamer-primed amplification of circular DNA templates. Figure 6A. Enhanced amplification of native p53 plasmid DNA in the presence of RNA polymerase (RNAP). Amplification times in hours are indicated above the lanes. Figure 6B. (Top and bottom) Quantification of DNA amplification over time and relative activity of RNA polymerase. Figure 6C. Enhanced amplification of denatured p53 plasmid DNA mediated by RNA polymerase in the presence of the indicated hexamer concentrations (in micromolar). The template used was 0.5 ng / μl of denatured p53 plasmid DNA, which does not contain T7, T3, or SP6 promoter sequences. [Figure 6C] RNA polymerase enhances and increases the yield of hexamer-primed amplification of circular DNA templates. Figure 6A. Enhanced amplification of native p53 plasmid DNA in the presence of RNA polymerase (RNAP). Amplification times in hours are indicated above the lanes. Figure 6B. (Top and bottom) Quantification of DNA amplification over time and relative activity of RNA polymerase. Figure 6C. Enhanced amplification of denatured p53 plasmid DNA mediated by RNA polymerase in the presence of the indicated hexamer concentrations (in micromolar). The template used was 0.5 ng / μl of denatured p53 plasmid DNA, which does not contain T7, T3, or SP6 promoter sequences. [Figure 7A]DNA amplification mediated by RNA polymerases and Phi29 obtained from different commercial suppliers. Figure 7A. RNA polymerases and phi29 DNA polymerase from different suppliers. Top panel: Phi29 from Biolab Innovative Research Technologies (supplier B); middle panel: Phi29 from New England Biolabs (supplier N); bottom panel: Phi29 from Thermo Fisher (supplier T). Lane - represents no priming control, and lane H represents hexamer priming. T7, T3, and SP6 RNA polymerases obtained from New England Biolabs (N) or Thermo Fisher (T) were used as indicated on each lane. The template used in each reaction was 0.5 ng / μl p53 plasmid DNA, which does not contain the T7, T3, or SP6 promoter sequence. Figure 7B. Amplification in the presence of different buffer systems. [Figure 7B] DNA amplification mediated by RNA polymerases and Phi29 obtained from different commercial suppliers. Figure 7A. RNA polymerases and phi29 DNA polymerase from different suppliers. Top panel: Phi29 from Biolab Innovative Research Technologies (supplier B); middle panel: Phi29 from New England Biolabs (supplier N); bottom panel: Phi29 from Thermo Fisher (supplier T). Lane - represents no priming control, and lane H represents hexamer priming. T7, T3, and SP6 RNA polymerases obtained from New England Biolabs (N) or Thermo Fisher (T) were used as indicated on each lane. The template used in each reaction was 0.5 ng / μl p53 plasmid DNA, which does not contain the T7, T3, or SP6 promoter sequence. Figure 7B. Amplification in the presence of different buffer systems. [Figure 8]Transfection efficiency and expression levels of DNA amplified under the indicated conditions. The upper panel shows the transfection efficiency (percentage of GFP-expressing cells in the total cell population) and mean fluorescence intensity (MFI) for human HEK293 cells, and the lower panel shows the results for mouse B16F10 cells. DNA amplification was primed with random hexamers in the absence (striped white bars) or presence (striped gray bars) of ribonucleotides, or with T7 RNA polymerase (black bars), T3 RNA polymerase (gray bars), or SP6 RNA polymerase (white bars) in the presence of ribonucleotides. [Figure 9] Luciferase activity over time in mice injected with plasmid DNA or linear DNA versus control mice. Means and standard deviations are shown for four mice per group. [Figure 10] Vaccination with plasmid DNA or linear DNA. Figure 10A. OVA epitope-specific T cells as a percentage of the total number of CD8 positive T cells induced by plasmid DNA or linear DNA versus control. Figure 10B. Kaplan-Meier plot of inoculated mice challenged with B16-OVA tumor cells 21 days after vaccination. DETAILED DESCRIPTION OF THE INVENTION

[0025] 4. Detailed Description of the Invention 4.1 Definition The term "template DNA molecule" or "DNA template," as used herein, refers to a DNA molecule to be replicated. The DNA template can be any DNA molecule, including single-stranded or double-stranded DNA molecules, linear or circular DNA molecules, and small or large DNA molecules ranging from linear or circular plasmid molecules of less than 10 kilobases to large DNA molecules such as genomic DNA molecules or BAC plasmids.

[0026] The term "suitable buffer" as used herein refers to an aqueous buffer whose pH is approximately constant. Buffers suitable for replication reactions contain a divalent metal salt, preferably a magnesium salt such as magnesium chloride, magnesium sulfate, and / or magnesium acetate.

[0027] The term "RNA polymerase," as used herein, refers to a DNA-dependent RNA polymerase enzyme that generates DNA-templated RNA transcripts. The RNA polymerase is preferably a member of the single-subunit RNA polymerase family, which includes many phage RNA polymerases (T7, T3, K11, SP6, N4, etc.) and mitochondrial RNA polymerase (McAllister and Raskin, 1993. Mol Microbiol. 10: 1-6). Preferred RNA polymerases are selected from the group consisting of T7 RNA polymerase, T3 RNA polymerase, SP6 RNA polymerase, or variants thereof. Such mutants include, for example, mutant T7 RNA polymerases that can utilize both canonical and non-canonical ribonucleotides and deoxyribonucleotides as substrates (Kostyuk et al., 1995. FEBS Lett. 369: 165-168; Sousa et al., 1995. EMBO J. 14(18): 4609-4621; Gudima et al., 1998. FEBS Lett. 439: 302-306; Padilla et al., 2002. Nucl. Acids Res. 30(24): el38), RNA polymerase mutants that exhibit increased thermostability (Hi-T7™ RNA Polymerase from New England Biolabs; Boulain et al., 2013. Protein Eng Des Sel. 26(11): 725-734), or mutant RNA polymerases that have reduced promoter specificity (Ikeda et al., 1993. Biochemistry 32(35):9115-9124).

[0028] The term "DNA polymerase," as used herein, refers to a DNA-dependent DNA polymerase enzyme that generates a DNA template DNA molecule by adding deoxyribonucleotides to the 3' end of a DNA strand.

[0029] The term "strand-displacing DNA polymerase," as used herein, refers to a DNA polymerase that can displace the downstream DNA strand. Thus, the enzyme can unwind a double-stranded DNA molecule during replication of the molecule. The polymerase preferably lacks 5' to 3' exonuclease activity. Suitable strand-displacing DNA-dependent DNA polymerases include the Klenow fragment of DNA polymerase I, the Klenow fragment of Bacillus stearothermophilus DNA polymerase, designated Bst polymerase (New England Biolabs, Ipswich, MA), Bsm DNA polymerase, large fragment (Thermo Fisher Scientific, Waltham, MA), BcaBEST DNA polymerase (Takara Bio, Kusatsu, Japan), Thermoanaerobacter thermohydrosulfuricus (Tts) DNA polymerase (US 5,744,312), DNA polymerases from Thermus aquaticus, Thermus flavus, or Thermus thermophiles, SEQUENASE (Thermo Fisher Scientific, Waltham, MA), and the like. These include a variant of T7 DNA-dependent DNA polymerase named T5 DNA-dependent DNA polymerase (Fujimura and Roop, 1976. J Biol Chem 251:2168-2174), T4 DNA polymerase holoenzyme (Kaboord and Benkovic, 1995. Curr Biol 5:149-157), a DNA-dependent DNA polymerase from Thermococcus litoralis named Bent polymerase (New England Biolabs, Ipswich, MA), phage M2 DNA polymerase (Matsumoto et al., 1989. Gene 84:247), and phage PRD1 DNA polymerase (Jung et al., 1987).Proc Natl Aced Sci USA 84: 8287; Zhu and Ito, 1994. Biochim Biophys Acta 1219: 267-276), Phi29 DNA polymerase, and any strand-displacing DNA-dependent DNA polymerase variants thereof, including, for example, fusions of DNA-binding proteins with the polymerase (de Vega et al., 2010. PNAS 107: 16506-16511).

[0030] The term "ribonucleotide," as used herein, refers to a molecule comprising a ribose sugar group, a nucleobase, and at least one phosphate group. The nucleobases include adenine, guanine, cytosine, uracil, and any modifications thereof. The nucleobases adenine and guanine are collectively referred to as purines, and cytosine and uracil are collectively referred to as pyrimidines. The term ribonucleotide includes reference to analogs of ribonucleotides, such as fluorescent molecules, e.g., 1,3-diaza-2-oxophenothiazine-ribose-5'-triphosphate (tCTP), and / or other analogs, such as inosine, xanthosine, N4-hydroxycytosine, N4-methoxycytosine, and 6H,8H-3,4-dihydropyrimido[4,5-c][1,2]oxazin-7-one (Suzuki et al., 2005. Nucleic Acids Symp Series 49: 97-98).

