Linear DNA with enhanced resistance against exonucleases and methods for the production thereof

Abstract

The present invention relates to methods for producing a linear deoxyribonucleic acid (DNA) product (e.g. a closed linear DNA product) with enhanced resistance to nuclease digestion. The present invention relates to methods comprising the steps: (a) contacting a double-stranded DNA molecule with an endonuclease, a ligase and first and second terminal adaptor molecules and intermediate adaptor molecules, to form a single contiguous aqueous volume; and (b) incubating the single contiguous aqueous volume to generate a linear DNA product (e.g. a closed linear DNA product). The present invention also relates to linear deoxyribonucleic acid (DNA) products (e.g. a closed linear DNA products) with enhanced resistance to nuclease digestion and uses thereof.

Description

[0001] LINEAR DNA WITH ENHANCED RESISTANCE AGAINST EXONUCLEASES AND METHODS FOR THE PRODUCTION THEREOF

[0002] TECHNICAL FIELD

[0003] The present invention relates to methods for producing a linear deoxyribonucleic acid (DNA) product (e.g. a closed linear DNA product) with enhanced resistance to nuclease digestion. The present invention relates to methods comprising the steps: (a) contacting a double-stranded DNA molecule with an endonuclease, a ligase and first and second terminal adaptor molecules and intermediate adaptor molecules, to form a single contiguous aqueous volume; and (b) incubating the single contiguous aqueous volume to generate a linear DNA product (e.g. a closed linear DNA product). The present invention also relates to linear deoxyribonucleic acid (DNA) products (e.g. a closed linear DNA products) with enhanced resistance to nuclease digestion and uses thereof.

[0004] BACKGROUND

[0005] DNA is susceptible to degradation by nucleases which are naturally occurring enzymes within organisms and which have a vital role in the regulation of many cellular processes, while also protecting against foreign DNA species. Enzymatic DNA degradation can render gene therapies ineffective and is a substantial consideration when developing gene therapies or DNA vaccines.

[0006] Considerable efforts have been made to extend the effective molecular lifetime of nucleic acids by increasing resistance of the nucleic acid molecules to both extracellular and intracellular nucleases.

[0007] For linear molecules, one of the proposed solutions includes the use of phosphorothioated nucleotides (i.e. 2'-deoxynucleotides-5'-(a-thio)-triphosphate).

[0008] Phosphorothioated nucleotides comprise a sulphur atom instead of a non-bridging oxygen atom. These modified nucleotides show comparable physical and chemical characteristics to corresponding unmodified nucleotides, but are resistant to exonuclease digestion. As such, the incorporation of the phosphoroth ioate functional group can prolong the half-life of the nucleic acid molecule.

[0009] Phosphorothioate modifications are used in nucleic acid drug development programmes. In therapeutic nucleic acids, the phosphorothioated nucleotides are incorporated into short, single-stranded polynucleotide chains. For example, an antisense oligonucleotide fomivirsen is a 21 -mer phosphorothioate oligodeoxynucleotide used to treat cytomegalovirus retinitis (Stein and Castanotto, "FDA-approved oligonucleotide therapies in 2017." Molecular Therapy 25.5 (2017): 1069-1075). Similarly, pegaptanib (brand name Macugen) is a short (27-nucleotides) aptamer with a phosphorothioate 3’-3’ deoxythymidine cap used for treating age-related macular degeneration of the retina. Phosphorothioate modifications have also been used in the context of a linear double-stranded polynucleotide chain (e.g. double-stranded DNA) to cap the ends of the polynucleotide chain to increase resistance to exonuclease digestion (Putney et al. "A DNA fragment with an alpha-phosphorothioate nucleotide at one end is asymmetrically blocked from digestion by exonuclease III and can be replicated in vivo." Proceedings of the National Academy of Sciences 78.12 (1981 ): 7350-7354). To cap the ends of the polynucleotide chain, the ends are digested with a restriction enzyme and treated with a DNA polymerase and a mixture of deoxyribonucleotide triphosphates (dNTPs), at least one type of which is a phosphorothioated nucleotide complementary to a nucleotide in the overhanging strand. Since DNA polymerases add nucleotides in the 5’ to 3’ direction, the result of this treatment is a blunt-ended polynucleotide fragment with a phosphorothioated nucleotide located at the 3’-end of each strand (i.e. in “the cap”).

[0010] Resistance to nuclease digestion can also be accomplished by using closed DNA molecules, such as plasmids or minicircles. However, plasmids and minicircles have limited utility in vivo due to their frequent contamination with toxic agents derived from cell components, fidelity issues that alter the sequence of interest, and presence of different species (supercoiled, linear and open circular).

[0011] Alternatively, resistance to nuclease digestion may be accomplished by producing closed linear DNA molecules. For example, WO2010 / 086626 A1 describes a method for producing a closed linear DNA by utilizing a protelomerase. However, this method is limited in that the action of protelomerase produces the same sequence at both ends of the closed linear DNA molecules, allowing for little flexibility.

[0012] W02008 / 095927 describes a system known as “Golden Gate” assembly, whereby separately digested fragments of a desired construct are ligated into a vector. This method is inefficient due to the number of ligation events that must occur and having to use multiple different restriction sites. Furthermore, the resulting yield is low and the desired DNA product must be amplified using bacteria to increase the amount of DNA, requiring the presence of, e.g. antibiotic resistance genes, meaning commercial scalability of the “Golden Gate” process is not possible without the use of fermentation.

[0013] Thus, a need exists for a more flexible method for producing a linear DNA product with an enhanced resistance to nuclease (e.g. exonuclease) digestion, whilst allowing for additional features to be incorporated for different applications.

[0014] DESCRIPTION

[0015] The invention provides a method for producing a linear deoxyribonucleic acid (DNA) product (e.g. a closed linear DNA product) with enhanced resistance to nuclease digestion. The invention is based on the addition of adaptor molecules to a double-stranded DNA molecule. The method of the invention relies on the addition of the adaptor molecules, an endonuclease and a ligase to the double-stranded DNA molecule in a single reaction volume (or single contiguous aqueous volume). Thus, the method for producing a linear DNA product comprises the steps: (a) contacting a double-stranded DNA molecule with an endonuclease, a ligase and first and second terminal adaptor molecules and n+m intermediate adaptor molecules and n+m intermediate adaptor molecules, to form a single contiguous aqueous volume, wherein n and m are each 0 or an integer of at least 1 , and wherein n+m is at least 1 , wherein n and m are each 0 or an integer of at least 1 , and wherein n + m is at least 1 ; and (b) incubating the single contiguous aqueous volume to generate the linear DNA product. Preferably, the linear DNA product has enhanced resistance to exonuclease (e.g. exonuclease I, exonuclease III and / or exonuclease VIII) digestion. The linear DNA product may be a closed linear DNA product. The linear DNA product may comprise nuclease-resistant nucleotides (i.e. protected nucleotides), such as phosphorothioated nucleotides. The linear DNA product may be a partially closed linear DNA product. The partially closed linear DNA product may comprise nuclease-resistant nucleotides (i.e. protected nucleotides), such as phosphorothioated nucleotides. The linear DNA product may comprise a cassette. The cassette may comprise a coding sequence.

[0016] The invention provides a method for producing a linear DNA product, wherein the method comprises: (a) contacting a double-stranded DNA molecule with an endonuclease and first and second terminal adaptor molecules and n + m intermediate adaptor molecules, , to form a single contiguous aqueous volume, wherein n and m are each 0 or an integer of at least 1 , and wherein n+m is at least 1 ; and (b) incubating the single contiguous aqueous volume to generate the linear DNA product, wherein the linear DNA product comprises a linear double-stranded region, wherein the linear double-stranded region comprises a portion of the double-stranded DNA molecule, and wherein n intermediate adaptor molecules are sequentially appended to a first end of the linear double stranded region and m intermediate adaptor molecules are sequentially appended to a second end of the linear doublestranded region, and wherein the first terminal adaptor molecule is appended to the nth intermediate adaptor molecule or, when n is 0, to the first end of the linear double stranded region, and the second terminal adaptor molecule is appended to the mth intermediate adaptor molecule, or when m is 0, to the second end of the linear double stranded region, n may be 0, 1 , 2, 3, 4, 5, 6, 7 or 8. m may be 0, 1 , 2, 3, 4, 5, 6, 7 or 8. Preferably, n is 0, 1 , 2, 3 or 4, and m is 0, 1 , 2, 3 or 4. .

[0017] The first and second terminal adaptor molecules may be identical molecules or they may be different molecules. For example, the first terminal adaptor molecule and / or the second terminal adaptor molecule may comprise a hairpin. The first terminal adaptor molecule and / or the second terminal adaptor molecule may double-stranded linear nucleic acid molecules comprising one or more nuclease- resistant nucleotides. The first terminal adaptor molecule may comprise a hairpin and the second terminal adaptor molecule may be a double-stranded linear nucleic acid molecule comprising one or more nuclease-resistant nucleotides. Thus, the linear DNA product produced by the methods described herein is resistant to nuclease (e.g. exonuclease) digestion.

[0018] The intermediate adaptor molecules may be the same or they may be different. One or more of the n+m intermediate adaptor molecules may comprise a spacer or a linker sequence. One or more of the n+m intermediate adaptor molecules may comprise a fluorophore, a barcode, a polyA signal, a biotinylated nucleotide, a protected nucleotide, a modified nucleotide, a spacer, polyA sequence, a promotor, an open reading frame, a UTR (untranslated region), a transcription factor binding site, or a terminator sequence. The n intermediate adaptor molecules sequentially appended to a first end of the doublestranded DNA molecule may be the same or may be different from the m intermediate adaptor molecules sequentially appended to the second end of the double-stranded DNA molecule.

[0019] A modified nucleotide may be 2-MethoxyEthoxy A, 2-MethoxyEthoxy MeC, 2-MethoxyEthoxy G, 2- MethoxyEthoxy T, 2'-O-Methyl RNA Bases, Fluoro Bases, 2-Aminopurine, 5-Bromo dU, deoxyUridine, 2,6-Diaminopurine (2-Amino-dA), Dideoxy-C, deoxyinosine, Hydroxymethyl dC, Iso-dG, Iso-dC, 5- Methyl dC or 5-Nitroindole.

[0020] The step of contacting the double-stranded DNA molecule with the endonuclease and first and second terminal adaptor molecules is preferably performed in the presence of a ligase. Thus, the invention provides a method for producing a linear deoxyribonucleic acid (DNA) product, wherein the method comprises:

[0021] (a) contacting a double-stranded DNA molecule with an endonuclease, a ligase and first and second terminal adaptor molecules and n+m intermediate adaptor molecules, to form a single contiguous aqueous volume, wherein n and m are each 0 or an integer of at least 1 , and wherein n+m is at least 1 ; and

[0022] (b) incubating the single contiguous aqueous volume to generate the linear DNA product, wherein the linear DNA product comprises a linear double-stranded region, wherein the linear double-stranded region comprises a linear portion of the double-stranded DNA molecule, and wherein n intermediate adaptor molecules are sequentially appended to a first end of the linear double stranded region and m intermediate adaptor molecules are sequentially appended to a second end of the linear doublestranded region, and wherein the first terminal adaptor molecule is appended to the nth intermediate adaptor molecule or, when n is 0, to the first end of the linear double stranded region, and the second terminal adaptor molecule is appended to the mth intermediate adaptor molecule, or when m is 0, to the second end of the linear double stranded region.

[0023] The appending (or linking or closing) of the first terminal adaptor molecule and / or the second terminal adaptor molecule and / or the intermediate adaptor molecules may be performed by hybridization or ligation of the adaptor molecules to the ends of the linear double-stranded region and / or to sequential adaptor molecules. Thus, the n intermediate adaptor molecules may be hybridized to the first end of the linear double-stranded region. The m intermediate adaptor molecules may be hybridized to the second end of the linear double-stranded region. The n intermediate adaptor molecules may be ligated to the first end of the linear double-stranded region. The m intermediate adaptor molecules may be ligated to the second end of the linear double-stranded region. The appending of the n+m intermediate adaptor molecules, first terminal adaptor molecule and the second terminal adaptor molecule may be performed by both hybridization and ligation of the adaptor molecules to the ends of the linear doublestranded region and / or to sequential adjacent adaptor molecules. Thus, n intermediate adaptor molecules may be hybridized and ligated to the first end of the linear double-stranded region. The m intermediate adaptor molecules may be hybridized and ligated to the second end of the linear doublestranded region. The appending may be performed via a linker or spacer molecule which facilitates joining of the adaptor molecule to the first and / or second end of the linear double-stranded region or to another adaptor molecule. The first terminal adaptor molecules may be hybridized, ligated or hybridized and ligated to the nth intermediate adaptor molecule or to the first end of the double-stranded DNA molecule. The second terminal adaptor molecule may hybridized, ligated or hybridized and ligated to the mth intermediate adaptor molecule or the second end of the double stranded DNA molecule, n may be 0, 1 , 2, 3, 4, 5, 6, 7 or 8. m may be 0, 1 , 2, 3, 4, 5, 6, 7 or 8. Preferably, n is 0, 1 , 2, 3 or 4, and m is 0, 1 , 2, 3 or 4. .

[0024] The method may further comprise, before step (a) (i.e. the step of contacting the double-stranded DNA molecule with the endonuclease, the ligase and the first and second terminal adaptor molecules and the n+m intermediate adaptor molecules), a step of amplification of a DNA template molecule to produce the double-stranded DNA molecule. Thus, the invention provides a method for producing a linear DNA product, the method comprises:

[0025] (a) amplification of a DNA template molecule comprising at least one cleavable (e.g. endonuclease) target sequence to generate a double-stranded DNA molecule;

[0026] (b) contacting the double-stranded DNA molecule with an endonuclease, a ligase and first and second terminal adaptor molecules and n+m intermediate adaptor molecules, to form a single contiguous aqueous volume, wherein n and m are each 0 or an integer of at least 1 , and wherein n+m is at least 1 ; and

[0027] (c) incubating the single contiguous aqueous volume to generate the linear DNA product, wherein the linear DNA product comprises a linear double-stranded region, and wherein n intermediate adaptor molecules are sequentially ligated to a first end of the linear double stranded region and m intermediate adaptor molecules are sequentially ligated to a second end of the linear double-stranded region, and wherein the first terminal adaptor molecule is ligated to the nth intermediate adaptor molecule or, when n is 0 , to the first end of the linear double stranded region, and the second terminal adaptor molecule is ligated to the mth intermediate adaptor molecule, or when m is 0, to the second end of the linear double stranded region.

[0028] The amplification may be an in vitro or in vivo amplification. Preferably, the amplification is an in vitro amplification. For example, the amplification may be performed by rolling circle amplification (RCA), MALBAC method, traditional polymerase chain reaction (PCR), nucleic acid sequence-based amplification (NASBA), loop-mediated isothermal amplification (LAMP), helicase-dependent amplification (HDA), multiple displacement amplification (MDA) and recombinase polymerase amplification (RPA). Preferably, the amplification is rolling circle amplification. Thus, the invention provides a method for producing a linear deoxyribonucleic acid (DNA) product, the method comprises: (a) rolling circle amplification of a DNA template molecule comprising at least one cleavable (e.g. endonuclease) target sequence to generate a double-stranded DNA molecule; (b) contacting the double-stranded DNA molecule with an endonuclease, a ligase and first and second terminal adaptor molecules and n+m intermediate adaptor molecules, to form a single contiguous aqueous volume, wherein n and m are each 0 or an integer of at least 1 , and n+m is at least 1 ; and

[0029] (c) incubating the single contiguous aqueous volume to generate the linear DNA product, wherein the linear DNA product comprises a linear double-stranded region, wherein the linear double-stranded region comprises a linear portion of the double-stranded DNA molecule, and wherein n intermediate adaptor molecules are sequentially ligated to a first end of the linear double stranded region and m intermediate adaptor molecules are sequentially ligated to a second end of the linear double-stranded region, and wherein the first terminal adaptor molecule is ligated to the nth intermediate adaptor molecule or, when n is 0 , to the first end of the linear double stranded region, and the second terminal adaptor molecule is ligated to the mth intermediate adaptor molecule, or when m is 0, to the second end of the linear double stranded region.

[0030] The method may further comprise (after the step of amplification and before the step of contacting the double-stranded DNA molecule with an endonuclease, a ligase and first and second terminal adaptor molecules) a step of heat-deactivation. Thus, the invention provides a method for producing a linear DNA product, the method comprises:

[0031] (a) amplification of a DNA template molecule comprising at least one cleavable (e.g. endonuclease) target sequence to generate a double-stranded DNA molecule;

[0032] (b) heat-deactivation of the reaction of step (a);

[0033] (c) contacting the double-stranded DNA molecule with an endonuclease, a ligase and first and second terminal adaptor molecules and n+m intermediate adaptor molecules, to form a single contiguous aqueous volume, wherein n and m are each 0 or an integer of at least 1 and n+m is at least 1 ; and

[0034] (d) incubating the single contiguous aqueous volume to generate the linear DNA product, wherein the linear DNA product comprises a linear double-stranded region, wherein the linear double-stranded region comprises a linear portion of the double-stranded DNA molecule, and wherein n intermediate adaptor molecules are sequentially ligated to a first end of the linear double stranded region and m intermediate adaptor molecules are sequentially ligated to a second end of the linear double-stranded region, and wherein the first terminal adaptor molecule is ligated to the nth intermediate adaptor molecule or, when n is 0 , to the first end of the linear double stranded region, and the second terminal adaptor molecule is ligated to the mth intermediate adaptor molecule, or when m is 0, to the second end of the linear double stranded region.

[0035] Preferably, amplification is rolling-circle amplification.

[0036] The step of heat-deactivation may be performed under conditions sufficient to inactive the reagents used during the amplification reaction. The step of heat-deactivation may be performed at a temperature of at least 50°C, at least 55°C, at least 60°C, at least 65°C, at least 70°C, at least 75°C, at least 80°C, at least 85°C, at least 90°C, at least 95°C, or at least 100°C. The step of heat-deactivation may be performed for at least 1 min, at least 3 mins, at least 5 mins, at least 10 mins, at least 15 mins, or at least 20 mins.

[0037] The inventors of the present application have surprisingly discovered that large concatemeric products of the rolling circle amplification reaction can be used to produce the DNA products described herein. This is surprising as the product of the rolling circle amplification has high viscosity and typically has to undergo purification steps before it can be utilized for downstream applications.

[0038] In the method described herein, after the step of amplification, the step of contacting the doublestranded DNA molecule with an endonuclease, a ligase and first and second terminal adaptor molecules and n+m intermediate adaptor molecules, to form a single contiguous aqueous volume, may be performed without purifying the product of the amplification reaction. That is to say that the step of contacting the double-stranded DNA molecule with an endonuclease, a ligase and first and second terminal adaptor molecules and n+m intermediate adaptor molecules may be performed directly after the step of amplification. The step of contacting the double-stranded DNA molecule with an endonuclease, a ligase and first and second terminal adaptor molecules and n+m intermediate adaptor molecules may be performed directly after the step of heat-deactivation.

[0039] The inventors of the present application have discovered a method which requires a very few steps to produce the DNA product described herein. The methods described herein are very time efficient. This is, in part, due to the fact that a step of purification is not required after the amplification reaction. Optionally, the product of the amplification reaction may be heat-deactivated. Surprising, the method described herein where the step of contacting the double-stranded DNA molecule with an endonuclease, a ligase and first and second terminal adaptor molecules and intermediate adaptor molecules is performed directly after the step of amplification (i.e. without the step of purification) produces higher yields of the DNA product described herein when compared to a method in which the two steps are separated by purification of the amplification product.

[0040] The method may further comprise, after the step of incubating the single contiguous aqueous volume, a step of purification of the linear DNA product.

[0041] The method may further comprise, after the step of incubating the single contiguous aqueous volume, a step of nuclease digestion. The nuclease digestion may be exonuclease digestion, such as exonuclease I and / or exonuclease III digestion. The step of nuclease digestion may take place before or after the step of purification. The step of nuclease digestion may allow for removal of any doublestranded DNA molecules and / or adaptor molecules which have not been used to produce linear DNA products. Thus, the method for producing a linear DNA product may comprise the steps:

[0042] (a) contacting a double-stranded DNA molecule with an endonuclease, a ligase and first and second terminal adaptor molecules and n+m intermediate adaptor molecules, to form a single contiguous aqueous volume, wherein n and m are each 0 or an integer of at least 1 , and wherein n+m is at least 1 ;

[0043] (b) incubating the single contiguous aqueous volume to generate the linear DNA product, wherein the linear DNA product comprises a linear double-stranded region, wherein the linear double-stranded region comprises a linear portion of the double-stranded DNA molecule, and wherein n intermediate adaptor molecules are sequentially appended to a first end of the linear double stranded region and m intermediate adaptor molecules are sequentially appended to a second end of the linear doublestranded region, and wherein the first terminal adaptor molecule is appended to the nth intermediate adaptor molecule or, when n is 0 , to the first end of the linear double stranded region, and the second terminal adaptor molecule is appended to the mth intermediate adaptor molecule, or when m is 0, to the second end of the linear double stranded region; and

[0044] (c) incubating the single contiguous aqueous volume with a nuclease (e.g. an exonuclease). n may be 0, 1 , 2, 3, 4, 5, 6, 7 or 8. m may be 0, 1 , 2, 3, 4, 5, 6, 7 or 8. Preferably, n is 0, 1 , 2, 3 or 4, and m is 0, 1 , 2, 3 or 4. .

[0045] The method for producing a linear DNA product may comprise the steps:

[0046] (a) amplification of a DNA template molecule comprising at least one cleavable (e.g. endonuclease) target sequence to generate a double-stranded DNA molecule;

[0047] (b) contacting the double-stranded DNA molecule with an endonuclease, a ligase and first and second terminal adaptor molecules and n+m intermediate adaptor molecules, to form a single contiguous aqueous volume, wherein n and m are each 0 or an integer of at least 1 , and wherein n+m is at least 1 ;

[0048] (c) incubating the single contiguous aqueous volume to generate the linear DNA product, wherein the linear DNA product comprises a linear double-stranded region, wherein the linear double-stranded region comprises a linear portion of the double-stranded DNA molecule, and wherein n intermediate adaptor molecules are sequentially ligated to a first end of the linear double stranded region and m intermediate adaptor molecules are sequentially ligated to a second end of the linear double-stranded region, and wherein the first terminal adaptor molecule is ligated to the nth intermediate adaptor molecule or, when n is 0 , to the first end of the linear double stranded region, and the second terminal adaptor molecule is ligated to the mth intermediate adaptor molecule, or when m is 0, to the second end of the linear double stranded region; and

[0049] (d) incubating the single contiguous aqueous volume with a nuclease (e.g. an exonuclease).

[0050] The method for producing a linear DNA product may comprise the steps:

[0051] (a) amplification of a DNA template molecule comprising at least one cleavable (e.g. endonuclease) target sequence to generate a double-stranded DNA molecule;

[0052] (b) contacting the double-stranded DNA molecule with an endonuclease, a ligase and first and second terminal adaptor molecules and n+m intermediate adaptor molecules, to form a single contiguous aqueous volume, wherein n and m are each 0 or an integer of at least 1 , and wherein n+m is at least 1 ;

[0053] (c) incubating the single contiguous aqueous volume to generate the linear DNA product, wherein the linear DNA product comprises a linear double-stranded region, wherein the linear double-stranded region comprises a linear portion of the double-stranded DNA molecule, and wherein n intermediate adaptor molecules are sequentially appended to a first end of the linear double stranded region and m intermediate adaptor molecules are sequentially appended to a second end of the linear doublestranded region, and wherein the first terminal adaptor molecule is appended to the nth intermediate adaptor molecule or, when n is 0 , to the first end of the linear double stranded region, and the second terminal adaptor molecule is appended to the mth intermediate adaptor molecule, or when m is 0, to the second end of the linear double stranded region;

[0054] (d) purification of the linear DNA product; and

[0055] (e) incubating the product of step (d) with a nuclease (e.g. an exonuclease).

[0056] The method for producing a linear DNA product may comprise the steps:

[0057] (a) amplification of a DNA template molecule comprising at least one cleavable (e.g. endonuclease) target sequence to generate a double-stranded DNA molecule;

[0058] (b) contacting the double-stranded DNA molecule with an endonuclease, a ligase and first and second terminal adaptor molecules and n+m intermediate adaptor molecules, to form a single contiguous aqueous volume, wherein n and m are each 0 or an integer of at least 1 , and wherein n+m is at least 1 ;

[0059] (c) incubating the single contiguous aqueous volume to generate the linear DNA product, wherein the linear DNA product comprises a linear double-stranded region, wherein the linear double-stranded region comprises a linear portion of the double-stranded DNA molecule, and wherein n intermediate adaptor molecules are sequentially appended to a first end of the linear double stranded region and m intermediate adaptor molecules are sequentially appended to a second end of the linear doublestranded region, and wherein the first terminal adaptor molecule is appended to the nth intermediate adaptor molecule or, when n is 0 , to the first end of the linear double stranded region, and the second terminal adaptor molecule is appended to the mth intermediate adaptor molecule, or when m is 0, to the second end of the linear double stranded region;

[0060] (d) incubating the single contiguous aqueous volume with a nuclease (e.g. an exonuclease); and

[0061] (e) purification of the linear DNA product.

[0062] The endonuclease may be a restriction enzyme endonuclease. The endonuclease may be a Type IIS restriction enzyme. The endonuclease may be any enzyme that recognizes a DNA sequence and cleaves outside of the recognition sequence. For example, the endonuclease may be a Bbsl, Bsal, BsmBI, BspQI, BtgZI, Esp3l,Sapl, Aarl, Acc36l, AcIWI, Acul, Ajul, Alol, Alw26l, Alwl, Arsl, AsuHPI, Bael, Bari, Bbvl, Bccl, BceAl, Bcgl, BciVI, BcoDI, BfuAI, Bful, Bmrl, Bmsl, Bmul, Bpil, Bpml, BpuEl, BsaXI, Bsel l, Bse3DI, BseGI, BseMI, BseMII, BseNI, BseRI, BseXI, Bsgl, BsIFI, BsmAI, BsmFI, Bsml, Bso31 l, BspCNI, BspMI, BspPI, BspQI, BspTNI, BsrDI, Bsrl, Bst6l, BstF5l, BstMAI, BstV11, BstV2l, Bsul, BtgZI, BtsCI, Btsl-v2, BtsMutl, Bvel, Csel, CspCI, Eam1 104l, Earl, Ecil, Eco31 l, Eco57l, Esp3l, Faql, Faul, Fokl, Gsul, Hgal, Hphl, HpyAV, Lgul, Lmnl, Lsp1 109I, Lwel, Mboll, Mlyl, Mmel, Mnll, Mva1269l, NmeAIII, PaqCI, PciSI, Pctl, Piel, Ppsl, Psrl, Schl, SfaNI, Taqll, TspDTI and / or TspGWI restriction enzyme.

[0063] Type IIS restriction endonucleases cleave the double-stranded DNA molecule outside of the recognition sequence (i.e. an endonuclease target sequence), which means that the recognition sequence (i.e. endonuclease target sequence) is not included in the linear DNA product.

[0064] The present inventors have surprisingly discovered methods for production of a linear DNA product of enhanced resistance to nuclease digestion. Specifically, the linear DNA product produced by the methods described herein has enhanced resistance to exonuclease digestion (e.g. exonuclease III digestion). The enhanced resistance to exonuclease digestion extends the life of the linear DNA product in a cell (i.e. the linear DNA product has enhanced resistance to intracellular exonucleases) and in a cell-free system (i.e. the linear DNA product has enhanced resistance to extracellular exonucleases). The present inventors have developed a method which relies on the addition of adaptor molecules (i.e. a first terminal adaptor molecule and a second terminal adaptor molecule and intermediate adaptor molecules) to one or both ends of a double-stranded DNA molecule. The terminal adaptor molecules may comprise a hairpin or a loop to form a closed linear DNA product with enhanced resistance to nuclease (e.g. exonuclease) digestion. In addition, the present inventors have discovered a method for efficient introduction of protected nucleotides in the form of a first terminal adaptor molecule and a second terminal adaptor molecule at both ends of a linear double-stranded region of the linear DNA product. The methods described herein may use a hairpin or a loop adaptor on one end of the DNA product and a linear adaptor comprising protected nucleotides on the other end of the DNA product. That is to say that any type of adaptors described herein may be used as long as the final DNA product is protected from nuclease (e.g. exonuclease) digestion. The methods of the invention provide protection from digestion by exonucleases that cleave the 3’-end nucleotides (e.g. exonuclease III) and exonucleases that cleave the 5’-end nucleotides (e.g. exonuclease VIII). Thus, the linear DNA product produced by the methods of the invention has prolonged in vivo expression when compared to a linear DNA product that does not comprise an adaptor molecule described herein.

[0065] The use of the intermediate adaptor molecules, that are appended sequentially, that is to say each intermediate adaptor molecule is appended to an adjacent intermediate adaptor molecule or to the first or second terminal adaptor molecule, gives greater flexibility to the structure, function and / or length of the linear DNA product. For example, the intermediate adaptor molecules may comprise a label, a targeting sequence, a coding sequence, or may be used to provide greater length to the DNA product.

[0066] As used herein the term “protected nucleotide” or “nuclease-resistant nucleotide” is intended to encompass any type of molecule that provides or enhances resistance to nuclease digestion (especially exonuclease digestion). Although the adaptor molecules are described herein as comprising phosphorothioated nucleotides, the skilled person would appreciate that the adaptor molecules may instead comprise any molecules that provide resistance to nuclease digestion (e.g. exonuclease III digestion). For example, the adaptor molecules may comprise nuclease resistant nucleotides i.e. modified nucleotides that provide or increase resistance to nucleases (e.g. exonucleases). The adaptor molecules may comprise a peptide, polypeptide or protein that provides or increases resistance to nucleases (e.g. exonucleases) digestion. The adaptor molecules may comprise 2'-O-methyl nucleotides or 2'-O-methoxyethyl (MOE) nucleotides.

[0067] As used herein, the term "phosphorothioated nucleotide" refers to a nucleotide that has an altered phosphate backbone, wherein, the sugar moieties are linked by a phosphorothioate bond. In the phosphate backbone of an oligonucleotide sequence, the phosphorothioate bond contains a sulphur atom as a substitute for a non-bridging oxygen atom. This modification renders the internucleotide linkage resistant to nuclease degradation.

[0068] As used herein, “terminal adaptor molecule” refers to an adaptor molecule that is appended to one end to the linear DNA region, directly or indirectly via one or more intermediate adaptor molecules, to form the end of the linear DNA product. A terminal adaptor molecule is nuclease-resistant once it is appended to the linear double-stranded region, directly or indirectly. A terminal adaptor molecule confers nuclease-resistance to the linear DNA product.

[0069] As used herein an “intermediate adaptor molecule” refers to an adaptor molecule that is appended to an end of another intermediate adaptor molecule or to an end of the linear double-stranded region by its first end, and has another intermediate adaptor molecule or a terminal adaptor molecule appended to its second end; that is, an intermediate adaptor molecule is hybridized or ligated to another molecule at both ends.

[0070] As used herein “sequentially”, means one after the other. That is to say, the n intermediate adaptor molecules are appended end-to-end from the first end of the double-stranded DNA molecule such that the n-(n-1 )th adaptor molecule is appended to the first end of the double-stranded DNA molecule, the nth adaptor is appended to the terminal adaptor molecule; and the m intermediate adaptor molecules are appended end-to-end from the second end of the double-stranded DNA molecule such that the m- (m-1 )th adaptor molecule is appended to the first end of the double-stranded DNA molecule, the mth adaptor is appended to the terminal adaptor molecule. For example, in the case where n is two and m is one, the linear DNA molecule comprises, from one end to the other end, a first terminal adaptor molecule-n2 intermediate adaptor molecule-n1 intermediate adaptor molecule-double-stranded linear region-mi intermediate adaptor molecule-second terminal adaptor molecule.

[0071] Each adaptor molecule may comprise a sequence at its first end that is compatible with a sequence of a second end of the next adaptor molecule to be sequentially appended or ligated. For example, when n=2, the first n intermediate adaptor will have a sequence at its second end that is complementary to a sequence at the first end of the linear double-stranded region. The second (or nth) intermediate adaptor molecule may comprise a sequence at its second end that is complementary to the sequence at the first end of the first n intermediate adaptor molecule and a sequence at its first end that is complementary to a sequence at an end of the first terminal adaptor molecule. When each adaptor molecule comprises a different complementary sequence, the adaptor molecules will be appended, or ligated, in sequence. The different complementary sequences may be generated by using TypellS restriction endonuclease that cuts at a target sequence at a separate location from the overhang that results from the cleaving by the endonuclease, e.g., Bsal. Up to 256 different 4 nucleotide overhangs can be created using typellS endonucleases such that the intermediate and terminal adaptor molecules sequentially and directionally append.

[0072] As used herein, “adjacent intermediate adaptor molecule” or “adjacent adaptor molecule” refers to an adaptor molecule that is appended or ligated directly to an end of another adaptor molecule.

[0073] The closed DNA product comprises n intermediate adaptor molecules between the first terminal adaptor molecule and the first end of the linear double stranded region, and comprises m intermediate adaptor molecules between the second terminal adaptor molecule and a second end of the linear doublestranded region, where n and m are each 0 or an integer of at least 1 , and n+m is at least 1 .

