GB2638211A - A nucleic acid assembly method using Type IIB enzymes - Google Patents

Abstract

The present invention relates to a method for assembling nucleic acid, comprising providing an extended oligonucleotide comprising at least two repeating units, a fragment of the sequence of interest and a recognition site of a Type IIB or IIB-like restriction enzyme, digesting said oligonucleotide to generate cleaved sequences of interest with single-stranded overhangs and annealing two or more cleaved sequences where the overhangs are complementary. Preferably the overhangs are generated at each end, are three or more base in length and non-palindromic. The method is plasmid-free. The digestion and annealing steps are repeated. The annealed sequences are ligated to form a longer length linear DNA or assembled using polymerase cycling assembly. The method can be performed in a single pot reaction.

Description

A nucleic acid assembly method using Type IIB enzymes

Technical Field

The present invention relates to a method and system for assembling DNA. In particular, it allows for efficient and ordered assembly of a nucleic acid of interest from a number of oligonucleotide fragments that contain repeating units of restriction enzyme restriction sites and sequences of interest. The method uses Type IIB enzymes to digest the oligonucleotide fragments in a manner which produces units of the sequences of interest with single stranded overhangs, referred to as sticky ends, that can then anneal together in a predetermined order.

Background

DNA synthesis plays a pivotal role in numerous fields including molecular biology, biotechnology, and medicine, where the ability to construct custom-designed DNA sequences is fundamental to advancing research and technological applications.

Advancements in DNA synthesis methodologies have enabled the fabrication of increasingly complex genetic constructs, facilitating a wide array of applications such as gene editing, protein engineering, synthetic biology, and the development of therapeutic agents. Chemical DNA synthesis for example involves the chemical assembly of DNA sequences using synthetic chemistry techniques. A typical form of chemical synthesis employs phosphoramidite chemistry on solid-phase supports, where nucleotides are sequentially added and chemically activated for coupling. Chemical synthesis allows for the production of DNA fragments but it is often limited to shorter sequences due to practical constraints.

Enzymatic DNA synthesis methods have also been developed. These enzymatic methods use DNA polymerases or other enzymatic systems to synthesize DNA in a template-directed manner. Cloning methods based on Type IIS restriction enzymes have been developed (see for example WO 2008/095927). Engler et al. PLoS ONE 4 (2009) e5553) described a protocol to assemble in one step and one tube at least nine separate DNA fragments together into an acceptor vector using Type IIS restriction enzymes by simply subjecting a mix of 10 undigested input plasmids to a restriction-ligation reaction and transforming the resulting mix into competent cells. This protocol was named "Golden Gate" synthesis. Golden Gate DNA synthesis is an enzymatic synthesis technique used for the rapid and efficient assembly of DNA fragments into larger constructs. It relies on plasmids and Type 115 restriction enzymes, such as Bsal or BsmBI, which cleave DNA outside and at one side of their recognition sequence, generating single-stranded overhangs. In Golden Gate synthesis the DNA fragments to be assembled are designed with complementary overhangs that match the recognition sites of the Type 115 restriction enzyme. Typically, these overhangs are 4-6 base pairs in length.

Each DNA fragment is incubated with the Type 115 restriction enzyme, which cleaves the DNA sequence outside of its recognition site, leaving behind the desired overhangs.

The digested DNA fragments are mixed together in a single reaction tube along with a DNA ligase enzyme and a reaction buffer. The complementary overhangs of the DNA fragments anneal to each other through base pairing, guided by the specific sequences that were precisely designed. A DNA ligase enzyme then catalyses the formation of phosphodiester bonds between the adjacent DNA fragments, resulting in their covalent linkage.

After the assembly reaction, the Type IIS restriction enzyme is heat-inactivated to prevent further digestion of the assembled DNA constructs.

The assembled DNA constructs are typically transformed into a suitable host organism, such as bacteria, for propagation and further analysis. Depending on the application, the transformed organisms may be used for gene expression, protein production, or other downstream experiments.