[0031] The term "deoxyribonucleotide," as used herein, refers to a molecule comprising a deoxyribose sugar group, a nucleobase, and at least one phosphate group. The nucleobases include the naturally occurring adenine, guanine, cytosine, thymidine, and any modifications thereof. The nucleobases adenine and guanine are collectively referred to as purines, and cytosine and thymidine are collectively referred to as pyrimidines. The term deoxyribonucleotide includes reference to analogs of deoxyribonucleotides, such as deoxyuridine, deoxyinosine, and / or deoxyxanthosine.

[0032] The term "nucleotide," as used herein, refers to a ribonucleotide, deoxyribonucleotide, or any variant or modification thereof. It is recognized that a wide variety of nucleotide modifications have been created and described, serving many useful purposes known to those of skill in the art. The term nucleotide, as used herein, encompasses such chemically, enzymatically, or metabolically modified forms of nucleotides.

[0033] The term "primer," as used herein, refers to an oligonucleotide that anneals to a template DNA and is effective in priming replication of the template DNA by a DNA polymerase. The oligonucleotide may be added externally to the template DNA and may contain deoxyribonucleotides, ribonucleotides, or combinations or variants thereof. Alternatively, the primer may be generated from a DNA template by an RNA polymerase. The primer can be generated by an RNA polymerase or a variant thereof in the presence of one or more nucleotides. The nucleotides preferably contain ribonucleotides, preferably one to three selected from GTP, ATP, and CTP, and more preferably contain at least one purine nucleobase, such as adenine or guanine, or both adenine and guanine nucleobases. Such variants include synthetic oligonucleotide analogs such as phosphorothioates, phosphotriesters, phosphorothioate dialkylated and phosphoramidate analogs, analogs with modifications at the 2' position of the nucleoside sugar ring such as 2'-fluoro, O-methyl, or methoxyethyl, peptide nucleic acids, bridged nucleic acids, and / or locked nucleic acid molecules.

[0034] The term "padlock probe," as used herein, refers to an oligonucleotide whose ends are complementary to adjacent sequences on a target template RNA or DNA molecule. Hybridization of a padlock probe to its target allows for circularization by ligation. The circularized padlock probe can then serve as a template DNA for amplification.

[0035] 4.2 Methods of the Invention The present invention is based on the unexpected finding that RNA polymerases, such as T7 RNA polymerase, can efficiently mediate priming of both natural and denatured DNA templates for subsequent amplification by DNA polymerases in the absence of a consensus T7 promoter sequence and an exogenously added oligonucleotide primer. Subsequently, it was discovered that other RNA polymerases, such as SP6 and T3 RNA polymerases, can also initiate DNA amplification in the absence of a consensus SP6 or T3 promoter sequence, respectively. Furthermore, the RNA polymerases have been shown to initiate priming on single-stranded DNA templates. The surprise of this finding lies in the fact that the transcriptional activity of DNA-dependent RNA polymerases, such as T7, T3, and SP6 RNA polymerases, has been widely described as being highly specific to their respective promoter sequences, with binding of the RNA polymerases occurring only on double-stranded DNA promoter sequences (Golomb et al., 1977. J Virol 21: 743-752; Stump and Hall, 1993. Nucleic Acids Res 21: 5480-5484; Maslak and Martin, 1993. Biochemistry 32: 4281-4285; Li et al., 1996. Biochemistry 35: 3722-3727).

[0036] Priming by the RNA polymerase according to the method of the present invention relies on the presence of at least one ribonucleotide. To prevent long-term polymerization of the RNA transcript, the reaction is preferably carried out in the presence of three or fewer ribonucleotides. The three or fewer ribonucleotides preferably include at least one purine ribonucleotide, such as ATP or GTP, or a combination thereof. Preferably, either CTP and / or UTP is omitted from the reaction mixture.

[0037] Accordingly, the present invention provides a method for amplifying a template DNA molecule, comprising the steps of: a) providing a template DNA molecule; b) providing an RNA polymerase, a DNA polymerase, and a combination of ribonucleotides and deoxyribonucleotides; and c) incubating the materials in a suitable buffer for a suitable period of time to allow replication and amplification of the template DNA molecule; and, optionally, d) detecting the amplified product.

[0038] The RNA polymerase can be any RNA polymerase that mediates priming for subsequent elongation by a DNA polymerase. Without being bound by theory, the RNA polymerase binds to a template DNA and adds a single RNA nucleotide that can serve as a primer for extension by a DNA-dependent DNA polymerase. Alternatively, or additionally, the RNA polymerase generates short stretches of RNA by a process termed abortive transcription, in which the RNA polymerase binds to a DNA molecule and initiates the synthesis of short mRNA transcripts. The abortive transcription can involve DNA scrunching (Revyakin et al., 2006. Science 314: 1139-1143). The short abortive mRNA transcripts can be used as ribonucleotide primers and extended by a DNA-dependent DNA polymerase.

[0039] Preferred RNA polymerases are T3 RNA polymerase, SP6 RNA polymerase and T7 RNA polymerase. A highly preferred RNA polymerase is T7 RNA polymerase.

[0040] The strand-displacing DNA-dependent DNA polymerase is preferably Phi29, Bst, and / or Bent DNA polymerase. Phi29 polymerase has an optimum temperature of approximately 30°C. Bst polymerase has an optimum temperature of 60-65°C, and Bent polymerase has an optimum temperature of approximately 72°C. Because most natural RNA polymerases are active between 30-37°C, Phi29 polymerase is the enzyme of choice for combination with T3 RNA polymerase, SP6 RNA polymerase, or T7 RNA polymerase. However, it will be apparent to those skilled in the art that more thermostable DNA-dependent RNA polymerases can be combined with Bst polymerase or Bent polymerase at higher incubation temperatures.

[0041] Phi29 DNA polymerase has higher processivity and strand displacement ability compared to Bst and / or Bent DNA polymerase. Furthermore, Phi29 DNA polymerase has proofreading activity, resulting in highly accurate replication (Garmendia et al., 1992. J. Biol. Chem. 267, pp. 2594-2599). Therefore, a highly preferred strand-displacing DNA-dependent DNA polymerase is Phi29 polymerase. However, other strand-displacing DNA-dependent DNA polymerases known or known in the future may also be suitable for use in the methods of the present invention.

[0042] The method of the present invention is carried out for a time suitable for amplifying the DNA template, which is preferably 0.5 to 48 hours, more preferably 1 to 24 hours, and even more preferably 2 to 20 hours, for example, at least 5 hours, at least 8 hours, at least 12 hours, or at least 16 hours.

[0043] The method of the present invention is carried out in a suitable buffer, preferably containing a buffering agent that maintains a nearly constant pH. The buffer may contain phosphate, borate, N-cyclohexyl-2-aminoethanesulfonic acid (CHES), tris(hydroxymethyl)aminoethane (Tris), 2-(N-morpholino)ethanesulfonic acid (MES), glycine, and / or [tris(hydroxymethyl)methylamino]propanesulfonic acid (TAPS). The preferred reaction pH for the polymerase is between 6 and 10, preferably between 7 and 9, e.g., 7.2, 7.5, 7.8, 8.0, 8.1, 8.5, 8.6, or 8.8.

[0044] A preferred buffer is or contains Tris, preferably 10-100 mM Tris, which is preferably adjusted to the desired pH by the addition of an acid such as acetic acid and / or hydrochloric acid.

[0045] A suitable buffer for replication reactions is Mg 2+ or Mn 2+ The divalent metal ion may be provided as a salt thereof, such as magnesium chloride, magnesium sulfate, and / or magnesium acetate. The concentration is preferably between 0.5 and 20 mM, for example, between 1 and 10 mM, preferably about 5-10 mM.

[0046] Additional components of the buffer may include potassium ions such as potassium chloride, other salts such as ammonium sulfate, and / or betaine, ethylene glycol, 1,2-propanediol and / or spermidine, e.g., 2-10% DMSO or 2-10% glycerol, which are known in the art to enhance replication of certain DNA templates (Cheng et al., 1994. Proc Natl Acad Sci 91: 5695-5699).

[0047] Preferably, the reaction buffer contains additional components that stabilize enzyme activity, including gelatin, albumin, a reducing agent such as beta-mercaptoethanol, dithiothreitol (DTT), and / or tris(2-carboxyethyl)phosphine (TCEP), and a mild detergent such as TWEEN 20 or Triton-X100.