[0074] The first end of the linear double stranded region and the first terminal adaptor molecule may be at the 3’ or 5’ end of the closed linear DNA product. The first end of the linear double-stranded DNA product and the first terminal adaptor molecule may be upstream or downstream of the DNA sequence of the linear double-stranded region.

[0075] The linear DNA product (e.g. the closed linear DNA product) produced by the methods of the present invention has additional advantageous properties, such as a substantial lack of a bacterial backbone and / or antibiotic resistant genes. Lack of these features is particularly beneficial in the production of a cell delivery system, such as a viral vector or a nanoparticle, for example, for cell therapy. Lack of these features makes the linear DNA product produced by the methods of the present invention particularly suitable for use in a pharmaceutical composition.

[0076] Unexpectedly, the present inventors have discovered a method for production of a linear DNA product of enhanced resistance to nuclease digestion which allows for efficient production of large quantities of the linear DNA product with enhanced resistance to exonuclease digestion together with the ability to add certain features to the linear DNA product. The large-scale manufacture of the product may be in a cell-free system, which results in the production of a pure sample comprising the linear DNA product substantively free of bacterial contaminants (e.g. remaining after cell lysis). 1. Methods for producing closed linear DNA product

[0077] The methods described herein may be used to produce a closed linear DNA product e.g. a covalently closed linear DNA product.

[0078] The invention provides a method for producing a closed linear DNA product, the method comprises:

[0079] (a) contacting a double-stranded DNA molecule with an endonuclease and first and second terminal adaptor molecules and n+m intermediate adaptor molecules, to form a single contiguous aqueous volume, wherein n and m are each 0 or an integer of at least 1 , and wherein n+m is at least 1 ; and

[0080] (b) incubating the single contiguous aqueous volume to generate the closed linear DNA product, wherein the closed linear DNA product comprises a linear double-stranded region, wherein the linear double-stranded region comprises a linear portion of the double-stranded DNA molecule, and wherein n intermediate adaptor molecules are sequentially appended to a first end of the linear double stranded region and m intermediate adaptor molecules are sequentially appended to a second end of the linear double-stranded region, and wherein the closed linear DNA product is closed at a first end by the first terminal adaptor molecule appended to the nth intermediate adaptor molecule or, when n is 0 , to the first end of the linear double stranded region, and closed at a second end by the second terminal adaptor molecule appended to the mth intermediate adaptor molecule, or when m is 0, to the second end of the linear double stranded region. n may be 0, 1 , 2, 3, 4, 5, 6, 7 or 8. m may be 0, 1 , 2, 3, 4, 5, 6, 7 or 8. Preferably, n is 0, 1 , 2, 3 or 4, and m is 0, 1 , 2, 3 or 4. .

[0081] The step of contacting the double-stranded DNA molecule with the endonuclease and first and second terminal adaptor molecules is preferably performed in the presence of a ligase. Thus, the invention provides a method for producing a closed linear DNA product, wherein the method comprises:

[0082] (a) contacting a double-stranded DNA molecule with an endonuclease, a ligase and first and second terminal adaptor molecules and n+m intermediate adaptor molecules, to form a single contiguous aqueous volume, wherein n and m are each 0 or an integer of at least 1 , and wherein n+m is at least 1 ; and

[0083] (b) incubating the single contiguous aqueous volume to generate the closed linear DNA product, wherein the closed linear DNA product comprises a linear double-stranded region, wherein the linear double-stranded region comprises a linear portion of the double-stranded DNA molecule, and wherein n intermediate adaptor molecules are sequentially appended to a first end of the linear double stranded region and m intermediate adaptor molecules are sequentially appended to a second end of the linear double-stranded region, and wherein the closed linear DNA product is closed at a first end by the first terminal adaptor molecule appended to the nth intermediate adaptor molecule or, when n is 0 , to the first end of the linear double stranded region, and closed at a second end by the second terminal adaptor molecule appended to the mth intermediate adaptor molecule, or when m is 0, to the second end of the linear double stranded region.

[0084] The linear double-stranded region may be the linear portion of the double-stranded DNA molecule.

[0085] The invention provides a method for producing a closed linear DNA product, wherein the method comprises:

[0086] (a) contacting a double-stranded DNA molecule with an endonuclease, a ligase and first and second terminal adaptor molecules and n+m intermediate adaptor molecules, to form a single contiguous aqueous volume, wherein n and m are each 0 or an integer of at least 1 , and wherein n+m is at least 1 ; and

[0087] (b) incubating the single contiguous aqueous volume to generate the closed linear DNA product, wherein the closed linear DNA product comprises a linear portion of the double-stranded DNA molecule, and wherein n intermediate adaptor molecules are sequentially appended to a first end of the linear double stranded region and m intermediate adaptor molecules are sequentially appended to a second end of the linear double-stranded region, and wherein the closed linear DNA product is closed at a first end by the first terminal adaptor molecule appended to the nth intermediate adaptor molecule or, when n is 0 , to the first end of the linear double stranded region, and closed at a second end by the second terminal adaptor molecule appended to the mth intermediate adaptor molecule, or when m is 0, to the second end of the linear double stranded region.

[0088] A closed linear DNA product has particular utility as a therapeutic agent (i.e. DNA therapeutic) which can be used to express a gene product in vivo. This is because its closed structure (e.g. covalently closed structure) prevents attack by enzymes such as exonucleases, leading to enhanced stability and longevity of gene expression as compared to "open" DNA molecules with exposed DNA ends. Linear double-stranded open-ended cassettes have been demonstrated to be inefficient with respect to gene expression when introduced into host tissue. This has been attributed to cassette instability due to the action of exonucleases in the extracellular space.

[0089] Sequestering DNA ends inside closed structures also has other advantages. The DNA ends are prevented from integrating with genomic DNA and so closed linear DNA products offer improved safety. In addition, the closed linear structure reduces concatamerisation of DNA products inside host cells and thus expression levels of the gene product can be regulated in a more sensitive manner.

[0090] The method of the invention may be used for production of DNA for in vitro expression in a host cell, for example, in DNA vaccines. DNA vaccines typically encode a modified form of an infectious organism's DNA. DNA vaccines are administered to a subject where they then express the selected protein of the infectious organism, initiating an immune response against that protein which is typically protective. DNA vaccines may also encode a tumour antigen in a cancer immunotherapy approach. The method may produce other types of therapeutic DNA molecules e.g. those used in gene therapy. For example, such DNA molecules can be used to express a functional gene where a subject has a genetic disorder caused by a dysfunctional version of that gene. Examples of such diseases include sickle cell anaemia, cystic fibrosis, Huntington disease, Duchenne’s Muscular Dystrophy, Haemophilia A, a1 -antitrypsin deficiency, primary ciliary dyskinesia, or respiratory distress syndrome of prematurity. Other diseases where gene therapy may be useful include metabolic diseases, respiratory diseases, inflammatory diseases, autoimmune, chronic and infectious diseases, including such disorders as AIDS, cancer, neurological diseases, cardiovascular disease, hypercholesterolemia, various blood disorders including various anaemias, thalassemia and haemophilia, and emphysema. For the treatment of solid tumours, genes encoding toxic peptides (i.e., chemotherapeutic agents such as ricin, diptheria toxin and cobra venom factor), tumour suppressor genes such as p53, genes coding for mRNA sequences which are antisense to transforming oncogenes, antineoplastic peptides such as tumor necrosis factor (TNF) and other cytokines, or transdominant negative mutants of transforming oncogenes, may be expressed.

[0091] The addition of intermediate adaptor molecules allows for additional flexibility in the production of the closed linear DNA products of the invention. The intermediate adaptor molecules may allow for longer closed linear DNA products to be produced. The intermediate adaptor molecules allow for bar codes, promotor sequences, labels, poly A signals and / or open reading frames to be incorporated into the closed linear DNA product of the invention. An intermediate adaptor molecule may comprise a cassette. For example, if it is advantageous to include several genes of interest, or open reading frames, in the closed linear DNA product, intermediate adaptor molecules may comprise one or more cassettes, and the linear DNA region may comprise one or more cassettes. An intermediate adaptor molecule may comprise a label, a signalling sequence, a targeting sequence, modified nucleotides, or a binding moiety. An intermediate adaptor molecule may comprise a promotor, a UTR, a transcription factor binding site, or a terminator sequence. For example, the n or m intermediate adaptor molecules that are appended or ligated upstream of the linear double-stranded region may be used to add or change a promotor sequence, add or change UTRs. The m or n intermediate adaptor molecules appended or ligated downstream of the linear double-stranded region may be used to add polyA signals or sequences, terminators, change or add UTRs. n or m intermediate adaptor molecules may be used to add barcodes, fluorophores, modified nucleotides, upstream or downstream of the linear double-stranded region.

[0092] A modified nucleotide may be 2-MethoxyEthoxy A, 2-MethoxyEthoxy MeC, 2-MethoxyEthoxy G, 2- MethoxyEthoxy T, 2'-O-Methyl RNA Bases, Fluoro Bases, 2-Aminopurine, 5-Bromo dU, deoxyUridine, 2,6-Diaminopurine (2-Amino-dA), Dideoxy-C, deoxyinosine, Hydroxymethyl dC, Iso-dG, Iso-dC, 5- Methyl dC or 5-Nitroindole.

[0093] As well as providing nuclease-resistance to the closed linear DNA product, the terminal adaptor molecules may also provide further functional features. A terminal adaptor molecule may allow for longer closed linear DNA products to be produced. Terminal adaptor molecules may allow for bar codes, promotor sequences, labels, poly A signals and / or open reading frames to be incorporated into the closed linear DNA product of the invention. A terminal adaptor molecule may comprise a label, a signalling sequence, a targeting sequence or a binding moiety. A terminal adaptor molecule may comprise a UTR or a terminator sequence.

[0094] The closing of the linear double-stranded region by the first terminal adaptor molecule and / or the second terminal adaptor molecule (directly or indirectly via one or more intermediate adaptor molecules) may be performed by hybridization or ligation of the adaptor molecules to the ends of the linear doublestranded region and / or to sequential adaptor molecules. Thus, the n intermediate adaptor molecules may be sequentially hybridized to the first end of the linear double-stranded region. The m intermediate adaptor molecules may be sequentially hybridized to the second end of the linear double-stranded region. The n intermediate adaptor molecules may be sequentially ligated to the first end of the linear double-stranded region. The m intermediate adaptor molecules may be sequentially ligated to the second end of the linear double-stranded region. The appending of the n+m intermediate adaptor molecules, first terminal adaptor molecule and the second terminal adaptor molecule may be performed by both hybridization and ligation of the adaptor molecules to the ends of the linear double-stranded region and / or to adjacent adaptor molecules. Thus, n intermediate adaptor molecules may be sequentially hybridized and ligated to the first end of the linear double-stranded region. The m intermediate adaptor molecules may be sequentially hybridized and ligated to the second end of the linear double-stranded region. The appending may be performed via a linker or spacer molecule which facilitates joining of the adaptor molecule to the first and / or second end of the linear double-stranded region or to an adjacent adaptor molecule. The first terminal adaptor molecules may be hybridized, ligated or hybridized and ligated to the nth intermediate adaptor molecule or to the first end of the double-stranded DNA molecule. The second terminal adaptor molecule may be hybridized, ligated or hybridized and ligated to the mth intermediate adaptor molecule or the second end of the double stranded DNA molecule, n may be 0, 1 , 2, 3, 4, 5, 6, 7 or 8. m may be 0, 1 , 2, 3, 4, 5, 6, 7 or 8. Preferably, n is 0, 1 , 2, 3 or 4, and m is 0, 1 , 2, 3 or 4. .

[0095] The step of incubating the single contiguous aqueous volume to generate the closed linear DNA product may comprise generating the linear portion of the double-stranded DNA molecule by digesting the double-stranded DNA molecule with the endonuclease.

[0096] The step of incubating the single contiguous aqueous volume may be performed under conditions that promote appending (or linking) of the first and second terminal adaptor molecules and / or intermediate adaptor molecules to the linear double-stranded region to produce the closed linear DNA product. The appending may be performed by creating a covalent link between the first and / or second terminal adaptor molecule and the first and / or second end of the linear double-stranded region. The appending may be performed by creating a covalent link between the first and / or second terminal adaptor molecule and an nth and / or mth intermediate adaptor molecule. The appending may be performed by creating a covalent link between an intermediate adaptor molecule and the first or second end of the linear double-stranded region. The appending may be performed by creating a covalent link between an intermediate adaptor molecule and an adjacent adaptor molecule. The step of incubating the single contiguous aqueous volume may be performed under conditions that promote digestion of the double-stranded DNA molecule to produce the linear portion of the doublestranded DNA molecule. The digestion of the double-stranded DNA molecule to produce the linear portion of the double-stranded DNA molecule may be performed at a first temperature of 1°C-100°C, 1°C -80°C, 5°C-70°C, 10°C-60°C, 15°C-55°C, 20°C-50°C, 25°C -45°C, 30°C-40°C, 35°C-39°C, 36°C- 38°C, or at about 37°C. The digestion may be endonuclease digestion, preferably Type IIS endonuclease digestion.

[0097] The step of incubating the single contiguous aqueous volume may be performed under conditions that promote ligation of the linear double-stranded region to the first and second terminal adaptor molecules and / or to an intermediate adaptor molecules. The step of incubating the single contiguous aqueous volume may be performed under conditions that promote ligation of the intermediate adaptor molecules to the first and second terminal adaptor molecules and / or adjacent intermediate adaptor molecules and / or the first and / or second end of the linear double-stranded region. The ligation may be at least 5%, at least 10%, at least 15, at least 20%, at least 25%, at least 30%, at least 35%, at least 40, at least 45%, at least 50%, at least 55%, at least 60, at least 65, at least 70%, at least 75, at least 80%, at least 82%, at least 85%, at least 90%, or at least 95% efficient. For example, at least 5%, at least 10%, at least 15, at least 20%, at least 25%, at least 30%, at least 35%, at least 40, at least 45%, at least 50%, at least 55%, at least 60, at least 65, at least 70%, at least 75, at least 80%, at least 82%, at least 85%, at least 90%, or at least 95% of the linear double-stranded regions (or the portions of the double-stranded DNA molecules) may be incorporated into closed linear DNA products. Preferably, the ligation is at least 15% efficient.

[0098] Ligation efficiency may be established based on DNA quantification values before and after the digestion / ligation reaction. Thus, ligation efficiency may be established based on the equation: (starting amplified DNA amount) / (final linear DNA amount) x 100%.

[0099] Ligation efficiency may also be established based on DNA quantification values before and after the digestion / ligation reaction and the subsequent exonuclease treatment to remove remaining open DNA constructs and adaptor molecules excess.

[0100] For example, the double-stranded DNA molecule generated by the rolling circle amplification is first quantified so that the amount of the double-stranded DNA molecule used as the starting material during the digestion / ligation reaction is known. After all the enzymatic reactions, the linear DNA product is quantified to calculate the ligation efficiency as per the equation above.

[0101] DNA quantification methods are known to a person skilled in the art. For example, DNA quantifications may be carried out using the Qubit dsDNA BR assay from ThermoFisher (https: / / www.thermofisher.eom / order / catalog / product / Q32850# / Q32850). The step of ligation of the linear double-stranded region to the first and second terminal adaptor molecules may be performed at a second temperature of 1 °C -90°C, 2°C -70°C, 5°C-60°C, 8°C-55°C, 9°C-50°C, 10°C-45°C, 1 1 °C-40°C, 12°C-37°C, 13°C-30°C, 14°C-25°C, 15°C-20°C or at about 16°C.

[0102] The step of incubating the single contiguous aqueous volume may comprise incubating at a first temperature and then incubating at a second temperature. The first temperature may be 1 °C-100°C, 1 °C-80°C, 5°C-70°C, 10°C-60°C, 15°C-55°C, 20°C-50°C, 25°C-45°C, 30°C-40°C, 35°C-39°C, 36°C- 38°C, or about 37°C. The second temperature may be 1 °C -90°C, 2°C -70°C, 5°C-60°C, 8°C-55°C, 9°C-50°C, 10°C-45°C, 1 1 °C-40°C, 12°C-37°C, 13°C-30°C, 14°C-25°C, 15°C-20°C or at about 16°C. Preferably, the first temperature is 35°C-39°C and the second temperature is 14°C-18°C. Using these conditions the endonuclease may be a Type IIS restriction endonuclease (e.g. Bsal) and the ligase may be T4 DNA ligase, T7 DNA ligase, mammalian DNA ligase I, III and IV; Taq DNA ligase, Tth DNA ligase, or E. coli DNA ligase.

[0103] The step of incubating the single contiguous aqueous volume may be performed isothermally. The step of incubating the single contiguous aqueous volume may comprise incubating at a constant temperature. The constant temperature promotes simultaneous digestion of the double-stranded DNA molecule to produce the linear portion of the double-stranded DNA molecule and ligation of the linear double-stranded region to the n + m intermediate adaptor molecules and / or first and second terminal adaptor molecules. For example, the constant temperature may be 20°C, 21 °C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31 °C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, or 40°C. Preferably, the constant temperature is 30°C. The constant temperature is intended to mean that the temperature does not significantly change during the reaction. The constant temperature is intended to mean that the temperature variation during the step of incubating the single contiguous aqueous volume is less than 10°C, less than 9°C, less than 8°C, less than 7°C, less than 6°C, less than 5°C, less than 4°C, less than 3°C, less than 2°C, or less than 1 °C. In a preferred embodiment the temperature during the step of incubating the single contiguous aqueous does not deviate by more than 5°C, preferably by not more than 3°C, even more preferably not more than 1 °C. Thus, the constant temperature may be a temperature in a range of 20°C-30°C, 22°C-32°C, 24°C-34°C, 26°C- 36°C, 28°C-38°C, 30°C-40°C, 22°C-28°C, 32°C-38°C, 25°C-35°C, 26°C-34°C, 27°C- 33°C, 27.5°C- 32.5°C, 28°C-32°C, 28.5°C-31 ,5°C, 29°C-31 °C, or 29.5°C-30.5°C. Preferably, the constant temperature is a temperature in a range of 27.5°C-32.5°C. Alternatively, the constant temperature may be a temperature in a range of 32°C-42°C, 33°C-41 °C, 34°C-40°C, 35°C-39°C, 36°C-38°C. Preferably, the constant temperature is a temperature in a range of 34.5°C-39.5°C.

[0104] The step of incubating the single contiguous aqueous volume may comprise cycling between the first temperature and the second temperature. The step of incubating the single contiguous aqueous volume may comprise cycling between the first temperature and the second temperature at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 80, at least 90, or at least 100 times, preferably at least 20 times. The step of incubating the single contiguous aqueous volume may comprise cycling between the first temperature and the second temperature less than 40, less than 35, less than 30 times, less than 29, less than 25 times. The step of incubating the single contiguous aqueous volume may comprise cycling between the first temperature and the second temperature 2-100, 5-80, 10-70, 20-60, or 30-60 times. The step of incubating the single contiguous aqueous volume may comprise cycling between the first temperature and the second temperature 2-20, 5-29, 61 -100, or 65-80 times.

[0105] The method may further comprise, before step (a) (i.e. the step of contacting the double-stranded DNA molecule with the endonuclease, the ligase and the first and second terminal adaptor molecules), a step of amplifying a DNA template molecule to produce the double-stranded DNA molecule. Thus, the invention provides a method for producing a closed linear DNA product, the method comprises:

[0106] (a) amplifying a DNA template molecule comprising at least one cleavable (e.g. endonuclease) target sequence to generate a double-stranded DNA molecule;

[0107] (b) contacting the double-stranded DNA molecule with an endonuclease, a ligase and first and second terminal adaptor molecules and n+m intermediate adaptor molecules, to form a single contiguous aqueous volume, wherein n and m are each 0 or an integer of at least 1 , and wherein n+m is at least 1 ; and

[0108] (c) incubating the single contiguous aqueous volume to generate the closed linear DNA product, wherein the closed linear DNA product comprises a linear double-stranded region, wherein the linear double-stranded region comprises a linear portion of the double-stranded DNA molecule , and wherein n intermediate adaptor molecules are sequentially appended to a first end of the linear double stranded region and m intermediate adaptor molecules are sequentially appended to a second end of the linear double-stranded region, and wherein the closed linear DNA product is closed at a first end by the first terminal adaptor molecule appended to the nth intermediate adaptor molecule or, when n is 0 , to the first end of the linear double stranded region, and closed at a second end by the second terminal adaptor molecule appended to the mth intermediate adaptor molecule, or when m is 0, to the second end of the linear double stranded region.

[0109] The step of amplifying may be performed by in vitro or in vivo amplification. Preferably, the step of amplifying is performed by in vitro amplification. For example, the step of amplifying may be performed by rolling circle amplification (RCA), MALBAC method, traditional polymerase chain reaction (PCR), nucleic acid sequence-based amplification (NASBA), loop-mediated isothermal amplification (LAMP), helicase-dependent amplification (HDA), multiple displacement amplification (MDA) and recombinase polymerase amplification (RPA). Preferably, the step of amplifying is performed by rolling circle amplification. Thus, the invention provides a method for producing a closed linear DNA product, the method comprises: (a) amplifying a DNA template molecule comprising at least one cleavable (e.g. endonuclease) target sequence to generate a double-stranded DNA molecule, wherein the DNA template molecule is amplified by rolling circle amplification;

[0110] (b) contacting the double-stranded DNA molecule with an endonuclease, a ligase and first and second terminal adaptor molecules and n+m intermediate adaptor molecules, to form a single contiguous aqueous volume, wherein n and m are each 0 or an integer of at least 1 , and wherein n+m is at least 1 ; and

[0111] (c) incubating the single contiguous aqueous volume to generate the closed linear DNA product, wherein the closed linear DNA product comprises a linear double-stranded region, wherein the linear double-stranded region comprises a linear portion of the double-stranded DNA molecule, and wherein n intermediate adaptor molecules are sequentially appended to a first end of the linear double stranded region and m intermediate adaptor molecules are sequentially appended to a second end of the linear double-stranded region, and wherein the closed linear DNA product is closed at a first end by the first terminal adaptor molecule appended to the nth intermediate adaptor molecule or, when n is 0 , to the first end of the linear double stranded region, and closed at a second end by the second terminal adaptor molecule appended to the mth intermediate adaptor molecule, or when m is 0, to the second end of the linear double stranded region.

[0112] Rolling circle amplification may be performed without any primers, or in the presence of a primer or multiple primers. For example, the primer may be a synthetic primer. The primers may be random primers. Rolling circle amplification may be performed in the presence of a primase. The primase may be TthPrimPol. Preferably, if the rolling circle amplification is performed without any primers, it is performed in the presence of a primase, such as TthPrimPol. Similarly, if a primer is used during amplification reaction, a primase is not used. The double-stranded DNA product may be generated by the rolling circle amplification in vitro under isothermal conditions using a suitable nucleic acid polymerase, such as Phi29 DNA polymerase.

[0113] In the methods described herein, the DNA template molecule may comprise at least one cleavable target sequence. The cleavable target sequence may be an endonuclease target sequence. Thus, the DNA template molecule may comprise at least one endonuclease target sequence. Preferably, the DNA template molecule comprises at least two endonuclease target sequences. The endonuclease target sequences may be the same or different. Preferably, the at least one endonuclease target sequence is a restriction endonuclease target sequence. Different restriction endonuclease target sequences would be known to the skilled person. The cleavable target sequence may be a Type IIS restriction endonuclease target sequence. For example, the restriction endonuclease target sequence may be a Bbsl, Bsal, BsmBI, BspQI, BtgZI, Esp3l,Sapl, Aarl, Acc36l, AcIWI, Acul, Ajul, Alol, Alw26l, Alwl, Arsl, AsuHPI, Bael, Bari, Bbvl, Bccl, BceAl, Bcgl, BciVI, BcoDI, BfuAI, Bful, Bmrl, Bmsl, Bmul, Bpil, Bpml, BpuEl, BsaXI, Bse1 1, Bse3DI, BseGI, BseMI, BseMII, BseNI, BseRI, BseXI, Bsgl, BsIFI, BsmAI, BsmFI, Bsml, Bso31 l, BspCNI, BspMI, BspPI, BspQI, BspTNI, BsrDI, Bsrl, Bst6l, BstF5l, BstMAI, BstV1 1, BstV2l, Bsul, BtgZI, BtsCI, Btsl-v2, BtsMutl, Bvel, Csel, CspCI, Eam1 104l, Earl, Ecil, Eco31 l, Eco57l, Esp3l, Faql, Faul, Fokl, Gsul, Hgal, Hphl, HpyAV, Lgul, Lmnl, Lsp1 109I, Lwel, Mboll, Mly I , Mmel, Mn 11 , Mva1269l, NmeAIII, PaqCI, PciSI, Pctl, Piel, Ppsl, Psrl, Schl, SfaNI, Taqll, TspDTI and / or TspGWI target sequence. The at least one cleavable sequence (e.g. endonuclease target sequence) may be a native cleavable sequence (i.e. a cleavable sequence present in the template molecule). Alternatively, the at least one cleavable sequence (e.g. endonuclease target sequence) may be introduced to the DNA template molecule prior to the production of the closed linear DNA product.

[0114] The endonuclease may be a restriction enzyme endonuclease. The endonuclease may be a Type IIS restriction enzyme. The endonuclease may be any enzyme that recognizes a DNA sequence and cleaves outside of the recognition sequence. For example, the endonuclease may be a Bbsl, Bsal, BsmBI, BspQI, BtgZI, Esp3l,Sapl, Aarl, Acc36l, AcIWI, Acul, Ajul, Alol, Alw26l, Alwl, Arsl, AsuHPI, Bael, Bari, Bbvl, Bccl, BceAl, Bcgl, BciVI, BcoDI, BfuAI, Bful, Bmrl, Bmsl, Bmul, Bpil, Bpml, BpuEl, BsaXI, Bsel l, Bse3DI, BseGI, BseMI, BseMII, BseNI, BseRI, BseXI, Bsgl, BsIFI, BsmAI, BsmFI, Bsml, Bso31 l, BspCNI, BspMI, BspPI, BspQI, BspTNI, BsrDI, Bsrl, Bst6l, BstF5l, BstMAI, BstV1 1, BstV2l, Bsul, BtgZI, BtsCI, Btsl-v2, BtsMutl, Bvel, Csel, CspCI, Eam1 104l, Earl, Ecil, Eco31 l, Eco57l, Esp3l, Faql, Faul, Fokl, Gsul, Hgal, Hphl, HpyAV, Lgul, Lmnl, Lsp1 109I, Lwel, Mboll, Mlyl, Mmel, Mnll, Mva1269l, NmeAIII, PaqCI, PciSI, Pctl, Piel, Ppsl, Psrl, Schl, SfaNI, Taqll, TspDTI and / or TspGWI restriction enzyme.

[0115] The ligase may be a DNA ligase, such as a T4 DNA ligase, T7 DNA ligase, mammalian DNA ligase I, III and IV; Taq DNA ligase, Tth DNA ligase, or E. coli DNA ligase.

[0116] The DNA template molecule used in the methods described herein may be single-stranded or doublestranded. Preferably, the DNA template molecule is double-stranded. The DNA template molecule may be a natural circular DNA molecule. For example, the DNA template molecule may be (i) a plasmid, (ii) a minicircle, (Hi) a cosmid, (iv) a bacterial artificial chromosome (BAC), or (v) a molecular inversion probe (MIP). The DNA template molecule may be an enzymatically produced circular DNA molecule. For example, the DNA template molecule may be (i) a circular DNA molecule obtained from recombinase reaction, preferably Cre recombinase reaction, or (ii) a circular DNA molecule obtained from ligase reaction, preferably using the golden gate assembly. The DNA template molecule may be an enzymatically produced covalently-closed linear DNA molecule. For example, the DNA template molecule may be (i) a DNA molecule processed with TelN protelomerase; or (ii) a DNA molecule generated by ligation of the DNA ends with an adaptor. The DNA template molecule may comprise an element that is double-stranded and an element that is single-stranded. For example, the template DNA molecule may comprise a double-stranded DNA and a single-stranded hairpin loop.

[0117] The DNA template molecule may be linear. If the DNA template molecule is linear, prior to amplification (e.g. rolling circle amplification), a DNA template molecule may be circularized to produce a DNA template molecule suitable for use in the methods described herein. The template DNA molecule may comprise a cassette. The cassette may be a mammalian expression cassette. The cassette may further comprise a promoter. The promoter may be a CMV promoter. The cassette may further comprise an enhancer. The cassette may further comprise a reporter gene, such as an eGFP reporter gene or a luciferase reporter gene. The cassette may further comprise a homopolymeric sequence. The cassette may further comprise a LoxP sequence, preferably two LoxP sequences. If the two LoxP sequences are oriented in the same direction, the DNA sequence between the two LoxP sequences is excised as a circular loop of DNA. If the two LoxP sequences are oriented in the opposite direction, the DNA sequence between the two LoxP sequences is inverted. Thus, preferably, the two LoxP sequences are in the same orientation (i.e. the same direction) in the template DNA molecule.

[0118] The DNA template molecule may comprise a homopolymeric sequence at a 5’-end or a 3’-end or both a 5’-end and a 3’-end. The homopolymeric sequence may be added to the DNA template molecule before circularization. The homopolymeric sequence may be a polyA, a polyC, a polyG, or a polyT sequence. The homopolymeric sequence may be between 3-200 nucleotides in length. The homopolymeric sequence may be used to facilitate purification of the linear DNA product, in which case, the homopolymeric sequence may be between 4-12 nucleotides in length, or between 5-10 nucleotides in length. The homopolymeric sequence may be used to improve mRNA expression, in which case, the homopolymeric sequence may be between 10-200 nucleotides in length, preferably between 80-150 nucleotides in length. The homopolymeric sequence may be at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 1 10, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, or at least 200 nucleotides in length. Preferably, the homopolymeric sequence is at least 100 nucleotides in length. More preferably still, the homopolymeric sequence is at least 120 nucleotides in length. For example, the homopolymeric sequence may comprise a polyA sequence of at least 120 nucleotides.

[0119] The method may further comprise (after the step of amplification and before the step of contacting the double-stranded DNA molecule with an endonuclease, a ligase and first and second terminal adaptor molecules) a step of heat-deactivation. Thus, the invention provides a method for producing a closed linear DNA product, the method comprises:

[0120] (a) amplification of a DNA template molecule comprising at least one cleavable (e.g. endonuclease) target sequence to generate a double-stranded DNA molecule;

[0121] (b) heat-deactivation of the reaction of step (a);

[0122] (c) contacting the double-stranded DNA molecule with an endonuclease, a ligase and first and second terminal adaptor molecules and n+m intermediate adaptor molecules, to form a single contiguous aqueous volume, wherein n and m are each 0 or an integer of at least 1 , and wherein n+m is at least 1 ; and

[0123] (d) incubating the single contiguous aqueous volume to generate the closed linear DNA product, wherein the closed linear DNA product comprises a linear double-stranded region, wherein the linear double-stranded region comprises a linear portion of the double-stranded DNA molecule, and wherein n intermediate adaptor molecules are sequentially appended to a first end of the linear double stranded region and m intermediate adaptor molecules are sequentially appended to a second end of the linear double-stranded region, and wherein the closed linear DNA product is closed at a first end by the first terminal adaptor molecule appended to the nth intermediate adaptor molecule or, when n is 0 , to the first end of the linear double stranded region, and closed at a second end by the second terminal adaptor molecule appended to the mth intermediate adaptor molecule, or when m is 0, to the second end of the linear double stranded region.

[0124] Preferably, amplification is rolling-circle amplification.

[0125] The step of heat-deactivation may be performed under conditions sufficient to inactive the reagents used during the amplification reaction. The step of heat-deactivation may be performed at a temperature of at least 50°C, at least 55°C, at least 60°C, at least 65°C, at least 70°C, at least 75°C, at least 80°C, at least 85°C, at least 90°C, at least 95°C, or at least 100°C. The step of heat-deactivation may be performed for at least 1 min, at least 3 mins, at least 5 mins, at least 10 mins, at least 15 mins, or at least 20 mins.

[0126] In the method described herein, after the step of amplification, the step of contacting the doublestranded DNA molecule with an endonuclease, a ligase and first and second terminal adaptor molecules and n+m intermediate adaptor molecules, to form a single contiguous aqueous volume, may be performed without purifying the product of the amplification reaction. That is to say that the step of contacting the double-stranded DNA molecule with an endonuclease, a ligase and first and second terminal adaptor molecules and n+m intermediate adaptor molecules may be performed directly after the step of amplification. The step of contacting the double-stranded DNA molecule with an endonuclease, a ligase and first and second terminal adaptor molecules and nm intermediate adaptor molecules may be performed directly after the step of heat-deactivation.

[0127] The method may further comprise, after the step of incubating the single contiguous aqueous volume, a step of purification of the closed linear DNA product.