Whilst Golden Gate synthesis has many benefits, it relies on the use of Type 115 restriction enzymes, which require specific recognition sequences for cleavage. This can limit the choice of DNA sequences that can be assembled, particularly if the desired sequences contain restriction sites that interfere with the assembly process. Some DNA sequences may also be incompatible with Golden Gate assembly due to the presence of secondary structures, repetitive elements, or other structural features that interfere with the assembly process. In such cases, alternative assembly methods may be required. Golden Gate synthesis also typically uses plasmids as the scaffold or backbone onto which the desired DNA fragments are ligated. The plasmid backbone usually contains the necessary regulatory elements (such as promoters, terminators, and selection markers) for gene expression or other functional elements required for the intended application.

Use of Golden Gate predominantly uses bacterial cloning to amplify ligated sequence in a plasmid. Bacterial cloning has may drawbacks including introduction of replication errors into the target sequence, difficulties in replication of particular sequences, time consuming and costly. This is particularly relevant in synthesis of DNA for therapeutic applications whereby sequence integrity is vital for patient safety.

Another drawback of known DNA synthesis and assembly methods is the need for further processing of the target sequence before it can be used in downstream applications. For example, in Golden Gate assembly whereby the target sequence is assembled in a plasmid, the target needs to be excised from the plasmid, isolated from the linearised plasmid DNA and purified before it can be used in downstream applications.

Existing methods of DNA synthesis often suffer from limitations such as high cost, limited sca la bility, high complexity, and restricted sequence length.

Summary of the Invention

According to the present invention there is provided a nucleic acid assembly method comprising: providing an extended oligonucleotide comprising at least two repeating units, the first unit being a nucleic acid fragment sequence of interest and the second unit being a recognition site for a Type IIB or Type IIB-like restriction enzyme; - digesting said oligonucleotide with a Type IIB or Type IIB-like restriction enzyme to provide cleaved nucleic acid fragment sequences of interest with single-stranded overhangs; - annealing two or more cleaved sequence portions where the single-stranded overhangs are complementary.

Advantageously, this method allows the directed assembly of a target nucleic acid e.g. a target DNA.

Preferably the step of digesting said extended oligonucleotide with a Type II B or Type IIB-like restriction enzyme to provide cleaved nucleic acid fragment sequences of interest with single-stranded overhangs occurs at a digesting temperature. Preferably, the digesting temperature is the activation temperature of the Type II B or Type IIB-like restriction enzyme.

Preferably the step of annealing two or more cleaved sequence portions where the single-stranded overhangs are complementary occurs at an annealing temperature. Preferably, the annealing temperature is lower than the digesting temperature.

Preferably, the step of digesting said oligonucleotide with a Type I IB or Type IIB-like restriction enzyme provides cleaved nucleic acid fragment sequences of interest with single-stranded overhangs on each end.

Being complementary means that single-stranded overhangs, said overhangs produced by restriction with a type ilB restriction endonuclease recognising the recognition sites of the cleavage sites, are complementary such that the single stranded overhangs can anneal.

Preferably, the annealed sequence portions are ligated, after annealing, to form a longer length linear DNA.

It is preferred that the overhangs are fully complementary when ligation is to occur, after annealing, to form a longer length linear DNA. it is preferred that the overhangs fully overlap when ligation is to occur, after annealing, to form a longer length linear DNA.

Preferably, a DNA ligase enzyme catalyses the formation of phosphodiester bonds between the adjacent DNA fragments, resulting in their covalent linkage.

Preferably, the extended oligonucleotide digestion, and the ligation and/or annealing of two or more cleaved sequence portions where the single-stranded overhangs are complementary, occur in a 'single pot reaction' or a 'one pot reaction'.

One-pot or single-pot reaction is hereinafter synonymous and means two or more processes are conducted in a single reaction vessel without isolating or purifying the resulting intermediates.