[0048] In an embodiment, a preferred buffer comprises Tris acetate, magnesium acetate, and potassium acetate, more preferably 33 mM Tris acetate, pH 7.9 at 37°C, 10 mM magnesium acetate, 66 mM potassium acetate, 0.1% (w / w) Tween 20, and 1 mM DTT.

[0049] In an embodiment, a preferred buffer comprises Tris-chloride, magnesium chloride, and ammonium sulfate, more preferably 50 mM Tris-HCl (pH 7.5 at 25° C.), 10 mM MgCl 2 , 10 mM (NH 4 ) 2 SO 4 , and 4 mM DTT.

[0050] The method of the present invention may further comprise providing at least one exogenous oligonucleotide as a primer, the oligonucleotide being complementary to a stretch of nucleotides on the template DNA molecule, a mixture of random oligonucleotides, or a combination thereof. The oligonucleotide or mixture of oligonucleotides preferably comprises or is one or more single-stranded nucleic acid molecules, preferably DNA molecules, RNA molecules, or mixtures or analogs thereof. The single-stranded nucleic acid molecules preferably have a length of 6 to 100 bases, for example, 9 to 30 bases. The single-stranded nucleic acid molecules may serve as additional starting points for replication and amplification of the template DNA molecule.

[0051] The amplification products generated by the method of the present invention can be detected and visualized, for example, by gel electrophoresis. Gel electrophoresis is a technique used to separate DNA molecules based on their size, since all DNA molecules essentially have the same charge. Electrophoresis involves passing an electric current through a gel containing the molecules of interest. Molecules move at different speeds through the gel based on their size, causing the molecules to separate from each other.

[0052] The gels are typically agarose or polyacrylamide gels. Polyacrylamide gels are usually used for small DNA fragments, typically up to 500-1000 base pairs (bp). Agarose gels can be used for DNA fragments of 100-20 kbp, but pulsed-field gel electrophoresis (PFGE) can achieve resolutions of over 6 Mb.

[0053] Alternatively, or additionally, amplification products produced by the methods of the invention may be detected and visualized by DNA binding or intercalating agents or dyes such as ethidium bromide, crystal violet, Haist stain or DAPI (4',6-diamidino-2-phenylindole), cyanine dyes such as SYBR Green or Eva Green. Alternatively, amplification products produced by the methods of the invention may be detected by using conjugated or fluorescent probes, such as, for example, by biotin- or fluorophore-tethered complementary oligonucleotides or fluorescently labeled molecular beacons, and / or by amplification in the presence of modified or conjugated dNTPs.

[0054] Examples of suitable fluorophores include Atto425 (ATTO-TEC GmbH, Siegen, Germany), Atto 647N (ATTO-TEC GmbH, Siegen, Germany), Yakima Yellow (Epoch Biosciences, Bothell, WA, USA), Cal610 (BioSearch Technologies, Petaluma, CA, USA), Cal635 (BioSearch Technologies, Petaluma, CA, USA), FAM (Thermo Fisher Scientific, Waltham, MA, USA), TET (Thermo Fisher Scientific, Waltham, MA, USA), HEX (Thermo Fisher Scientific, Waltham, MA, USA), cyanine dyes such as Cy5, Cy5.5, Cy3, Cy3.5, and Cy7 (Thermo Fisher Scientific, Waltham, MA, USA), and Alexa dyes (Thermo Fisher Scientific, Waltham, MA, USA). Examples of suitable anti-cancer agents include, but are not limited to, fluorescein isothiocyanate (FITC, Thermo Fisher Scientific, Waltham, MA USA), Tamra (Thermo Fisher Scientific, Waltham, MA USA), ROX (Thermo Fisher Scientific, Waltham, MA USA), JOE (Thermo Fisher Scientific, Waltham, MA USA), fluorescein isothiocyanate (FITC, Thermo Fisher Scientific, Waltham, MA USA), and tetramethylrhodamine (TRITC, Thermo Fisher Scientific, Waltham, MA USA).

[0055] As known to those skilled in the art, the fluorophores can be detected using any suitable method known in the art, for example, by exciting the fluorophore with an appropriate wavelength of light and detecting the emitted fluorescence.

[0056] The template DNA molecule for amplification by the methods of the present invention can be any linear or circular DNA molecule, including mitochondrial DNA and genomic DNA from bacteriophages, viruses, bacteria, including archaea; protists, such as amoebozoa, choanozoa, and excavata; chromistas, including algae and diatoms; plants; fungi; and animals, including humans. Amplification of such genomic templates, preferably whole genome templates, can be used in biomedical and forensic applications. Genomic analyses, such as comparative genomic hybridization, genotyping of polymorphic loci, and detection of disease gene mutations, are important in genetic medicine and forensic science.

[0057] Many biomedical and forensic DNA analysis techniques require nanogram to microgram quantities of genomic DNA. DNA samples often must be amplified before genomic analysis can be performed. The methods of the present invention may be used for such whole genome amplification of genomic template DNA molecules.

[0058] Alternatively, or additionally, the methods of the present invention can be used to amplify circular template DNA molecules. The circular DNA template can be, for example, a recombinant DNA plasmid, a naturally occurring plasmid, a mitochondrial genome, or the circular genomes of some bacteriophages and viruses. The circular DNA template can be artificially generated, for example, by gene synthesis using a padlock probe, or by recombinant DNA techniques including enzymatic ligation with T4 DNA ligase, template-free ligation using specialized DNA ligases such as those capable of template independence, or intramolecular ligation of single-stranded DNA sequences to generate single-stranded DNA circles, e.g., CircLigase (Lucigen, Middleton, WI). As those skilled in the art will recognize, linear DNA template products can also be converted to circular templates by self-ligation or ligation of terminal hairpin loops, for example, using DNA recombinases, protelomerases, resolvases, or integrases.

[0059] Amplification methods such as those of the present invention are ideally suited to generating DNA templates for RNA synthesis or in vitro transcription, including linear templates. For example, a circular DNA construct comprising a promoter for an RNA polymerase, such as an SP6, T3, or T7 promoter, and optionally a poly(A) sequence, can be amplified in accordance with the present invention in the presence of a DNA polymerase and an RNA polymerase (either compatible or incompatible with the promoter site). A restriction enzyme may be present in the amplification reaction to digest the amplification product and generate individual linearized amplification products suitable for in vitro or in vivo transcription. The restriction enzyme is preferably selective for the amplification product but does not restrict the template DNA molecule. Such selectivity can be provided, for example, by a methylation-sensitive restriction enzyme, such as Dam. + Template DNA molecules generated in E. coli strains (which methylate the A in the recognition sequence GATC) cannot be restricted with MboI, while the amplification products are unmethylated and can be restricted by this enzyme. As is well known in the art, methylation sites that overlap the recognition sites for certain endonucleases (e.g., XbaI, ClaI) can also be used. For more examples, see international.neb.com / tools-and-resources / usage-guidelines / dam-and-dcm-methylases-of-e-coli or the restriction enzyme database REBASE (at rebase.neb.com / rebase / rebms.html). Similarly, Dcm + Template DNA molecules produced in E. coli strains (which methylate the C in CCAGG and CCTGG) cannot be restricted at the methylated sites by certain restriction enzymes such as StyD4I, ApaI, or FseI, whereas the amplification products are unmethylated and can be restricted by these enzymes.

[0060] Furthermore, amplification methods such as those of the present invention are ideally suited to generating linear viral templates. For example, a circular construct containing a DNA copy of a viral genome can be amplified in the presence of a DNA polymerase and an RNA polymerase, such as T7 polymerase, nucleotides, and ribonucleotides. The circular construct contains a restriction enzyme recognition sequence, preferably a Dam, Dcm, or EcoKI methylase-sensitive restriction enzyme recognition sequence, in the middle of the terminal repeat sequence. The presence of the restriction enzyme during the amplification reaction restricts the amplification product, resulting in a linearized, amplified genomic DNA copy of the viral genome with terminal repeat sequences present at both ends.

[0061] 4.3 Use of the Invention As shown hereinabove, RNA polymerases such as T7 RNA polymerase can mediate priming on template DNA for subsequent extension by DNA-dependent DNA polymerases. Surprisingly, this phenomenon occurred even when the DNA template did not contain a consensus T7 promoter sequence. Other DNA-dependent RNA polymerases, such as SP6 and T3 RNA polymerases, were also found to initiate replication of DNA templates in the absence of consensus SP6 or T3 promoter sequences, respectively.

[0062] Thus, the present invention provides the use of an RNA polymerase for providing a primer onto a DNA template, said use according to the present invention further comprising the step of amplifying said DNA template from the primer by a strand-displacing DNA-dependent DNA polymerase, preferably in the presence of deoxyribonucleotides, preferably in the presence of all four deoxyribonucleotides.

[0063] As indicated herein above, said strand-displacing DNA-dependent DNA polymerase is preferably Phi29, Bst, and / or Bent DNA polymerase.