[0128] The method may further comprise, after the step of incubating the single contiguous aqueous volume, a step of nuclease digestion. The nuclease digestion may be exonuclease digestion, such as exonuclease I and / or exonuclease III digestion. The step of nuclease digestion may take place before or after the step of purification. This step allows for removal of any double-stranded DNA molecules and / or adaptor molecules which have not been used in the course of performing the method. Thus, the method may comprise the steps:

[0129] (a) amplifying a DNA template molecule comprising at least one cleavable (e.g. endonuclease) target sequence to generate a double-stranded DNA molecule; (b) contacting the double-stranded DNA molecule with an endonuclease, a ligase and first and second terminal adaptor molecules and n+m intermediate adaptor molecules, to form a single contiguous aqueous volume, wherein n and m are each 0 or an integer of at least 1 , and wherein n+m is at least 1 ;

[0130] (c) incubating the single contiguous aqueous volume to generate the closed linear DNA product, wherein the closed linear DNA product comprises a linear double-stranded region, wherein the linear double-stranded region comprises a linear portion of the double-stranded DNA molecule, and wherein n intermediate adaptor molecules are sequentially appended to a first end of the linear double stranded region and m intermediate adaptor molecules are sequentially appended to a second end of the linear double-stranded region, and wherein the closed linear DNA product is closed at a first end by the first terminal adaptor molecule appended to the nth intermediate adaptor molecule or, when n is 0 , to the first end of the linear double stranded region, and closed at a second end by the second terminal adaptor molecule appended to the mth intermediate adaptor molecule, or when m is 0, to the second end of the linear double stranded region; and

[0131] (d) incubating the single contiguous aqueous volume with a nuclease (e.g. exonuclease).

[0132] In the method described herein, after the step of amplification, the step of contacting the doublestranded DNA molecule with an endonuclease, a ligase and first and second terminal adaptor molecules and n+m intermediate adaptor molecules, to form a single contiguous aqueous volume, may be performed without purifying the product of the amplification reaction. That is to say that the step of contacting the double-stranded DNA molecule with an endonuclease, a ligase and first and second terminal adaptor molecules and n+m intermediate adaptor molecules may be performed directly after the step of amplification. The step of contacting the double-stranded DNA molecule with an endonuclease, a ligase and first and second terminal adaptor molecules and n+m intermediate adaptor molecules may be performed directly after the step of heat-deactivation.

[0133] The method may comprise the steps:

[0134] (a) amplifying a DNA template molecule comprising at least one cleavable (e.g. endonuclease) target sequence to generate a double-stranded DNA molecule;

[0135] (b) contacting the double-stranded DNA molecule with an endonuclease, a ligase and first and second terminal adaptor molecules and n+m intermediate adaptor molecules, to form a single contiguous aqueous volume, wherein n and m are each 0 or an integer of at least 1 , and wherein n+m is at least 1 ;

[0136] (c) incubating the single contiguous aqueous volume to generate the closed linear DNA product, wherein the closed linear DNA product comprises a linear double-stranded region, wherein the linear double-stranded region comprises a linear portion of the double-stranded DNA molecule, and wherein n intermediate adaptor molecules are sequentially appended to a first end of the linear double stranded region and m intermediate adaptor molecules are sequentially appended to a second end of the linear double-stranded region, and wherein the closed linear DNA product is closed at a first end by the first terminal adaptor molecule appended to the nth intermediate adaptor molecule or, when n is 0 , to the first end of the linear double stranded region, and closed at a second end by the second terminal adaptor molecule appended to the mth intermediate adaptor molecule, or when m is 0, to the second end of the linear double stranded region;

[0137] (d) purifying the closed linear DNA product; and

[0138] (e) incubating the purified product of step (d) with a nuclease (e.g. exonuclease).

[0139] The method may comprise the steps:

[0140] (a) amplifying a DNA template molecule comprising at least one cleavable (e.g. endonuclease) target sequence to generate a double-stranded DNA molecule;

[0141] (b) heat-deactivation of the reaction of step (a);

[0142] (c) contacting the double-stranded DNA molecule with an endonuclease, a ligase and first and second terminal adaptor molecules and n+m intermediate adaptor molecules, to form a single contiguous aqueous volume, wherein n and m are each 0 or an integer of at least 1 , and wherein n+m is at least 1 ;

[0143] (d) incubating the single contiguous aqueous volume to generate the closed linear DNA product, wherein the closed linear DNA product comprises a linear double-stranded region, wherein the linear double-stranded region comprises a linear portion of the double-stranded DNA molecule, and wherein n intermediate adaptor molecules are sequentially appended to a first end of the linear double stranded region and m intermediate adaptor molecules are sequentially appended to a second end of the linear double-stranded region, and wherein the closed linear DNA product is closed at a first end by the first terminal adaptor molecule appended to the nth intermediate adaptor molecule or, when n is 0 , to the first end of the linear double stranded region, and closed at a second end by the second terminal adaptor molecule appended to the mth intermediate adaptor molecule, or when m is 0, to the second end of the linear double stranded region;

[0144] (e) purifying the closed linear DNA product; and

[0145] (f) incubating the purified product of step (d) with a nuclease (e.g. exonuclease).

[0146] The method may comprise the steps:

[0147] (a) amplifying a DNA template molecule comprising at least one cleavable (e.g. endonuclease) target sequence to generate a double-stranded DNA molecule;

[0148] (b) contacting the double-stranded DNA molecule with an endonuclease, a ligase and first and second terminal adaptor molecules and n+m intermediate adaptor molecules, to form a single contiguous aqueous volume, wherein n and m are each 0 or an integer of at least 1 , and wherein n+m is at least 1 ;

[0149] (c) incubating the single contiguous aqueous volume to generate the closed linear DNA product, wherein the closed linear DNA product comprises a linear double-stranded region, wherein the linear double-stranded region comprises a linear portion of the double-stranded DNA molecule, and wherein n intermediate adaptor molecules are sequentially appended to a first end of the linear double stranded region and m intermediate adaptor molecules are sequentially appended to a second end of the linear double-stranded region, and wherein the closed linear DNA product is closed at a first end by the first terminal adaptor molecule appended to the nth intermediate adaptor molecule or, when n is 0 , to the first end of the linear double stranded region, and closed at a second end by the second terminal adaptor molecule appended to the mth intermediate adaptor molecule, or when m is 0, to the second end of the linear double stranded region;

[0150] (d) incubating the single contiguous aqueous volume with a nuclease (e.g. exonuclease); and

[0151] (e) purifying the closed linear DNA product.

[0152] The method may comprise the steps:

[0153] (a) amplifying a DNA template molecule comprising at least one cleavable (e.g. endonuclease) target sequence to generate a double-stranded DNA molecule;

[0154] (b) heat-deactivation of the reaction of step (a);

[0155] (c) contacting the double-stranded DNA molecule with an endonuclease, a ligase and first and second terminal adaptor molecules and n+m intermediate adaptor molecules, to form a single contiguous aqueous volume, wherein n and m are each 0 or an integer of at least 1 , and wherein n+m is at least 1 ;

[0156] (d) incubating the single contiguous aqueous volume to generate the closed linear DNA product, wherein the closed linear DNA product comprises a linear double-stranded region, wherein the linear double-stranded region comprises a linear portion of the double-stranded DNA molecule, and wherein the closed linear DNA product is closed at a first end by the first terminal adaptor molecule appended to the nth intermediate adaptor molecule or, when n is 0 , to the first end of the linear double stranded region, and closed at a second end by the second terminal adaptor molecule appended to the mth intermediate adaptor molecule, or when m is 0, to the second end of the linear double stranded region;

[0157] (e) incubating the single contiguous aqueous volume with a nuclease (e.g. exonuclease); and

[0158] (f) purifying the closed linear DNA product.

[0159] The step of incubating the single contiguous aqueous volume (or the purified product of step (d)) with a nuclease may be performed at a temperature of 5-90°C, 10-80°C, 15-70°C, 20-60°C, 25-50°C, 30- 45°C or 35-40°C. The step of incubating the single contiguous aqueous volume (or the purified product of step (d)) with a nuclease may be performed for at least 10, at least 20, at least 30, at least 40, at least 50, or at least 60 min. The step of incubating the single contiguous aqueous volume (or the purified product of step (d)) may be performed at two different temperatures. For example, the step of incubating the single contiguous aqueous volume (or the purified product of step (d)) may be performed at 15- 40°C for 10-60 minutes followed by a temperature of 60-90°C for 10-30 min. The higher temperature typically inactivates the nuclease (e.g. exonuclease). Thus, the method further provides a step of inactivating the nuclease (e.g. exonuclease). The step of incubating the single contiguous aqueous volume (or the purified product of step (d)) may be performed at 37°C for 30 min and 80°C for 20 min. Preferably, the step of inactivating the nuclease (e.g. exonuclease) is performed at a temperature of 70-80°C. The step of inactivating the nuclease (e.g. exonuclease) may be performed for at least 1 , at least 5, at least 10, at least 20 or at least 30 minutes. Preferably, the step of inactivating the nuclease (e.g. exonuclease) is performed for at least 5 minutes.

[0160] The method may be a cell-free method.

[0161] The closed linear DNA product may be partially double-stranded and / or partially single-stranded. The closed linear DNA product may comprise a portion that is double-stranded and a portion that is singlestranded.

[0162] The closed linear DNA product may comprise a cassette. The cassette may comprise a coding sequence. The coding sequence may encode a gene of interest, for example a gene encoding a protein. The cassette may comprise at least a portion of a promoter and a coding sequence. The cassette may comprise a promoter and a coding sequence. The cassette may comprise a promoter, a coding sequence, a ribosomal binding site and a translational termination sequence. The cassette may additionally comprise sequences aiding protein expression, such as a cap-independent translation element. The cassette may comprise (or encode) a repair template (or editing template). The repair template (or editing template) may be for use in CRISPR-Cas mediated homology directed repair (HDR). The cassette may encode CRISPR guide RNA. The cassette may be a mammalian expression cassette. The promoter may be a CMV promoter. The cassette may further comprise an enhancer. The cassette may further comprise a reporter gene, such as an eGFP reporter gene or a luciferase reporter gene. The cassette may further comprise a homopolymeric sequence, such as a polyA, poly C, polyT or polyG sequence. The homopolymeric sequence may be between 3-200 nucleotides in length. The homopolymeric sequence may be used to facilitate purification of the cassette, in which case, the homopolymeric sequence may be between 4-12 nucleotides in length, or between 5-10 nucleotides in length. The homopolymeric sequence may be used to improve mRNA expression, in which case, the homopolymeric sequence may be between 10-200 nucleotides in length, preferably between 80-150 nucleotides in length. The homopolymeric sequence may be at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 1 10, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, or at least 200 nucleotides in length. For example, the homopolymeric sequence may comprise a polyA sequence of at least 120 nucleotides.

[0163] The closed linear DNA product may comprise a spacer. The spacer may be at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 125, at least 150, at least 175, or at least 200 base pairs long.

[0164] The closed linear DNA product may comprise an inverted terminal repeat sequence.

[0165] The closed linear DNA product may be at least 50, at least 100, at least 250, at least 500, at least 1000, at least 2000, at least 3000, at least 4000, at least 5000, at least 6000, at least 7000, at least 8000, at least 9000, at least 10,000, at least 1 1 ,000, at least 12,000, at least 13,000, at least 14,000, or at least 15,000 base pairs long. Preferably, the closed linear DNA product is at least 50 base pairs long.

[0166] The double-stranded DNA molecule may be circular, or branched.

[0167] The double-stranded DNA molecule may not comprise an adaptor. The double-stranded DNA molecule may not comprise a hairpin, a loop or a stem-loop structure.

[0168] The double-stranded DNA molecule may comprise a cassette. The cassette may comprise a coding sequence. The coding sequence may encode a gene of interest, for example a gene encoding a protein. The cassette may comprise at least a portion of a promoter and a coding sequence. The cassette may comprise a promoter and a coding sequence. The cassette may comprise a promoter, a coding sequence, a ribosomal binding site and a translational termination sequence. The cassette may additionally comprise sequences aiding protein expression, such as a cap-independent translation element. The cassette may comprise (or encode) a repair template (or editing template). The repair template (or editing template) may be for use in CRISPR-Cas mediated homology directed repair (HDR). The cassette may encode CRISPR guide RNA. The cassette may be a mammalian expression cassette. The promoter may be a CMV promoter. The cassette may further comprise an enhancer. The cassette may further comprise a reporter gene, such as an eGFP reporter gene or a luciferase reporter gene. The cassette may further comprise a homopolymeric sequence, such as a polyA, poly C, polyT or polyG sequence. The homopolymeric sequence may be between 3-200 nucleotides in length. The homopolymeric sequence may be used to facilitate purification of the cassette, in which case, the homopolymeric sequence may be between 4-12 nucleotides in length, or between 5-10 nucleotides in length. The homopolymeric sequence may be used to improve mRNA expression, in which case, the homopolymeric sequence may be between 10-200 nucleotides in length, preferably between 80-150 nucleotides in length. The homopolymeric sequence may be at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 1 10, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, or at least 200 nucleotides in length. Preferably, the homopolymeric sequence is at least 100 nucleotides in length. More preferably still, the homopolymeric sequence is at least 120 nucleotides in length. For example, the homopolymeric sequence may comprise a polyA sequence of at least 120 nucleotides.

[0169] The double-stranded DNA molecule may comprise a spacer. The spacer may be at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 125, at least 150, at least 175, or at least 200 base pairs long. The spacer may improve an amplification yield of the double-stranded DNA molecule.

[0170] The double-stranded DNA molecule may be at least 50, at least 100, at least 250, at least 500, at least 1000, at least 2000, at least 3000, at least 4000, at least 5000, at least 6000, at least 7000, at least 8000, at least 9000, at least 10,000, at least 1 1 ,000, at least 12,000, at least 13,000, at least 14,000, or at least 15,000 base pairs long. Preferably, the double-stranded DNA molecule is at least 50 base pairs long.

[0171] The double-stranded DNA molecule may comprise one or more cleavable (e.g. endonuclease) target sequences. The double-stranded DNA molecule may comprise two cleavable (e.g. endonuclease) target sequences. The one or more cleavable (e.g. endonuclease) target sequences may be Type IIS endonuclease target sequences. The one or more cleavable (e.g. endonuclease) target sequences may be Bbsl, Bsal, BsmBI, BspQI, BtgZI, Esp3l,Sapl, Aarl, Acc36l, AcIWI, Acul, Ajul, Alol, Alw26l, Alwl, Arsl, AsuHPI, Bael, Bari, Bbvl, Bccl, BceAl, Bcgl, BciVI, BcoDI, BfuAI, Bful, Bmrl, Bmsl, Bmul, Bpil, Bpml, BpuEl, BsaXI, Bsel l, Bse3DI, BseGI, BseMI, BseMII, BseNI, BseRI, BseXI, Bsgl, BsIFI, BsmAI, BsmFI, Bsml, Bso31 l, BspCNI, BspMI, BspPI, BspQI, BspTNI, BsrDI, Bsrl, Bst6l, BstF5l, BstMAI, BstV11, BstV2l, Bsul, BtgZI, BtsCI, Btsl-v2, BtsMutl, Bvel, Csel, CspCI, Eam1 104l, Earl, Ecil, Eco31 l, Eco57l, Esp3l, Faql, Faul, Fokl, Gsul, Hgal, Hphl, HpyAV, Lgul, Lmnl, Lsp1 109I, Lwel, Mboll, Mlyl, Mmel, Mnll, Mva1269l, NmeAIII, PaqCI, PciSI, Pctl, Piel, Ppsl, Psrl, Schl, SfaNI, Taqll, TspDTI and / or TspGWI target sequences.

[0172] The double-stranded DNA molecule may be a product of amplification. Preferably, the amplification is rolling circle amplification.

[0173] The linear double-stranded region (e.g. the linear portion of the double-stranded molecule) may be at least 50, at least 100, at least 250, at least 500, at least 1000, at least 2000, at least 3000, at least 4000, at least 5000, at least 6000, at least 7000, at least 8000, at least 9000, at least 10,000, at least 1 1 ,000, at least 12,000, at least 13,000, at least 14,000, or at least 15,000 base pairs long. Preferably, the double-stranded DNA molecule is at least 50 base pairs long.

[0174] The linear double-stranded region (e.g. the linear portion of the double-stranded molecule) may comprise a sequence that is at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to the sequence of the double-stranded DNA molecule.

[0175] The first end and the second end of the linear double-stranded region (e.g. the linear portion of the double-stranded molecule) may be resistant to nuclease digestion. Preferably, the first end and the second end of the linear double-stranded region are resistant to the exonuclease digestion, such as exonuclease III digestion and / or exonuclease I digestion.

[0176] The linear double-stranded region may comprise a 3’-OH group at first and / or second ends. The 3’-OH group may facilitate ligation to the first and / or second terminal adaptor molecule(s) and / or n+m intermediate adaptor molecules (which may comprise a 5’ phosphate). The linear double-stranded region may comprise a 5’ phosphate at first and / or second ends. The 5’ phosphate may facilitate ligation to the first and / or second terminal adaptor molecule(s) (which may comprise a 3’-OH group). The linear double-stranded region (e.g. the linear portion of the double-stranded molecule) may comprise an overhang. For example, the linear double-stranded region may comprise a 5’ overhang or a 3’ overhang. The linear double-stranded region may comprise a blunt end or blunt ends. The linear double-stranded region may comprise: a 5’ overhang and a blunt end, two 5’ overhangs, a 3’ overhang and a blunt end, two 3’ overhangs, or a 5’ overhang and a 3’ overhang. The overhang may have at least 3 nucleotides (preferably from 4 to 8 nucleotides). The overhang may be in the sense strand or the antisense strand of the linear double-stranded region.

[0177] The linear portion of the double-stranded DNA molecule (e.g. the linear portion of the double-stranded molecule) may be at least 50, at least 100, at least 250, at least 500, at least 1000, at least 2000, at least 3000, at least 4000, at least 5000, at least 6000, at least 7000, at least 8000, at least 9000, at least 10,000, at least 1 1 ,000, at least 12,000, at least 13,000, at least 14,000, or at least 15,000 base pairs long. Preferably, double-stranded DNA molecule is at least 50 base pairs long.

[0178] The first terminal adaptor molecule and / or the second terminal adaptor molecule and / or one or more or n+m of the n+m intermediate adaptor molecules may be a synthetic adaptor molecule.

[0179] The first terminal adaptor molecule may be a nucleic acid adaptor molecule. The second terminal adaptor molecule may be a nucleic acid adaptor molecule. One or more or n+m of the n+m intermediate adaptor molecules may be a nucleic acid adaptor molecule. The first terminal adaptor molecule and / or the second terminal adaptor molecule may comprise a self-complementary element which creates a loop, such as a hairpin loop or a stem loop. Thus, the first terminal adaptor molecule may comprise a hairpin or a stem-loop. The second terminal adaptor molecule may comprise a hairpin or a stem-loop. Both the first and second terminal adaptor molecules may comprise a hairpin or a stem-loop. The terminal or intermediate adaptor molecules may each comprise a doublestranded portion comprising a sense strand and an antisense strand, wherein the sense strand and the antisense strand are linked together in a hairpin such that the sense strand is hybridized to the antisense strand. The double-stranded portion of an adaptor may comprise a 3’ overhang or a 5’ overhang of at least 1 , at least 2, at least 3, at least 4, or at least 5 nucleotides. Preferably the 3’ overhang or the 5’ overhang is 4-8 nucleotides. Each end of the linear double-stranded region (or linear portion of the double-stranded DNA molecule) may comprise a 3’ or a 5’ overhang.

[0180] A portion of the first terminal adaptor molecule (e.g. the overhang) may be complementary to the first end of the linear double-stranded region or complementary to a first end of an intermediate adaptor molecule, preferably the nth intermediate adaptor molecule. A portion of the second terminal adaptor molecule (e.g. the overhang) may be complementary to the second end of the linear double-stranded region or complementary to a first end of an intermediate adaptor molecule, preferably the mth adaptor molecule. A portion of the first end of the linear double-stranded region maybe complementary to a first end of an intermediate adaptor molecule, preferably n-(n-1 ) intermediate adaptor molecule. A portion of the second end of the linear double-stranded region maybe complementary to a first end of an intermediate adaptor molecule, preferably m-(m-1 ) intermediate adaptor molecule. A portion of a second end of an intermediate adaptor molecule may be complementary to a first end of an adjacent intermediate adaptor molecule, for example a portion of the second end of the nth intermediate adaptor molecule may be complementary to a portion of the first end of the n-1 th intermediate adaptor molecule, and a portion of the second end of the n-1 th adaptor molecule may be complementary to a portion of the first end of the n-2th intermediate adaptor molecule, and so on. A portion of a second end of an intermediate adaptor molecule may be complementary to a first end of an adjacent intermediate adaptor molecule, for example a portion of the second end of the mth intermediate adaptor molecule may be complementary to a portion of the first end of the m-1th intermediate adaptor molecule, and a portion of the second end of the m-1 th adaptor molecule may be complementary to a portion of the first end of the m-2th intermediate adaptor molecule, and so on. n may be 0, 1 , 2, 3, 4, 5, 6, 7 or 8. m may be 0, 1 , 2, 3, 4, 5, 6, 7 or 8. Preferably, n is 0, 1 , 2, 3 or 4, and m is 0, 1 , 2, 3 or 4. .

[0181] The closed linear DNA product may be a covalently closed linear DNA product. Thus, in embodiments where the terminal adaptor molecules comprise a loop (e.g. a hairpin), the terminal adaptor molecules close the ends of the linear double-stranded region (or close the ends of the nth and mth intermediate adaptor molecules) forming a covalently closed linear DNA product.

[0182] The first and / or second terminal adaptor molecule may comprise or consist of the sequence of SEQ ID NO: 9 or a portion thereof. The first and / or second terminal adaptor molecule may comprise at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, or at least 19 contiguous nucleotides of SEQ ID NO: 9. The double-stranded portion of the first and / or second terminal adaptor molecule may comprise the sequence of SEQ ID NO: 10 or a portion thereof. The double-stranded portion of the first and / or second terminal adaptor molecule may comprise at least 8, at least 9, at least 10, at least 1 1 , at least 12, at least 13, at least 14 or at least 15 contiguous nucleotides of SEQ ID NO: 10. The singlestranded portion of the first and / or second terminal adaptor molecule may comprise a sequence of ACTCA. The single-stranded portion of the first terminal adaptor molecule and / or the second terminal adaptor molecule may comprise at least 1 , at least 2, at least 3, at least 4 or at least 5 contiguous nucleotides of the sequence ACTCA. The first and / or second terminal adaptor molecule may comprise the sequence of SEQ ID NO: 12. The first and / or second adaptor molecule may comprise at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, or at least 19 contiguous nucleotides of SEQ ID NO:12.

[0183] The invention provides a method for producing a covalently closed linear DNA product, wherein the method comprises:

[0184] (a) contacting a double-stranded DNA molecule with an endonuclease, a ligase and first and second terminal adaptor molecules and n+m intermediate adaptor molecules, to form a single contiguous aqueous volume, wherein n and m are each 0 or an integer of at least 1 , and wherein n+m is at least 1 , wherein the first and second terminal adaptor molecules are nucleic acid adaptor molecules that each comprise a hairpin; and

[0185] (b) incubating the single contiguous aqueous volume to generate the covalently closed linear DNA product, wherein the covalently closed linear DNA product comprises a linear double-stranded region, wherein the linear double-stranded region comprises a linear portion of the double-stranded DNA molecule, wherein n intermediate adaptor molecules are sequentially appended to a first end of the linear double stranded region and m intermediate adaptor molecules are sequentially appended to a second end of the linear double-stranded region, wherein the closed linear DNA product is closed at a first end by the first terminal adaptor molecule appended to the nth intermediate adaptor molecule or, when n is 0 , to the first end of the linear double stranded region, and closed at a second end by the second terminal adaptor molecule appended to the mth intermediate adaptor molecule, or when m is 0, to the second end of the linear double stranded region, wherein (i) the first terminal adaptor molecule comprises an overhang that is complementary to and anneals to an overhang at the first end of the linear double-stranded region or the nth intermediate adaptor molecule thereby closing the first end of the linear double-stranded region and (ii) the second terminal adaptor molecule comprises an overhang that is complementary to and anneals to an overhang at the second end of the linear double-stranded region or the mth intermediate adaptor molecule thereby closing the second end of the linear double-stranded region.

[0186] The invention provides a method for producing a covalently closed linear DNA product, wherein the method comprises:

[0187] (a) contacting a double-stranded DNA molecule with an endonuclease, a ligase and first and second terminal adaptor molecules and n+m intermediate adaptor molecules, to form a single contiguous aqueous volume, wherein n and m are each 0 or an integer of at least 1 , and wherein n+m is at least 1 , wherein the first and second terminal adaptor molecules are nucleic acid adaptor molecules that each comprise a hairpin; and

[0188] (b) incubating the single contiguous aqueous volume to generate the covalently closed linear DNA product, wherein the covalently closed linear DNA product comprises a linear double-stranded region, wherein the linear double-stranded region comprises a linear portion of the double-stranded DNA molecule, and wherein n intermediate adaptor molecules are sequentially appended to a first end of the linear double stranded region and m intermediate adaptor molecules are sequentially appended to a second end of the linear double-stranded region, and wherein the closed linear DNA product is closed at a first end by the first terminal adaptor molecule appended to the nth intermediate adaptor molecule or, when n is 0 , to the first end of the linear double stranded region, and closed at a second end by the second terminal adaptor molecule appended to the mth intermediate adaptor molecule, or when m is 0, to the second end of the linear double stranded region, wherein (i) the first terminal adaptor molecule comprises an overhang that is complementary to and anneals to an overhang at the first end of the linear double-stranded region or to the nth intermediate adaptor molecule thereby closing the first end of the linear double-stranded region and (ii) the second terminal adaptor molecule comprises an overhang that is complementary to and anneals to an overhang at the second end of the linear double-stranded region or to the mth intermediate adaptor molecule thereby closing the second end of the linear double-stranded region, and wherein the first terminal adaptor molecule is ligated to the first end of the linear double-stranded region and the second terminal adaptor molecule is ligated to the second end of the linear double-stranded region.

[0189] The invention provides a method for producing a covalently closed linear DNA product, wherein the method comprises:

[0190] (a) contacting a double-stranded DNA molecule with an endonuclease, a ligase and first and second terminal adaptor molecules and n+m intermediate adaptor molecules, to form a single contiguous aqueous volume, wherein n and m are each 0 or an integer of at least 1 , and wherein n+m is at least 1 , wherein the first and second terminal adaptor molecules are nucleic acid adaptor molecules that each comprise a hairpin; and

[0191] (b) incubating the single contiguous aqueous volume to generate the covalently closed linear DNA product, wherein the covalently closed linear DNA product comprises a linear portion of the double-stranded DNA molecule, and wherein n intermediate adaptor molecules are sequentially appended to a first end of the linear double stranded region and m intermediate adaptor molecules are sequentially appended to a second end of the linear double-stranded region, and wherein the closed linear DNA product is closed at a first end by the first terminal adaptor molecule appended to the nth intermediate adaptor molecule or, when n is 0 , to the first end of the linear double stranded region, and closed at a second end by the second terminal adaptor molecule appended to the mth intermediate adaptor molecule, or when m is 0, to the second end of the linear double stranded region, wherein (i) the first terminal adaptor molecule comprises an overhang that is complementary to and anneals to an overhang at the first end of the linear portion of the double-stranded DNA molecule or to the nth intermediate adaptor molecule, thereby closing the first end of the linear portion of the double-stranded DNA molecule and (ii) the second terminal adaptor molecule comprises an overhang that is complementary to and anneals to an overhang at the second end of the linear portion of the double-stranded DNA molecule or to the mth intermediate adaptor molecule, thereby closing the second end of the linear portion of the double-stranded DNA molecule, and wherein the first terminal adaptor molecule is ligated to the first end of the linear portion of the double-stranded DNA molecule or to the nth intermediate adaptor molecule, and the second terminal adaptor molecule is ligated to the second end of the linear portion of the double-stranded DNA molecule or to the mth intermediate adaptor molecule, n may be 0, 1 , 2, 3, 4, 5, 6, 7 or 8. m may be 0, 1 , 2, 3, 4, 5, 6, 7 or 8. Preferably, n is 0, 1 , 2, 3 or 4, and m is 0, 1 , 2, 3 or 4. .

[0192] The first terminal adaptor molecule and / or the second terminal adaptor molecule and / or the n+m intermediate adaptor molecules may not be a plasmid or a vector DNA.

[0193] The first terminal adaptor molecule and / or the second terminal adaptor molecule may comprise a single-stranded portion. The single-stranded portion may form a hairpin or a stem-loop. Thus, the first terminal adaptor molecule and / or the second terminal adaptor molecule may comprise a loop portion. The single-stranded portion may comprise less than 10, 9, 8, 7, 6, 5, 4, 3, 2 nucleotides. Preferably, the single-stranded portion comprises 5 nucleotides.

[0194] The first terminal adaptor molecule and / or the second terminal adaptor molecule and / or one or more of the n+m intermediate adaptor molecules may comprise a double-stranded portion. The doublestranded portion may comprise less than 50, less than 45, less than 40, less than 35, less than 30, less than 25, less than 20, less than 15, or less than 10 base pairs. The double-stranded portion may comprise at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11 , at least 12, at least 13, at least 14, or at least 15 base pairs.

[0195] The first terminal adaptor molecule and / or the second terminal adaptor molecule and / or one or more of the n+m intermediate adaptor molecules may comprise a 5’ phosphate. The 5’ phosphate may facilitate ligation to the linear double-stranded region or to adjacent terminal or intermediate adaptor molecules (which may comprise a 3’-OH group at first and / or second ends). The first terminal adaptor molecule and / or the second terminal adaptor molecule and / or one or more of the n+m intermediate adaptor molecules may comprise a 3’-OH. The 3’-OH may facilitate ligation to the linear doublestranded region and / or to adjacent intermediate adaptor molecules (which may comprise a 5’ phosphate at first and / or second ends).

[0196] The first terminal adaptor molecule and / or the second terminal adaptor molecule may comprise the sequence of SEQ ID NO: 1 or a portion thereof. The first terminal adaptor molecule and / or the second terminal adaptor molecule may comprise at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, or at least 19 contiguous nucleotides of SEQ ID NO: 1 . The double-stranded portion of the first terminal adaptor molecule and / or the second terminal adaptor molecule may comprise the sequence of SEQ ID NO: 2 or a portion thereof. The double-stranded portion of the first terminal adaptor molecule and / or the second terminal adaptor molecule may comprise at least 8, at least 9, at least 10, at least 11 , at least 12, at least 13, at least 14 or at least 15 contiguous nucleotides of SEQ ID NO: 2. The single-stranded portion of the first terminal adaptor molecule and / or the second terminal adaptor molecule may comprise a sequence of ACTCA. The single-stranded portion of the first terminal adaptor molecule and / or the second terminal adaptor molecule may comprise at least 1 , at least 2, at least 3, at least 4 or at least 5 contiguous nucleotides of the sequence ACTCA. The first and second adapter molecules may comprise an identical nucleic acid sequence. The first and second adapter molecules may comprise a different nucleic acid sequence.

[0197] The first terminal adaptor molecule may comprise a portion that is complementary to the first end of the linear double-stranded region (or the linear portion of the double-stranded DNA molecule). The second terminal adaptor molecule may comprise a portion that is complementary to the second end of the linear double-stranded region (or the linear portion of the double-stranded DNA molecule). The first terminal adaptor molecule may comprise a portion that anneals to the first end of the linear double-stranded region (or the linear portion of the double-stranded DNA molecule). The second terminal adaptor molecule may comprise a portion that anneals to the second end of the linear double-stranded region (or the linear portion of the double-stranded DNA molecule). The first terminal adaptor molecule may comprise a portion that is complementary and anneals to the first end of the linear double-stranded region (or the linear portion of the double-stranded DNA molecule). The second terminal adaptor molecule may comprise a portion that is complementary and anneals to the second end of the linear double-stranded region (or the linear portion of the double-stranded DNA molecule).

[0198] The portion that is complementary or anneals to the first or second end of the linear double-stranded region (or the linear portion of the double-stranded DNA molecule) may be a 5’ overhang or a 3’ overhang of the first and / or second terminal adaptor molecule. The overhang of the first terminal adaptor molecule may be complementary to the first end of the linear double-stranded region and / or the overhang of the second terminal adaptor molecule may be complementary to the second end of the double-stranded region. The overhang of the first terminal adaptor molecule may anneal to the first end of the linear double-stranded region and / or the overhang of the second terminal adaptor molecule may anneal to the second end of the linear double-stranded region. The overhang of the first terminal adaptor molecule may be complementary to and anneal to the first end of the linear double-stranded region and / or the overhang of the second terminal adaptor molecule may be complementary to and anneal to the second end of the linear double-stranded region.

[0199] The first terminal adaptor molecule may comprise a portion that is complementary to the first end of the nth intermediate adaptor molecule. The second terminal adaptor molecule may comprise a portion that is complementary to the first end of the mth intermediate adaptor molecule. The first terminal adaptor molecule may comprise a portion that anneals to the first end of the nth intermediate adaptor molecule. The second terminal adaptor molecule may comprise a portion that anneals to first end of the mth intermediate adaptor molecule. The first terminal adaptor molecule may comprise a portion that is complementary and anneals to first end of the nth intermediate adaptor molecule. The second terminal adaptor molecule may comprise a portion that is complementary and anneals to first end of the mth intermediate adaptor molecule.