Preferably, the Type IIB restriction enzyme and DNA ligase are provided in the same reaction pot and the temperature is cycled between the digesting temperature and the annealing temperature.

Preferably, the method comprises providing a plurality or set of extended oligonucleotides comprising at least two repeating units. Most preferably, different extended oligonucleotides contain different nucleic acid fragment sequences. Most preferably the single-stranded overhangs, said overhangs produced by restriction with a type IIC3 restriction endonuclease recognising the recognition sites of the cleavage sites, differ between different extended oligonucleotides in the plurality or set, Optionally, the repeating first units within an extended nucleotide may comprise different nucleic acid fragment sequences of interest.

Optionally, the single-stranded overhangs are 2 or more bases in length. Preferably, the single-stranded overhangs are 3 or more bases in length.

Preferably the single-stranded overhangs are non-palindromic to ensure directional assembly.

Advantageously, designing non-palindromic single-stranded overhangs ensures directional assembly of cleaved nucleic acid fragment sequences of interest.

The method does not require vectors. In a preferred embodiment the method is plasmid-free.

Preferably, the method is for forming a target polynucleotide and comprises the step of determining, for the target polynucleotide, an optimized overhang sequence for each nucleic acid fragment sequence of interest for joining into an ordered set to assemble the target polynucleotide.

Importantly, the overhangs can be precisely engineered by designing the recognition site within the DNA fragment.

Preferably the digestion and annealing steps are repeated or cycled.

As digesting and then annealing (and preferably ligation) can result in the binding of a unit with a sequence of interest to another unit with a sequence of interest in the desired fashion OR can result in annealing (and optional ligation) of a sequence of interest unit back to the original restriction site unit from which it was cleaved/digested (which is undesirable) the method includes the step of cycling by digesting the annealed (and optionally ligated) products again. Only DNA products that include a sequence of interest unit re-ligated back to a restriction site will be digested in this later cycle (as only they now have the necessary restriction site -a sequence of interest unit that has annealed (and optionally ligated) to another sequence of interest unit would not have a restriction site and would not therefore be digested in this cycle).

Preferably the digestion and annealing steps are repeated or cycled multiple times. Preferably, there are 10 or more repetitions of the digestion and annealing steps. Optionally, there are 20 or more repetitions of the digestion and annealing steps. In one embodiment, there are 30 or more repetitions of the digestion and annealing steps.

Advantageously there is no need to purify after every step or after every cycle -this gives improved yield compared to many other methods as DNA is typically lost during purification steps.

Optionally, the ligation step may be included in the cycling e.g. the digestion, annealing and ligation steps are cycled.

Optionally, after cycling of the digestion and annealing steps (and preferably after the ligation step) the DNA is separated e.g. by HPLC.

The Type IIB or Type IIB-like restriction is a restriction endonuclease.

Optionally, the Type IIB or Type IIB-like restriction enzymes are selected from BsaXl and Bpl-I restriction enzymes.

Alternatively, the Type IIB or Type IIB-like restriction enzymes are selected from the following list (where N can be any base; Y can be t/u or C; V can be a or g or c): Name Sequence Length (without sticky end) Sticky end overhang Restriction enzyme recognition site CspCl (10-11/12-13)CAANNNNNGTGG(12-13/10-11) 33 2 Alol (7/12-13)GAACNNNNNNTCC(12-13/7) 27 5 Ppil (7/12)GAACNNNNNCTC(13/8) 28 5 Psrl (7/12)GAACNNNNNNTAC(12/7) 27 5 BO (8/13)GAGNNNNNCTC(13/8) 27 5 Fall (8/13)AAGNNNNNCTT(13/8) 27 5 Bsp241 (8/13)GACNNNNNNTGG(12/7) 27 5 BsaXl (9/12)ACNNNNNCTCC(10/7) 27 3 HaelV (7/13)GAYNNNNNRTC(14/9) 27 6 Cjel (8/14)CCANNNNNNGT(15/9) 28 6 CjePI (7/13)CCANNNNNNNTC(14/8) 27 6 Hin4I (8/13)GAYNNNNNVTC(13/8) 27 5 Bael (10/15) ACNNNNGTAYC(12/7) 28 5 Alf! (10/12)GCANNNNNNTGC(12/10) 32 2 Bcgl (10/12)CGANNNNNNTGC(12/10) 32 2 BsIFI (6/10)GGGAC(10/14) 21 4 Type IIB restriction endonucleases cleave DNA outside of the recognition sequence and at both sides of the recognition sequence, for example Bcgl which recognizes an asymmetric sequence, or 8p11 which recognizes a symmetric sequence. This differs from Type Ils restriction endonucleases cleave DNA at one side of the recognition sequence.