[0064] The DNA template can be any linear or circular DNA molecule, including plasmid DNA, synthetic DNA, mitochondrial DNA, and genomic DNA from bacteriophages, viruses, prokaryotes, and eukaryotes, including humans.

[0065] Amplification of the DNA template may be used for biomedical, diagnostic, forensic, or therapeutic purposes or applications. Genomic analyses, such as comparative genomic hybridization, genotyping of polymorphic loci, detection of disease gene mutations, and DNA sequencing, are important in biomedical research, genomic medicine, diagnostics, and forensic science. Diagnostic applications also include amplification of DNA templates from viruses, bacteria, and other microbial pathogens by the methods of the invention, prior to detection of the amplified products by DNA sequencing or an appropriate method.

[0066] Alternatively, or additionally, the use of an RNA polymerase to deposit a primer onto a DNA template according to the present invention is in DNA sequencing, which includes any method, technique, or process that determines the precise ordering of nucleotides in the template DNA.

[0067] Therapeutic applications of DNA, such as gene therapy or DNA vaccines, rely on DNA-based therapeutics, including expression vectors, oligonucleotides for antisense or exon-skipping applications, DNA decoys, DNA aptamers, and DNAzymes. Clinical use of such DNA-based therapeutics requires quality-controlled, highly pure DNA preparations. While relatively short oligonucleotides can be chemically synthesized to high purity, longer DNA molecules are typically isolated as plasmids from bacterial fermentation cultures. The plasmids must be isolated through multiple distinct processing steps, including extensive purification procedures to remove any traces of bacterial genomic DNA, RNA, proteins, and endotoxins. Synthetic cell-free DNA amplification by the methods of the present invention provides a simple, scalable, and affordable alternative for the production of DNA-based therapeutics. Furthermore, the methods of the present invention could be readily adapted to incorporate modified nucleotides into DNA-based therapeutics, which could enhance their therapeutic efficacy.

[0068] Alternatively, the use according to the present invention is in liquid-phase immunoassays and / or immunohistochemistry. Priming with a DNA-dependent RNA polymerase and subsequent amplification with a strand-displacing DNA-dependent RNA polymerase can be performed in free solution and on immobilized targets (solid-phase amplification). Immunohistochemistry is a method that can provide diagnostic and prognostic information to morphological observations and soluble assays. Signal amplification, which uses conjugation to components of a DNA template in a hybridization reaction, followed by priming with a DNA-dependent RNA polymerase and subsequent amplification with a strand-displacing DNA-dependent RNA polymerase, can be applied to increase the sensitivity and specificity of the hybridization reaction.

[0069] Attachment of the target template to the solid support can be convenient and can be achieved through the use of a polymer that serves to attach the target template to the solid support. Such solid-state substrates useful in the described methods can include any solid material to which nucleotides can be attached. This includes materials such as acrylamide, cellulose, nitrocellulose, glass, polystyrene, polyethylene vinyl acetate, polypropylene, polymethacrylic acid, polyethylene, polyethylene oxide, glass, polysilicates, polycarbonate, Teflon, fluorocarbons, nylon, silicone rubber, polyanhydrides, polyglycolic acid, polylactic acid, polyorthoesters, polypropylfumerate, collagen, glycosaminoglycans, and polyamino acids. Solid-state substrates can have any useful form, including thin films or membranes, beads, bottles, dishes, fibers, fabrics, molded polymers, particles, and microparticles. Preferred forms of solid-state substrates are glass slides or microtiter plates, such as standard 96-well plates. Preferred embodiments utilize glass or plastic as the support.

[0070] The DNA template is preferably a circular DNA template, which allows rolling circle amplification to enhance the sensitivity and specificity of immunoassays and / or immunohistochemistry.Due to the high signal-to-noise ratio thus obtained, the circular DNA template is suitable for detecting, quantifying, and visualizing low-abundance proteinaceous markers, viral and bacterial DNA in clinical samples, and as an on-chip signal amplification method for nucleic acid hybridization, for example, in DNA and RNA microarray assays.

[0071] Furthermore, the amplification techniques presented herein can be applied to the construction of DNA nanostructures and DNA hydrogels.

[0072] 4.4 Parts kit The present invention further provides a kit of parts comprising parts carrying a DNA-dependent RNA polymerase, parts carrying a strand-displacing DNA-dependent DNA polymerase, or a combination of said enzymes in a single ready-to-use solution, each of which can be replaced by a similar protein known in the art that functions in substantially the same way.

[0073] The kit of parts may further comprise a suitable reaction buffer, deoxynucleotides and / or ribonucleotides, at least one of which is a purine ribonucleotide. The ribonucleotides preferably include ATP, GTP, or both ATP and GTP.

[0074] The kit optionally further comprises providing a primer comprising at least one oligonucleotide complementary to a stretch of nucleotides on the template DNA molecule, a mixture of random oligonucleotides, or a combination thereof.

[0075] The kit preferably further comprises instructions for using a DNA-dependent RNA polymerase and a strand-displacing DNA-dependent DNA polymerase for priming and subsequent amplification of a DNA template.

[0076] The present invention further provides the use of a kit of parts according to the present invention for the amplification of a DNA template. The DNA template may be any linear or circular DNA molecule, including plasmid DNA, mitochondrial DNA, and genomic DNA from bacteriophages, viruses, bacteria including Archaea, protists such as Amoebozoa, Choanozoa, and Excavata, Chromistas including algae and diatoms, plants, fungi, and animals including humans. Amplification of the genomic template, preferably a whole genome template, may be used in biomedical and forensic applications, including diagnostic applications.

[0077] The present invention is further described by reference to the following examples, which are presented herein for illustrative purposes only and should not be construed as limiting the invention in any way. [Example]

[0078] 5. Working Example Example 1 General Materials and Methods Reagent suppliers: T7 RNA polymerase (M0251), T3 RNA polymerase (M0378), SP6 RNA polymerase (M0207), Phi 29 DNA polymerase (M0269), ribonucleotides (N0450), dNTP mix (N0447), XbaI (R0145, 20 U / μl), BsaI-HFv2 (R3733, 20 U / μl), and PvuI-HF (R3150, 20 U / μl) were from New England Biolabs (NEB; Ipswich, MA, USA); phi29 DNA polymerase (EN-20) was from Biolab Innovative Research Technologies (BLIRT; Gdansk, Poland); T7 RNA polymerase (EP0111), T3 RNA polymerase (EP0101), and SP6 RNA polymerase (EP0131) and Phi 29 DNA polymerase (EP0091) were purchased from Thermo Fisher Scientific (TF, Bleiswijk, the Netherlands); commercial stock buffers supplemented with enzymes and used in some of the examples are shown below. Random hexamers containing two 3' phosphorothioate linkages were purchased from Integrated DNA Technologies (IDT, Coralville, OH, USA).

[0079] DNA template: Double-stranded circular DNA plasmids were purified from E. coli cultures using Nucleobond or NucleoSpin columns (Macherey-Nagel, Düren, Germany). Plasmid p3 is a 5-kb CMV-GFP expression construct containing a single T7 promoter consensus sequence (5' TAATACGACTCACTATAG 3'). pGEM-7Zf+ (Promega Corporation, Madison, WI, USA, hereafter referred to as pGEM) is an empty 3-kb standard cloning vector containing the T7 and SP6 promoter consensus sequences (5' ATTTAGGTGACACTATAG 3') in reverse orientation. pBlueScript II SK+ (Stratagene, La Jolla, CA, USA, hereafter referred to as pBSK) is a widely used 3-kb phagemid cloning vector harboring the T7 and T3 promoter consensus sequences (5' AATTAACCCTCACTAAAG 3') in reverse orientation. Plasmid p53 is a 3.7-kb CMV-GFP mammalian expression vector lacking any T7, T3, or SP6 promoter sequences. Plasmid B327 (3.8 kilobases) was constructed from p53 by inserting a BsaI restriction site and a T7 promoter consensus sequence in reverse orientation on either side of the CMV-GFP expression cassette. All plasmids contain a single, unique XbaI recognition sequence for linearization. HeLa genomic DNA (referred to herein as gDNA) and single-stranded circular DNA derived from M13mp18 were purchased from NEB (catalog numbers N40065 and N4040S, respectively).