[0200] The portion that is complementary or anneals to the first end of the nth or mth intermediate adaptor molecule may be a 5’ overhang or a 3’ overhang of the first and / or second terminal adaptor molecule. The overhang of the first terminal adaptor molecule may be complementary to the first end of the nth intermediate adaptor molecule and / or the overhang of the second terminal adaptor molecule may be complementary to first end of the mth intermediate adaptor molecule. The overhang of the first terminal adaptor molecule may anneal to first end of the nth intermediate adaptor molecule and / or the overhang of the second terminal adaptor molecule may anneal to first end of the mth intermediate adaptor molecule. The overhang of the first terminal adaptor molecule may be complementary to and anneal to first end of the nth intermediate adaptor molecule and / or the overhang of the second terminal adaptor molecule may be complementary to and anneal to first end of the mth intermediate adaptor molecule.

[0201] The first terminal adaptor molecule and / or the second terminal adaptor molecule and / or one or more of the n+m intermediate adaptor molecules may not comprise a Type IIS endonuclease target sequence. The first terminal adaptor molecule and / or the second terminal adaptor molecule may not comprise Bbsl, Bsal, BsmBI, BspQI, BtgZI, Esp3l,Sapl, Aarl, Acc36l, AcIWI, Acul, Ajul, Alol, Alw26l, Alwl, Arsl, AsuHPI, Bael, Bari, Bbvl, Bccl, BceAl, Bcgl, BciVI, BcoDI, BfuAI, Bful, Bmrl, Bmsl, Bmul, Bpil, Bpml, BpuEl, BsaXI, Bsel l, Bse3DI, BseGI, BseMI, BseMII, BseNI, BseRI, BseXI, Bsgl, BsIFI, BsmAI, BsmFI, Bsml, Bso31 l, BspCNI, BspMI, BspPI, BspQI, BspTNI, BsrDI, Bsrl, Bst6l, BstF5l, BstMAI, BstV11, BstV2l, Bsul, BtgZI, BtsCI, Btsl-v2, BtsMutl, Bvel, Csel, CspCI, Eam1104l, Earl, Ecil, Eco31 l, Eco57l, Esp3l, Faql, Faul, Fokl, Gsul, Hgal, Hphl, HpyAV, Lgul, Lmnl, Lsp1109I, Lwel, Mboll, Mlyl, Mmel, Mnll, Mva1269l, NmeAIII, PaqCI, PciSI, Pctl, Piel, Ppsl, Psrl, Schl, SfaNI, Taqll, TspDTI and / or TspGWI target sequences.

[0202] The first terminal adaptor molecule and / or the second terminal adaptor molecule and / or one or more of the n+m intermediate adaptor molecules may comprise one or more locked nucleic acids (LNAs).

[0203] The first terminal adaptor molecule and / or the second terminal adaptor molecule and / or one or more of the n+m intermediate adaptor molecules may comprise one or more protected nucleotides (i.e. nuclease-resistant nucleotides), such as phosphorothioated nucleotides. The protected nucleotides may be located in the single-stranded portion (e.g. hairpin portion) of the terminal adaptor molecules or the double-stranded portion of the terminal or intermediate adaptor molecules. The protected nucleotides may be located in the overhang portion of the terminal or intermediate adaptor molecules.

[0204] The closed linear DNA product may comprise a plurality of phosphorothioated nucleotides at internal positions in each strand. For example, the closed linear DNA product may comprise at least 2, at least 4, at least 6, at least 8, at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 125, at least 150, at least 175, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, or at least 500 protected nucleotides (e.g. phosphorothioated nucleotides) at internal positions in each strand. Preferably, the closed linear DNA product comprises at least 2 protected nucleotides (e.g. phosphorothioated nucleotides) at internal positions in each strand.

[0205] The terminal and / or intermediate adaptor molecules may comprise at least two types of phosphorothioated nucleotides. For example, the at least two types of phosphorothioated nucleotides are: a-S-dATP and a-S-dCTP, a-S-dATP and a-S-dGTP, a-S-dATP and a-S-dTTP, a-S-dCTP and a- S-dGTP, a-S-dCTP and a-S-dTTP, or a-S-dGTP and a-S-dTTP. The terminal and / or intermediate adaptor molecules may comprise at least three types of phosphorothioated nucleotides. For example, the at least three types of phosphorothioated nucleotides are:

[0206] (a) a-S-dATP, a-S-dCTP and a-S-dGTP;

[0207] (b) a-S-dATP, a-S-dCTP and a-S-dTTP;

[0208] (c) a-S-dATP, a-S-dGTP and a-S-dTTP; or

[0209] (d) a-S-dCTP, a-S-dGTP and a-S-dTTP.

[0210] The terminal and / or intermediate adaptor molecules may comprise at least four types of phosphorothioated nucleotides. For example, the at least four types of protected nucleotides are a-S- dATP, a-S-dCTP, a-S-dGTP and a-S-dTTP

[0211] The internal positions may not be located between the second and penultimate nucleotide of the closed linear DNA product.

[0212] The linear double-stranded region (or linear portion of the double-stranded molecule) may comprise a plurality of phosphorothioated nucleotides at internal positions in each strand. For example, the linear double-stranded region (or linear portion of the double-stranded molecule) may comprise at least 2, at least 4, at least 6, at least 8, at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 125, at least 150, at least 175, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, at least or 500 protected nucleotides (e.g. phosphorothioated nucleotides) at internal positions in each strand. Preferably, the linear double-stranded region (or linear portion of the doublestranded molecule) comprises at least 2 protected nucleotides (e.g. phosphorothioated nucleotides) at internal positions in each strand. The internal positions may not be located between the second and penultimate nucleotide of the linear double-stranded region (or linear portion of the double-stranded molecule).

[0213] One or more of the n+m intermediate adaptor molecules may comprise a plurality of phosphorothioated nucleotides at internal positions in each strand. For example, the one or more of the n+m intermediate adaptor molecules may comprise at least 2, at least 4, at least 6, at least 8, at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 125, at least 150, at least 175, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, at least or 500 protected nucleotides (e.g. phosphorothioated nucleotides) at internal positions in each strand.

[0214] The nucleotides resistant to exonuclease digestion (i.e. protected nucleotides) suitable for use in the methods described herein may be phosphorothioated nucleotides. For example, phosphorothioated nucleotides may be a-S-dATP (i.e. 2’-deoxyadenosine-5’-(a-thio)-triphosphate), a-S-dCTP (i.e. 2’- deoxycytidine-5’-(a-thio)-triphosphate), a-S-dGTP (i.e. 2’-deoxyguanosine-5’-(a-thio)-triphosphate), a- S-dTTP (i.e. 2’-deoxythymidine-5’-(a-thio)-triphosphate), a-S-dUTP (i.e. 2’-deoxyuridine-5’-(a-thio)- triphosphate), and / or uridine 2’, 3’-cyclophosphorothioate.

[0215] The phosphorothioated nucleotides may be Sp-isomers, Rp-isomers or a mixture of both Sp- and Rp- isomers.

[0216] The nucleotides resistant to exonuclease digestion (i.e. protected nucleotides) may be 2'-O-methyl nucleotides or 2'-O-methoxyethyl (MOE) nucleotides. For example, the MOE nucleotides may be 2’-O- methoxy-ethyl guanosine, 2’-O-methoxy-ethyl cytidine, 2’-O-methoxy-ethyl adenosine, and / or 2’-O- methoxy-ethyl thymidine.

[0217] The first end of the linear double-stranded region may be complementary to a portion of the first terminal adaptor molecule or to a portion of an intermediate adaptor molecule, preferably the n-(n-1 )th adaptor molecule. The second end of the linear double-stranded region may be complementary to a portion of the second terminal adaptor molecule or to a portion of an intermediate adaptor molecule, preferably the m-(m-1 )th adaptor molecule. The first end and / or the second end of the linear doublestranded region may be generated by endonuclease digestion.

[0218] The first terminal adaptor molecule and / or the second terminal adaptor molecule and / or one or more of the n+m intermediate adaptor molecules may comprise a functional portion. The functional portion may be a binding molecule, a targeting sequence, a signal sequence or a probe. The functional portion may be a cassette, an open reading frame or a coding sequence. The functional portion may be a promotor, and enhancer, NLS sequence, modified nucleotides, a terminator, a transcription factor binding cite, a bar code or a fluorophore.

[0219] The functional portion may be a probe. As used herein, the term “probe” refers to a fragment of DNA, RNA or DNA / RNA chimera of variable length (e.g. 3-1000 bases long), which is used to detect the presence of target nucleotide sequences that are complementary to the sequence in the probe. Typically, the probe hybridizes to single-stranded nucleic acid whose base sequence allows probetarget base pairing due to complementarity between the probe and target. Thus, the functional portion may be a DNA sequence, a RNA sequence or a DNA / RNA chimera sequence. As used herein, the term “complementary” refers to the pairing of nucleotide sequences according to Watson / Crick pairing rules. For example, a sequence 5’-GCGGTCCCA-3’ has the complementary sequence of 5’-TGGGACCGC- 3’. A complement sequence can also be a sequence of RNA complementary to the DNA sequence.

[0220] The functional portion may be a binding molecule. The term “binding molecule” refers to any molecule capable of binding to the linear DNA product described herein and / or that is capable of binding to a further molecule or target. The binding molecule may be a protein, a polypeptide, or a peptide. The binding molecule may be an antibody, such as a monoclonal antibody or a polyclonal antibody. The binding molecule may be an antibody fragment. The functional portion may facilitate detection of the DNA product by binding to capture molecules (e.g. capture antibodies bound by protein-protein interactions). The functional portion may bind to a cell target, for example, a cell receptor.

[0221] The functional portion may be a label. The ‘label’ can be any chemical entity which enable the detection of the double-stranded nucleic acid molecule via, physical, chemical and / or biological means. The label may be a chromophore, a fluorophore and / or a radioactive molecule.

[0222] The functional portion may be a targeting sequence. The targeting sequence may be a fragment of DNA or RNA of variable length, which is used to target the DNA product to a specific location in a cell. The targeting sequence may be used to increased transfection efficiency of non-viral gene delivery by virtue of enhanced nuclear import of the closed linear DNA product. For example, the targeting sequence may be a DNA nuclear targeting sequences (i.e. a recognition sequence for endogenous DNA-binding proteins), such as SV40 enhancer sequence (preferably downstream from the cassette). The targeting sequence may be a protelomerase targeting sequence.

[0223] To facilitate detection and / or quantification of the DNA product, the functional portion may comprise a fluorophore, a radioactive compound or a barcode.

[0224] A signal corresponding to the presence, absence and / or level of the closed linear DNA product may be measured using a barcode. The barcode may comprise at least one binding moiety linked to a barcoded portion, wherein the barcoded portion comprises at least one nucleotide (i.e. wherein the barcoded portion comprises a nucleotide sequence at least one nucleotide in length), and wherein the binding moiety is capable of binding to the 3’ overhang, the 5’ overhang or the blunt end of the closed linear DNA product. The binding moiety is capable of binding to 3’ and / or 5’ end of the closed linear DNA product. The signal may be measured by determining the presence, absence and / or level of the barcoded portion of the barcode (e.g. by sequencing or PCR). The barcoded portion may comprise at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 nucleotides. The barcode may comprise at least 2 binding moieties (e.g. a first binding moiety and a second binding moiety). For example, the first binding moiety linked to the first barcoded portion may bind to the 3’ end of the closed linear DNA product and the second binding moiety linked to the second barcoded portion may bind to the 5’ end of the closed linear DNA product. The 3’ and 5’ ends may comprise a 3’ overhang, a 5’ overhang or a blunt end.

[0225] A signal corresponding to the presence, absence and / or level of the closed linear DNA product may be measured using a fluorophore (i.e. a fluorescently-labelled molecule), which is attached or bound to the 3’ overhang, the 5’ overhang or the blunt end of the closed linear DNA product. The signal may be measured by flow cytometry and / or fluorescence-activated cell sorting. The functional portion may also facilitate DNA sequencing. For example, the functional portion may be a sequencing adapter. The term “sequencing adapter” is intended to encompass one or more nucleic acid domains that include at least a portion of a nucleic acid sequence (or complement thereof) utilized by a sequencing platform of interest, such as a sequencing platform provided by Illumina® (e.g. the HiSeq™, MiSeq™ and / or Genome Analyzer™ sequencing systems), Oxford Nanopore™ Technologies (e.g. the MinlON sequencing system), Ion Torrent™ (e.g. the Ion PGM™ and / or Ion Proton™ sequencing systems), Pacific Biosciences (e.g. the PACBIO RS II sequencing system); Life Technologies™ (e.g. a SOLiD sequencing system), Roche (e.g. the 454 GS FLX+ and / or GS Junior sequencing systems), or any other sequencing platform of interest.

[0226] The first terminal adaptor molecule and / or the second terminal adaptor molecule and / or one or more of the n+m intermediate adaptor molecules may comprise an inverted terminal repeat sequence. The inverted terminal repeat sequences of the first terminal adaptor molecule and the second terminal adaptor molecule may be symmetrical (i.e. have the same symmetrical three-dimensional organization with respect to each other) or asymmetrical (i.e. have different three-dimensional organization with respect to each other). The inverted terminal repeat sequences of the first terminal adaptor molecule and the second terminal adaptor molecule and / or one or more of the n+m intermediate adaptor molecules may be from the same or different serotypes. An inverted terminal repeat sequence may comprise a terminal resolution site and a Rep binding site.

[0227] One or more of the n+m intermediate adaptor molecules (e.g. 1 , 2, 3, 4, 5, 6, 7 or 8 intermediate adaptor molecules) may comprise a cassette. The cassette may comprise a coding sequence. The coding sequence may encode a gene of interest, for example a gene encoding a protein. The cassette may comprise at least a portion of a promoter and a coding sequence. The cassette may comprise a promoter and a coding sequence. The cassette may comprise a promoter, a coding sequence, a ribosomal binding site and a translational termination sequence. The cassette may additionally comprise sequences aiding protein expression, such as a cap-independent translation element. The cassette may comprise (or encode) a repair template (or editing template). The repair template (or editing template) may be for use in CRISPR-Cas mediated homology directed repair (HDR). The cassette may encode CRISPR guide RNA. The cassette may be a mammalian expression cassette. The promoter may be a CMV promoter. The cassette may further comprise an enhancer. The cassette may further comprise a reporter gene, such as an eGFP reporter gene or a luciferase reporter gene. The cassette may further comprise a homopolymeric sequence, such as a polyA, poly C, polyT or polyG sequence. The homopolymeric sequence may be between 3-200 nucleotides in length. The homopolymeric sequence may be used to facilitate purification of the cassette, in which case, the homopolymeric sequence may be between 4-12 nucleotides in length, or between 5-10 nucleotides in length. The homopolymeric sequence may be used to improve mRNA expression, in which case, the homopolymeric sequence may be between 10-200 nucleotides in length, preferably between 80-150 nucleotides in length. The homopolymeric sequence may be at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 1 10, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, or at least 200 nucleotides in length. Preferably, the homopolymeric sequence is at least 100 nucleotides in length. More preferably still, the homopolymeric sequence is at least 120 nucleotides in length. For example, the homopolymeric sequence may comprise a polyA sequence of at least 120 nucleotides.

[0228] One or more of the n+m intermediate adaptor molecules (e.g. 1 , 2, 3, 4, 5, 6, 7 or 8 intermediate adaptor molecules) may comprise a spacer. The spacer may be at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 125, at least 150, at least 175, at least 200, or at least 300 base pairs long.

[0229] One or more of the n+m intermediate adaptor molecules (e.g. 1 , 2, 3, 4, 5, 6, 7 or 8 intermediate adaptor molecules) may be at least, 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11 , at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 100, at least 250, at least 500, at least 1000, at least 2000, at least 3000, at least 4000, at least 5000, at least 6000, at least 7000, at least 8000, at least 9000, at least 10,000, at least 11 ,000, at least 12,000, at least 13,000, at least 14,000, or at least 15,000 base pairs long. Preferably, the one or more of the n+m intermediate adaptor molecules (e.g. 1 , 2, 3, 4, 5, 6, 7 or 8 intermediate adaptor molecules) is at least 10 base pairs long. A terminator adaptor molecule may be may be at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11 , at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 100 base pairs long.

[0230] The first terminal adaptor molecule and / or the second terminal adaptor molecule and / or one or more of the n+m intermediate adaptor molecules may comprise a polyA signal sequence.

[0231] The first terminal adaptor molecule and / or the second terminal adaptor molecule and / or one or more of the n+m intermediate adaptor molecules may comprise an aptamer.

[0232] The first terminal adaptor molecule and / or the second terminal adaptor molecule and / or one or more of the n+m intermediate adaptor molecules may confer resistance to the nuclease digestion, such as exonuclease digestion (e.g. exonuclease I and / or exonuclease III digestion).

[0233] The closing of the linear double-stranded region (or the linear portion of the double-stranded DNA molecule) at the first end may generate a first closed end of the closed linear DNA product. The closing of the linear double-stranded region (or the linear portion of the double-stranded DNA molecule) at the second end may generate a second closed end of the closed linear DNA product. The first closed end and the second closed end of the closed linear DNA product may be resistant to nuclease digestion. The nuclease digestion may be exonuclease digestion. Preferably, the nuclease digestion is exonuclease III digestion and / or exonuclease I digestion. 2. Methods for producing a linear DNA product comprising nuclease-resistant nucleotides

[0234] The methods described herein may be used to produce a linear DNA product comprising nuclease- resistant (i.e. protected nucleotides).

[0235] The invention provides a method for producing a linear deoxyribonucleic acid (DNA) product, wherein the method comprises:

[0236] (a) contacting a double-stranded DNA molecule with an endonuclease and first and second terminal adaptor molecules and n+m intermediate adaptor molecules, to form a single contiguous aqueous volume, wherein n and m are each 0 or an integer of at least 1 , and wherein n+m is at least 1 ; and

[0237] (b) incubating the single contiguous aqueous volume to generate the linear DNA product, wherein the linear DNA product comprises a linear double-stranded region, wherein the linear doublestranded region comprises a linear portion of the double-stranded DNA molecule, and wherein n intermediate adaptor molecules are sequentially appended to a first end of the linear double stranded region and m intermediate adaptor molecules are sequentially appended to a second end of the linear double-stranded region, and wherein the first terminal adaptor molecule is appended to a first end of the linear double-stranded region and the second terminal adaptor molecule is appended to a second end of the linear double-stranded region, and wherein the first and second terminal adaptor molecules are nucleic acid molecules that comprise one or more nuclease-resistant nucleotides (i.e. protected nucleotides).

[0238] The step of contacting the double-stranded DNA molecule with the endonuclease and first and second terminal adaptor molecules is preferably performed in the presence of a ligase. Thus, the method for producing a linear DNA product may comprise the steps:

[0239] (a) contacting a double-stranded DNA molecule with an endonuclease, a ligase and first and second terminal adaptor molecules and n+m intermediate adaptor molecules, to form a single contiguous aqueous volume, wherein n and m are each 0 or an integer of at least 1 , and wherein n+m is at least 1 ; and

[0240] (b) incubating the single contiguous aqueous volume to generate the linear DNA product, wherein the linear DNA product comprises a linear double-stranded region, wherein the linear doublestranded region comprises a linear portion of the double-stranded DNA molecule, and wherein n intermediate adaptor molecules are sequentially appended to a first end of the linear double stranded region and m intermediate adaptor molecules are sequentially appended to a second end of the linear double-stranded region, and wherein the first terminal adaptor molecule is appended to a first end of the linear double-stranded region and the second terminal adaptor molecule is appended to a second end of the linear double-stranded region, and wherein the first and second terminal adaptor molecules are nucleic acid molecules that comprise one or more nuclease-resistant nucleotides (i.e. protected nucleotides). The linear DNA product produced by the methods described herein has enhanced resistance to nuclease (e.g. exonuclease) digestion. For example, the linear DNA product has prolonged in vivo expression when compared to a linear DNA product that does not contain protected nucleotides.

[0241] The addition of intermediate adaptor molecules allows for additional flexibility in the production of the linear DNA products of the invention. The intermediate adaptor molecules may allow for longer linear DNA products to be produced. The intermediate adaptor molecules allow for bar codes, promotor sequences, labels, poly A signals and / or open reading frames to be incorporated into the linear DNA product of the invention. An intermediate adaptor molecule may comprise a cassette. For example, if it is advantageous to include several genes of interest, or open reading frames, in the linear DNA product, intermediate adaptor molecules may comprise one or more cassettes, and the linear doublestranded region may comprise one or more cassettes. An intermediate adaptor molecule may comprise a label, a signalling sequence, a targeting sequence, a modified nucleotide, or a binding moiety. An intermediate adaptor molecule may comprise a promotor, a UTR, a transcription factor binding site, or a terminator sequence.

[0242] A modified nucleotide may be 2-MethoxyEthoxy A, 2-MethoxyEthoxy MeC, 2-MethoxyEthoxy G, 2- MethoxyEthoxy T, 2'-O-Methyl RNA Bases, Fluoro Bases, 2-Aminopurine, 5-Bromo dU, deoxyUridine, 2,6-Diaminopurine (2-Amino-dA), Dideoxy-C, deoxyinosine, Hydroxymethyl dC, Iso-dG, Iso-dC, 5- Methyl dC or 5-Nitroindole.

[0243] As well as providing nuclease-resistance to the linear DNA product, the terminal adaptor molecules may also provide further functional features. A terminator adaptor molecule may allow for longer linear DNA products to be produced. Terminator adaptor molecules may allow for bar codes, promotor sequences, labels, poly A signals and / or open reading frames to be incorporated into the linear DNA product of the invention. A terminal adaptor molecule may comprise a label, a signalling sequence (e.g. a NLS), a targeting sequence or a binding moiety. A terminal adaptor molecule may comprise a promotor, a UTR or a terminator sequence.

[0244] The appending of the first terminal adaptor molecule and / or the second terminal adaptor molecule to the first and / or second end of the linear double-stranded region (directly or indirectly via one or more intermediate adaptor molecules) may be performed by hybridization or ligation of the adaptor molecules to the ends of the linear double-stranded region and / or to sequential adaptor molecules. Thus, the n intermediate adaptor molecules may be sequentially hybridized to the first end of the linear doublestranded region. The m intermediate adaptor molecules may be sequentially hybridized to the second end of the linear double-stranded region. The n intermediate adaptor molecules may be sequentially ligated to the first end of the linear double-stranded region. The m intermediate adaptor molecules may be sequentially ligated to the second end of the linear double-stranded region. The appending of the n+m intermediate adaptor molecules, first terminal adaptor molecule and the second terminal adaptor molecule may be performed by both hybridization and ligation of the adaptor molecules to the ends of the linear double-stranded region and / or to adjacent adaptor molecules. Thus, n intermediate adaptor molecules may be sequentially hybridized and ligated to the first end of the linear double-stranded region. The m intermediate adaptor molecules may be sequentially hybridized and ligated to the second end of the linear double-stranded region. The appending may be performed via a linker or spacer molecule which facilitates joining of the adaptor molecule to the first and / or second end of the linear double-stranded region or to an adjacent adaptor molecule. The first terminal adaptor molecules may be hybridized, ligated or hybridized and ligated to the nth intermediate adaptor molecule or to the first end of the double-stranded DNA molecule. The second terminal adaptor molecule may hybridized, ligated or hybridized and ligated to the mth intermediate adaptor molecule or the second end of the double stranded DNA molecule. The hybridization is based on complementarity of a portion of the first and / or second terminal adaptor molecules and / or a portion of the first and / or second ends of the n+m intermediate adaptor molecules to the first and / or second end of the linear double-stranded region.

[0245] The method for producing a linear DNA product may comprise the steps:

[0246] (a) contacting a double-stranded DNA molecule with an endonuclease, a ligase and first and second terminal adaptor molecules and n+m intermediate adaptor molecules, to form a single contiguous aqueous volume, wherein n and m are each 0 or an integer of at least 1 , and wherein n+m is at least 1 ; and

[0247] (b) incubating the single contiguous aqueous volume to generate the linear DNA product, wherein the linear DNA product comprises a linear double-stranded region, wherein the linear doublestranded region comprises a linear portion of the double-stranded DNA molecule, and wherein n intermediate adaptor molecules are sequentially appended to a first end of the linear double stranded region and m intermediate adaptor molecules are sequentially appended to a second end of the linear double-stranded region, and wherein the first terminal adaptor molecule is appended to the nth intermediate adaptor molecule or, when n is 0, to the first end of the linear double stranded region, and the second terminal adaptor molecule is appended to the mth intermediate adaptor molecule, or when m is 0, to the second end of the linear double stranded region, and wherein the first terminal adaptor molecule and second terminal adaptor molecules are nucleic acid molecules that comprise one or more nuclease-resistant nucleotides (i.e. protected nucleotides).

[0248] The method for producing a linear DNA product may comprise the steps:

[0249] (a) contacting a double-stranded DNA molecule with an endonuclease, a ligase and first and second terminal adaptor molecules and n+m intermediate adaptor molecules, to form a single contiguous aqueous volume, wherein n and m are each 0 or an integer of at least 1 , and wherein n+m is at least 1 ; and

[0250] (b) incubating the single contiguous aqueous volume to generate the linear DNA product, wherein the linear DNA product comprises a linear portion of the double-stranded DNA molecule, and wherein n intermediate adaptor molecules are sequentially ligated to a first end of the linear double stranded region and m intermediate adaptor molecules are sequentially ligated to a second end of the linear double-stranded region, and wherein the first terminal adaptor molecule is ligated to the nth intermediate adaptor molecule or, when n is 0, to the first end of the linear double stranded region, and the second terminal adaptor molecule is ligated to the mth intermediate adaptor molecule, or when m is 0, to the second end of the linear double stranded region, and wherein the first terminal adaptor molecule and second terminal adaptor molecules are nucleic acid molecules that comprise one or more nuclease-resistant nucleotides (i.e. protected nucleotides), n may be 0, 1 , 2, 3, 4, 5, 6, 7 or 8. m may be 0, 1 , 2, 3, 4, 5, 6, 7 or 8. Preferably, n is 0, 1 , 2, 3 or 4, and m is 0, 1 , 2, 3 or 4. .

[0251] As used herein, the term “complementary” refers to the pairing of nucleotide sequences according to Watson / Crick pairing rules. For example, a sequence 5’-GCGGTCCCA-3’ has the complementary sequence of 5’-TGGGACCGC-3’. A complement sequence can also be a sequence of RNA complementary to the DNA sequence.

[0252] Preferably, the step of contacting the double-stranded DNA molecule with the endonuclease, the ligase and first and second terminal adaptor molecules is performed in a single reaction (i.e. a single step).

[0253] The step of incubating the single contiguous aqueous volume to generate the linear DNA product may comprise generating the linear portion of the double-stranded DNA molecule by digesting the doublestranded DNA molecule with the endonuclease.

[0254] The step of incubating the single contiguous aqueous volume may be performed under conditions that promote appending (or linking) of the first and second terminal adaptor molecules and / or intermediate adaptor molecules to the linear double-stranded region to produce the linear DNA product. The appending may be performed by creating a covalent link between the first and / or second terminal adaptor molecule and the first and / or second end of the linear double-stranded region. The appending may be performed by creating a covalent link between the first and / or second terminal adaptor molecule and an nth and / or mth intermediate adaptor molecule. The appending may be performed by creating a covalent link between an intermediate adaptor molecule and the first or second end of the linear double-stranded region. The appending may be performed by creating a covalent link between an intermediate adaptor molecule and an adjacent adaptor molecule.

[0255] The step of incubating the single contiguous aqueous volume may be performed under conditions that promote digestion of the double-stranded DNA molecule to produce the linear portion of the doublestranded DNA molecule. The digestion of the double-stranded DNA molecule to produce the linear portion of the double-stranded DNA molecule may be performed at a first temperature of 1°C-100°C, 1°C -80°C, 5°C-70°C, 10°C-60°C, 15°C-55°C, 20°C-50°C, 25°C -45°C, 30°C-40°C, 35°C-39°C, 36°C- 38°C, or at about 37°C. The digestion may be endonuclease digestion, preferably Type IIS endonuclease digestion. The step of incubating the single contiguous aqueous volume may be performed under conditions that promote ligation of the linear double-stranded region to the first and second terminal adaptor molecules and / or to an intermediate adaptor molecules. The step of incubating the single contiguous aqueous volume may be performed under conditions that promote ligation of the intermediate adaptor molecules to the first and second terminal adaptor molecules and / or adjacent intermediate adaptor molecules and / or the first and / or second end of the linear double-stranded region. The ligation may be at least 5%, at least 10%, at least 15, at least 20%, at least 25%, at least 30%, at least 35%, at least 40, at least 45%, at least 50%, at least 55%, at least 60, at least 65, at least 70%, at least 75, at least 80%, at least 82%, at least 85%, at least 90%, or at least 95% efficient. For example, at least 5%, at least 10%, at least 15, at least 20%, at least 25%, at least 30%, at least 35%, at least 40, at least 45%, at least 50%, at least 55%, at least 60, at least 65, at least 70%, at least 75, at least 80%, at least 82%, at least 85%, at least 90%, or at least 95% of the linear double-stranded regions (or the portions of the double-stranded DNA molecules) may be incorporated into closed linear DNA products. Preferably, the ligation is at least 15% efficient.

[0256] The step of ligation of the linear double-stranded region to the first and second terminal adaptor molecules and / or n+m intermediate adaptor molecules may be performed at a second temperature of 1 °C -90°C, 2°C -70°C, 5°C-60°C, 8°C-55°C, 9°C-50°C, 10°C-45°C, 1 1 °C-40°C, 12°C-37°C, 13°C-30°C, 14°C-25°C, 15°C-20°C or at about 16°C.

[0257] The step of incubating the single contiguous aqueous volume may comprise incubating at a first temperature and then incubating at a second temperature. The first temperature may be 1 °C-100°C, 1 °C-80°C, 5°C-70°C, 10°C-60°C, 15°C-55°C, 20°C-50°C, 25°C-45°C, 30°C-40°C, 35°C-39°C, 36°C- 38°C, or about 37°C. The second temperature may be 1 °C -90°C, 2°C -70°C, 5°C-60°C, 8°C-55°C, 9°C-50°C, 10°C-45°C, 1 1 °C-40°C, 12°C-37°C, 13°C-30°C, 14°C-25°C, 15°C-20°C or at about 16°C. Preferably, the first temperature is 35°C-39°C. Preferably, the second temperature is 14°C-18°C.

[0258] The step of incubating the single contiguous aqueous volume may comprise cycling between the first temperature and the second temperature. The step of incubating the single contiguous aqueous volume may comprise cycling between the first temperature and the second temperature at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 80, at least 90, or at least 100 times, preferably at least 20 times. The step of incubating the single contiguous aqueous volume may comprise cycling between the first temperature and the second temperature less than 40, less than 35, less than 30 times, less than 29, less than 25 times. The step of incubating the single contiguous aqueous volume may comprise cycling between the first temperature and the second temperature 2-100, 5-80, 10-70, 20-60, or 30-60 times. The step of incubating the single contiguous aqueous volume may comprise cycling between the first temperature and the second temperature 2-20, 5-29, 61 -100, or 65-80 times. The step of incubating the single contiguous aqueous volume may be performed isothermally. The step of incubating the single contiguous aqueous volume may comprise incubating at a constant temperature. The constant temperature promotes simultaneous digestion of the double-stranded DNA molecule to produce the linear portion of the double-stranded DNA molecule and ligation of the linear double-stranded region to the n+m intermediate adaptor molecules and / or the first and second terminal adaptor molecules. For example, the constant temperature may be 20°C, 21 °C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31 °C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, or 40°C. Preferably, the constant temperature is 30°C. The constant temperature is intended to mean that the temperature does not significantly change during the reaction. The constant temperature is intended to mean that the temperature variation during the step of incubating the single contiguous aqueous volume is less than 10°C, less than 9°C, less than 8°C, less than 7°C, less than 6°C, less than 5°C, less than 4°C, less than 3°C, less than 2°C, or less than 1 °C. In a preferred embodiment the temperature during the step of incubating the single contiguous aqueous does not deviate by more than 5°C, preferably by not more than 3°C, even more preferably not more than 1 °C. Thus, the constant temperature may be a temperature in a range of 20°C-30°C, 22°C-32°C, 24°C-34°C, 26°C- 36°C, 28°C-38°C, 30°C-40°C, 22°C-28°C, 32°C-38°C, 25°C-35°C, 26°C-34°C, 27°C- 33°C, 27.5°C- 32.5°C, 28°C-32°C, 28.5°C-31 ,5°C, 29°C-31 °C, or 29.5°C-30.5°C. Preferably, the constant temperature is a temperature in a range of 27.5°C-32.5°C. Alternatively, the constant temperature may be a temperature in a range of 32°C-42°C, 33°C-41 °C, 34°C-40°C, 35°C-39°C, 36°C-38°C. Preferably, the constant temperature is a temperature in a range of 34.5°C-39.5°C.

[0259] The first and second terminal adaptor molecules and / or one or more of the n+m intermediate adaptor molecules may comprise one or more phosphorothioated nucleotides, such that, once the terminal adaptor molecules are appended (e.g. ligated) to the linear double-stranded region, the linear DNA product is resistant to nuclease digestion or has improved or enhanced resistance to nuclease digestion. The linear DNA product may be resistant to 3’-end exonuclease digestion (e.g. by exonuclease III) and / or 5’-end exonuclease digestion (e.g. by exonuclease VIII).