Preferably, the DNA ligase is a Type 2 ligase, or variant thereof such as a thermostable variant or high salt tolerant ligase.

Optionally, the step of providing the extended oligonucleotide comprising at least two repeating units includes synthesising the extended oligonucleotide comprising at least two repeating units.

Synthesising the extended oligonucleotide comprising at least two repeating units may occur in solution or on a solid support. Synthesis can occur through known methods such as chemical synthesis e.g. phosphoramidite synthesis, or most preferably by enzymatic methods such as those described in EP3759253 (incorporated herein by reference) or C. J. Whitfield et al Chem. Eur. 1 2018, 24, 15267 which are particularly well suited to synthesising longer sequences which incorporate repeat sequences.

According to a related aspect of the present invention there is provided a system for producing a target nucleic acid comprising: providing a set of extended oligonucleotides, each extended oligonucleotide comprising: (i) a recognition site for a Type IIB or Type IIB-like restriction enzyme, the cleavage sites for said restriction enzyme being provided upstream and downstream of said recognition site; (ii) a fragment sequence of interest between the upstream cleavage site of the recognition site of (i) and the downstream cleavage site of a further recognition site of Type IIB or Type IIB-like restriction enzyme; (iii) said further recognition site for a Type IIB or Type IIB-like restriction enzyme, the cleavage sites for said restriction enzyme being provided upstream and downstream of said further recognition site, wherein in a first extended oligonucleotide, a Type IIB or Type II B-like restriction enzyme can produce at least one first cleaved fragment sequence of interest (ii) with single-stranded overhangs on each end by cleaving both the upstream cleavage site of item (i) and the downstream cleavage site of item (Hi); and in a second extended oligonucleotide, a Type IIB or Type IIB-like restriction enzyme can produce at least one second cleaved fragment sequence of interest (ii) with single-stranded overhangs on each end by cleaving both the upstream cleavage site of item (i) and the downstream cleavage site of item (Hi); a single-stranded overhang of the first cleaved fragment sequence of interest from the first extended oligonucleotide being complementary to a single-stranded overhang of the second cleaved fragment sequence of interest from the second extended oligonucleotide; such that, under appropriate conditions, the first cleaved fragment sequence of interest and second cleaved fragment sequence of interest will anneal, and be preferably ligated after annealing, to form a linear DNA.

In an alternative embodiment, rather than ligating, after annealing, to form a longer length linear DNA, the annealed sequence portions are assembled using polymerase cycling assembly (PCA).

Optionally the single stranded overhangs that are produced by restriction with type IIB restriction endonuclease recognising the recognition sites of the cleavage sites; partially overlap such that portions of single stranded DNA remain between annealed sections.

Optionally, the annealed sequence portions are extended by DNA polymerase such that single stranded DNA remain between annealed sections is filled in.

DNA polymerase and deoxynucleoside tri phosphates (dNTPs) are provided.

One skilled in the art would understand the basic requirements of extension by PCA including appropriate buffers and conditions.

Where appropriate the same optional and preferable features apply to the system as described for the method.