[0080] Standard amplification conditions: Unless otherwise specified, standard amplification conditions were performed in 0.5 ml or 0.2 ml PCR tubes containing a 20 μl reaction volume as follows: Reactions were set up in 1× Phi29 buffer B containing 33 mM Tris acetate (pH 7.9 at 37°C), 66 mM potassium acetate, 10 mM magnesium acetate, 0.1% Tween-20, and 1 mM DTT, or in 1× Phi29 buffer N containing 50 mM Tris-HCl (pH 7.5 at 25°C), 10 mM MgCl2, 10 mM (NH4)2SO4, and 4 mM DTT. Reaction mixtures were supplemented with 1 mM dNTPs (NEB, N0447) and 0.25 U / μl Phi29 DNA polymerase (Biolab Innovative Research Technologies, EN-20). A premix containing all common components was used in all experiments to minimize sample-to-sample variation. Template DNA, hexamers, ribonucleotides, and / or RNA polymerase were added to the premix or to individual reactions as indicated, as needed. All pipetting steps were performed on ice. Reaction mixtures were incubated at 30°C for 16 hours or the indicated time, followed by heat inactivation at 65°C for 10 minutes in a PTC-200 Peltier Thermal Cycler DNA Engine (MJ Research) equipped with two 30-well alpha units for 0.5 mL tubes or a 96-well T100 Thermocycler (Bio-Rad) for 0.2 mL tubes.

[0081] Analysis of DNA amplification products: Following incubation and heat inactivation for the indicated times, the reaction mixtures were diluted 5-fold with 5 mM EDTA (pH 8.0) and incubated at 65°C for 30 min to solubilize magnesium pyrophosphate and high-molecular-weight DNA, which often form gel-like precipitates due to excessive nucleotide conversion and DNA amplification. The solubilized reaction products were then diluted once with water to reach a final concentration of 2 mM EDTA and stored at -20°C for further analysis.

[0082] Restriction analysis: To verify the amplification products, 3–5 μl of the diluted reaction mixture was digested with 5 units of XbaI or BsaI in CutSmart buffer (New England Biolabs, 50 mM potassium acetate, 20 mM Tris acetate, 10 mM magnesium acetate, 100 μg / ml BSA, pH 7.9 at 25°C). Following a 1-h incubation at 37°C, 4 μl of 6× loading solution (NEB; Ipswitch, MA, USA) was added, and the digestion products were analyzed on an agarose gel.

[0083] Gel electrophoresis: Samples were loaded onto a 1% agarose gel containing ethidium bromide and subjected to electrophoretic analysis in 1x TAE buffer (40 mM Tris, 20 mM acetic acid, 1 mM EDTA). A 1 kb DNA ladder (NEB, N3232) was used for reference and DNA fragment size estimation. Amplification and / or restriction digestion products were visualized using a UV transilluminator and captured on a ProXima 10 Phi imaging platform.

[0084] Example 2 To examine whether RNA polymerases can initiate specific priming of DNA amplification by binding to their cognate promoter sequences, different plasmid DNA templates harboring zero, one, or two consensus promoter sequences for different RNA polymerases shown in Table 1 were tested in standard amplification reactions.

[0085] Standard amplification reactions were set up in 1x Phi29 buffer B containing 0.25 U / μl Phi29, 1 mM dNTPs, 0.5 mM ribonucleotides GTP / ATP, 10 ng of template DNA or water, and 50 μM hexamer (upper left part of Figure 1A), 0.5 U / μl T7 RNA polymerase (upper right part), 0.5 U / μl T3 RNA polymerase (lower left part), or 0.5 U / μl SP6 RNA polymerase (lower right part of Figure 1A). Following a 16-hour incubation at 30°C, the reaction products were diluted and linearized by XbaI restriction enzyme digestion prior to analysis by gel electrophoresis.

[0086] Unexpectedly, despite the widely described high specificity of T7 RNA polymerase for its cognate promoter sequence, we found that T7 RNA polymerase efficiently enabled priming of DNA amplification against DNA templates without such a promoter sequence (plasmid p53, lane 2). Plasmids containing a single T7 promoter sequence (p3, lane 3) or two T7 promoter sequences in opposite orientations (pB327, lane 4) were amplified equally well in the presence of T7 RNA polymerase, suggesting that priming occurs independently of the T7 promoter sequence. Surprisingly, T3 and SP6 RNA polymerases, also known to be strictly specific for their respective promoter sequences, were found to efficiently enable priming of all plasmid DNA templates used, regardless of the presence of their cognate promoter sequences.

[0087] Thus, this experiment, in addition to other examples described below, demonstrates that RNA polymerase enables primerless amplification of DNA. Given the simplicity of the method and the wide availability of the enzymes and reaction components used, the DNA amplification method of the present invention may prove to be a valuable tool in a variety of applications, such as for research, diagnostic, and therapeutic purposes.

[0088] [Table 1]

[0089] To further analyze and confirm whether RNA polymerase priming actually occurs regardless of its promoter sequence, plasmid pB327 (containing two T7 promoters) was amplified under standard amplification conditions in the presence of random hexamers, T7 RNA polymerase, T3 RNA polymerase, or SP6 RNA polymerase, in the absence or presence of purinergic ribonucleotides. Each reaction contained the following components: 1 mM dNTPs (NEB, N0447) and 0.25 U / μl Phi29 (Blirt, EN20) in 1× Phi29 Reaction Buffer B. Reactions 2 and 3 (corresponding to lane numbering in Figure 1B) were supplemented with 50 μM random hexamers, reactions 4 and 7 with 0.5 U / μl T7 RNA polymerase, and reactions 5 and 6 with 0.5 U / μl T3 RNA polymerase or 0.5 U / μl SP6 RNA polymerase, respectively. Reactions 2 through 5 were further supplemented with the ribonucleotides GTP and ATP (0.5 mM each). Finally, all reactions received 10 ng of pB327 template DNA. Reactions were incubated at 30°C for 16 hours, followed by BsaI restriction analysis and gel electrophoresis. For reference, 200 ng of unamplified template DNA pB327 was included in the restriction analysis to verify the resulting restriction pattern (lane 8 in Figure 1B).

[0090] As described, both hexamers and RNA polymerases are capable of priming template DNA for amplification, but hexamers certainly appear to be very effective at priming native plasmid DNA. Although the template DNA used, pB327, contains only two promoter consensus sequences for T7 RNA polymerase, we found that T3 and SP6 RNA polymerases also efficiently primed pB327 DNA amplification. Given the reversed T7 promoter site, one would expect exponential amplification if T7 RNA polymerase were expected to bind to its cognate promoter sequence and prime DNA amplification from the promoter site. However, clearly, the intensity of the amplification product in the T7 RNA polymerase-primed reaction did not exceed that of the T3- or SP6 RNA polymerase reactions (compare lanes 4, 5, and 6), again demonstrating that priming by RNA polymerase occurs independently of its cognate promoter sequence. Interestingly, in the absence of ribonucleotides, no amplification product was observed in the reaction mixture with T7 RNA polymerase (compare lanes 4 and 7), which led us to further investigate the dependence on ribonucleotides.

[0091] Example 3 To investigate whether the observed priming of DNA amplification by RNA polymerases depends on specific ribonucleotides or their concentrations, standard amplification reactions were set up in which only the type of RNA polymerase or the type of ribonucleotide was varied (Figure 2A). Each reaction contained 1 mM dNTPs, 10 ng of p53 template DNA, and 0.25 U / μl Phi29 (BLIRT EN20) in 1x Phi29 Reaction Buffer B. T7-, T3-, or SP6-RNA polymerase was added to the reaction mixture at 0.5 U / μl. Ribonucleotides were added to individual reactions as indicated in Figure 2A. To analyze the results, 0.5 μl of each reaction product was run on an agarose gel. As can be seen in Figure 2A, all three RNA polymerases enable priming of DNA amplification in the absence of ribonucleotides. Interestingly, UTP appears to be highly ineffective for priming by RNA polymerase, while the highest yields are obtained using GTP and / or ATP. Therefore, it is preferred to include at least one purinergic ribonucleotide in the methods of the invention.

[0092] The optimal concentration of purinergic ribonucleotides for priming DNA amplification by RNA polymerase was established by titrating a mixture of GTP and ATP from 1 mM to 4 μM each (Figure 2B). Each reaction contained 1 mM dNTPs, 10 ng of pB327 template DNA, 0.25 U / μl Phi29 (BLIRT EN20), and 0.5 U / μl T7-, T3-, or SP6-RNA polymerase in 1× Phi29 Reaction Buffer B. As a negative control, water was added instead of ribonucleotides. DNA amplification was still readily observed at GTP / ATP concentrations as low as 15 micromolar, demonstrating that ribonucleotides, while essential, can be used at much lower concentrations than deoxyribonucleotides in the method of the present invention.