[0260] The first and second adaptor molecules and / or one or more of the n+m intermediate adaptor molecules may comprise a plurality of phosphorothioated nucleotides. For example, the terminal or intermediate adaptor molecules may comprise at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 1 1 , at least 12, at least 13, at least 14, at least 15 or at least 16 phosphorothioated nucleotides in each strand.

[0261] A terminal and / or intermediate adaptor molecule may be a nucleic acid adaptor molecule. The terminal or intermediate adaptor molecule may be double-stranded. The terminal or intermediate adaptor molecule may comprise a portion that is double-stranded. The first and / or second terminal adaptor molecules may comprise at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 1 1 , at least 12, at least 13, at least 14, at least 15, or at least 16 base pairs.

[0262] The terminal and / or intermediate adaptor molecules may comprise a plurality of phosphorothioated nucleotides in each strand. For example, the adaptor molecules may comprise at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 1 1 , at least 12, at least 13, at least 14, at least 15 or at least 16 phosphorothioated nucleotides in each strand. One or more of the n+m intermediate adaptor molecules may comprise a plurality of phosphorothioated nucleotides at internal positions in each strand. For example, the one or more of the n+m intermediate adaptor molecules may comprise at least 2, at least 4, at least 6, at least 8, at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 125, at least 150, at least 175, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, at least or 500 protected nucleotides (e.g. phosphorothioated nucleotides) at internal positions in each strand.

[0263] The terminal adaptor molecules may comprise a plurality of phosphorothioated nucleotides at internal positions in each strand. For example, the adaptor molecules may comprise at least 1 , at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 1 1 , at least 12, at least 13, at least 14, at least 15 or at least 16 phosphorothioated nucleotides at internal positions in each strand. Preferably, the adaptor molecule comprises at least 2 phosphorothioated nucleotides at internal positions in each strand.

[0264] The internal positions may not be located between the second and penultimate nucleotide of the adaptor molecule. The internal positions may be any position in the adaptor molecules other than the last nucleotide at the end of each strand.

[0265] The terminal or intermediate adaptor molecules may comprise at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 100% of protected nucleotides.

[0266] Once the terminal adaptor molecules are appended to the linear double-stranded region (directly or indirectly via n or m intermediate adaptor molecules), the linear DNA product may comprise a protected nucleotide (e.g. phosphorothioated nucleotide) at the 5’-end (or at the 5’-end region) of one or both strands. Preferably, the linear DNA product comprises a phosphorothioated nucleotide at the 5’-end (or at the 5’-end region) of one or both strands. The linear DNA product may comprise a phosphorothioated nucleotide at the 5’-end (or at the 5’-end region) of one or both strands. As most exonucleases, for example exonuclease III, remove nucleotides from the 3’-end of the polynucleotide chain, the linear DNA product may comprise a protected nucleotide at the 3’-end (or at the 3’-end region) of one or both strands. Preferably, the linear DNA product comprises a phosphorothioated nucleotide at the 3’-end (or the 3’-end region) of one or both strands. The linear DNA product may comprise at least one phosphorothioated nucleotide at the 3’-end (or the 3’-end region) and at least one phosphorothioated nucleotide at the 5’-end (or the 5’-end region) of one or both strands. The linear DNA product may comprise a phosphorothioated nucleotide at the 3’-end (or the 3’-end region) and the 5’-end (or the 5’-end region) of one or both strands.

[0267] The linear DNA product may additionally comprise a plurality of protected nucleotides (e.g. phosphorothioated nucleotides) at internal positions in each strand. For example, the linear DNA product may comprise at least 2, at least 4, at least 6, at least 8, at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 125, at least 150, at least 175, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, or at least 500 protected nucleotides (e.g. phosphorothioated nucleotides) at internal positions in each strand. Preferably, the linear DNA product comprises at least 2 protected nucleotides (e.g. phosphorothioated nucleotides) at internal positions in each strand.

[0268] The internal positions may not be located between the second and penultimate nucleotide of the linear DNA product. The internal positions may be any position in the adaptor molecules other than the last nucleotide at the end of each strand.

[0269] The linear double-stranded region (or linear portion of the double-stranded molecule) may comprise a plurality of phosphorothioated nucleotides at internal positions in each strand. For example, the linear double-stranded region (or linear portion of the double-stranded molecule) may comprise at least 2, at least 4, at least 6, at least 8, at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 125, at least 150, at least 175, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, or at least 500 protected nucleotides (e.g. phosphorothioated nucleotides) at internal positions in each strand. Preferably, the linear double-stranded region (or linear portion of the double-stranded molecule) comprises at least 2 protected nucleotides (e.g. phosphorothioated nucleotides) at internal positions in each strand. The internal positions may not be located between the second and penultimate nucleotide of the linear double-stranded region (or linear portion of the double-stranded molecule).

[0270] The nucleotides resistant to exonuclease digestion (i.e. protected nucleotides) may be phosphorothioated nucleotides of at least one type. For example, the at least one type of phosphorothioated nucleotides is a-S-dATP (i.e. 2’-deoxyadenosine-5’-(a-thio)-triphosphate), a-S- dCTP (i.e. 2’-deoxycytidine-5’-(a-thio)-triphosphate), a-S-dGTP (i.e. 2’-deoxyguanosine-5’-(a-thio)- triphosphate), a-S-dTTP (i.e. 2’-deoxythymidine-5’-(a-thio)-triphosphate), a-S-dUTP (i.e. 2’- deoxyuridine-5’-(a-thio)-triphosphate), and / or uridine 2’, 3’-cyclophosphorothioate.

[0271] The terminal and / or intermediate adaptor molecules may comprise at least two types of phosphorothioated nucleotides. For example, the at least two types of phosphorothioated nucleotides are: a-S-dATP and a-S-dCTP, a-S-dATP and a-S-dGTP, a-S-dATP and a-S-dTTP, a-S-dCTP and a- S-dGTP, a-S-dCTP and a-S-dTTP, or a-S-dGTP and a-S-dTTP.

[0272] The terminal and / or intermediate adaptor molecules may comprise at least three types of phosphorothioated nucleotides. For example, the at least three types of phosphorothioated nucleotides are:

[0273] (e) a-S-dATP, a-S-dCTP and a-S-dGTP;

[0274] (f) a-S-dATP, a-S-dCTP and a-S-dTTP;

[0275] (g) a-S-dATP, a-S-dGTP and a-S-dTTP; or

[0276] (h) a-S-dCTP, a-S-dGTP and a-S-dTTP.

[0277] The terminal and / or intermediate adaptor molecules may comprise at least four types of phosphorothioated nucleotides. For example, the at least four types of protected nucleotides are a-S- dATP, a-S-dCTP, a-S-dGTP and a-S-dTTP.

[0278] The phosphorothioated nucleotides may be Sp-isomers, Rp-isomers or a mixture of both Sp- and Rp- isomers.

[0279] The nucleotides resistant to exonuclease digestion (i.e. protected nucleotides) may be MOE nucleotides of at least one type, or at least two, three or four types. For example, the MOE nucleotides may be 2’- O-methoxy-ethyl guanosine, 2’-O-methoxy-ethyl cytidine, 2’-O-methoxy-ethyl adenosine, and / or 2’-O- methoxy-ethyl thymidine.

[0280] The method may further comprise, before step (a) (i.e. the step of contacting the double-stranded DNA molecule with the endonuclease, the ligase and the first and second terminal adaptor molecules and n+m intermediate adaptor molecules), a step of amplifying a DNA template molecule to produce the double-stranded DNA molecule. Thus, the invention provides a method for producing a linear DNA product, the method comprises:

[0281] (a) amplifying a DNA template molecule comprising at least one cleavable (e.g. endonuclease) target sequence to generate a double-stranded DNA molecule;

[0282] (b) contacting the double-stranded DNA molecule with an endonuclease, a ligase and first and second terminal adaptor molecules and n+m intermediate adaptor molecules, to form a single contiguous aqueous volume, wherein n and m are each 0 or an integer of at least 1 , and wherein n+m is at least 1 ; and

[0283] (c) incubating the single contiguous aqueous volume to generate the linear DNA product, wherein the linear DNA product comprises a linear double-stranded region, wherein the linear doublestranded region comprises a linear portion of the double-stranded DNA molecule, and wherein n intermediate adaptor molecules are sequentially appended to a first end of the linear double stranded region and m intermediate adaptor molecules are sequentially appended to a second end of the linear double-stranded region, and wherein the first terminal adaptor molecule is appended to the nth intermediate adaptor molecule or, when n is 0, to the first end of the linear double stranded region, and the second terminal adaptor molecule is appended to the mth intermediate adaptor molecule, or when m is 0, to the second end of the linear double stranded region, and wherein the first terminal adaptor molecule and second terminal adaptor molecules are nucleic acid molecules that comprise one or more nuclease-resistant nucleotides (i.e. protected nucleotides).

[0284] The step of amplifying may be performed by in vitro or in vivo amplification. Preferably, the step of amplifying is performed by in vitro amplification. For example, the step of amplifying may be performed by rolling circle amplification (RCA), MALBAC method, traditional polymerase chain reaction (PCR), nucleic acid sequence-based amplification (NASBA), loop-mediated isothermal amplification (LAMP), helicase-dependent amplification (HDA), multiple displacement amplification (MDA) and recombinase polymerase amplification (RPA). Preferably, the step of amplifying is performed by rolling circle amplification. Thus, the invention provides a method for producing a linear DNA product, the method comprises:

[0285] (a) amplifying a DNA template molecule comprising at least one cleavable (e.g. endonuclease) target sequence to generate a double-stranded DNA molecule, wherein the DNA template molecule is amplified by rolling circle amplification;

[0286] (b) contacting the double-stranded DNA molecule with an endonuclease, a ligase and first and second terminal adaptor molecules and n+m intermediate adaptor molecules, to form a single contiguous aqueous volume, wherein n and m are each 0 or an integer of at least 1 , and wherein n+m is at least 1 ; and

[0287] (c) incubating the single contiguous aqueous volume to generate the linear DNA product, wherein the linear DNA product comprises a linear double-stranded region, wherein the linear doublestranded region comprises a linear portion of the double-stranded DNA molecule, and wherein n intermediate adaptor molecules are sequentially appended to a first end of the linear double stranded region and m intermediate adaptor molecules are sequentially appended to a second end of the linear double-stranded region, and wherein the first terminal adaptor molecule is appended to the nth intermediate adaptor molecule or, when n is 0, to the first end of the linear double stranded region, and the second terminal adaptor molecule is appended to the mth intermediate adaptor molecule, or when m is 0, to the second end of the linear double stranded region, and wherein the first terminal adaptor molecule and second terminal adaptor molecules are nucleic acid molecules that comprise one or more nuclease-resistant nucleotides (i.e. protected nucleotides).

[0288] Rolling circle amplification may be performed without any primers, or in the presence of a primer or multiple primers. For example, the primer may be a synthetic primer. The primers may be random primers. Rolling circle amplification may be performed in the presence of a primase. The primase may be Tt / iPrimPol. Preferably, if the rolling circle amplification is performed without any primers, it is performed in the presence of a primase, such as Tt / iPrimPol. Similarly, if a primer is used during amplification reaction, a primase is not used. The double-stranded DNA product may be generated by the rolling circle amplification in vitro under isothermal conditions using a suitable nucleic acid polymerase, such as Phi29 DNA polymerase.

[0289] In the methods described herein, the DNA template molecule may comprise at least one cleavable target sequence. The cleavable target sequence may be an endonuclease target sequence. Thus, the DNA template molecule may comprise at least one endonuclease target sequence. Preferably, the DNA template molecule comprises at least two endonuclease target sequences. The endonuclease target sequences may be the same or different. Preferably, the at least one endonuclease target sequence is a restriction endonuclease target sequence. Different restriction endonuclease target sequences would be known to the skilled person. The cleavable target sequence may be a Type IIS restriction endonuclease target sequence. For example, the restriction endonuclease target sequence may be a Bbsl, Bsal, BsmBI, BspQI, BtgZI, Esp3l,Sapl, Aarl, Acc36l, AcIWI, Acul, Ajul, Alol, Alw26l, Alwl, Arsl, AsuHPI, Bael, Bari, Bbvl, Bccl, BceAl, Bcgl, BciVI, BcoDI, BfuAI, Bful, Bmrl, Bmsl, Bmul, Bpil, Bpml, BpuEl, BsaXI, Bse1 1, Bse3DI, BseGI, BseMI, BseMII, BseNI, BseRI, BseXI, Bsgl, BsIFI, BsmAI, BsmFI, Bsml, Bso31 l, BspCNI, BspMI, BspPI, BspQI, BspTNI, BsrDI, Bsrl, Bst6l, BstF5l, BstMAI, BstV1 1, BstV2l, Bsul, BtgZI, BtsCI, Btsl-v2, BtsMutl, Bvel, Csel, CspCI, Eam1 104l, Earl, Ecil, Eco31 l, Eco57l, Esp3l, Faql, Faul, Fokl, Gsul, Hgal, Hphl, HpyAV, Lgul, Lmnl, Lsp1 109I, Lwel, Mboll, Mly I , Mmel, Mn 11 , Mva1269l, NmeAIII, PaqCI, PciSI, Pctl, Piel, Ppsl, Psrl, Schl, SfaNI, Taqll, TspDTI and / or TspGWI target sequence. The at least one cleavable sequence (e.g. endonuclease target sequence) may be a native cleavable sequence (i.e. a cleavable sequence present in the template molecule). Alternatively, the at least one cleavable sequence (e.g. endonuclease target sequence) may be introduced to the DNA template molecule prior to the production of the linear DNA product.

[0290] The endonuclease may be a restriction enzyme endonuclease. The endonuclease may be a Type IIS restriction enzyme. The endonuclease may be any enzyme that recognizes a DNA sequence and cleaves outside of the recognition sequence. For example, the endonuclease may be a Bbsl, Bsal, BsmBI, BspQI, BtgZI, Esp3l,Sapl, Aarl, Acc36l, AcIWI, Acul, Ajul, Alol, Alw26l, Alwl, Arsl, AsuHPI, Bael, Bari, Bbvl, Bccl, BceAl, Bcgl, BciVI, BcoDI, BfuAI, Bful, Bmrl, Bmsl, Bmul, Bpil, Bpml, BpuEl, BsaXI, Bsel l, Bse3DI, BseGI, BseMI, BseMII, BseNI, BseRI, BseXI, Bsgl, BsIFI, BsmAI, BsmFI, Bsml, Bso31 l, BspCNI, BspMI, BspPI, BspQI, BspTNI, BsrDI, Bsrl, Bst6l, BstF5l, BstMAI, BstV1 1, BstV2l, Bsul, BtgZI, BtsCI, Btsl-v2, BtsMutl, Bvel, Csel, CspCI, Eam1 104l, Earl, Ecil, Eco31 l, Eco57l, Esp3l, Faql, Faul, Fokl, Gsul, Hgal, Hphl, HpyAV, Lgul, Lmnl, Lsp1 109I, Lwel, Mboll, Mlyl, Mmel, Mnll, Mva1269l, NmeAIII, PaqCI, PciSI, Pctl, Piel, Ppsl, Psrl, Schl, SfaNI, Taqll, TspDTI and / or TspGWI restriction enzyme.

[0291] The ligase may be a DNA ligase, such as a T4 DNA ligase, T7 DNA ligase, mammalian DNA ligase I, III and IV; Taq DNA ligase, Tth DNA ligase, or E. coli DNA ligase.

[0292] The DNA template molecule used in the methods described herein may be single-stranded or doublestranded. Preferably, the DNA template molecule is double-stranded. The DNA template molecule may be a natural circular DNA molecule. For example, the DNA template molecule may be (i) a plasmid, (ii) a minicircle, (Hi) a cosmid, (iv) a bacterial artificial chromosome (BAC), or (v) a molecular inversion probe (MIP). The DNA template molecule may be an enzymatically produced circular DNA molecule. For example, the DNA template molecule may be (i) a circular DNA molecule obtained from recombinase reaction, preferably Cre recombinase reaction, or (ii) a circular DNA molecule obtained from ligase reaction, preferably using the golden gate assembly. The DNA template molecule may be an enzymatically produced covalently-closed linear DNA molecule. For example, the DNA template molecule may be (i) a DNA molecule processed with TelN protelomerase; or (ii) a DNA molecule generated by ligation of the DNA ends with an adaptor. The DNA template molecule may comprise an element that is double-stranded and an element that is single-stranded. For example, the template DNA molecule may comprise a double-stranded DNA and a single-stranded hairpin loop.

[0293] The DNA template molecule may be linear. If the DNA template molecule is linear, prior to amplification (e.g. rolling circle amplification), a DNA template molecule may be circularized to produce a DNA template molecule suitable for use in the methods described herein.

[0294] The template DNA molecule may comprise a cassette. The cassette may be a mammalian expression cassette. The cassette may further comprise a promoter. The promoter may be a CMV promoter. The cassette may further comprise an enhancer. The cassette may further comprise a reporter gene, such as an eGFP reporter gene or a luciferase reporter gene. The cassette may further comprise a homopolymeric sequence. The cassette may further comprise a LoxP sequence, preferably two LoxP sequences. If the two LoxP sequences are oriented in the same direction, the DNA sequence between the two LoxP sequences is excised as a circular loop of DNA. If the two LoxP sequences are oriented in the opposite direction, the DNA sequence between the two LoxP sequences is inverted. Thus, preferably, the two LoxP sequences are in the same orientation in the template DNA molecule.

[0295] The DNA template molecule may comprise a homopolymeric sequence at a 5’-end or a 3’-end or both a 5’-end and a 3’-end. The homopolymeric sequence may be added to the DNA template molecule before circularization. The homopolymeric sequence may be a polyA, a polyC, a polyG, or a polyT sequence. The homopolymeric sequence may be between 3-200 nucleotides in length. The homopolymeric sequence may be used to facilitate purification of the linear DNA product, in which case, the homopolymeric sequence may be between 4-12 nucleotides in length, or between 5-10 nucleotides in length. The homopolymeric sequence may be used to improve mRNA expression, in which case, the homopolymeric sequence may be between 10-200 nucleotides in length, preferably between 80-150 nucleotides in length. The homopolymeric sequence may be at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 1 10, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, or at least 200 nucleotides in length. Preferably, the homopolymeric sequence is at least 100 nucleotides in length. More preferably still, the homopolymeric sequence is at least 120 nucleotides in length. For example, the homopolymeric sequence may comprise a polyA sequence of at least 120 nucleotides. The method may further comprise, after the step of incubating the single contiguous aqueous volume, a step of purification of the linear DNA product.

[0296] The method may further comprise, after the step of incubating the single contiguous aqueous volume, a step of nuclease digestion. The nuclease digestion may be exonuclease digestion, such as exonuclease I and / or exonuclease III digestion. The step of nuclease digestion may take place before or after the step of purification. This step allows for removal of any double-stranded DNA molecules and / or adaptor molecules which have not been used in the course of performing the method. Thus, the method may comprise the steps:

[0297] (a) amplifying a DNA template molecule comprising at least one cleavable (e.g. endonuclease) target sequence to generate a double-stranded DNA molecule;

[0298] (b) contacting the double-stranded DNA molecule with an endonuclease, a ligase and first and second terminal adaptor molecules and n+m intermediate adaptor molecules, to form a single contiguous aqueous volume, wherein n and m are each 0 or an integer of at least 1 , and wherein n+m is at least;

[0299] (c) incubating the single contiguous aqueous volume to generate the linear DNA product, wherein the linear DNA product comprises a linear double-stranded region, wherein the linear doublestranded region comprises a linear portion of the double-stranded DNA molecule, and wherein n intermediate adaptor molecules are sequentially appended to a first end of the linear double stranded region and m intermediate adaptor molecules are sequentially appended to a second end of the linear double-stranded region, and wherein the first terminal adaptor molecule is appended to the nth intermediate adaptor molecule or, when n is 0, to the first end of the linear double stranded region, and the second terminal adaptor molecule is appended to the mth intermediate adaptor molecule, or when m is 0, to the second end of the linear double stranded region, and wherein the first terminal adaptor molecule and second terminal adaptor molecules are nucleic acid molecules that comprise one or more nuclease-resistant nucleotides (i.e. protected nucleotides); and

[0300] (d) incubating the single contiguous aqueous volume with a nuclease (e.g. exonuclease).

[0301] The method may comprise the steps:

[0302] (a) amplifying a DNA template molecule comprising at least one cleavable (e.g. endonuclease) target sequence to generate a double-stranded DNA molecule;

[0303] (b) contacting the double-stranded DNA molecule with an endonuclease, a ligase and first and second terminal adaptor molecules and n+m intermediate adaptor molecules, to form a single contiguous aqueous volume, wherein n and m are each 0 or an integer of at least 1 , and wherein n+m is at least 1 ;

[0304] (c) incubating the single contiguous aqueous volume to generate the linear DNA product, wherein the linear DNA product comprises a linear double-stranded region, wherein the linear doublestranded region comprises a linear portion of the double-stranded DNA molecule, and wherein n intermediate adaptor molecules are sequentially appended to a first end of the linear double stranded region and m intermediate adaptor molecules are sequentially appended to a second end of the linear double-stranded region, and wherein the first terminal adaptor molecule is appended to the nth intermediate adaptor molecule or, when n is 0, to the first end of the linear double stranded region, and the second terminal adaptor molecule is appended to the mth intermediate adaptor molecule, or when m is 0, to the second end of the linear double stranded region, and wherein the first terminal adaptor molecule and second terminal adaptor molecules are nucleic acid molecules that comprise one or more nuclease-resistant nucleotides (i.e. protected nucleotides);

[0305] (d) purifying the closed linear DNA product; and

[0306] (e) incubating the purified product of step (d) with a nuclease (e.g. exonuclease).

[0307] The method may comprise the steps:

[0308] (a) amplifying a DNA template molecule comprising at least one cleavable (e.g. endonuclease) target sequence to generate a double-stranded DNA molecule;

[0309] (b) contacting the double-stranded DNA molecule with an endonuclease, a ligase and first and second terminal adaptor molecules and n+m intermediate adaptor molecules, to form a single contiguous aqueous volume, wherein n and m are each 0 or an integer of at least 1 , and wherein n+m is at least 1 ;

[0310] (c) incubating the single contiguous aqueous volume to generate the linear DNA product, wherein the linear DNA product comprises a linear double-stranded region, wherein the linear doublestranded region comprises a linear portion of the double-stranded DNA molecule, and wherein n intermediate adaptor molecules are sequentially appended to a first end of the linear double stranded region and m intermediate adaptor molecules are sequentially appended to a second end of the linear double-stranded region, and wherein the first terminal adaptor molecule is appended to the nth intermediate adaptor molecule or, when n is 0, to the first end of the linear double stranded region, and the second terminal adaptor molecule is appended to the mth intermediate adaptor molecule, or when m is 0, to the second end of the linear double stranded region, and wherein the first terminal adaptor molecule and second terminal adaptor molecules are nucleic acid molecules that comprise one or more nuclease-resistant nucleotides (i.e. protected nucleotides);

[0311] (d) incubating the single contiguous aqueous volume with a nuclease (e.g. exonuclease); and

[0312] (e) purifying the closed linear DNA product.

[0313] The step of incubating the single contiguous aqueous volume (or the purified product of step (d)) with a nuclease may be performed at a temperature of 5-90°C, 10-80°C, 15-70°C, 20-60°C, 25-50°C, 30- 45°C or 35-40°C. The step of incubating the single contiguous aqueous volume (or the purified product of step (d)) with a nuclease may be performed for at least 10, at least 20, at least 30, at least 40, at least 50, or at least 60 min. The step of incubating the single contiguous aqueous volume (or the purified product of step (d)) may be performed at two different temperatures. For example, the step of incubating the single contiguous aqueous volume (or the purified product of step (d)) may be performed at 15- 40°C for 10-60 minutes followed by a temperature of 60-90°C for 10-30 min. The higher temperature typically inactivates the nuclease (e.g. exonuclease). Thus, the method further provides a step of inactivating the nuclease (e.g. exonuclease). The step of incubating the single contiguous aqueous volume (or the purified product of step (d)) may be performed at 37°C for 30 min and 80°C for 20 min. Preferably, the step of inactivating the nuclease (e.g. exonuclease) is performed at a temperature of 70-80°C. The step of inactivating the nuclease (e.g. exonuclease) may be performed for at least 1 , at least 5, at least 10, at least 20 or at least 30 minutes. Preferably, the step of inactivating the nuclease (e.g. exonuclease) is performed for at least 5 minutes.

[0314] A portion of the first terminal adaptor molecule (e.g. the overhang) may be complementary to the first end of the linear double-stranded region or complementary to a first end of an intermediate adaptor molecule, preferably the nth intermediate adaptor molecule. A portion of the second terminal adaptor molecule (e.g. the overhang) may be complementary to the second end of the linear double-stranded region or complementary to a first end of an intermediate adaptor molecule, preferably the mth adaptor molecule. A portion of the first end of the linear double-stranded region maybe complementary to a first end of an intermediate adaptor molecule, preferably n-(n-1 ) intermediate adaptor molecule. A portion of the second end of the linear double-stranded region maybe complementary to a first end of an intermediate adaptor molecule, preferably m-(m-1 ) intermediate adaptor molecule. A portion of a second end of an intermediate adaptor molecule may be complementary to a first end of an adjacent intermediate adaptor molecule, for example a portion of the second end of the nth intermediate adaptor molecule may be complementary to a portion of the first end of the n-1 th intermediate adaptor molecule, and a portion of the second end of the n-1 th adaptor molecule may be complementary to a portion of the first end of the n-2th intermediate adaptor molecule, and so on. A portion of a second end of an intermediate adaptor molecule may be complementary to a first end of an adjacent intermediate adaptor molecule, for example a portion of the second end of the mth intermediate adaptor molecule may be complementary to a portion of the first end of the m-1th intermediate adaptor molecule, and a portion of the second end of the m-1 th adaptor molecule may be complementary to a portion of the first end of the m-2th intermediate adaptor molecule, and so on. n may be 0, 1 , 2, 3, 4, 5, 6, 7 or 8. m may be 0, 1 , 2, 3, 4, 5, 6, 7 or 8. Preferably, n is 0, 1 , 2, 3 or 4, and m is 0, 1 , 2, 3 or 4. .

[0315] The first end and the second end of the linear double-stranded region may be resistant to nuclease digestion. Preferably, the first end and the second end of the linear double-stranded region are resistant to the exonuclease digestion, such as exonuclease III digestion and / or exonuclease I digestion.

[0316] The linear DNA product may be partially double-stranded and / or partially single-stranded. The linear DNA product may comprise a portion that is double-stranded and a portion that is single-stranded.

[0317] The linear DNA product may comprise a cassette. The cassette may comprise a coding sequence. The coding sequence may encode a gene of interest, for example a gene encoding a protein. The cassette may comprise at least a portion of a promoter and a coding sequence. The cassette may comprise a promoter and a coding sequence. The cassette may comprise a promoter, a coding sequence, a ribosomal binding site and a translational termination sequence. The cassette may additionally comprise sequences aiding protein expression, such as a cap-independent translation element. The cassette may comprise (or encode) a repair template (or editing template). The repair template (or editing template) may be for use in CRISPR-Cas mediated homology directed repair (HDR). The cassette may encode CRISPR guide RNA. The cassette may be a mammalian expression cassette. The promoter may be a CMV promoter. The cassette may further comprise an enhancer. The cassette may further comprise a reporter gene, such as an eGFP reporter gene or a luciferase reporter gene. The cassette may further comprise a homopolymeric sequence, such as a polyA, poly C, polyT or polyG sequence. The homopolymeric sequence may be between 3-200 nucleotides in length. The homopolymeric sequence may be used to facilitate purification of the cassette, in which case, the homopolymeric sequence may be between 4-12 nucleotides in length, or between 5-10 nucleotides in length. The homopolymeric sequence may be used to improve mRNA expression, in which case, the homopolymeric sequence may be between 10-200 nucleotides in length, preferably between 80-150 nucleotides in length. The homopolymeric sequence may be at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 1 10, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, or at least 200 nucleotides in length. Preferably, the homopolymeric sequence is at least 100 nucleotides in length. More preferably still, the homopolymeric sequence is at least 120 nucleotides in length. For example, the homopolymeric sequence may comprise a polyA sequence of at least 120 nucleotides.

[0318] The linear DNA product may comprise a spacer. The spacer may be at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 125, at least 150, at least 175, or at least 200 base pairs long. The spacer may improve ligation efficiency of the first and second terminal adaptor molecules to the linear double-stranded region. The spacer may improve a cell transfection yields.

[0319] The linear DNA product may comprise an inverted terminal repeat sequence.

[0320] The linear DNA product may be at least 50, at least 100, at least 250, at least 500, at least 1000, at least 2000, at least 3000, at least 4000, at least 5000, at least 6000, at least 7000, at least 8000, at least 9000, at least 10,000, at least 1 1 ,000, at least 12,000, at least 13,000, at least 14,000, or at least 15,000 base pairs long. Preferably, the linear DNA product is at least 50 base pairs long.

[0321] The double-stranded DNA molecule may be circular, or branched.

[0322] The double-stranded DNA molecule may not comprise an adaptor molecule. The double-stranded DNA molecule may not comprise a hairpin, a loop or a stem-loop structure. The double-stranded DNA molecule may comprise a cassette. The cassette may comprise a coding sequence. The coding sequence may encode a gene of interest, for example a gene encoding a protein. The cassette may comprise at least a portion of a promoter and a coding sequence. The cassette may comprise a promoter and a coding sequence. The cassette may comprise a promoter, a coding sequence, a ribosomal binding site and a translational termination sequence. The cassette may additionally comprise sequences aiding protein expression, such as a cap-independent translation element. The cassette may comprise (or encode) a repair template (or editing template). The repair template (or editing template) may be for use in CRISPR-Cas mediated homology directed repair (HDR). The cassette may encode CRISPR guide RNA. The cassette may be a mammalian expression cassette. The promoter may be a CMV promoter. The cassette may further comprise an enhancer. The cassette may further comprise a reporter gene, such as an eGFP reporter gene or a luciferase reporter gene. The cassette may further comprise a homopolymeric sequence, such as a polyA, poly C, polyT or polyG sequence. The homopolymeric sequence may be between 3-200 nucleotides in length. The homopolymeric sequence may be used to facilitate purification of the cassette, in which case, the homopolymeric sequence may be between 4-12 nucleotides in length, or between 5-10 nucleotides in length. The homopolymeric sequence may be used to improve mRNA expression, in which case, the homopolymeric sequence may be between 10-200 nucleotides in length, preferably between 80-150 nucleotides in length. The homopolymeric sequence may be at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 1 10, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, or at least 200 nucleotides in length. Preferably, the homopolymeric sequence is at least 100 nucleotides in length. More preferably still, the homopolymeric sequence is at least 120 nucleotides in length. For example, the homopolymeric sequence may comprise a polyA sequence of at least 120 nucleotides.

[0323] The double-stranded DNA molecule may comprise a spacer. The spacer may be at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 125, at least 150, at least 175, or at least 200 base pairs long. The spacer may improve an amplification yield of the double-stranded DNA molecule. The spacer may improve ligation efficiency of the first and second terminal adaptor molecules to the linear double-stranded region. The spacer may improve a cell transfection yields.

[0324] The double-stranded DNA molecule may be at least 50, at least 100, at least 250, at least 500, at least 1000, at least 2000, at least 3000, at least 4000, at least 5000, at least 6000, at least 7000, at least 8000, at least 9000, at least 10,000, at least 1 1 ,000, at least 12,000, at least 13,000, at least 14,000, or at least 15,000 base pairs long. Preferably, the double-stranded DNA molecule is at least 50 base pairs long.

[0325] The double-stranded DNA molecule may comprise one or more cleavable (e.g. endonuclease) target sequences. The double-stranded DNA molecule may comprise two cleavable (e.g. endonuclease) target sequences. The one or more endonuclease target sequences may be Type IIS endonuclease target sequences. The one or more endonuclease target sequences may be Bbsl, Bsal, BsmBI, BspQI, BtgZI, Esp3l,Sapl, Aarl, Acc36l, AcIWI, Acul, Ajul, Alol, Alw26l, Alwl, Arsl, AsuHPI, Bael, Bari, Bbvl, Bccl, BceAl, Bcgl, BciVI, BcoDI, BfuAI, Bful, Bmrl, Bmsl, Bmul, Bpil, Bpml, BpuEl, BsaXI, Bsel l, Bse3DI, BseGI, BseMI, BseMII, BseNI, BseRI, BseXI, Bsgl, BsIFI, BsmAI, BsmFI, Bsml, Bso31 l, BspCNI, BspMI, BspPI, BspQI, BspTNI, BsrDI, Bsrl, Bst6l, BstF5l, BstMAI, BstV11, BstV2l, Bsul, BtgZI, BtsCI, Btsl-v2, BtsMutl, Bvel, Csel, CspCI, Eam1 104l, Earl, Ecil, Eco31 l, Eco57l, Esp3l, Faql, Faul, Fokl, Gsul, Hgal, Hphl, HpyAV, Lgul, Lmnl, Lspt 109I, Lwel, Mboll, Mlyl, Mmel, Mnll, Mva1269l, NmeAIII, PaqCI, PciSI, Pctl, Piel, Ppsl, Psrl, Schl, SfaNI, Taqll, TspDTI and / or TspGWI target sequences.

[0326] The double-stranded DNA molecule may be a product of amplification. Preferably, the amplification is rolling circle amplification.