Various further features and aspects of the invention are defined in the claims.

Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs.

As used herein, Type IIB and Type IIB-like enzymes refer to restriction enzymes which cleave both DNA strands at specified locations distant from their recognition sequences, they cleave on both sides of the recognition site such that they liberate the recognition site from the remainder of the DNA. The cleavage produces staggered/overhanging ends of two or more bases.

As used herein "nucleic acid sequence", "oligonucleotide", "polynucleotide", "nucleic acid molecule" and variations thereof are used interchangeably to refer to plurality of nucleotides in either a regular or irregular sequence. Polynucleotides are typically single-stranded or double-stranded (duplex), but may adopt higher-order structures that contain three (triplex) or four (quadruplex/i-motif) strands, or may contain a mix of these configurations in different loci under suitable conditions. They have at least two adjacent nucleotides.

As used herein, "upstream" means in the direction of an oligonucleotide's 5' end, and "downstream" means in the direction of an oligonucleotide's 3' end.

Nucleic acid sequences may be of genomic, synthetic or recombinant origin, and may be double-stranded or single-stranded (representing the sense or antisense strand). The term "nucleotide sequence" or "nucleic acid sequence" includes genomic DNA, cDNA, synthetic DNA, and RNA (e.g. mRNA) and analogs of the DNA or RNA generated, e.g., by the use of nucleotide analogs. In other words, modified DNA or RNA bases are also encompassed. A polynucleotide may therefore include one or a plurality of modified DNA or RNA bases.

Polynucleotides bearing multiple modifications at specific sites have applications in synthetic biology, nanomaterial fabrication, bioanalytical, and sequencing applications. For example, DNA can be chemically modified at any, or all, of its three component parts -the phosphate linkage, the sugar ring, or the nucleobase. A variety of modified nucleotides can be obtained commercially as deoxynucleotidetriphosphates (dNTPs) or as phosphoramidite derivatives. These and other modified nucleotides can be synthesised and inserted into DNA or RNA either enzymatically as dNTPs, or through automated DNA synthesis as phosphoramidites.

Nucleotide residues are usually derived from the naturally occurring purine bases, namely adenine (A), guanine (G), hypoxanthine (I), and xanthine (X), and pyrimidine bases, namely cytosine (C), thymine (T), and uracil (U). Nucleotide analogues may be used at one or more of the positions within the polynucleotide sequence, such nucleotide analogues being modified in e.g. the base portion and/or the sugar portion and/or the phosphate linkage. Any nucleotide analogue can be used provided that it does not prevent the polynucleotide from hybridising and that it is accepted by polymerase as both a template and a substrate.

Nucleic acid sequences presented herein are conventionally written 5' to 3' (left to right). A "linear polynucleotide" refers to a polynucleotide which is not branched or circularised (i.e. the 3' end is not circularised with the 5' end).

"Nuclease" refers to an enzyme that cleaves nucleic acids at a phosphodiester linkage or other linkage between nucleosides. Endonucleases cleave a substrate nucleic acid at one or more internal linkages to produce two or more products.

In preferred examples, the target nucleic acid is DNA. Alternatively the target nucleic acid could be RNA. The DNA or DNA may comprise natural or modified bases, including a combination thereof. Several modified bases are known in the art and suitable modified bases can therefore be readily identified by a person of skill in the art.

Brief Description of the Drawings

Embodiments of the present invention will now be described by way of example only with reference to the accompanying drawings where like parts are provided with corresponding reference numerals and in which: Figure 1 is a scheme to illustrate the extension and diegstion of sequences that contain the recognition site for Type IIB restriction endonuclease BsaXl (U2) and a target sequence (U1). This extension uses the non-perfect self-priming DNA extension approach of not starting with repeating units of the same sequence but instead with an overhang in the starting oligoseed.