[0093] Typically, binding of oligonucleotides or random hexamer primers to double-stranded DNA requires an additional denaturation step in which the template DNA is denatured by heat or alkali treatment to allow primer annealing. Such steps require additional equipment and / or buffers, but more importantly, these additional steps increase the risk of contamination with exogenous DNA and can severely affect the quality of the template DNA. To clarify whether priming with RNA polymerase also requires template DNA denaturation, pB327 plasmid was mixed with excess random hexamers and left in its native double-stranded form, or denatured at 95°C for 5 min and slowly cooled to allow hexamer primer annealing to the template DNA. To minimize the confounding effects of possible self-priming due to nicks in the template DNA, the pB327 plasmid was first treated with T5 exonuclease. For this purpose, 3 μg of pB327 was incubated with 30 units of T5 exonuclease (NEB, M0363) in CutSmart buffer for 2 hours at 37°C and purified using a NucleoSpin column (Macherey-Nagel, Düren, Germany). Reactions were set up under standard conditions, with final concentrations of 0.5 ng / μl template DNA, 50 μM random hexamers, and 0.5 mM GTP / ATP in each reaction. RNA polymerase was added to 0.5 U / μl as indicated above each lane in Figure 3. Separate aliquots of the complete reaction mixture were incubated at 30°C, and amplification reactions were terminated at the indicated time points by heat inactivation at 65°C for 10 minutes and stored at -20°C until further analysis. Reaction products were digested with XbaI prior to analysis by gel electrophoresis. The digested reaction product migrates at the expected size of linearized pB327 (3.8 kb), and the band intensity increases over time. As can be concluded from Figure 3, efficient amplification with random hexamers requires denaturation of the template DNA (compare lanes 1 and 5), and the addition of RNA polymerase allows priming and amplification of both native and denatured template DNA.This implies that the use of RNA polymerase in DNA amplification eliminates the need for a denaturation step, thereby making the process truly isothermal.

[0094] Example 4 The transcriptional activity of DNA-dependent RNA polymerases such as T7-, T3-, and SP6 RNA polymerases is highly specific to their respective promoter sequences, and this binding region is recognized as a double-stranded duplex (Golomb et al., 1977, J Virol 21: 743-752; Stump and Hall, 1993, Nucleic Acids Res 21: 5480-5484; Maslak and Martin, 1993, Biochemistry 32: 4281-4285; Li et al., 1996, Biochemistry 35: 3722-3727). The present invention demonstrates that RNA polymerases can also function to prime DNA amplification, and that this unexpectedly occurs in the absence of promoter sequences. To clarify whether RNA polymerase priming also occurs on single-stranded DNA templates, a standard amplification reaction was set up using single-stranded M13mp18 phage DNA as a template. M13mp18 (NEB, N4040) is a 7.2-kilobase circular, single-stranded DNA molecule that contains a single, unique XbaI recognition site, allowing M13mp18 to linearize after replication into a double-stranded molecule. The M13mp18 sequence (Genbank accession number X02513) lacks any T7, T3, or SP6 promoter sequences. To test DNA amplification, standard reaction conditions were set up using 1 ng / μl single-stranded M13mp18 template DNA and 0.5 mM GTP / ATP in 1× Phi29 buffer (New England Biolabs). Reaction products were digested with XbaI and analyzed by gel electrophoresis. As shown in Figure 4, the digested reaction product migrated at the expected size of linearized M13mp18 (7.2 kb), indicating that the single-stranded M13mp18 template was faithfully replicated and amplified to double-stranded DNA by both hexamer and RNA polymerase priming. The upper band in Figure 4 corresponds to a hole in the agarose gel and most likely represents a single-stranded high-molecular-weight concatemer of the M13mp18 template. This example also illustrates that RNA polymerase, apart from functioning independently of its promoter sequence, can efficiently prime the replication and amplification of a single-stranded DNA template.

[0095] Example 5 Whole genome amplification (WGA) is a powerful technique for amplifying small amounts of genomic DNA for further processing, analysis, or sequencing. Most commercially available kits use PCR-based methods (e.g., SMARTer® PicoPLEX®, Takara Bio USA, Mountain View, CA; GenomePlex® WGA kits, Sigma-Aldrich, St. Louis, MO) or multiple displacement amplification (MDA), such as GenomiPhi™ (GE Healthcare, Chicago, IL) and REPLI-g (Qiagen, Hilden, Germany). A common feature of these kits is that they rely on the use of random primers or semi-degenerate oligonucleotides. One disadvantage of these methods is that the genomic DNA template must be denatured in order for the primers to anneal and prime amplification. In cases where only trace amounts or very old DNA are available (e.g., in forensic science or archaeology), denaturing the precious DNA sample can be detrimental to downstream analysis. Another disadvantage of using random or semi-random primers is biased amplification of template DNA. This can result in information loss, sequencing errors due to mispriming, or nonspecific amplification due to self-priming of random oligonucleotides (see, e.g., Hansen et al., 2010, Nucleic Acids Res 38: el31; van Gurp et al., 2013, PLoS ONE 8: e85583; Sabina and Leamon, 2015, Methods Mol Biol 1347: 15–41). A new method for genome amplification using the primase-polymerase (PrimPol) isolated from Thermus thermophilus HB27 has recently been reported (Picher et al., 2016, Nature Comm 7: 13296), which possesses endogenous primase activity without the need for exogenously added oligonucleotide primers. However, this method still requires denaturation of template DNA prior to amplification, and the enzyme used is not widely available.To analyze the ability of RNA polymerase to prime amplification of native, undenatured genomic DNA, native HeLa genomic DNA was used as a template. Amplification was performed under standard amplification conditions in 1x Phi29 buffer (New England Biolabs) containing 0.5 mM GTP / ATP and 50 μM random hexamers, 0.5 U / μl RNA polymerase as indicated, or a combination of 50 μM random hexamers and 0.5 U / μl RNA polymerase (Figures 5A and 5B). Identical reactions with and without template were set up using 1 ng / μl native HeLa genomic DNA (NEB, N4006) as the template for amplification (Figures 5A and 5B). Following amplification, 1 μl of undigested amplification product was analyzed on a 0.7% agarose gel. As shown in Figure 5A, all reactions except for the no-priming control in lane 1 yielded amplification products, with the major DNA band migrating approximately 50–70 kb, which is considered the maximum product length for Phi29-mediated DNA polymerization. Samples containing random hexamers (lanes 2, 6–8) exhibited higher intensities and appear to contain more amplified DNA. However, as concluded from the no-template control in Figure 5B, the majority of the products amplified in the presence of random hexamers appear to be artifacts, most likely resulting from self-priming and extension by the random hexamers. In contrast, the no-template control reactions amplified in the presence of RNA polymerase alone lacked any nonspecific amplification products (lanes 3–5). This strongly suggests that priming by RNA polymerase results in specific amplification of native genomic DNA, making this method an excellent alternative to existing WGA methods.

[0096] Example 6 We also investigated whether RNA polymerases could alter the kinetics of random hexamer-primed DNA amplification or enhance product yield. To this end, standard amplification reactions were performed in 1x Phi29 buffer (Biolab Innovative Research Technologies) containing 50 μM hexamers, 0.5 mM GTP / ATP, and 10 ng of pB327 plasmid per reaction. The reaction premix was divided into four portions and supplemented with water (hexamers only, upper left panel) or 0.5 U / μl of the indicated RNA polymerase (Figure 6A). At the indicated time points, reactions were stopped, and equal amounts of amplification product were analyzed by XbaI digestion and gel electrophoresis. To quantify kinetics, band intensities were determined using ImageJ software (Schneider et al., 2012. Nature Methods 9: 671–675) and analyzed graphically in Microsoft Excel (Figure 6B). Clearly, the addition of RNA polymerase dramatically increases the overall yield of the amplification reaction, reaching a maximum yield much more quickly than with hexamers alone. The maximum yield is determined by the depletion of dNTPs in the reaction mixture or by the inhibitory effect of pyrophosphate formed during dNTP polymerization, and appears to be reached between 8 and 16 hours for T7 and T3 RNA polymerases and somewhat earlier for SP6 RNA polymerase.

[0097] Commercially available isothermal DNA amplification kits typically use random hexamer primers at very high concentrations (50 or even 100 μM), which represent a vast molar excess over the amplification product. To determine whether this concentration could be reduced in the presence of RNA polymerase without loss of yield, a fixed amount of T7 RNA polymerase and varying amounts of random hexamers were tested in standard amplification reactions. To allow random hexamers to anneal to the template DNA, heat-denatured p53 plasmid DNA was used as the template in this experiment. Furthermore, this template does not contain any consensus promoter sequence for T7 RNA polymerase. Following a 16-hour incubation at 30°C, the reaction products were analyzed on a gel. In the absence of T7 RNA polymerase, no DNA amplification was observed without random hexamers (Figure 6C; first lane in the series). Furthermore, whereas maximum yield in the presence of T7 RNA polymerase appears to be reached at a hexamer concentration of 20 μM, in the absence of RNA polymerase, maximum amplification yield was not reached even in reactions containing 100 μM, indicating that the addition of RNA polymerase and ribonucleotides strongly enhances primer-dependent DNA amplification.