[0327] The linear double-stranded region may be at least 50, at least 100, at least 250, at least 500, at least 1000, at least 2000, at least 3000, at least 4000, at least 5000, at least 6000, at least 7000, at least 8000, at least 9000, at least 10,000, at least 1 1 ,000, at least 12,000, at least 13,000, at least 14,000, or at least 15,000 base pairs long. Preferably, the double-stranded DNA molecule is at least 50 base pairs long.

[0328] The linear double-stranded region may comprise a 3’-OH group at first and / or second ends. The 3’-OH group may facilitate ligation to the first and / or second terminal adaptor molecule(s) (which may comprise a 5’ phosphate). The linear double-stranded region may comprise a 5’ phosphate at first and / or second ends. The 5’ phosphate may facilitate ligation to the first and / or second terminal adaptor molecule(s) (which may comprise a 3’-OH group).

[0329] The linear double-stranded region (e.g. the linear portion of the double-stranded molecule) may comprise an overhang. For example, the linear double-stranded region may comprise a 5’ overhang or a 3’ overhang. The linear double-stranded region may comprise a blunt end or blunt ends. The linear double-stranded region may comprise: a 5’ overhang and a blunt end, two 5’ overhangs, a 3’ overhang and a blunt end, two 3’ overhangs, or a 5’ overhang and a 3’ overhang. The overhang may have at least 3 nucleotides (preferably from 4 to 8 nucleotides). The overhang may be in the sense strand or the antisense strand of the linear double-stranded region.

[0330] The linear portion of the double-stranded DNA molecule (e.g. the linear portion of the double-stranded molecule) may be at least 50, at least 100, at least 250, at least 500, at least 1000, at least 2000, at least 3000, at least 4000, at least 5000, at least 6000, at least 7000, at least 8000, at least 9000, at least 10,000, at least 1 1 ,000, at least 12,000, at least 13,000, at least 14,000, or at least 15,000 base pairs long. Preferably, double-stranded DNA molecule is at least 50 base pairs long. The first terminal adaptor molecule and / or the second terminal adaptor molecule and / or one or more of the n+m intermediate adaptor molecules may be a synthetic adaptor molecule.

[0331] The first terminal adaptor molecule and / or the second terminal adaptor molecule and / or one or more of n+m intermediate adaptor molecules may not be a plasmid or a vector DNA.

[0332] The first terminal adaptor molecule and / or the second terminal adaptor molecule and / or one or more of n+m intermediate adaptor molecules may comprise a single-stranded portion. The single-stranded portion may comprise less than 10, 9, 8, 7, 6, 5, 4, 3, 2 nucleotides. Preferably, the single-stranded portion comprises 5 nucleotides.

[0333] The first terminal adaptor molecule and / or the second terminal adaptor molecule and / or one or more of the n+m intermediate adaptor molecules may comprise a double-stranded portion. The doublestranded portion may comprise less than 50, less than 45, less than 40, less than 35, less than 30, less than 25, less than 20, less than 15, or less than 10 base pairs. The double-stranded portion may comprise at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 1 1 , at least 12, at least 13, at least 14, or at least 15 base pairs.

[0334] The first and / or second terminal adaptor molecule may comprise or consist of the sequences of SEQ ID NO:13 and / or SEQ ID NO:14. The first and / or second terminal adaptor molecule may comprise or at least 15, 14, 13, 12, 1 1 , 10, 19, 8, 7, 6, 5, contiguous nucleotides thereof.

[0335] The first terminal adaptor molecule and / or the second terminal adaptor molecule and / or one or more of the n+m intermediate adaptor molecules may comprise a 5’ phosphate. The 5’ phosphate may facilitate ligation to the linear double-stranded region or to adjacent terminal or intermediate adaptor molecules (which may comprise a 3’-OH group at first and / or second ends). The first terminal adaptor molecule and / or the second terminal adaptor molecule and / or one or more of the n+m intermediate adaptor molecules may comprise a 3’-OH. The 3’-OH may facilitate ligation to the linear doublestranded region and / or to adjacent intermediate adaptor molecules (which may comprise a 5’ phosphate at first and / or second ends).

[0336] The first terminal adaptor molecule may comprise a portion that is complementary to the first end of the linear double-stranded region (or the linear portion of the double-stranded DNA molecule). The second terminal adaptor molecule may comprise a portion that is complementary to the second end of the linear double-stranded region (or the linear portion of the double-stranded DNA molecule). The first terminal adaptor molecule may comprise a portion that anneals to the first end of the linear double-stranded region (or the linear portion of the double-stranded DNA molecule). The second terminal adaptor molecule may comprise a portion that anneals to the second end of the linear double-stranded region (or the linear portion of the double-stranded DNA molecule). The first terminal adaptor molecule may comprise a portion that is complementary and anneals to the first end of the linear double-stranded region (or the linear portion of the double-stranded DNA molecule). The second terminal adaptor molecule may comprise a portion that is complementary and anneals to the second end of the linear double-stranded region (or the linear portion of the double-stranded DNA molecule).

[0337] The portion that is complementary or anneals to the first or second end of the linear double-stranded region (or the linear portion of the double-stranded DNA molecule) may be a 5’ overhang or a 3’ overhang of the first and / or second terminal adaptor molecule. The overhang of the first terminal adaptor molecule may be complementary to the first end of the linear double-stranded region and / or the overhang of the second terminal adaptor molecule may be complementary to the second end of the double-stranded region. The overhang of the first terminal adaptor molecule may anneal to the first end of the linear double-stranded region and / or the overhang of the second terminal adaptor molecule may anneal to the second end of the linear double-stranded region. The overhang of the first terminal adaptor molecule may be complementary to and anneal to the first end of the linear double-stranded region and / or the overhang of the second terminal adaptor molecule may be complementary to and anneal to the second end of the linear double-stranded region.

[0338] The first terminal adaptor molecule may comprise a portion that is complementary to the first end of the nth intermediate adaptor molecule. The second terminal adaptor molecule may comprise a portion that is complementary to the first end of the mth intermediate adaptor molecule. The first terminal adaptor molecule may comprise a portion that anneals to the first end of the nth intermediate adaptor molecule. The second terminal adaptor molecule may comprise a portion that anneals to first end of the mth intermediate adaptor molecule. The first terminal adaptor molecule may comprise a portion that is complementary and anneals to first end of the nth intermediate adaptor molecule. The second terminal adaptor molecule may comprise a portion that is complementary and anneals to first end of the mth intermediate adaptor molecule, n may be 0, 1 , 2, 3, 4, 5, 6, 7 or 8. m may be 0, 1 , 2, 3, 4, 5, 6, 7 or 8. Preferably, n is 0, 1 , 2, 3 or 4, and m is 0, 1 , 2, 3 or 4. .

[0339] The portion that is complementary or anneals to the first end of the nth or mth intermediate adaptor molecule may be a 5’ overhang or a 3’ overhang of the first and / or second terminal adaptor molecule. The overhang of the first terminal adaptor molecule may be complementary to the first end of the nth intermediate adaptor molecule and / or the overhang of the second terminal adaptor molecule may be complementary to first end of the mth intermediate adaptor molecule. The overhang of the first terminal adaptor molecule may anneal to first end of the nth intermediate adaptor molecule and / or the overhang of the second terminal adaptor molecule may anneal to first end of the mth intermediate adaptor molecule. The overhang of the first terminal adaptor molecule may be complementary to and anneal to first end of the nth intermediate adaptor molecule and / or the overhang of the second terminal adaptor molecule may be complementary to and anneal to first end of the mth intermediate adaptor molecule. The first terminal adaptor molecule and / or the second terminal adaptor molecule may not comprise a Type IIS endonuclease target sequence. The first terminal adaptor molecule and / or the second terminal adaptor molecule may not comprise Bbsl, Bsal, BsmBI, BspQI, BtgZI, Esp3l,Sapl, Aarl, Acc36l, AcIWI, Acul, Ajul, Alol, Alw26l, Alwl, Arsl, AsuHPI, Bael, Bari, Bbvl, Bccl, BceAl, Bcgl, BciVI, BcoDI, BfuAI, Bful, Bmrl, Bmsl, Bmul, Bpil, Bpml, BpuEl, BsaXI, Bsel l, Bse3DI, BseGI, BseMI, BseMII, BseNI, BseRI, BseXI, Bsgl, BsIFI, BsmAI, BsmFI, Bsml, Bso31 l, BspCNI, BspMI, BspPI, BspQI, BspTNI, BsrDI, Bsrl, Bst6l, BstF5l, BstMAI, BstV11, BstV2l, Bsul, BtgZI, BtsCI, Btsl-v2, BtsMutl, Bvel, Csel, CspCI, Eam1 104l, Earl, Ecil, Eco31 l, Eco57l, Esp3l, Faql, Faul, Fokl, Gsul, Hgal, Hphl, HpyAV, Lgul, Lmnl, Lsp1109I, Lwel, Mboll, Mlyl, Mmel, Mnll, Mva1269l, NmeAIII, PaqCI, PciSI, Pctl, Piel, Ppsl, Psrl, Schl, SfaNI, Taqll, TspDTI and / or TspGWI Sapl target sequences.

[0340] The first terminal adaptor molecule and / or the second terminal adaptor molecule and / or one or more of the n+m intermediate adaptor molecules may comprise a functional portion. The functional portion may be a binding molecule, a targeting sequence, a signal sequence or a probe. The functional portion may be a cassette, an open reading frame or a coding sequence. The functional portion may be a promotor, and enhancer, NLS sequence, a UTR, ITR or other repeats, a terminator sequence, modified nucleotides or a fluorophore.

[0341] The functional portion may be a probe. As used herein, the term “probe” refers to a fragment of DNA, RNA or DNA / RNA chimera of variable length (e.g. 3-1000 bases long), which is used to detect the presence of target nucleotide sequences that are complementary to the sequence in the probe. Typically, the probe hybridizes to single-stranded nucleic acid whose base sequence allows probetarget base pairing due to complementarity between the probe and target. Thus, the functional portion may be a DNA sequence, a RNA sequence or a DNA / RNA chimera sequence. As used herein, the term “complementary” refers to the pairing of nucleotide sequences according to Watson / Crick pairing rules. For example, a sequence 5'-GCGGTCCCA-3' has the complementary sequence of 5'-TGGGACCGC- 3'. A complement sequence can also be a sequence of RNA complementary to the DNA sequence.

[0342] The functional portion may be a binding molecule. The term “binding molecule” refers to any molecule capable of binding to the linear DNA product described herein and / or that is capable of binding to a further molecule or target. The binding molecule may be a protein, a polypeptide, or a peptide. The binding molecule may be an antibody, such as a monoclonal antibody or a polyclonal antibody. The binding molecule may be an antibody fragment.

[0343] The functional portion may facilitate detection of the DNA product by binding to capture molecules (e.g. capture antibodies bound by protein-protein interactions). The functional portion may bind to a cell target, for example, a cell receptor. The functional portion may be a label. The ‘label’ can be any chemical entity which enable the detection of the double-stranded nucleic acid molecule via, physical, chemical and / or biological means. The label may be a chromophore, a fluorophore and / or a radioactive molecule.

[0344] The functional portion may be a targeting sequence. The targeting sequence may be a fragment of DNA or RNA of variable length, which is used to target the DNA product to a specific location in a cell. The targeting sequence may be used to increased transfection efficiency of non-viral gene delivery by virtue of enhanced nuclear import of the linear DNA product. For example, the targeting sequence may be a DNA nuclear targeting sequences (i.e. a recognition sequence for endogenous DNA-binding proteins), such as SV40 enhancer sequence (preferably downstream from the cassette). The targeting sequence may be a protelomerase targeting sequence or a truncated variant thereof.

[0345] To facilitate detection and / or quantification of the DNA product, the functional portion may comprise a fluorophore, a radioactive compound or a barcode.

[0346] A signal corresponding to the presence, absence and / or level of the linear DNA product may be measured using a barcode. The barcode may comprise at least one binding moiety linked to a barcoded portion, wherein the barcoded portion comprises at least one nucleotide (i.e. wherein the barcoded portion comprises a nucleotide sequence at least one nucleotide in length), and wherein the binding moiety is capable of binding to the 3’ overhang, the 5’ overhang or the blunt end of the linear DNA product. The binding moiety is capable of binding to 3’ and / or 5’ end of the linear DNA product. The signal may be measured by determining the presence, absence and / or level of the barcoded portion of the barcode (e.g. by sequencing or PCR). The barcoded portion may comprise at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 nucleotides. The barcode may comprise at least 2 binding moieties (e.g. a first binding moiety and a second binding moiety). For example, the first binding moiety linked to the first barcoded portion may bind to the 3’ end of the linear DNA product and the second binding moiety linked to the second barcoded portion may bind to the 5’ end of the linear DNA product. The 3’ and 5’ ends may comprise a 3’ overhang, a 5’ overhang or a blunt end.

[0347] A signal corresponding to the presence, absence and / or level of the linear DNA product may be measured using a fluorophore (i.e. a fluorescently-labelled molecule), which is attached or bound to the 3’ overhang, the 5’ overhang or the blunt end of the linear DNA product. The signal may be measured by flow cytometry and / or fluorescence-activated cell sorting.

[0348] The functional portion may also facilitate DNA sequencing. For example, the functional portion may be a sequencing adapter. The term “sequencing adapter” is intended to encompass one or more nucleic acid domains that include at least a portion of a nucleic acid sequence (or complement thereof) utilized by a sequencing platform of interest, such as a sequencing platform provided by Illumina® (e.g. the HiSeq™, MiSeq™ and / or Genome Analyzer™ sequencing systems), Oxford Nanopore™ Technologies (e.g. the MinlON sequencing system), Ion Torrent™ (e.g. the Ion PGM™ and / or Ion Proton™ sequencing systems), Pacific Biosciences (e.g. the PACBIO RS II sequencing system); Life Technologies™ (e.g. a SOLiD sequencing system), Roche (e.g. the 454 GS FLX+ and / or GS Junior sequencing systems), or any other sequencing platform of interest.

[0349] One or more of the n+m intermediate adaptor molecules (e.g. 1 , 2, 3, 4, 5, 6, 7 or 8 intermediate adaptor molecules) may comprise a cassette. The cassette may comprise a coding sequence. The coding sequence may encode a gene of interest, for example a gene encoding a protein. The cassette may comprise at least a portion of a promoter and a coding sequence. The cassette may comprise a promoter and a coding sequence. The cassette may comprise a promoter, a coding sequence, a ribosomal binding site and a translational termination sequence. The cassette may additionally comprise sequences aiding protein expression, such as a cap-independent translation element. The cassette may comprise (or encode) a repair template (or editing template). The repair template (or editing template) may be for use in CRISPR-Cas mediated homology directed repair (HDR). The cassette may encode CRISPR guide RNA. The cassette may be a mammalian expression cassette. The promoter may be a CMV promoter. The cassette may further comprise an enhancer. The cassette may further comprise a reporter gene, such as an eGFP reporter gene or a luciferase reporter gene. The cassette may further comprise a homopolymeric sequence, such as a polyA, poly C, polyT or polyG sequence. The homopolymeric sequence may be between 3-200 nucleotides in length. The homopolymeric sequence may be used to facilitate purification of the cassette, in which case, the homopolymeric sequence may be between 4-12 nucleotides in length, or between 5-10 nucleotides in length. The homopolymeric sequence may be used to improve mRNA expression, in which case, the homopolymeric sequence may be between 10-200 nucleotides in length, preferably between 80-150 nucleotides in length. The homopolymeric sequence may be at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 1 10, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, or at least 200 nucleotides in length. Preferably, the homopolymeric sequence is at least 100 nucleotides in length. More preferably still, the homopolymeric sequence is at least 120 nucleotides in length. For example, the homopolymeric sequence may comprise a polyA sequence of at least 120 nucleotides.

[0350] One or more of the n+m intermediate adaptor molecules (e.g. 1 , 2, 3, 4, 5, 6, 7 or 8 intermediate adaptor molecules) may comprise a spacer. The spacer may be at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 125, at least 150, at least 175, at least 200 or at least 300 base pairs long.

[0351] One or more of the n+m intermediate adaptor molecules (e.g. 1 , 2, 3, 4, 5, 6, 7 or 8 intermediate adaptor molecules) may be at least, 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 1 1 , at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 100, at least 250, at least 500, at least 1000, at least 2000, at least 3000, at least 4000, at least 5000, at least 6000, at least 7000, at least 8000, at least 9000, at least 10,000, at least 11 ,000, at least 12,000, at least 13,000, at least 14,000, or at least 15,000 base pairs long. Preferably, the one or more of the n+m intermediate adaptor molecules (e.g. 1 , 2, 3, 4, 5, 6, 7 or 8 intermediate adaptor molecules) is at least 10 base pairs long.

[0352] A terminator adaptor molecule may be may be at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11 , at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 100 base pairs long

[0353] The first terminal adaptor molecule and / or the second terminal adaptor molecule and / or one or more of the n+m intermediate adaptor molecules may comprise a polyA signal sequence.

[0354] The first terminal adaptor molecule and / or the second terminal adaptor molecule and / or one or more of the n+m intermediate adaptor molecules may comprise an aptamer.

[0355] The first terminal adaptor molecule and / or the second terminal adaptor molecule and / or one or more of the n+m intermediate adaptor molecules may confer resistance to the nuclease digestion, such as exonuclease digestion (e.g. exonuclease I and / or exonuclease III digestion).

[0356] 3. Methods for producing a partially closed linear DNA product comprising nuclease- resistant nucleotides

[0357] The methods described herein may be used to produce a partially closed linear DNA product comprising nuclease-resistant (i.e. protected nucleotides).

[0358] The invention provides a method for producing a partially closed deoxyribonucleic acid (DNA) product, wherein the method comprises:

[0359] (a) contacting a double-stranded DNA molecule with an endonuclease and first and second terminal adaptor molecules and n+m intermediate adaptor molecules, to form a single contiguous aqueous volume, wherein n and m are each 0 or an integer of at least 1 , and wherein n+m is at least 1 ; and

[0360] (b) incubating the single contiguous aqueous volume to generate the partially closed linear DNA product, wherein the partially closed linear DNA product comprises a linear double-stranded region, wherein the linear double-stranded region comprises a linear portion of the double-stranded DNA molecule, and wherein n intermediate adaptor molecules are sequentially appended to a first end of the linear double stranded region and m intermediate adaptor molecules are sequentially appended to a second end of the linear double-stranded region, and wherein the partially closed DNA product is closed at a first end by the first terminal adaptor molecule appended to the nth intermediate adaptor molecule or, when n is 0, to the first end of the linear double stranded region, and the second terminal adaptor molecule is appended to the mth intermediate adaptor molecule, or when m is 0, to the second end of the linear double stranded region, and wherein the second terminal adaptor molecule is a nucleic acid molecules that comprise one or more nuclease-resistant nucleotides.

[0361] The step of contacting the double-stranded DNA molecule with the endonuclease and first and second terminal adaptor molecules is preferably performed in the presence of a ligase. Thus, the method for producing a partially closed linear DNA product may comprise the steps:

[0362] (a) contacting a double-stranded DNA molecule with an endonuclease, a ligase and first and second terminal adaptor molecules and n+m intermediate adaptor molecules, to form a single contiguous aqueous volume, wherein n and m are each 0 or an integer of at least 1 , and wherein n+m is at least 1 ; and

[0363] (b) incubating the single contiguous aqueous volume to generate the partially closed linear DNA product, wherein the partially closed linear DNA product comprises a linear double-stranded region, wherein the linear double-stranded region comprises a linear portion of the double-stranded DNA molecule, and wherein n intermediate adaptor molecules are sequentially appended to a first end of the linear double stranded region and m intermediate adaptor molecules are sequentially appended to a second end of the linear double-stranded region, and wherein the partially closed DNA product is closed at a first end by the first terminal adaptor molecule appended to the nth intermediate adaptor molecule or, when n is 0, to the first end of the linear double stranded region, and the second terminal adaptor molecule is appended to the mth intermediate adaptor molecule, or when m is 0, to the second end of the linear double stranded region, and wherein the second terminal adaptor molecule is a nucleic acid molecules that comprise one or more nuclease-resistant nucleotides.

[0364] The partially closed linear DNA product produced by the methods described herein has enhanced resistance to nuclease (e.g. exonuclease) digestion.

[0365] The appending of the first terminal adaptor molecule and / or the second terminal adaptor molecule to the first and / or second end of the linear double-stranded region (directly or indirectly via one or more intermediate adaptor molecules) may be performed by hybridization or ligation of the adaptor molecules to the ends of the linear double-stranded region and / or to sequential adaptor molecules. Thus, the n intermediate adaptor molecules may be sequentially hybridized to the first end of the linear doublestranded region. The m intermediate adaptor molecules may be sequentially hybridized to the second end of the linear double-stranded region. The n intermediate adaptor molecules may be sequentially ligated to the first end of the linear double-stranded region. The m intermediate adaptor molecules may be sequentially ligated to the second end of the linear double-stranded region. The appending of the n+m intermediate adaptor molecules, first terminal adaptor molecule and the second terminal adaptor molecule may be performed by both hybridization and ligation of the adaptor molecules to the ends of the linear double-stranded region and / or to adjacent adaptor molecules. Thus, n intermediate adaptor molecules may be sequentially hybridized and ligated to the first end of the linear double-stranded region. The m intermediate adaptor molecules may be sequentially hybridized and ligated to the second end of the linear double-stranded region. The appending may be performed via a linker or spacer molecule which facilitates joining of the adaptor molecule to the first and / or second end of the linear double-stranded region or to an adjacent adaptor molecule. The first terminal adaptor molecules may be hybridized, ligated or hybridized and ligated to the nth intermediate adaptor molecule or to the first end of the double-stranded DNA molecule. The second terminal adaptor molecule may hybridized, ligated or hybridized and ligated to the mth intermediate adaptor molecule or the second end of the double stranded DNA molecule.

[0366] The method for producing a partially closed linear DNA product may comprise the steps:

[0367] (a) contacting a double-stranded DNA molecule with an endonuclease, a ligase and first and second terminal adaptor molecules and n+m intermediate adaptor molecules, to form a single contiguous aqueous volume, wherein n and m are each 0 or an integer of at least 1 , and wherein n+m is at least 1 ; and

[0368] (b) incubating the single contiguous aqueous volume to generate the partially closed linear DNA product, wherein the partially closed linear DNA product comprises a linear double-stranded region, wherein the linear double-stranded region comprises a linear portion of the double-stranded DNA molecule, and wherein n intermediate adaptor molecules are sequentially appended to a first end of the linear double stranded region and m intermediate adaptor molecules are sequentially appended to a second end of the linear double-stranded region, and wherein the partially closed DNA product is closed at a first end by the first terminal adaptor molecule appended to the nth intermediate adaptor molecule or, when n is 0, to the first end of the linear double stranded region, and the second terminal adaptor molecule is appended to the mth intermediate adaptor molecule, or when m is 0, to the second end of the linear double stranded region, and wherein the second terminal adaptor molecule is a nucleic acid molecules that comprise one or more nuclease-resistant nucleotides (i.e. protected nucleotides), n may be 0, 1 , 2, 3, 4, 5, 6, 7 or 8. m may be 0, 1 , 2, 3, 4, 5, 6, 7 or 8. Preferably, n is 0, 1 , 2, 3 or 4, and m is 0, 1 , 2, 3 or 4. .

[0369] The method for producing a partially closed linear DNA product may comprise the steps:

[0370] (a) contacting a double-stranded DNA molecule with an endonuclease, a ligase and first and second terminal adaptor molecules and n+m intermediate adaptor molecules, to form a single contiguous aqueous volume, wherein n and m are each 0 or an integer of at least 1 , and wherein n+m is at least 1 ; and

[0371] (b) incubating the single contiguous aqueous volume to generate the partially closed linear DNA product, wherein the partially closed linear DNA product comprises a linear portion of the doublestranded DNA molecule, and wherein n intermediate adaptor molecules are sequentially appended to a first end of the linear double stranded region and m intermediate adaptor molecules are sequentially appended to a second end of the linear double-stranded region, and wherein the partially closed DNA product is closed at a first end by the first terminal adaptor molecule appended to the nth intermediate adaptor molecule or, when n is 0, to the first end of the linear double stranded region, and the second terminal adaptor molecule is appended to the mth intermediate adaptor molecule, or when m is 0, to the second end of the linear double stranded region, and wherein the second terminal adaptor molecule is a nucleic acid molecules that comprise one or more nuclease-resistant nucleotides (i.e. protected nucleotides).

[0372] As used herein, the term “complementary” refers to the pairing of nucleotide sequences according to Watson / Crick pairing rules. For example, a sequence 5’-GCGGTCCCA-3’ has the complementary sequence of 5’-TGGGACCGC-3’. A complement sequence can also be a sequence of RNA complementary to the DNA sequence.

[0373] Preferably, the step of contacting the double-stranded DNA molecule with the endonuclease, the ligase and first and second terminal adaptor molecules is performed in a single reaction (i.e. a single step).

[0374] The step of incubating the single contiguous aqueous volume to generate the partially closed linear DNA product may comprise generating the linear portion of the double-stranded DNA molecule by digesting the double-stranded DNA molecule with the endonuclease.

[0375] The addition of intermediate adaptor molecules allows for additional flexibility in the production of the partially closed linear DNA products of the invention. The intermediate adaptor molecules may allow for longer partially closed linear DNA products to be produced. The intermediate adaptor molecules allow for bar codes, promotor sequences, labels, poly A signals and / or open reading frames to be incorporated into the partially closed linear DNA product of the invention. An intermediate adaptor molecule may comprise a cassette. For example, if it is advantageous to include several genes of interest, or open reading frames, in the partially closed linear DNA product, intermediate adaptor molecules may comprise one or more cassettes, and the linear DNA region may comprise one or more cassettes. An intermediate adaptor molecule may comprise a label, a signalling sequence, a targeting sequence or a binding moiety. An intermediate adaptor molecule may comprise a promotor, a UTR or a terminator sequence.

[0376] As well as providing nuclease-resistance to the partially closed linear DNA product, the terminal adaptor molecules may also provide further functional features. A terminator adaptor molecule may allow for longer partially closed linear DNA products to be produced. Terminator adaptor molecules may allow for bar codes, promotor sequences, labels, poly A signals and / or open reading frames to be incorporated into the partially closed linear DNA product of the invention. A terminal adaptor molecule may comprise a label, a signalling sequence, a targeting sequence or a binding moiety. A terminal adaptor molecule may comprise a UTR or a terminator sequence.

[0377] The step of incubating the single contiguous aqueous volume may be performed under conditions that promote appending (or linking) of the first and second terminal adaptor molecules and / or intermediate adaptor molecules to the linear double-stranded region to produce the partially closed linear DNA product. The appending may be performed by creating a covalent link between the first and / or second terminal adaptor molecule and / or n+m intermediate adaptor molecules and the first and / or second end of the linear double-stranded region. The appending may be performed by creating a covalent link between the first and / or second terminal adaptor molecule and an nth and / or mth intermediate adaptor molecule. The appending may be performed by creating a covalent link between an intermediate adaptor molecule and the first or second end of the linear double-stranded region. The appending may be performed by creating a covalent link between an intermediate adaptor molecule and an adjacent adaptor molecule.

[0378] The step of incubating the single contiguous aqueous volume may be performed under conditions that promote digestion of the double-stranded DNA molecule to produce the linear portion of the doublestranded DNA molecule. The digestion of the double-stranded DNA molecule to produce the linear portion of the double-stranded DNA molecule may be performed at a first temperature of 1°C-100°C, 1°C -80°C, 5°C-70°C, 10°C-60°C, 15°C-55°C, 20°C-50°C, 25°C -45°C, 30°C-40°C, 35°C-39°C, 36°C- 38°C, or at about 37°C. The digestion may be endonuclease digestion, preferably Type IIS endonuclease digestion.

[0379] The step of incubating the single contiguous aqueous volume may be performed under conditions that promote ligation of the linear double-stranded region to the first and second terminal adaptor molecules. The ligation may be at least 5%, at least 10%, at least 15, at least 20%, at least 25%, at least 30%, at least 35%, at least 40, at least 45%, at least 50%, at least 55%, at least 60, at least 65, at least 70%, at least 75, at least 80%, at least 82%, at least 85%, at least 90%, or at least 95% efficient. For example, at least 5%, at least 10%, at least 15, at least 20%, at least 25%, at least 30%, at least 35%, at least 40, at least 45%, at least 50%, at least 55%, at least 60, at least 65, at least 70%, at least 75, at least 80%, at least 82%, at least 85%, at least 90%, or at least 95% of the linear double-stranded regions (or the portions of the double-stranded DNA molecules) may be incorporated into closed linear DNA products. Preferably, the ligation is at least 15% efficient.

[0380] The step of ligation of the linear double-stranded region to the first and second terminal adaptor molecules may be performed at a second temperature of 1°C -90°C, 2°C -70°C, 5°C-60°C, 8°C-55°C, 9°C-50°C, 10°C-45°C, 11°C-40°C, 12°C-37°C, 13°C-30°C, 14°C-25°C, 15°C-20°C or at about 16°C.

[0381] The step of incubating the single contiguous aqueous volume may comprise incubating at a first temperature and then incubating at a second temperature. The first temperature may be 1°C-100°C, 1°C-80°C, 5°C-70°C, 10°C-60°C, 15°C-55°C, 20°C-50°C, 25°C-45°C, 30°C-40°C, 35°C-39°C, 36°C- 38°C, or about 37°C. The second temperature may be 1°C -90°C, 2°C -70°C, 5°C-60°C, 8°C-55°C, 9°C-50°C, 10°C-45°C, 11°C-40°C, 12°C-37°C, 13°C-30°C, 14°C-25°C, 15°C-20°C or at about 16°C. Preferably, the first temperature is 35°C-39°C. Preferably, the second temperature is 14°C-18°C. The step of incubating the single contiguous aqueous volume may comprise cycling between the first temperature and the second temperature. The step of incubating the single contiguous aqueous volume may comprise cycling between the first temperature and the second temperature at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 80, at least 90, or at least 100 times, preferably at least 20 times. The step of incubating the single contiguous aqueous volume may comprise cycling between the first temperature and the second temperature less than 40, less than 35, less than 30 times, less than 29, less than 25 times. The step of incubating the single contiguous aqueous volume may comprise cycling between the first temperature and the second temperature 2-100, 5-80, 10-70, 20-60, or 30-60 times. The step of incubating the single contiguous aqueous volume may comprise cycling between the first temperature and the second temperature 2-20, 5-29, 61 -100, or 65-80 times.

[0382] The step of incubating the single contiguous aqueous volume may be performed isothermally. The step of incubating the single contiguous aqueous volume may comprise incubating at a constant temperature. The constant temperature promotes simultaneous digestion of the double-stranded DNA molecule to produce the linear portion of the double-stranded DNA molecule and ligation of the linear double-stranded region to the first and second terminal adaptor molecules. For example, the constant temperature may be 20°C, 21 °C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31 °C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, or 40°C. Preferably, the constant temperature is 30°C. The constant temperature is intended to mean that the temperature does not significantly change during the reaction. The constant temperature is intended to mean that the temperature variation during the step of incubating the single contiguous aqueous volume is less than 10°C, less than 9°C, less than 8°C, less than 7°C, less than 6°C, less than 5°C, less than 4°C, less than 3°C, less than 2°C, or less than 1 °C. In a preferred embodiment the temperature during the step of incubating the single contiguous aqueous does not deviate by more than 5°C, preferably by not more than 3°C, even more preferably not more than 1 °C. Thus, the constant temperature may be a temperature in a range of 20°C-30°C, 22°C-32°C, 24°C-34°C, 26°C-36°C, 28°C-38°C, 30°C-40°C, 22°C-28°C, 32°C-38°C, 25°C- 35°C, 26°C-34°C, 27°C- 33°C, 27.5°C-32.5°C, 28°C-32°C, 28.5°C-31 ,5°C, 29°C-31 °C, or 29.5°C- 30.5°C. Preferably, the constant temperature is a temperature in a range of 27.5°C-32.5°C.

[0383] Alternatively, the constant temperature may be a temperature in a range of 32°C-42°C, 33°C-41 °C, 34°C-40°C, 35°C-39°C, 36°C-38°C. Preferably, the constant temperature is a temperature in a range of 34.5°C-39.5°C.

[0384] The first and second terminal adaptor molecules may comprise one or more phosphorothioated nucleotides, such that, once the adaptor molecules are appended (e.g. ligated) to the linear doublestranded region, the partially closed linear DNA product is resistant to nuclease digestion or has improved or enhanced resistance to nuclease digestion. The partially closed linear DNA product may be resistant to 3’-end exonuclease digestion (e.g. by exonuclease III) and / or 5’-end exonuclease digestion (e.g. by exonuclease VIII). A terminal or intermediate adaptor molecule may comprise a plurality of phosphorothioated nucleotides. For example, the terminal or intermediate adaptor molecule may comprise at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 1 1 , at least 12, at least 13, at least 14, at least 15 or at least 16 phosphorothioated nucleotides in each strand.

[0385] A terminal or intermediate adaptor molecule may be a nucleic acid adaptor molecule. The terminal or intermediate adaptor molecule may be double-stranded. The terminal or intermediate adaptor molecule may comprise a portion that is double-stranded.