Digestion of the extended DNA (e) gives both the target DNA and the BsaXl recognition site; and Figure 2 shows the characterisation of extension, i) 1% agarose gel electrophoresis of extended DNA, Lane L: Gene Ruler 1kB Plus, Lane 1: extension product of Oligo-20/Oligo-21, Lane 2: extension product of Oligo-22/Oligo-23, Lane 3: extension product of Oligo-24/Oligo25. ii) Image) plot of the gel electrophoresis; and Figure 3 Charatcerisation of the digest product from a mix of Oligo-20/Oligo-21, Oligo- 22/Oligo-23 and Oligo-24/Oligo-25. i) 4% MetaPhor agarose gel elextrophoresis, Lane L: TriDye Ultra Low Range Ladder, Lane 1: 20 ng/RL of DNA, Lane 2: 15 ng/p1 of DNA, Lane 3: 10 ng/RL of DNA, Lane 4: 5 ng/RL of DNA, Lane 5: 1 ng/LIL of DNA. ii) Image) plot of the gel electrophoresis; and Figure 4 Characterisation of digestion of extended Oligo-24/Oligo-25, i) 4% MetaPhor agarose gel electrophoresis of digested DNA, Lane L: TriDye Ultra Low Range Ladder, Lane 1: digestion of Oligo-24/Oligo-25 at 0.8 ng/p.L. ii) Image) plot of the gel electrophoresis; and Figure 5 -i) 4% MetaPhor agarose gel electrophoresis of the ligation of digest target sections Lane L: TriDye Ultra Low Range Ladder, Lane 1: ligation of 23mer-20/23mer-21, 23mer- 22/23mer-23 and 23mer-24/23mer-25 together, Lane 2: 23mer-20/23mer-21, Lane 3: 23mer-22/23mer-23, Lane 4: 23mer-24/23mer-25. ii) Image! plot of the gel electrophoresis; and Figure 6 is a scheme to illustrate the cycling of digestion and ligation to give a target product method; and Figure 7 shows characterisation of extended DNA after 30 M-Gate heat-cool cycles of 5 minutes of digestion (37 °C) and 5 minutes of ligation (16 °C) i) 4% MetaPhor agarose gel electrophoresis, Lane L: TriDye Ultra Low Range Ladder, Lane 1: M-Gate reaction with both ligase and restriction enzyme, Lane 2: M-Gate reaction control with only restriction enzyme, Lane 3: M-Gate reaction control with only ligase. ii) I mageJ plot of the gel electrophoresis; and Figure 8 shows characterisation of extended DNA after 30 M-Gate heat-cool cycles of 30 minutes of digestion (37 °C) and 15 minutes of ligation (16 °C) i) 4% MetaPhor agarose gel electrophoresis, Lane L: TriDye Ultra Low Range Ladder, Lane 1: M-Gate reaction with both ligase and restriction enzyme, Lane 2: M-Gate reaction control with only restriction enzyme, Lane 3: M-Gate reaction control with only ligase. ii) I mageJ plot of the gel electrophoresis; and Figure 9 shows Characterisation of extended DNA at a concentration of 0.8 ngkil_ after 30 M-Gate heat-cool cycles of 30 minutes of digestion (37 °C) and 15 minutes of ligation (16 °C) i) 4% MetaPhor agarose gel electrophoresis, Lane L: TriDye Ultra Low Range Ladder, Lane 1: M-Gate reaction with both ligase and restriction enzyme, Lane 2: M-Gate reaction control with only restriction enzyme, Lane 3: M-Gate reaction control with only ligase. ii) lmagal plot of the gel electrophoresis; and Figure 10 -shows characterisation of extended DNA at a concentration of 0.8 nettl_ after 30 M-Gate heat-cool cycles of 30 minutes of digestion (37 °C) and 15 minutes of ligation (16 °C), i) Tapestation D1000 screentape, Lane L: TriDye Ultra Low Range Ladder, Lane 1: M-Gate reaction with both ligase and restriction enzyme, Lane 2: M-Gate reaction control with only restriction enzyme, Lane 3: M-Gate reaction control with both restriction enzyme and ligase but no extended Oligo-22/Oligo-23. ii) Intensity plot of screentape; and Figure 11 shows a method of producing an extended oligonucleotide using an enzymatic slippage method; and Figure 12 shows a method of assembling a target DNA using the method of the present invention.