[0098] Example 7 To verify that the method of the present invention works with similar enzymes from other suppliers and to test the robustness of the invented method, standard reaction conditions were set up using 10 ng of p53 plasmid DNA as template and Phi29 DNA polymerase from three different suppliers. Additionally, RNA polymerases from two different suppliers were included to test all possible combinations (Figure 7A). Reactions in panel a contained 0.25 U / μl Phi29 polymerase from Blirt, reactions in panel b contained 0.25 U / μl Phi29 polymerase from New England Biolabs, and reactions in panel c contained 0.25 U / μl Phi29 polymerase from Thermo Fisher. All three DNA polymerases were used in combination with reaction buffers from the corresponding suppliers. Reaction products were digested with XbaI and analyzed by gel electrophoresis. As illustrated in Figure 7A, all six tested RNA polymerases enabled efficient priming of DNA amplification, comparable to priming with random hexamers (compare lane 2 with lanes 3-8 in each panel). Furthermore, Phi29 DNA polymerases from three different suppliers efficiently amplified template DNA (compare panels a-c), demonstrating that the method of the present invention functions with a variety of polymerases from different suppliers. The different intensities of amplification products between lanes suggest that certain combinations of RNA polymerase and DNA polymerase from the same or different suppliers may function better or worse. To further test the robustness of this method, RNA polymerase-primed DNA amplification was performed in two buffers with significantly different compositions: Phi29 buffer N (labeled "Tris-HCl buffer" in Figure 7B) containing 50 mM Tris-HCl (pH 7.5 at 25 °C), 10 mM MgCl, 10 mM (NH)SO, and 4 mM DTT; or Phi29 buffer B (labeled "Tris-Acetate Buffer" in Figure 7B) containing 33 mM Tris-Acetate (pH 7.9 at 37 °C), 66 mM potassium acetate, 10 mM magnesium acetate, 0.1% Tween-20, and 1 mM DTT.All other reaction components were kept the same: 1 mM dNTPs, 0.5 ng / μl p53 plasmid DNA, 0.25 U / μl Phi29 polymerase (Blirt, EN20), and 0.5 mM GTP / ATP. Reactions were primed by adding 50 μM random hexamers (lanes 1 and 5), 0.5 U / μl T7 RNA polymerase (T7R, Thermo Fisher Scientific) (lanes 2 and 6), 0.5 U / μl T3 RNA polymerase (T3R, Thermo Fisher Scientific) (lanes 3 and 7), or 0.5 U / μl SP6 RNA polymerase (T3R, Thermo Fisher Scientific) (lanes 4 and 8), and incubated at 30°C for 16 hours to allow DNA amplification. Reaction products were digested with XbaI and analyzed by gel electrophoresis. As depicted in Figure 7B, no significant differences in yield were observed among the different buffer series. Thus, the examples presented support a robust amplification method that is not dependent on any particular enzyme supplier or buffer composition.

[0099] Example 8 Therapeutic applications of DNA, such as gene therapy or DNA vaccines, require highly pure, contaminant-free, sequence-confirmed DNA preparations. Therefore, synthetic cell-free DNA amplification according to the present invention provides a simple and affordable alternative for the amplification and production of DNA-based therapeutics. Alternatively, the method of the present invention may be used in DNA sequencing processes, preferably when limited amounts of template DNA are present. The production of functional DNA was tested by amplifying a GFP-expressing vector under various conditions and transfecting the digested amplification products into cells for expression analysis. The DNA sequences of the amplification products were verified by Sanger sequencing.

[0100] Materials and Methods Amplification: DNA for transfection studies was generated under standard reaction conditions using 10 ng of p53 as template DNA. Amplification was initiated by adding 50 μM random hexamers with or without 0.5 mM GTP / ATP (to eliminate possible effects of ribonucleotides on expression studies), or by adding 0.5 mM GTP / ATP and 0.5 U / μl T7 RNA polymerase, 0.5 U / μl T3 RNA polymerase, or 0.5 U / μl SP6 RNA polymerase. P53 plasmid amplification products were digested with PvuI (NEB, R3150) for 2 hours at 37°C. Plasmid p53 contains a single, unique PvuI recognition site in the vector backbone; therefore, digestion with PvuI yields a linearized full-length plasmid containing an intact expression cassette driving GFP expression. Following digestion, linear DNA fragments were purified using NucleoSpin Gel and PCR Clean-up (Macherey-Nagel, Diiren, Germany), diluted in 10 mM Tris-HCl, 0.1 mM EDTA, and DNA concentrations were measured on a NanoDrop spectrophotometer (ThermoFisher).

[0101] Cell culture and transfection: Human embryonic kidney cells (HEK293, ATCC CRL-1573) and mouse melanoma cells (B16F10, ATCC CRL-6475) were maintained under standard tissue culture conditions in IMDM medium (ThermoFisher-Gibco, 21980-032) supplemented with 5% heat-inactivated fetal bovine serum (FBS, Sigma-Aldrich, F0804) and 1% penicillin-streptomycin mixture (Lonza, 17-602E). One day before transfection, 20 × 10 HEK293 or 4 × 10 B16-F10 cells were seeded per well in 100 μl of IMDM medium in 96-well flat-bottom tissue culture plates. The following day, transfections were performed in triplicate using Saint-DNA transfection reagent (SD-2001-01, Synvolux Products, Leiden, the Netherlands) according to the manufacturer's instructions. Briefly, Saint-DNA was brought to room temperature and vortexed for 30 seconds. For each well, 50 ng of PvuI-digested and purified RCA product was combined with 1 μl of Saint-DNA in a total volume of 10 μl of phosphate-buffered saline (PBS, Lonza, 17-516F) for 5 minutes at room temperature. The complex (10 μl) was added to each well, and the cells were placed in a CO2 incubator. After 48 hours, cells were rinsed once with PBS, harvested by trypsinization (ThermoFisher-Gibco), and washed twice with FACS buffer containing PBS and 0.5% bovine serum albumin (BSA, Biowest, P6154). GFP expression was analyzed on a Guava EasyCyte 5HT (Merck-Millipore, Burlington, MA, USA). Data were analyzed using GuavaSoft 3.3 (Merck).

[0102] DNA Sequencing: To verify the identity and fidelity of nucleotide incorporation during amplification, RCA products were sequenced using Sanger sequencing. For this purpose, plasmid pB327 was amplified under standard amplification conditions in the presence of random hexamers or RNA polymerase. Each reaction contained the following components: 1 mM dNTPs, 0.5 mM GTP / ATP, 0.25 U / μl Phi29 (Blirt, EN20), and 10 ng of pB327 plasmid DNA template in 1x Phi29 Reaction Buffer N. Priming was initiated with 50 μM random hexamers, 0.5 U / μl T7 RNA polymerase, 0.5 U / μl T3 RNA polymerase, or 0.5 U / μl SP6 RNA polymerase, as indicated in Table 2 below. The reaction was incubated at 30°C for 16 hours, followed by XbaI digestion and purification using NucleoSpin Gel and PCR Clean-up (Macherey-Nagel, Düren, Germany). DNA sequencing was performed by Baseclear BV (Leiden, The Netherlands) on an ABI 3730 Genetic Analyzer (Fisher Scientific, Landsmeer, The Netherlands) using pB327-specific forward sequencing primers and BigDye® Terminator v3.1 cycle sequencing (Fisher Scientific, Landsmeer, The Netherlands).

[0103] result To compare transfection efficiency and expression of the reaction products, p53 template DNA was amplified under standard conditions in the presence of random hexamers or RNA polymerase. The p53 plasmid contains an expression cassette with a CMV promoter, green fluorescent protein (GFP), and beta globin poly(A) signal, allowing single-cell expression monitoring by flow cytometry.

[0104] As illustrated in Figure 8, left panel, all amplification products were efficiently transfected in both HEK293 cells (up to 90%) and B16F10 cells (up to 70%), with no apparent difference in the number of transfected cells. Expression levels, as assessed by the level of GFP fluorescence per cell, did not show any significant differences between the transfected DNA products. This demonstrates that (1) the inclusion of ribonucleotides in the amplification reaction does not affect transcription of the amplification products, and (2) priming DNA amplification by RNA polymerase according to the methods of the present invention delivers pure and functional DNA suitable for cellular expression and potential therapeutic purposes.

[0105] [Table 2]

[0106] The sequencing results (see Table 2) revealed that RNA polymerase priming could faithfully replicate and amplify the template DNA, and the amplified products were 100% identical in DNA sequence to the original template DNA, arguing that the method of the present invention can also be used for DNA sequencing purposes.