[0386] The first and / or second terminal adaptor molecules and / or one or more of n+m intermediate adaptor molecules may comprise at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 1 1 , at least 12, at least 13, at least 14, at least 15, or at least 16 base pairs. The first and / or second terminal adaptor molecules and / or one or more of n+m intermediate adaptor molecules may comprise at least 1000 base pairs, at least 750 base pairs, at least 500 base pairs, at least 250 base pairs, at least 200 base pairs, at least 150 base pairs, at least 100 base pairs or at least 75 base pairs.

[0387] The terminal or intermediate adaptor molecule may comprise a plurality of phosphorothioated nucleotides in each strand. For example, the terminal or intermediate adaptor molecules may comprise at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 1 1 , at least 12, at least 13, at least 14, at least 15 or at least 16 phosphorothioated nucleotides in each strand.

[0388] The terminal or intermediate adaptor molecule may comprise a plurality of phosphorothioated nucleotides at internal positions in each strand. For example, the terminal or intermediate adaptor molecules may comprise at least 1 , at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 1 1 , at least 12, at least 13, at least 14, at least 15 or at least 16 phosphorothioated nucleotides at internal positions in each strand. Preferably, the terminal or intermediate adaptor molecule comprises at least 2 phosphorothioated nucleotides at internal positions in each strand.

[0389] The internal positions may not be located between the second and penultimate nucleotide of the adaptor molecule. The internal positions may be any position in the adaptor molecules other than the last nucleotide at the end of each strand.

[0390] The terminal or intermediate adaptor molecule may comprise at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 100% of protected nucleotides. Once the adaptor molecules are appended to the linear double-stranded region, the partially closed linear DNA product may comprise a protected nucleotide (e.g. phosphorothioated nucleotide) at the 5’-end (or at the 5’-end region) of one or both strands. Preferably, the partially closed linear DNA product comprises a phosphorothioated nucleotide at the 5’-end (or at the 5’-end region) of one strand. The partially closed linear DNA product may comprise a phosphorothioated nucleotide at the 5’-end (or at the 5’-end region) of one strand. As most exonucleases, for example exonuclease III, remove nucleotides from the 3’-end of the polynucleotide chain, the linear DNA product may comprise a protected nucleotide at the 3’-end (or at the 3’-end region) of one strand. Preferably, the partially closed linear DNA product comprises a phosphorothioated nucleotide at the 3’-end (or the 3’-end region) of one strand. The partially closed linear DNA product may comprise at least one phosphorothioated nucleotide at the 3’-end (or the 3’-end region) and at least one phosphorothioated nucleotide at the 5’-end (or the 5’-end region) of one strand. The partially closed linear DNA product may comprise at least one phosphorothioated nucleotide at the 3’-end (or the 3’-end region) of the sense strand and the 5’-end (or the 5’-end region) if the antisense strand. The partially closed linear DNA product may comprise at least one phosphorothioated nucleotide at the 5’-end (or the 5’-end region) of the sense strand and the 3’-end (or the 3’-end region) if the antisense strand. Thus, both the sense and antisense strands in the double stranded partially closed linear DNA product may be protected from nuclease digestion by using nuclease-resistant nucleotides at one end of the partially closed linear DNA product.

[0391] One of the terminal adaptor molecules used in the methods described herein may comprise a self- complementary element which creates a loop, such as a hairpin loop or a stem loop. Thus, the one of the terminal adaptor molecules may comprise a hairpin or a stem-loop. The terminal adaptor molecule may comprise a double-stranded portion comprising a sense strand and an antisense strand, wherein the sense strand and the antisense strand are linked together in a hairpin such that the sense strand is hybridized to the antisense strand. The double-stranded portion of a terminal adaptor may comprise a 3’ overhang or a 5’ overhang of at least 1 , at least 2, at least 3, at least 4, or at least 5 nucleotides. Preferably the 3’ overhang or the 5’ overhang is 4-8 nucleotides. Each end of the linear doublestranded region (or linear portion of the double-stranded DNA molecule) may comprise a 3’ or a 5’ overhang. A portion of the terminal adaptor molecule (e.g. the overhang) may be complementary to the first end or the second end of the linear double-stranded region.

[0392] The method described herein may use a first terminal adaptor molecule which comprises hairpin loop or stem loop, or any other structure which is able to close one end of the linear DNA molecule, and a second terminal adaptor molecule which is a linear nucleic acid molecule comprising nuclease- resistant nucleotides to produce a partially closed linear DNA product.

[0393] The partially closed linear DNA product may be a partially covalently closed DNA product. Thus, in embodiments where the first terminal adaptor molecule comprise a loop (e.g. a hairpin), the terminal adaptor molecule closes one end of the linear double-stranded region forming a partially covalently closed DNA product.

[0394] The first terminal adaptor molecule may comprise a single-stranded portion. The single-stranded portion may form a hairpin or a stem-loop. The single-stranded portion may comprise less than 10, 9, 8, 7, 6, 5, 4, 3, 2 nucleotides. Preferably, the single-stranded portion comprises 5 nucleotides.

[0395] The first terminal adaptor molecule may comprise or consist of the sequence of SEQ ID NO: 9 or a portion thereof. The first terminal adaptor molecule may comprise at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, or at least 19 contiguous nucleotides of SEQ ID NO: 9. The doublestranded portion of the first terminal adaptor molecule may comprise the sequence of SEQ ID NO: 10 or a portion thereof. The double-stranded portion of the first and / or second terminal adaptor molecule may comprise at least 8, at least 9, at least 10, at least 1 1 , at least 12, at least 13, at least 14 or at least 15 contiguous nucleotides of SEQ ID NO: 10. The single-stranded portion of the first terminal adaptor molecule may comprise a sequence of ACTCA. The single-stranded portion of the first terminal adaptor molecule may comprise at least 1 , at least 2, at least 3, at least 4 or at least 5 contiguous nucleotides of the sequence ACTCA. The first terminal adaptor molecule may comprise the sequence of SEQ ID NO: 12. The first terminal adaptor molecule may comprise at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, or at least 19 contiguous nucleotides of SEQ ID NO:12.

[0396] The second terminal adaptor molecule may comprise or consist of the sequences of SEQ ID NO:13 and / or SEQ ID NO:14. The second terminal adaptor molecule may comprise or at least 15, 14, 13, 12, 1 1 , 10, 19, 8, 7, 6, 5, contiguous nucleotides thereof.

[0397] The partially closed linear DNA product may additionally comprise a plurality of protected nucleotides (e.g. phosphorothioated nucleotides) at internal positions in each strand. For example, the linear DNA product may comprise at least 2, at least 4, at least 6, at least 8, at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 125, at least 150, at least 175, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, or at least 500 protected nucleotides (e.g. phosphorothioated nucleotides) at internal positions in each strand. Preferably, the partially closed linear DNA product comprises at least 2 protected nucleotides (e.g. phosphorothioated nucleotides) at internal positions in each strand.

[0398] The internal positions may not be located between the second and penultimate nucleotide of the partially closed linear DNA product. The internal positions may be any position in the adaptor molecules other than the last nucleotide at the end of each strand.

[0399] The linear double-stranded region (or linear portion of the double-stranded molecule) may comprise a plurality of phosphorothioated nucleotides at internal positions in each strand. For example, the linear double-stranded region (or linear portion of the double-stranded molecule) may comprise at least 2, at least 4, at least 6, at least 8, at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 125, at least 150, at least 175, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, or at least 500 protected nucleotides (e.g. phosphorothioated nucleotides) at internal positions in each strand. Preferably, the linear double-stranded region (or linear portion of the double-stranded molecule) comprises at least 2 protected nucleotides (e.g. phosphorothioated nucleotides) at internal positions in each strand. The internal positions may not be located between the second and penultimate nucleotide of the linear double-stranded region (or linear portion of the double-stranded molecule).

[0400] The nucleotides resistant to exonuclease digestion (i.e. protected nucleotides) may be phosphorothioated nucleotides of at least one type. For example, the at least one type of phosphorothioated nucleotides is a-S-dATP (i.e. 2’-deoxyadenosine-5’-(a-thio)-triphosphate), a-S- dCTP (i.e. 2’-deoxycytidine-5’-(a-thio)-triphosphate), a-S-dGTP (i.e. 2’-deoxyguanosine-5’-(a-thio)- triphosphate), a-S-dTTP (i.e. 2’-deoxythymidine-5’-(a-thio)-triphosphate), a-S-dUTP (i.e. 2’- deoxyuridine-5’-(a-thio)-triphosphate), and / or uridine 2’, 3’-cyclophosphorothioate.

[0401] The terminal or intermediate adaptor molecules may comprise at least two types of phosphorothioated nucleotides. For example, the at least two types of phosphorothioated nucleotides are: a-S-dATP and a-S-dCTP, a-S-dATP and a-S-dGTP, a-S-dATP and a-S-dTTP, a-S-dCTP and a-S-dGTP, a-S-dCTP and a-S-dTTP, or a-S-dGTP and a-S-dTTP.

[0402] The terminal or intermediate adaptor molecules may comprise at least three types of phosphorothioated nucleotides. For example, the at least three types of phosphorothioated nucleotides are:

[0403] (i) a-S-dATP, a-S-dCTP and a-S-dGTP;

[0404] (j) a-S-dATP, a-S-dCTP and a-S-dTTP;

[0405] (k) a-S-dATP, a-S-dGTP and a-S-dTTP; or

[0406] (l) a-S-dCTP, a-S-dGTP and a-S-dTTP.

[0407] The adaptor molecules may comprise at least four types of phosphorothioated nucleotides. For example, the at least four types of protected nucleotides are a-S-dATP, a-S-dCTP, a-S-dGTP and a-S- dTTP.

[0408] The phosphorothioated nucleotides may be Sp-isomers, Rp-isomers or a mixture of both Sp- and Rp- isomers.

[0409] The nucleotides resistant to exonuclease digestion (i.e. protected nucleotides) may be MOE nucleotides of at least one type, or at least two, three or four types. For example, the MOE nucleotides may be 2’- O-methoxy-ethyl guanosine, 2’-O-methoxy-ethyl cytidine, 2’-O-methoxy-ethyl adenosine, and / or 2’-O- methoxy-ethyl thymidine. The method may further comprise, before step (a) (i.e. the step of contacting the double-stranded DNA molecule with the endonuclease, the ligase and the first and second terminal adaptor molecules), a step of amplifying a DNA template molecule to produce the double-stranded DNA molecule. Thus, the invention provides a method for producing a partially closed linear DNA product, the method comprises:

[0410] (a) amplifying a DNA template molecule comprising at least one cleavable (e.g. endonuclease) target sequence to generate a double-stranded DNA molecule;

[0411] (b) contacting the double-stranded DNA molecule with an endonuclease, a ligase and first and second terminal adaptor molecules and n+m intermediate adaptor molecules, to form a single contiguous aqueous volume, wherein n and m are each 0 or an integer of at least 1 , and wherein n+m is at least 1 ; and

[0412] (c) incubating the single contiguous aqueous volume to generate the partially closed linear DNA product, wherein the partially closed linear DNA product comprises a linear double-stranded region, wherein the linear double-stranded region comprises a linear portion of the double-stranded DNA molecule, and wherein n intermediate adaptor molecules are sequentially appended to a first end of the linear double stranded region and m intermediate adaptor molecules are sequentially appended to a second end of the linear double-stranded region, and wherein the partially closed DNA product is closed at a first end by the first terminal adaptor molecule appended to the nth intermediate adaptor molecule or, when n is 0, to the first end of the linear double stranded region, and the second terminal adaptor molecule is appended to the mth intermediate adaptor molecule, or when m is 0, to the second end of the linear double stranded region, and wherein the second terminal adaptor molecule is a nucleic acid molecules that comprise one or more nuclease-resistant nucleotides (i.e. protected nucleotides).

[0413] The step of amplifying may be performed by in vitro or in vivo amplification. Preferably, the step of amplifying is performed by in vitro amplification. For example, the step of amplifying may be performed by rolling circle amplification (RCA), MALBAC method, traditional polymerase chain reaction (PCR), nucleic acid sequence-based amplification (NASBA), loop-mediated isothermal amplification (LAMP), helicase-dependent amplification (HDA), multiple displacement amplification (MDA) and recombinase polymerase amplification (RPA). Preferably, the step of amplifying is performed by rolling circle amplification. Thus, the invention provides a method for producing a partially closed linear DNA product, the method comprises:

[0414] (a) amplifying a DNA template molecule comprising at least one cleavable (e.g. endonuclease) target sequence to generate a double-stranded DNA molecule, wherein the DNA template molecule is amplified by rolling circle amplification;

[0415] (b) contacting the double-stranded DNA molecule with an endonuclease, a ligase and first and second terminal adaptor molecules and n+m intermediate adaptor molecules, to form a single contiguous aqueous volume, wherein n and m are each 0 or an integer of at least 1 , and wherein n+m is at least 1 ; and (c) incubating the single contiguous aqueous volume to generate the partially closed linear DNA product, wherein the partially closed linear DNA product comprises a linear double-stranded region, wherein the linear double-stranded region comprises a linear portion of the double-stranded DNA molecule, and wherein n intermediate adaptor molecules are sequentially appended to a first end of the linear double stranded region and m intermediate adaptor molecules are sequentially appended to a second end of the linear double-stranded region, and wherein the partially closed DNA product is closed at a first end by the first terminal adaptor molecule appended to the nth intermediate adaptor molecule or, when n is 0, to the first end of the linear double stranded region, and the second terminal adaptor molecule is appended to the mth intermediate adaptor molecule, or when m is 0, to the second end of the linear double stranded region, and wherein the second terminal adaptor molecule is a nucleic acid molecules that comprise one or more nuclease-resistant nucleotides (i.e. protected nucleotides).

[0416] Rolling circle amplification may be performed without any primers, or in the presence of a primer or multiple primers. For example, the primer may be a synthetic primer. The primers may be random primers. Rolling circle amplification may be performed in the presence of a primase. The primase may be Tt / iPrimPol. Preferably, if the rolling circle amplification is performed without any primers, it is performed in the presence of a primase, such as Tt / iPrimPol. Similarly, if a primer is used during amplification reaction, a primase is not used. The double-stranded DNA product may be generated by the rolling circle amplification in vitro under isothermal conditions using a suitable nucleic acid polymerase, such as Phi29 DNA polymerase.

[0417] In the methods described herein, the DNA template molecule may comprise at least one cleavable target sequence. The cleavable target sequence may be an endonuclease target sequence. Thus, the DNA template molecule may comprise at least one endonuclease target sequence. Preferably, the DNA template molecule comprises at least two endonuclease target sequences. The endonuclease target sequences may be the same or different. Preferably, the at least one endonuclease target sequence is a restriction endonuclease target sequence. Different restriction endonuclease target sequences would be known to the skilled person. The cleavable target sequence may be a Type IIS restriction endonuclease target sequence. For example, the restriction endonuclease target sequence may be a Bbsl, Bsal, BsmBI, BspQI, BtgZI, Esp3l,Sapl, Aarl, Acc36l, AcIWI, Acul, Ajul, Alol, Alw26l, Alwl, Arsl, AsuHPI, Bael, Bari, Bbvl, Bccl, BceAl, Bcgl, BciVI, BcoDI, BfuAI, Bful, Bmrl, Bmsl, Bmul, Bpil, Bpml, BpuEl, BsaXI, Bse1 1, Bse3DI, BseGI, BseMI, BseMII, BseNI, BseRI, BseXI, Bsgl, BsIFI, BsmAI, BsmFI, Bsml, Bso31 l, BspCNI, BspMI, BspPI, BspQI, BspTNI, BsrDI, Bsrl, Bst6l, BstF5l, BstMAI, BstV1 1, BstV2l, Bsul, BtgZI, BtsCI, Btsl-v2, BtsMutl, Bvel, Csel, CspCI, Eam1 104l, Earl, Ecil, Eco31 l, Eco57l, Esp3l, Faql, Faul, Fokl, Gsul, Hgal, Hphl, HpyAV, Lgul, Lmnl, Lsp1 109I, Lwel, Mboll, Mly I , Mmel, Mn 11 , Mva1269l, NmeAIII, PaqCI, PciSI, Pctl, Piel, Ppsl, Psrl, Schl, SfaNI, Taqll, TspDTI and / or TspGWI target sequence. The at least one cleavable sequence (e.g. endonuclease target sequence) may be a native cleavable sequence (i.e. a cleavable sequence present in the template molecule). Alternatively, the at least one cleavable sequence (e.g. endonuclease target sequence) may be introduced to the DNA template molecule prior to the production of the partially closed linear DNA product.

[0418] The endonuclease may be a restriction enzyme endonuclease. The endonuclease may be a Type IIS restriction enzyme. The endonuclease may be any enzyme that recognizes a DNA sequence and cleaves outside of the recognition sequence. For example, the endonuclease may be a Bbsl, Bsal, BsmBI, BspQI, BtgZI, Esp3l,Sapl, Aarl, Acc36l, AcIWI, Acul, Ajul, Alol, Alw26l, Alwl, Arsl, AsuHPI, Bael, Bari, Bbvl, Bccl, BceAl, Bcgl, BciVI, BcoDI, BfuAI, Bful, Bmrl, Bmsl, Bmul, Bpil, Bpml, BpuEl, BsaXI, Bsel l, Bse3DI, BseGI, BseMI, BseMII, BseNI, BseRI, BseXI, Bsgl, BsIFI, BsmAI, BsmFI, Bsml, Bso31 l, BspCNI, BspMI, BspPI, BspQI, BspTNI, BsrDI, Bsrl, Bst6l, BstF5l, BstMAI, BstV1 1, BstV2l, Bsul, BtgZI, BtsCI, Btsl-v2, BtsMutl, Bvel, Csel, CspCI, Eam1 104l, Earl, Ecil, Eco31 l, Eco57l, Esp3l, Faql, Faul, Fokl, Gsul, Hgal, Hphl, HpyAV, Lgul, Lmnl, Lspt 109I, Lwel, Mboll, Mlyl, Mmel, Mnll, Mva1269l, NmeAIII, PaqCI, PciSI, Pctl, Piel, Ppsl, Psrl, Schl, SfaNI, Taqll, TspDTI and / or TspGWI restriction enzyme.

[0419] The ligase may be a DNA ligase, such as a T4 DNA ligase, T7 DNA ligase, mammalian DNA ligase I, III and IV; Taq DNA ligase, Tth DNA ligase, or E. coli DNA ligase.

[0420] The DNA template molecule used in the methods described herein may be single-stranded or doublestranded. Preferably, the DNA template molecule is double-stranded. The DNA template molecule may be a natural circular DNA molecule. For example, the DNA template molecule may be (i) a plasmid, (ii) a minicircle, (Hi) a cosmid, (iv) a bacterial artificial chromosome (BAC), or (v) a molecular inversion probe (MIP). The DNA template molecule may be an enzymatically produced circular DNA molecule. For example, the DNA template molecule may be (i) a circular DNA molecule obtained from recombinase reaction, preferably Cre recombinase reaction, or (ii) a circular DNA molecule obtained from ligase reaction, preferably using the golden gate assembly. The DNA template molecule may be an enzymatically produced covalently-closed linear DNA molecule. For example, the DNA template molecule may be (i) a DNA molecule processed with TelN protelomerase; or (ii) a DNA molecule generated by ligation of the DNA ends with an adaptor. The DNA template molecule may comprise an element that is double-stranded and an element that is single-stranded. For example, the template DNA molecule may comprise a double-stranded DNA and a single-stranded hairpin loop.

[0421] The DNA template molecule may be linear. If the DNA template molecule is linear, prior to amplification (e.g. rolling circle amplification), a DNA template molecule may be circularized to produce a DNA template molecule suitable for use in the methods described herein.

[0422] The template DNA molecule may comprise a cassette. The cassette may be a mammalian expression cassette. The cassette may further comprise a promoter. The promoter may be a CMV promoter. The cassette may further comprise an enhancer. The cassette may further comprise a reporter gene, such as an eGFP reporter gene or a luciferase reporter gene. The cassette may further comprise a homopolymeric sequence. The cassette may further comprise a LoxP sequence, preferably two LoxP sequences. If the two LoxP sequences are oriented in the same direction, the DNA sequence between the two LoxP sequences is excised as a circular loop of DNA. If the two LoxP sequences are oriented in the opposite direction, the DNA sequence between the two LoxP sequences is inverted. Thus, preferably, the two LoxP sequences are in the same orientation in the template DNA molecule.

[0423] The DNA template molecule may comprise a homopolymeric sequence at a 5’-end or a 3’-end or both a 5’-end and a 3’-end. The homopolymeric sequence may be added to the DNA template molecule before circularization. The homopolymeric sequence may be a polyA, a polyC, a polyG, or a polyT sequence. The homopolymeric sequence may be between 3-200 nucleotides in length. The homopolymeric sequence may be used to facilitate purification of the partially closed linear DNA product, in which case, the homopolymeric sequence may be between 4-12 nucleotides in length, or between 5-10 nucleotides in length. The homopolymeric sequence may be used to improve mRNA expression, in which case, the homopolymeric sequence may be between 10-200 nucleotides in length, preferably between 80-150 nucleotides in length. The homopolymeric sequence may be at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 1 10, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, or at least 200 nucleotides in length. Preferably, the homopolymeric sequence is at least 100 nucleotides in length. More preferably still, the homopolymeric sequence is at least 120 nucleotides in length. For example, the homopolymeric sequence may comprise a polyA sequence of at least 120 nucleotides.

[0424] The method may further comprise, after the step of incubating the single contiguous aqueous volume, a step of purification of the partially closed linear DNA product.

[0425] The method may further comprise, after the step of incubating the single contiguous aqueous volume, a step of nuclease digestion. The nuclease digestion may be exonuclease digestion, such as exonuclease I and / or exonuclease III digestion. The step of nuclease digestion may take place before or after the step of purification. This step allows for removal of any double-stranded DNA molecules and / or adaptor molecules which have not been used in the course of performing the method. Thus, the method may comprise the steps:

[0426] (a) amplifying a DNA template molecule comprising at least one cleavable (e.g. endonuclease) target sequence to generate a double-stranded DNA molecule;

[0427] (b) contacting the double-stranded DNA molecule with an endonuclease, a ligase and first and second terminal adaptor molecules and n+m intermediate adaptor molecules, to form a single contiguous aqueous volume, wherein n and m are each 0 or an integer of at least 1 , and wherein n+m is at least 1 ;

[0428] (c) incubating the single contiguous aqueous volume to generate the partially closed linear DNA product, wherein the partially closed linear DNA product comprises a linear double-stranded region, wherein the linear double-stranded region comprises a linear portion of the double-stranded DNA molecule, and wherein n intermediate adaptor molecules are sequentially appended to a first end of the linear double stranded region and m intermediate adaptor molecules are sequentially appended to a second end of the linear double-stranded region, and wherein the partially closed DNA product is closed at a first end by the first terminal adaptor molecule appended to the nth intermediate adaptor molecule or, when n is 0, to the first end of the linear double stranded region, and the second terminal adaptor molecule is appended to the mth intermediate adaptor molecule, or when m is 0, to the second end of the linear double stranded region, and wherein the second terminal adaptor molecule is a nucleic acid molecules that comprise one or more nuclease-resistant nucleotides (i.e. protected nucleotides); and

[0429] (d) incubating the single contiguous aqueous volume with a nuclease (e.g. exonuclease).

[0430] The method may comprise the steps:

[0431] (a) amplifying a DNA template molecule comprising at least one cleavable (e.g. endonuclease) target sequence to generate a double-stranded DNA molecule;

[0432] (b) contacting the double-stranded DNA molecule with an endonuclease, a ligase and first and second terminal adaptor molecules and n+m intermediate adaptor molecules, to form a single contiguous aqueous volume, wherein n and m are each 0 or an integer of at least 1 , and wherein n+m is at least 1 ;

[0433] (c) incubating the single contiguous aqueous volume to generate the partially closed linear DNA product, wherein the partially closed linear DNA product comprises a linear double-stranded region, wherein the linear double-stranded region comprises a linear portion of the double-stranded DNA molecule, and wherein n intermediate adaptor molecules are sequentially appended to a first end of the linear double stranded region and m intermediate adaptor molecules are sequentially appended to a second end of the linear double-stranded region, and wherein the partially closed DNA product is closed at a first end by the first terminal adaptor molecule appended to the nth intermediate adaptor molecule or, when n is 0, to the first end of the linear double stranded region, and the second terminal adaptor molecule is appended to the mth intermediate adaptor molecule, or when m is 0, to the second end of the linear double stranded region, and wherein the second terminal adaptor molecule is a nucleic acid molecules that comprise one or more nuclease-resistant nucleotides (i.e. protected nucleotides);

[0434] (d) purifying the closed linear DNA product; and

[0435] (e) incubating the purified product of step (d) with a nuclease (e.g. exonuclease).

[0436] The method may comprise the steps:

[0437] (a) amplifying a DNA template molecule comprising at least one cleavable (e.g. endonuclease) target sequence to generate a double-stranded DNA molecule;

[0438] (b) contacting the double-stranded DNA molecule with an endonuclease, a ligase and first and second terminal adaptor molecules and n+m intermediate adaptor molecules, to form a single contiguous aqueous volume, wherein n and m are each 0 or an integer of at least 1 , and wherein n+m is at least 1 ; (c) incubating the single contiguous aqueous volume to generate the partially closed linear DNA product, wherein the partially closed linear DNA product comprises a linear double-stranded region, wherein the linear double-stranded region comprises a linear portion of the double-stranded DNA molecule, and wherein n intermediate adaptor molecules are sequentially appended to a first end of the linear double stranded region and m intermediate adaptor molecules are sequentially appended to a second end of the linear double-stranded region, and wherein the partially closed DNA product is closed at a first end by the first terminal adaptor molecule appended to the nth intermediate adaptor molecule or, when n is 0, to the first end of the linear double stranded region, and the second terminal adaptor molecule is appended to the mth intermediate adaptor molecule, or when m is 0, to the second end of the linear double stranded region, and wherein the second terminal adaptor molecule is a nucleic acid molecules that comprise one or more nuclease-resistant nucleotides (i.e. protected nucleotides);

[0439] (d) incubating the single contiguous aqueous volume with a nuclease (e.g. exonuclease); and

[0440] (e) purifying the closed linear DNA product.

[0441] The step of incubating the single contiguous aqueous volume (or the purified product of step (d)) with a nuclease may be performed at a temperature of 5-90°C, 10-80°C, 15-70°C, 20-60°C, 25-50°C, 30- 45°C or 35-40°C. The step of incubating the single contiguous aqueous volume (or the purified product of step (d)) with a nuclease may be performed for at least 10, at least 20, at least 30, at least 40, at least 50, or at least 60 min. The step of incubating the single contiguous aqueous volume (or the purified product of step (d)) may be performed at two different temperatures. For example, the step of incubating the single contiguous aqueous volume (or the purified product of step (d)) may be performed at 15- 40°C for 10-60 minutes followed by a temperature of 60-90°C for 10-30 min. The higher temperature typically inactivates the nuclease (e.g. exonuclease). Thus, the method further provides a step of inactivating the nuclease (e.g. exonuclease). The step of incubating the single contiguous aqueous volume (or the purified product of step (d)) may be performed at 37°C for 30 min and 80°C for 20 min. Preferably, the step of inactivating the nuclease (e.g. exonuclease) is performed at a temperature of 70-80°C. The step of inactivating the nuclease (e.g. exonuclease) may be performed for at least 1 , at least 5, at least 10, at least 20 or at least 30 minutes. Preferably, the step of inactivating the nuclease (e.g. exonuclease) is performed for at least 5 minutes.

[0442] The first terminal adaptor molecule and / or the second terminal adaptor molecule may comprise an overhang. The end of the linear double-stranded region may comprise a 3’ or a 5’ overhang. A portion of the first terminal adaptor molecule (e.g. the overhang) may be complementary to the first end of the linear double-stranded region. A portion of the second terminal adaptor molecule may be complementary to the second end of the linear double-stranded region.

[0443] The first end and the second end of the linear double-stranded region may be resistant to nuclease digestion. Preferably, the first end and the second end of the linear double-stranded region are resistant to the exonuclease digestion, such as exonuclease III digestion and / or exonuclease I digestion.

[0444] The linear DNA product may be partially double-stranded and / or partially single-stranded. The linear DNA product may comprise a portion that is double-stranded and a portion that is single-stranded.

[0445] The partially closed linear DNA product may comprise a cassette. The cassette may comprise a coding sequence. The coding sequence may encode a gene of interest, for example a gene encoding a protein. The cassette may comprise at least a portion of a promoter and a coding sequence. The cassette may comprise a promoter and a coding sequence. The cassette may comprise a promoter, a coding sequence, a ribosomal binding site and a translational termination sequence. The cassette may additionally comprise sequences aiding protein expression, such as a cap-independent translation element. The cassette may comprise (or encode) a repair template (or editing template). The repair template (or editing template) may be for use in CRISPR-Cas mediated homology directed repair (HDR). The cassette may encode CRISPR guide RNA. The cassette may be a mammalian expression cassette. The promoter may be a CMV promoter. The cassette may further comprise an enhancer. The cassette may further comprise a reporter gene, such as an eGFP reporter gene or a luciferase reporter gene. The cassette may further comprise a homopolymeric sequence, such as a polyA, poly C, polyT or polyG sequence. The homopolymeric sequence may be between 3-200 nucleotides in length. The homopolymeric sequence may be used to facilitate purification of the cassette, in which case, the homopolymeric sequence may be between 4-12 nucleotides in length, or between 5-10 nucleotides in length. The homopolymeric sequence may be used to improve mRNA expression, in which case, the homopolymeric sequence may be between 10-200 nucleotides in length, preferably between 80-150 nucleotides in length. The homopolymeric sequence may be at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 1 10, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, or at least 200 nucleotides in length. Preferably, the homopolymeric sequence is at least 100 nucleotides in length. More preferably still, the homopolymeric sequence is at least 120 nucleotides in length. For example, the homopolymeric sequence may comprise a polyA sequence of at least 120 nucleotides.

[0446] The partially closed linear DNA product may comprise a spacer. The spacer may be at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 125, at least 150, at least 175, or at least 200 base pairs long. The spacer may improve ligation efficiency of the first and second terminal adaptor molecules to the linear double-stranded region. The spacer may improve a cell transfection yields.

[0447] The partially closed linear DNA product may comprise an inverted terminal repeat sequence.

[0448] The partially closed linear DNA product may be at least 50, at least 100, at least 250, at least 500, at least 1000, at least 2000, at least 3000, at least 4000, at least 5000, at least 6000, at least 7000, at least 8000, at least 9000, at least 10,000, at least 1 1 ,000, at least 12,000, at least 13,000, at least 14,000, or at least 15,000 base pairs long. Preferably, the partially closed linear DNA product is at least 50 base pairs long.

[0449] The double-stranded DNA molecule may be circular or branched.

[0450] The double-stranded DNA molecule may not comprise an adaptor molecule. The double-stranded DNA molecule may not comprise a hairpin, a loop or a stem-loop structure.

[0451] The double-stranded DNA molecule may comprise a cassette. The cassette may comprise a coding sequence. The coding sequence may encode a gene of interest, for example a gene encoding a protein. The cassette may comprise at least a portion of a promoter and a coding sequence. The cassette may comprise a promoter and a coding sequence. The cassette may comprise a promoter, a coding sequence, a ribosomal binding site and a translational termination sequence. The cassette may additionally comprise sequences aiding protein expression, such as a cap-independent translation element. The cassette may comprise (or encode) a repair template (or editing template). The repair template (or editing template) may be for use in CRISPR-Cas mediated homology directed repair (HDR). The cassette may encode CRISPR guide RNA. The cassette may be a mammalian expression cassette. The promoter may be a CMV promoter. The cassette may further comprise an enhancer. The cassette may further comprise a reporter gene, such as an eGFP reporter gene or a luciferase reporter gene. The cassette may further comprise a homopolymeric sequence, such as a polyA, poly C, polyT or polyG sequence. The homopolymeric sequence may be between 3-200 nucleotides in length. The homopolymeric sequence may be used to facilitate purification of the cassette, in which case, the homopolymeric sequence may be between 4-12 nucleotides in length, or between 5-10 nucleotides in length. The homopolymeric sequence may be used to improve mRNA expression, in which case, the homopolymeric sequence may be between 10-200 nucleotides in length, preferably between 80-150 nucleotides in length. The homopolymeric sequence may be at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 1 10, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, or at least 200 nucleotides in length. Preferably, the homopolymeric sequence is at least 100 nucleotides in length. More preferably still, the homopolymeric sequence is at least 120 nucleotides in length. For example, the homopolymeric sequence may comprise a polyA sequence of at least 120 nucleotides.

[0452] The double-stranded DNA molecule may comprise a spacer. The spacer may be at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 125, at least 150, at least 175, or at least 200 base pairs long. The spacer may improve an amplification yield of the double-stranded DNA molecule. The spacer may improve ligation efficiency of the first and second terminal adaptor molecules to the linear double-stranded region. The spacer may improve a cell transfection yields. The double-stranded DNA molecule may be at least 50, at least 100, at least 250, at least 500, at least 1000, at least 2000, at least 3000, at least 4000, at least 5000, at least 6000, at least 7000, at least 8000, at least 9000, at least 10,000, at least 1 1 ,000, at least 12,000, at least 13,000, at least 14,000, or at least 15,000 base pairs long. Preferably, the double-stranded DNA molecule is at least 50 base pairs long.