Detailed Description

Embodiments of the present invention will be described along with experimental work confirming the suitability of the method for assembling target nucleic acids.

In preferred embodiments, volumes of target can be assembled in a cell free manner, without the need for plasmids or in-cell replication. Effectively, as the target is provided as separate component parts -i.e. fragment sequences of interest -multiple fragment sequences of interest can be provided in each extended DNA oligonucleotide. As the fragment sequences of interest are separated from each other by Type IIB restriction enzyme recognition sites they can then be separated (cleaved) and then ligated in to the desired target sequence order. As an extended DNA oligonucleotide can contain large numbers of fragment sequences of interest this results in similarly large numbers of target being produced. This negates the need for further amplification of the target after assembly. It also means that the assembled target is immediately available for use requiring no or minimal further processing.

Production of extended oligonucleotides Figure 11 shows a method of producing an extended oligonucleotide using an enzymatic slippage method.

The precursor steps show that a target 1 of e.g. 100bp can be split into five sections, being five 20bp fragment sequences of interest 2a, 2b, 2c, 2d and 2e. In a preferred embodiment, each of the fragment sequences of interest 2a-e are amplified into extended oligonucleotides 3, comprising repeat blocks of fragment sequences of interest 2a-e and Type IIB restriction enzyme recognition sites 4 (bounded by the cleavage sites which are the recognition sequence, and which are both upstream and downstream of the recognition site), using an enzymatic slippage reaction. Whilst the extended oligonucleotides could be synthesised by other means, enzymatic slippage reactions are particularly suitable as they can provide long lengths of repeat portions (in this case each portion having a fragment sequence of interest 2a-e and a Type IIB restriction enzyme recognition site 4) with high fidelity. Each extended oligonucleotide 3 produced can be greater than 200bp, or greater than 300bp, or greater than 500bp thus incorporating large multiples of fragment sequences of interest 2a-e. Whilst figure 11 shows enzymatic slippage occurring in solution, which may be preferable in some cases, it would be understood that it could also be carried out on a surface and the extended oligonucleotides cleaved from the surface.

Single stranded primers ('oligoseeds') are provided for each fragment sequence of interest 2a-e. Each primer comprises at least two tandem repeats of a portion comprising both one of the nucleic acid fragment sequences of interest 2a-e and a Type IIB restriction enzyme recognition site 4. The primer polynucleotides are contacted with a single stranded template oligonucleotide /polynucleotide comprising at least two tandem repeats that are complementary to the sequence of the primer polynucleotide. This occurs under hybridisation conditions that permit mismatched duplex formation between a sequence and its complement such that a 5' overhang of the template polynucleotide is generated, wherein the 5' overhang comprises at least one tandem repeat that is complementary to the sequence of the primer polynucleotide. The mismatched duplexes are then contacted with a thermostable 5' to 3' polymerase and nucleotides under extension conditions that permit polynucleotide extension in a 5' to 3' direction. The newly formed duplex -an extended oligonucleotide 3 -is then denatured under denaturing conditions to generate a single stranded immobilised polynucleotide; and the steps are repeated at least once, but preferably multiple times (preferably 10 or more times, or 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 85, at least 90, at least 95, at least 100 times, at least 110 times, at least 120 times, at least 140 times, at least 150 times, at least 160 times, at least 170 times, at least 180 times, at least 190 times, at least 200 times, or at least 250 times etc.), to increase the number of tandem repeats in the immobilised polynucleotide (e.g. it may result in an extended oligonucleotide with 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 85, at least 90, at least 95, at least 100 copies, at least 110 copies, at least 120 copies, at least 140 copies, at least 150 copies, at least 160 copies, at least 170 copies, at least 180 copies, at least 190 copies, at least 200 copies, or at least 250 copies etc of the nucleic acid fragment sequence of interest 2a-e each being separated from the next by a Type IIB restriction enzyme recognition site 4).