[0107] Example 9 For gene therapy purposes, sustained expression and reproducibility are critical factors for success. To examine and compare in vivo gene expression of synthetic linear DNA, mice were intradermally injected with equimolar amounts of plasmid DNA (pDNA, 10 μg) or linear DNA (InDNA, 5.4 μg) encoding firefly luciferase, or phosphate-buffered saline as a control, on days 0 and 41 (n=4 per group). Luciferase activity was measured with an IVIS Spectrum in vivo imaging system (Perkin Elmer) at the indicated time points. Luciferase activity was approximately 2.0 × 10 in mice injected with either plasmid DNA or linear DNA. 7 Starting at photons / second (p / s), after one month it is approximately 1.0×10 6p / s (Figure 9). A second injection of DNA yielded similar levels and kinetics of luciferase activity with both plasmid and linear DNA, demonstrating that the synthetic linear DNA produced by the methods of the present invention is active in vivo and comparable to plasmid DNA.

[0108] Example 10 Materials and Methods Mice and tumor cell lines: C57BL / 6 (Jico) mice and B6 albino mice (strain B6 / Rj-Tyrc / c) were purchased from Jackson Laboratory (Bar Harbor, ME, USA) and Janvier Labs (Le Genest-Saint-Isle, France), respectively. Mice were housed under FELASA-compliant conditions, and all animal experiments were approved by the Dutch Animal Ethics Committee in accordance with its guidelines. B16-OVA, a derivative of the B16-F10 melanoma cell line stably transfected with ovalbumin, was maintained in a culture medium consisting of IMDM (ThermoFisher-Gibco, Waltham, MA, USA) supplemented with 8% fetal bovine serum (Sigma-Aldrich, Zwijndrecht, the Netherlands) in the presence of L-glutamine, penicillin, and streptomycin (all ThermoFisher-Gibco) in a humidified CO2 incubator (37°C, 5% CO2).

[0109] Generation of linear DNA for in vivo studies: Circular plasmid DNA encoding firefly luciferase or the B16-OVA epitope flanked by BsaI restriction enzyme sites was amplified in vitro using the method of the present invention. Expression cassettes lacking the plasmid backbone sequence were obtained from the concatemer amplification product by digestion with BsaI (NEB, Ipswich, MA, USA) and ligated to nuclease-resistant hairpin oligos (IDT, Coralville, OH, USA) during eight cycles of 1 h at 37°C and 1 h at 16°C to generate linear DNA (InDNA) with closed ends. T5 exonuclease (NEB, Ipswich, MA, USA) was then added for 16 h at 37°C to remove the vector backbone and unligated product. Plasmid DNA and linear DNA produced by E. coli bacterial culture were purified twice using Nucleobond Xtra maxi EF columns (Macherey-Nagel, Duren, Germany) and resuspended in 10% Tris-EDTA buffer.

[0110] In vivo bioluminescence imaging: On days 0 and 41, B6 albino mice were injected intradermally at the base of their tails with an equimolar dose of luciferase-encoding plasmid DNA or linear DNA. The control group received the same volume of PBS. For in vivo bioluminescence imaging, mice were subcutaneously injected with 150 mg / kg D-luciferin (Synchem; catalog no. bc219). After 15 minutes, mice were anesthetized with isoflurane inhalation, and imaging was performed using an IVIS Spectrum small animal imaging system (PerkinElmer). Optical signals using an open filter and automatic acquisition time were quantified at the base of the mouse's tail using a fixed-size region of interest throughout the entire experiment. Image analysis and luminescence quantification were performed using Livinglmage software (PerkinElmer).

[0111] Mouse vaccination, immune response, and tumor burden: On day 0, male C57BL / 6 mice (8 mice in each of three groups) were intradermally injected with 30 μl of 0.9% NaCl solution containing equimolar amounts (4.3 pmol) of DNA molecules. The first group was vaccinated with 10 μg of circular plasmid DNA (3.6 kB) encoding multiple antigens, including OVA-specific epitopes. The second group was vaccinated with 5.4 μg of linear DNA (1.9 kb), amplified from the same plasmid vector used in the first group but lacking the bacterial backbone sequence; the third (control) group was left untreated. Two weeks after vaccination, all mice were bled, treated with red blood cell lysis buffer, and stained with PE-conjugated H2-Kb / SIINFEKL tetramer produced at the LUMC tetramer facility, Leiden, the Netherlands, to detect OVA-specific CD8 T cells. Samples were analyzed by flow cytometry using a BD LSRII (Becton Dickinson, San Jose, CA, USA) and FlowJo software (FlowJo LLC). Analyzed data were plotted using GraphPad Prism software (San Diego, CA, USA). On day 21 post-vaccination, mice were subcutaneously injected with 50,000 B16-OVA cells. Tumor growth was monitored every 3-4 days, and tumor size was calculated as (length × width × width) / 2. 1000 mm 3 Mice bearing tumors exceeding 100 μg / kg or with bleeding ulcers were sacrificed by CO2 asphyxiation.

[0112] result The method of the present invention is particularly suitable for the rapid and inexpensive production of pure DNA, e.g., for the production of personalized cancer vaccines. To examine whether synthetic linear DNA induces T cell activation and tumor protection, naive 6- to 8-week-old C57BL / 6 mice received a single intradermal vaccination with equimolar amounts of plasmid DNA (pDNA, 10 μg) or linear DNA (InDNA, 5.4 μg) encoding an OVA epitope. Control mice were left untreated (n = 8 per group). Tetramer staining and flow cytometry were performed on peripheral blood 14 days after vaccination to detect the induction of OVA-specific T cells. As shown in Figure 10A, vaccination with either plasmid DNA or linear DNA resulted in a significant and comparable induction of T cells specific for the OVA epitope encoded within the vaccine. The same mice were then challenged with B16-OVA tumor cells (50,000 cells / mouse) 21 days after vaccination, and tumor growth was monitored. Figure 10B shows that all untreated mice (with one exception) died within one month due to tumor growth. In contrast, vaccination with plasmid DNA (10 μg) or linear DNA (5.4 μg) provided nearly complete protection against tumor growth. Surviving mice remained tumor-free for the remainder of the experiment (well over 100 days).

Claims

1. 1. A method for amplifying a template DNA molecule, comprising: a) providing a template DNA molecule; b) providing a promoter-dependent RNA polymerase of the family of single-subunit RNA polymerases, a strand-displacing DNA polymerase, and a combination of ribonucleotides and deoxyribonucleotides; c) incubating the material in a suitable buffer for a suitable period of time to allow replication and amplification of the template DNA molecule. Including, priming by the promoter-dependent RNA polymerase occurs independently of consensus promoter sequences; method.

2. 2. The method of claim 1, wherein the single-subunit RNA polymerase is selected from the group consisting of T7 RNA polymerase, T3 RNA polymerase, SP6 RNA polymerase, and mutants thereof.

3. 3. The method of claim 1 or claim 2, wherein the strand-displacing DNA polymerase is selected from Phi29, Bst, Bent DNA polymerase, and combinations thereof.

4. 4. The method of claim 1, wherein the ribonucleotides comprise at least one ribonucleotide having a purine nucleobase.

5. 5. The method of claim 4, wherein the purine nucleobase is adenine or guanine, or a combination thereof.

6. 6. The method of claim 1, wherein the replication and amplification further comprises providing at least one oligonucleotide complementary to the template DNA molecule, a mixture of random oligonucleotides, or a combination thereof.

7. 7. The method of claim 1, wherein at least one of the nucleotides or ribonucleotides is modified or labeled.

8. The method of claim 7, wherein the at least one species is modified or labeled with a detectable label.

9. 9. The method according to claim 1, wherein the amplification product is detected by fluorescence and / or by chemical means.

10. 10. The method of any one of claims 1 to 9, wherein the amplification products are detected by biotin or fluorophore-labeled probes and / or by amplification in the presence of fluorophore-conjugated dNTPs.

11. 1. Use of a promoter-dependent RNA polymerase to deposit a primer onto a double-stranded (ds) DNA template, wherein the RNA polymerase is from the family of single-subunit RNA polymerases.

12. 12. The use according to claim 11, wherein the double-stranded DNA template is further amplified from the primers by a strand-displacing DNA polymerase in the presence of deoxyribonucleotides.

13. 13. The use according to claim 12, wherein said providing of a primer comprises providing at least one ribonucleotide having a purine nucleobase.

14. 14. The use according to claim 13, wherein the provision of a primer comprises providing two or three ribonucleotides selected from GTP, ATP, and CTP.

15. 15. Use according to any one of claims 11 to 14 for amplifying linear or circular single- or double-stranded DNA molecules, including genomic DNA.

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