[0453] The double-stranded DNA molecule may comprise one or more cleavable (e.g. endonuclease) target sequences. The double-stranded DNA molecule may comprise two cleavable (e.g. endonuclease) target sequences. The one or more endonuclease target sequences may be Type IIS endonuclease target sequences. The one or more endonuclease target sequences may be Bbsl, Bsal, BsmBI, BspQI, BtgZI, Esp3l,Sapl, Aarl, Acc36l, AcIWI, Acul, Ajul, Alol, Alw26l, Alwl, Arsl, AsuHPI, Bael, Bari, Bbvl, Bccl, BceAl, Bcgl, BciVI, BcoDI, BfuAI, Bful, Bmrl, Bmsl, Bmul, Bpil, Bpml, BpuEl, BsaXI, Bsel l, Bse3DI, BseGI, BseMI, BseMII, BseNI, BseRI, BseXI, Bsgl, BsIFI, BsmAI, BsmFI, Bsml, Bso31 l, BspCNI, BspMI, BspPI, BspQI, BspTNI, BsrDI, Bsrl, Bst6l, BstF5l, BstMAI, BstV11, BstV2l, Bsul, BtgZI, BtsCI, Btsl-v2, BtsMutl, Bvel, Csel, CspCI, Eam1 104l, Earl, Ecil, Eco31 l, Eco57l, Esp3l, Faql, Faul, Fokl, Gsul, Hgal, Hphl, HpyAV, Lgul, Lmnl, Lsp1 109I, Lwel, Mboll, Mlyl, Mmel, Mnll, Mva1269l, NmeAIII, PaqCI, PciSI, Pctl, Piel, Ppsl, Psrl, Schl, SfaNI, Taqll, TspDTI and / or TspGWI target sequences.

[0454] The double-stranded DNA molecule may be a product of amplification. Preferably, the amplification is rolling circle amplification.

[0455] The linear double-stranded region may be at least 50, at least 100, at least 250, at least 500, at least 1000, at least 2000, at least 3000, at least 4000, at least 5000, at least 6000, at least 7000, at least 8000, at least 9000, at least 10,000, at least 1 1 ,000, at least 12,000, at least 13,000, at least 14,000, or at least 15,000 base pairs long. Preferably, the double-stranded DNA molecule is at least 50 base pairs long.

[0456] The linear double-stranded region may comprise a 3’-OH group at first and / or second ends. The 3’-OH group may facilitate ligation to the first and / or second terminal adaptor molecule(s) and / or n+M intermediate adaptor molecules (which may comprise a 5’ phosphate). The linear double-stranded region may comprise a 5’ phosphate at first and / or second ends. The 5’ phosphate may facilitate ligation to the first and / or second terminal adaptor molecule(s) and / or intermediate adaptor molecules (which may comprise a 3’-OH group).

[0457] The linear double-stranded region (e.g. the linear portion of the double-stranded molecule) may comprise an overhang. For example, the linear double-stranded region may comprise a 5’ overhang or a 3’ overhang. The linear double-stranded region may comprise a blunt end or blunt ends. The linear double-stranded region may comprise: a 5’ overhang and a blunt end, two 5’ overhangs, a 3’ overhang and a blunt end, two 3’ overhangs, or a 5’ overhang and a 3’ overhang. The overhang may have at least 3 nucleotides (preferably from 4 to 8 nucleotides). The overhang may be in the sense strand or the antisense strand of the linear double-stranded region.

[0458] The linear portion of the double-stranded DNA molecule (e.g. the linear portion of the double-stranded molecule) may be at least 50, at least 100, at least 250, at least 500, at least 1000, at least 2000, at least 3000, at least 4000, at least 5000, at least 6000, at least 7000, at least 8000, at least 9000, at least 10,000, at least 11 ,000, at least 12,000, at least 13,000, at least 14,000, or at least 15,000 base pairs long. Preferably, double-stranded DNA molecule is at least 50 base pairs long.

[0459] The first terminal adaptor molecule and / or the second terminal adaptor molecule or one or more of n+m intermediate adaptor molecules may be a synthetic adaptor molecule.

[0460] The first terminal adaptor molecule and / or the second terminal adaptor molecule and / or one or more of n+m intermediate adaptor molecules may not be a plasmid or a vector DNA.

[0461] The adaptor molecule may comprise a double-stranded portion. The double-stranded portion may comprise less than 50, less than 45, less than 40, less than 35, less than 30, less than 25, less than 20, less than 15, or less than 10 base pairs. The double-stranded portion may comprise at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11 , at least 12, at least 13, at least 14, or at least 15 base pairs.

[0462] The adaptor molecule may comprise a 5’ phosphate. The 5’ phosphate may facilitate ligation to the linear double-stranded region.

[0463] The first terminal adaptor molecule may comprise a portion that is complementary to the first end of the linear double-stranded region (or the linear portion of the double-stranded DNA molecule). The second terminal adaptor molecule may comprise a portion that is complementary to the second end of the linear double-stranded region (or the linear portion of the double-stranded DNA molecule). The first terminal adaptor molecule may comprise a portion that anneals to the first end of the linear double-stranded region (or the linear portion of the double-stranded DNA molecule). The second terminal adaptor molecule may comprise a portion that anneals to the second end of the linear double-stranded region (or the linear portion of the double-stranded DNA molecule). The first terminal adaptor molecule may comprise a portion that is complementary and anneals to the first end of the linear double-stranded region (or the linear portion of the double-stranded DNA molecule). The second terminal adaptor molecule may comprise a portion that is complementary and anneals to the second end of the linear double-stranded region (or the linear portion of the double-stranded DNA molecule).

[0464] The portion that is complementary or anneals to the first or second end of the linear double-stranded region (or the linear portion of the double-stranded DNA molecule) may be a 5’ overhang or a 3’ overhang of the first and / or second terminal adaptor molecule. The overhang of the first terminal adaptor molecule may be complementary to the first end of the linear double-stranded region and / or the overhang of the second terminal adaptor molecule may be complementary to the second end of the double-stranded region. The overhang of the first terminal adaptor molecule may anneal to the first end of the linear double-stranded region and / or the overhang of the second terminal adaptor molecule may anneal to the second end of the linear double-stranded region. The overhang of the first terminal adaptor molecule may be complementary to and anneal to the first end of the linear double-stranded region and / or the overhang of the second terminal adaptor molecule may be complementary to and anneal to the second end of the linear double-stranded region.

[0465] The first terminal adaptor molecule may comprise a portion that is complementary to the first end of the nth intermediate adaptor molecule. The second terminal adaptor molecule may comprise a portion that is complementary to the first end of the mth intermediate adaptor molecule. The first terminal adaptor molecule may comprise a portion that anneals to the first end of the nth intermediate adaptor molecule. The second terminal adaptor molecule may comprise a portion that anneals to first end of the mth intermediate adaptor molecule. The first terminal adaptor molecule may comprise a portion that is complementary and anneals to first end of the nth intermediate adaptor molecule. The second terminal adaptor molecule may comprise a portion that is complementary and anneals to first end of the mth intermediate adaptor molecule.

[0466] The portion that is complementary or anneals to the first end of the nth or mth intermediate adaptor molecule may be a 5’ overhang or a 3’ overhang of the first and / or second terminal adaptor molecule. The overhang of the first terminal adaptor molecule may be complementary to the first end of the nth intermediate adaptor molecule and / or the overhang of the second terminal adaptor molecule may be complementary to first end of the mth intermediate adaptor molecule. The overhang of the first terminal adaptor molecule may anneal to first end of the nth intermediate adaptor molecule and / or the overhang of the second terminal adaptor molecule may anneal to first end of the mth intermediate adaptor molecule. The overhang of the first terminal adaptor molecule may be complementary to and anneal to first end of the nth intermediate adaptor molecule and / or the overhang of the second terminal adaptor molecule may be complementary to and anneal to first end of the mth intermediate adaptor molecule.

[0467] The first terminal adaptor molecule and / or the second terminal adaptor molecule and / or one or more of the n+m intermediate adaptor molecules may comprise a 5’ phosphate. The 5’ phosphate may facilitate ligation to the linear double-stranded region or to adjacent terminal or intermediate adaptor molecules (which may comprise a 3’-OH group at first and / or second ends). The first terminal adaptor molecule and / or the second terminal adaptor molecule and / or one or more of the n+m intermediate adaptor molecules may comprise a 3’-OH. The 3’-OH may facilitate ligation to the linear doublestranded region and / or to adjacent intermediate adaptor molecules (which may comprise a 5’ phosphate at first and / or second ends). A portion of the first terminal adaptor molecule (e.g. the overhang) may be complementary to the first end of the linear double-stranded region or complementary to a first end of an intermediate adaptor molecule, preferably the nth intermediate adaptor molecule. A portion of the second terminal adaptor molecule (e.g. the overhang) may be complementary to the second end of the linear double-stranded region or complementary to a first end of an intermediate adaptor molecule, preferably the mth adaptor molecule. A portion of the first end of the linear double-stranded region maybe complementary to a first end of an intermediate adaptor molecule, preferably n-(n-1 ) intermediate adaptor molecule. A portion of the second end of the linear double-stranded region maybe complementary to a first end of an intermediate adaptor molecule, preferably m-(m-1) intermediate adaptor molecule. A portion of a second end of an intermediate adaptor molecule may be complementary to a first end of an adjacent intermediate adaptor molecule, for example a portion of the second end of the nth intermediate adaptor molecule may be complementary to a portion of the first end of the n-1 th intermediate adaptor molecule, and a portion of the second end of the n-1 th adaptor molecule may be complementary to a portion of the first end of the n-2th intermediate adaptor molecule, and so on. A portion of a second end of an intermediate adaptor molecule may be complementary to a first end of an adjacent intermediate adaptor molecule, for example a portion of the second end of the mth intermediate adaptor molecule may be complementary to a portion of the first end of the m-1th intermediate adaptor molecule, and a portion of the second end of the m-1th adaptor molecule may be complementary to a portion of the first end of the m-2th intermediate adaptor molecule, and so on. n may be 0, 1 , 2, 3, 4, 5, 6, 7 or 8. m may be 0, 1 , 2, 3, 4, 5, 6, 7 or 8. Preferably, n is 0, 1 , 2, 3 or 4, and m is 0, 1 , 2, 3 or 4. .

[0468] The first terminal adaptor molecule and / or the second terminal adaptor molecule may not comprise a Type IIS endonuclease target sequence. The first terminal adaptor molecule and / or the second terminal adaptor molecule may not comprise Bbsl, Bsal, BsmBI, BspQI, BtgZI, Esp3l,Sapl, Aarl, Acc36l, AcIWI, Acul, Ajul, Alol, Alw26l, Alwl, Arsl, AsuHPI, Bael, Bari, Bbvl, Bccl, BceAl, Bcgl, BciVI, BcoDI, BfuAI, Bful, Bmrl, Bmsl, Bmul, Bpil, Bpml, BpuEl, BsaXI, Bsel l, Bse3DI, BseGI, BseMI, BseMII, BseNI, BseRI, BseXI, Bsgl, BsIFI, BsmAI, BsmFI, Bsml, Bso31 l, BspCNI, BspMI, BspPI, BspQI, BspTNI, BsrDI, Bsrl, Bst6l, BstF5l, BstMAI, BstV11, BstV2l, Bsul, BtgZI, BtsCI, Btsl-v2, BtsMutl, Bvel, Csel, CspCI, Eam1104l, Earl, Ecil, Eco31 l, Eco57l, Esp3l, Faql, Faul, Fokl, Gsul, Hgal, Hphl, HpyAV, Lgul, Lmnl, Lsp1109I, Lwel, Mboll, Mlyl, Mmel, Mnll, Mva1269l, NmeAIII, PaqCI, PciSI, Pctl, Piel, Ppsl, Psrl, Schl, SfaNI, Taqll, TspDTI and / or TspGWI Sapl target sequences.

[0469] The first terminal adaptor molecule and / or the second terminal adaptor molecule and / or one or more of the n+m intermediate adaptor molecules may comprise a functional portion. The functional portion may be a binding molecule, a targeting sequence, or a probe. The functional portion may be a cassette, an open reading frame or a coding sequence. The functional portion may be a promotor, and enhancer, NLS sequence, a UTR, ITR or other repeats, a terminator sequence, modified nucleotides or a fluorophore. The functional portion may be a probe. As used herein, the term “probe” refers to a fragment of DNA, RNA or DNA / RNA chimera of variable length (e.g. 3-1000 bases long), which is used to detect the presence of target nucleotide sequences that are complementary to the sequence in the probe. Typically, the probe hybridizes to single-stranded nucleic acid whose base sequence allows probetarget base pairing due to complementarity between the probe and target. Thus, the functional portion may be a DNA sequence, a RNA sequence or a DNA / RNA chimera sequence. As used herein, the term “complementary” refers to the pairing of nucleotide sequences according to Watson / Crick pairing rules. For example, a sequence 5'-GCGGTCCCA-3' has the complementary sequence of 5'-TGGGACCGC- 3'. A complement sequence can also be a sequence of RNA complementary to the DNA sequence.

[0470] The functional portion may be a binding molecule. The term “binding molecule” refers to any molecule capable of binding to the linear DNA product described herein and / or that is capable of binding to a further molecule or target. The binding molecule may be a protein, a polypeptide, or a peptide. The binding molecule may be an antibody, such as a monoclonal antibody or a polyclonal antibody. The binding molecule may be an antibody fragment.

[0471] The functional portion may facilitate detection of the DNA product by binding to capture molecules (e.g. capture antibodies bound by protein-protein interactions). The functional portion may bind to a cell target, for example, a cell receptor.

[0472] The functional portion may be a label. The ‘label’ can be any chemical entity which enable the detection of the double-stranded nucleic acid molecule via, physical, chemical and / or biological means. The label may be a chromophore, a fluorophore and / or a radioactive molecule.

[0473] The functional portion may be a targeting sequence. The targeting sequence may be a fragment of DNA or RNA of variable length, which is used to target the DNA product to a specific location in a cell. The targeting sequence may be used to increased transfection efficiency of non-viral gene delivery by virtue of enhanced nuclear import of the partially closed linear DNA product. For example, the targeting sequence may be a DNA nuclear targeting sequences (i.e. a recognition sequence for endogenous DNA-binding proteins), such as SV40 enhancer sequence (preferably downstream from the cassette). The targeting sequence may be a protelomerase targeting sequence, or a truncated variant thereof.

[0474] To facilitate detection and / or quantification of the DNA product, the functional portion may comprise a fluorophore, a radioactive compound or a barcode.

[0475] A signal corresponding to the presence, absence and / or level of the partially closed linear DNA product may be measured using a barcode. The barcode may comprise at least one binding moiety linked to a barcoded portion, wherein the barcoded portion comprises at least one nucleotide (i.e. wherein the barcoded portion comprises a nucleotide sequence at least one nucleotide in length), and wherein the binding moiety is capable of binding to the 3’ overhang, the 5’ overhang or the blunt end of the partially closed linear DNA product. The binding moiety is capable of binding to 3’ and / or 5’ end of the partially closed linear DNA product. The signal may be measured by determining the presence, absence and / or level of the barcoded portion of the barcode (e.g. by sequencing or PCR). The barcoded portion may comprise at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 nucleotides. The barcode may comprise at least 2 binding moieties (e.g. a first binding moiety and a second binding moiety). For example, the first binding moiety linked to the first barcoded portion may bind to the 3’ end of the partially closed linear DNA product and / or the second binding moiety linked to the second barcoded portion may bind to the 5’ end of the partially closed linear DNA product.

[0476] A signal corresponding to the presence, absence and / or level of the partially closed linear DNA product may be measured using a fluorophore (i.e. a fluorescently-labelled molecule), which is attached or bound to the 3’ overhang, the 5’ overhang or the blunt end of the partially closed linear DNA product. The signal may be measured by flow cytometry and / or fluorescence-activated cell sorting.

[0477] The functional portion may also facilitate DNA sequencing. For example, the functional portion may be a sequencing adapter. The term “sequencing adapter” is intended to encompass one or more nucleic acid domains that include at least a portion of a nucleic acid sequence (or complement thereof) utilized by a sequencing platform of interest, such as a sequencing platform provided by Illumina® (e.g. the HiSeq™, MiSeq™ and / or Genome Analyzer™ sequencing systems), Oxford Nanopore™ Technologies (e.g. the MinlON sequencing system), Ion Torrent™ (e.g. the Ion PGM™ and / or Ion Proton™ sequencing systems), Pacific Biosciences (e.g. the PACBIO RS II sequencing system); Life Technologies™ (e.g. a SOLiD sequencing system), Roche (e.g. the 454 GS FLX+ and / or GS Junior sequencing systems), or any other sequencing platform of interest.

[0478] One or more of the n+m intermediate adaptor molecules (e.g. 1 , 2, 3, 4, 5, 6, 7 or 8 intermediate adaptor molecules) may comprise a cassette. The cassette may comprise a coding sequence. The coding sequence may encode a gene of interest, for example a gene encoding a protein. The cassette may comprise at least a portion of a promoter and a coding sequence. The cassette may comprise a promoter and a coding sequence. The cassette may comprise a promoter, a coding sequence, a ribosomal binding site and a translational termination sequence. The cassette may additionally comprise sequences aiding protein expression, such as a cap-independent translation element. The cassette may comprise (or encode) a repair template (or editing template). The repair template (or editing template) may be for use in CRISPR-Cas mediated homology directed repair (HDR). The cassette may encode CRISPR guide RNA. The cassette may be a mammalian expression cassette. The promoter may be a CMV promoter. The cassette may further comprise an enhancer. The cassette may further comprise a reporter gene, such as an eGFP reporter gene or a luciferase reporter gene. The cassette may further comprise a homopolymeric sequence, such as a polyA, poly C, polyT or polyG sequence. The homopolymeric sequence may be between 3-200 nucleotides in length. The homopolymeric sequence may be used to facilitate purification of the cassette, in which case, the homopolymeric sequence may be between 4-12 nucleotides in length, or between 5-10 nucleotides in length. The homopolymeric sequence may be used to improve mRNA expression, in which case, the homopolymeric sequence may be between 10-200 nucleotides in length, preferably between 80-150 nucleotides in length. The homopolymeric sequence may be at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 1 10, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, or at least 200 nucleotides in length. Preferably, the homopolymeric sequence is at least 100 nucleotides in length. More preferably still, the homopolymeric sequence is at least 120 nucleotides in length. For example, the homopolymeric sequence may comprise a polyA sequence of at least 120 nucleotides.

[0479] One or more of the n+m intermediate adaptor molecules (e.g. 1 , 2, 3, 4, 5, 6, 7 or 8 intermediate adaptor molecules) may comprise a spacer. The spacer may be at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 125, at least 150, at least 175, or at least 200 base pairs long.

[0480] One or more of the n+m intermediate adaptor molecules (e.g. 1 , 2, 3, 4, 5, 6, 7 or 8 intermediate adaptor molecules) may be at least, 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 1 1 , at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 100, at least 250, at least 500, at least 1000, at least 2000, at least 3000, at least 4000, at least 5000, at least 6000, at least 7000, at least 8000, at least 9000, at least 10,000, at least 1 1 ,000, at least 12,000, at least 13,000, at least 14,000, or at least 15,000 base pairs long. Preferably, the one or more of the n+m intermediate adaptor molecules (e.g. 1 , 2, 3, 4, 5, 6, 7 or 8 intermediate adaptor molecules) is at least 10 base pairs long.

[0481] The first terminal adaptor molecule and / or the second terminal adaptor molecule and / or one or more of the n+m intermediate adaptor molecules may comprise a polyA signal sequence. One or more of the n+m intermediate adaptor molecules may comprise a polyA signal downstream of a barcode.

[0482] The first terminal adaptor molecule and / or the second terminal adaptor molecule and / or one or more of the n+m intermediate adaptor molecules may comprise an aptamer.

[0483] 4. Methods for transcription and protein expression

[0484] The invention provides a method for in vitro transcription of a linear DNA product (e.g. a closed linear DNA product or a partially closed linear DNA product), wherein the method comprises contacting the linear DNA product (e.g. a closed linear DNA product or a partially closed linear DNA product), produced by the methods described herein, with a polymerase and producing a transcription product encoded by the linear DNA product (e.g. the closed linear DNA product or a partially closed linear DNA product). The invention provides a method for in vitro transcription of a linear DNA product (e.g. a closed linear DNA product or a partially closed linear DNA product), wherein the method comprises:

[0485] (a) producing a linear DNA product (e.g. a closed linear DNA product or a partially closed linear DNA product) by any of the methods described herein;

[0486] (b) contacting the linear DNA product (e.g. the closed linear DNA product or a partially closed linear DNA product), with a polymerase; and

[0487] (c) producing a transcription product encoded by the linear DNA product (e.g. the closed linear DNA product or a partially closed linear DNA product).

[0488] The invention provides a method for in vitro transcription of a linear DNA product, wherein the method comprises:

[0489] (a) contacting a double-stranded DNA molecule with an endonuclease, a ligase and first and second terminal adaptor molecules and n+m intermediate adaptor molecules, to form a single contiguous aqueous volume, wherein n and m are each 0 or an integer of at least 1 , and wherein n+m is at least 1 ; and

[0490] (b) incubating the single contiguous aqueous volume to generate the linear DNA product, wherein the linear DNA product comprises a linear double-stranded region, wherein the linear double-stranded region comprises a linear portion of the double-stranded DNA molecule, and wherein n intermediate adaptor molecules are sequentially appended to a first end of the linear double stranded region and m intermediate adaptor molecules are sequentially appended to a second end of the linear doublestranded region, and wherein the first terminal adaptor molecule is appended to the nth intermediate adaptor molecule or, when n is 0 , to the first end of the linear double stranded region, and the second terminal adaptor molecule is appended to the mth intermediate adaptor molecule, or when m is 0, to the second end of the linear double stranded region; and

[0491] (c) contacting the linear DNA product with a polymerase; and

[0492] (d) producing a transcription product encoded by the linear DNA product.

[0493] The method may use adaptor molecules which generate a closed linear DNA product, such as adaptor molecules described herein. A method for in vitro transcription of a closed linear DNA product may comprise:

[0494] (a) contacting a double-stranded DNA molecule with an endonuclease, a ligase and first and second terminal adaptor molecules and n+m intermediate adaptor molecules, to form a single contiguous aqueous volume, wherein n and m are each 0 or an integer of at least 1 , and wherein n+m is at least 1 ;

[0495] (b) incubating the single contiguous aqueous volume to generate the closed linear DNA product, wherein the closed linear DNA product comprises a linear double-stranded region, and wherein n intermediate adaptor molecules are sequentially appended to a first end of the linear double stranded region and m intermediate adaptor molecules are sequentially appended to a second end of the linear double-stranded region, and wherein the closed linear DNA product is closed at a first end by the first terminal adaptor molecule appended to the nth intermediate adaptor molecule or, when n is 0 , to the first end of the linear double stranded region, and closed at a second end by the second terminal adaptor molecule appended to the mth intermediate adaptor molecule, or when m is 0, to the second end of the linear double stranded region;

[0496] (c) contacting the closed linear DNA product with a polymerase; and

[0497] (d) producing a transcription product encoded by the closed linear DNA product.

[0498] The method may use adaptor molecules which comprise protected nucleotides, such as the adaptor molecules described herein. A method for in vitro transcription of a linear DNA product may comprise:

[0499] (a) contacting a double-stranded DNA molecule with an endonuclease, a ligase and first and second terminal adaptor molecules and n+m intermediate adaptor molecules, to form a single contiguous aqueous volume, wherein n and m are each 0 or an integer of at least 1 , and wherein n+m is at least 1 ;

[0500] (b) incubating the single contiguous aqueous volume to generate the linear DNA product, wherein the linear DNA product comprises a linear double-stranded region, wherein the linear double-stranded region comprises a linear portion of the double-stranded DNA molecule, and wherein n intermediate adaptor molecules are sequentially appended to a first end of the linear double stranded region and m intermediate adaptor molecules are sequentially appended to a second end of the linear doublestranded region, and wherein the first terminal adaptor molecule is appended to the nth intermediate adaptor molecule or, when n is 0, to the first end of the linear double stranded region, and the second terminal adaptor molecule is appended to the mth intermediate adaptor molecule, or when m is 0, to the second end of the linear double stranded region, and wherein the first terminal adaptor molecule and second terminal adaptor molecules are nucleic acid molecules that comprise one or more nuclease-resistant nucleotides (i.e. protected nucleotides);

[0501] (c) contacting the linear DNA product with a polymerase; and

[0502] (d) producing a transcription product encoded by the linear DNA product.

[0503] The method may use adaptor molecules which comprise protected nucleotides and adaptor molecules which comprise a hairpin or a stem loop, such as the adaptor molecules described herein. A method for in vitro transcription of a partially closed linear DNA product may comprise:

[0504] (a) contacting a double-stranded DNA molecule with an endonuclease, a ligase and first and second terminal adaptor molecules and n+m intermediate adaptor molecules, to form a single contiguous aqueous volume, wherein n and m are each 0 or an integer of at least 1 , and wherein n+m is at least 1 ;

[0505] (b) incubating the single contiguous aqueous volume to generate the partially closed linear DNA product, wherein the partially closed linear DNA product comprises a linear double-stranded region, wherein the linear double-stranded region comprises a linear portion of the double-stranded DNA molecule, and wherein n intermediate adaptor molecules are sequentially appended to a first end of the linear double stranded region and m intermediate adaptor molecules are sequentially appended to a second end of the linear double-stranded region, and wherein the partially closed DNA product is closed at a first end by the first terminal adaptor molecule appended to the nth intermediate adaptor molecule or, when n is 0, to the first end of the linear double stranded region, and the second terminal adaptor molecule is appended to the mth intermediate adaptor molecule, or when m is 0, to the second end of the linear double stranded region, and wherein the second terminal adaptor molecule is a nucleic acid molecules that comprise one or more nuclease-resistant nucleotides;

[0506] (c) contacting the partially closed linear DNA product with a polymerase; and

[0507] (d) producing a transcription product encoded by the partially closed linear DNA product.

[0508] The invention provides a method for producing a protein, wherein the method comprises introducing the linear DNA product (e.g. the closed linear DNA product or the partially closed linear DNA product), produced by the methods described herein, into a cell (e.g. a prokaryotic cell or a eukaryotic cell) or a cell-free expression system to generate a protein encoded by the linear DNA product (e.g. the closed linear DNA product or the partially closed linear DNA product).

[0509] The invention provides a method for producing a protein, wherein the method comprises:

[0510] (a) producing a linear DNA product (e.g. a closed linear DNA product or partially closed linear DNA product) by any of the methods described herein; and

[0511] (b) introducing the linear DNA product (e.g. the closed linear DNA product or partially closed linear DNA product) into a cell (e.g. a prokaryotic cell or a eukaryotic cell) or a cell-free expression system to generate a protein encoded by the linear DNA product (e.g. the closed linear DNA product or partially closed line...

Claims

CLAIMS1 . A method for producing a closed linear deoxyribonucleic acid (DNA) product, wherein the method comprises:(a) contacting a double-stranded DNA molecule with an endonuclease, a first terminal adaptor molecule, a second terminal adaptor molecule and n + m intermediate adaptor molecules, to form a single contiguous aqueous volume, wherein n and m are each 0 or an integer of at least 1 , and wherein n + m is at least 1 ; and(b) incubating the single contiguous aqueous volume to generate the closed linear DNA product, wherein the closed linear DNA product comprises a linear double-stranded region, wherein the linear double-stranded region comprises a linear portion of the double-stranded DNA molecule, and wherein n intermediate adaptor molecules are sequentially appended to a first end of the linear double stranded region and m intermediate adaptor molecules are sequentially appended to a second end of the linear double-stranded region, and wherein the closed linear DNA product is closed at a first end by the first terminal adaptor molecule appended to the nth intermediate adaptor molecule or, when n is 0 , to the first end of the linear double stranded region, and closed at a second end by the second terminal adaptor molecule appended to the mth intermediate adaptor molecule, or when m is 0, to the second end of the linear double stranded region.

2. The method of claim 1 , wherein the first and / or second terminal adaptor molecules comprise a hairpin.

3. A method for producing a linear deoxyribonucleic acid (DNA) product, wherein the method comprises:(a) contacting a double-stranded DNA molecule with an endonuclease, a first terminal adaptor molecule, a second terminal adaptor molecule and n + m intermediate adaptor molecules, to form a single contiguous aqueous volume, wherein n and m are each 0 or an integer of at least 1 , and wherein n + m is at least 1 ; and(b) incubating the single contiguous aqueous volume to generate the linear DNA product, wherein the linear DNA product comprises a linear double-stranded region, wherein the linear double-stranded region comprises a linear portion of the double-stranded DNA molecule, and wherein n intermediate adaptor molecules are sequentially appended to a first end of the linear double stranded region and m intermediate adaptor molecules are sequentially appended to a second end of the linear double-stranded region, and wherein the first terminal adaptor molecule is appended to the nth intermediate adaptor molecule or, when n is 0, to the first end of the linear double stranded region, and the second terminal adaptor molecule is appended to the mth intermediate adaptor molecule, or when m is 0, to the second end of the linear double stranded region, and wherein the first terminal adaptor molecule and secondterminal adaptor molecules are nucleic acid molecules that comprise one or more nuclease- resistant nucleotides.

4. A method for producing a partially closed deoxyribonucleic acid (DNA) product, wherein the method comprises:(a) contacting a double-stranded DNA molecule with an endonuclease, a first terminal adapter molecule, a second terminal adaptor molecule and n + m intermediate adaptor molecules, to form a single contiguous aqueous volume, wherein n and m are each 0 or an integer of at least 1 , and wherein n + m is at least 1 ; and(b) incubating the single contiguous aqueous volume to generate the partially closed linear DNA product, wherein the closed linear DNA product comprises a linear double-stranded region, wherein the linear double-stranded region comprises a linear portion of the double-stranded DNA molecule, and wherein n intermediate adaptor molecules are sequentially appended to a first end of the linear double stranded region and m intermediate adaptor molecules are sequentially appended to a second end of the linear double-stranded region, and wherein the partially closed DNA product is closed at a first end by the first terminal adaptor molecule appended to the nth intermediate adaptor molecule or, when n is 0, to the first end of the linear double stranded region, and the second terminal adaptor molecule is appended to the mth intermediate adaptor molecule, or when m is 0, to the second end of the linear double stranded region, and wherein the second terminal adaptor molecule is a nucleic acid molecules that comprise one or more nuclease-resistant nucleotides.

5. The method of claim 4, wherein the first terminal adaptor molecule comprises a hairpin6. The method of any one of claims 1 -5, wherein the endonuclease is a Type IIS restriction endonuclease, optionally wherein the endonuclease is Bbsl, Bsal, BsmBI, BspQI, BtgZI, Esp3l,Sapl, Aarl, Acc36l, AcIWI, Acul, Ajul, Alol, Alw26l, Alwl, Arsl, AsuHPI, Bael, Bari, Bbvl, Bccl, BceAl, Bcgl, BciVI, BcoDI, BfuAI, Bful, Bmrl, Bmsl, Bmul, Bpil, Bpml, BpuEl, BsaXI, Bsel l, Bse3DI, BseGI, BseMI, BseMII, BseNI, BseRI, BseXI, Bsgl, BsIFI, BsmAI, BsmFI, Bsml, Bso31 l, BspCNI, BspMI, BspPI, BspQI, BspTNI, BsrDI, Bsrl, Bst6l, BstF5l, BstMAI, BstV11, BstV2l, Bsul, BtgZI, BtsCI, Btsl-v2, BtsMutl, Bvel, Csel, CspCI, Eam1 104l, Earl, Ecil, Eco31 l, Eco57l, Esp3l, Faql, Faul, Fokl, Gsul, Hgal, Hphl, HpyAV, Lgul, Lmnl, Lsp1109I, Lwel, Mboll, Mlyl, Mmel, Mnll, Mva1269l, NmeAIII, PaqCI, PciSI, Pctl, Piel, Ppsl, Psrl, Schl, SfaNI, Taqll, TspDTI and / or TspGWI restriction endonuclease.

7. The method of any one of claims 1 -6, wherein the first terminal adaptor molecule and / or the second terminal adaptor molecule and / or one or more intermediate adaptor molecules are nucleic acid adaptor molecules8. The method of any one of claims 1 -7 wherein step a) further comprises contacting the double stranded DNA molecule with a ligase.

9. The method of any one of claims 1 -8, wherein the first end-adaptor molecule and / or the second end-adaptor molecule and / or one or more intermediate adaptor molecules comprises a doublestranded region with an overhang.

10. The method of anyone of claims 3-9, wherein the one or more nuclease-resistant nucleotides are one or more phosphorothioated nucleotides.11 . The method of any one of claims 1 -10, wherein one or more of the n+m intermediate adaptor molecules are 10-500, 50-400, 100-300 or 200-250 base pairs in length.

12. The method of any one of claims 1 -11 , wherein one or more of the n+m intermediate adaptor molecules comprises a fluorophore, a barcode, a polyA signal, a biotinylated nucleotide, protected nucleotides, spacers, polyA sequence, a promotor, an open reading frame, a targeting sequence or a localisation signal.

13. The method of any one of claims 1 to 12, wherein n is 0, 1 , 2 or 3.14 . The method of any one of claims 1 to 13, wherein m is 0, 1 , 2 or 3.

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