In this embodiment an extended oligonucleotide 3a-e is provided for each nucleic acid fragment sequence of interest 2a-e. If desired the extended nucleotide(s) 3a-e can be removed from the surface.

In an alternative embodiment the single stranded primers ('oligoseeds') are immobilised to a surface by an optional linker such as a silane linker molecule, a biotin-streptavidin complex, a thiol-Au linker, covalent Si-C bonds to silicon, covalent Si-0 bonds to silicon, covalent Si-N bonds to silicon, a nanoparticle linker, or a dynamic covalent bond.

In the above context "contacting" refers to direct contact between the primer polynucleotide and the template polynucleotide, for example in an appropriate buffer and container for the subsequent thermocycling steps of the method. Suitable buffers and containers are well known and include e.g. PCR buffers and Eppendorf tubes.

M-Gate assembly Figure 12 shows a method of assembling a target DNA. Advantageously, as the assembly can be carried out with a starting point of multiples of nucleic acid fragment sequence of interest 2a-e, this results in multiple final products being assembled. As such it is not necessary to carry out later amplification steps (commonly carried out in cells for example -making the method easily cell free) as the 'amplification' of building blocks of the end product e.g. the nucleic acid fragment sequences of interest 2a-e has already occurred.

Each of the extended oligonucleotides 3a-e from the above 'production of extended oligonucleotides' method are provided in a single reaction pot, container or vessel. This may be the same pot, container or vessel that was used to produce the extended oligonucleotides or may be a different pot, container or vessel. Extended oligonucleotide 3a comprises multiple copies of nucleic acid fragment sequence of interest 2a, each separated from the next by a Type IIB restriction recognition site 4; extended oligonucleotide 3b comprises multiple copies of nucleic acid fragment sequence of interest 2b, each separated from the next by a Type IIB restriction recognition site 4; extended oligonucleotide 3c comprises multiple copies of nucleic acid fragment sequence of interest 2c, each separated from the next by a Type IIB restriction recognition site 4; extended oligonucleotide 3d comprises multiple copies of nucleic acid fragment sequence of interest 2d, each separated from the next by a Type IIB restriction recognition site 4; extended oligonucleotide 3e comprises multiple copies of nucleic acid fragment sequence of interest 2e, each separated from the next by a Type IIB restriction recognition site 4.

A Type IIB restriction enzyme 5 that recognises the Type IIB restriction recognition site 4 is provided, as is a DNA ligase enzyme 6. In this preferred embodiment the restriction enzyme is Bpl-l.

The temperature is taken to the activation temperature ('digestion temperature') of the Type IIB restriction enzyme to initiate a digest of extended oligonucleotides 3a-e. The extended oligonucleotides 3a-e are digested with the Type IIB restriction enzyme to provide multiple cleaved nucleic acid fragment sequences of interest 2a-e with single-stranded overhangs, as well as separate Type IIB restriction recognition sites 4. The temperature is then lowered to an annealing temperature such that cleaved nucleic acid sequences of interest 2a-e which have complementary single-stranded overhangs then anneal and the DNA ligase ligates the portions to form a longer length linear DNA. The temperature is then cycled (thermocycled) between the digestion temperature and the annealing temperature multiple times to reduce the amount of end product that has re-ligated to a restriction recognition site 4. As the restriction enzyme cuts at either side of the restriction site and removes it entirely, only product that has re-ligated to a restriction recognition site 4 will be digested in a subsequent cycle. As such, nucleic acid fragment sequences of interest 2a-e which have ligated together are not cleaved in subsequent rounds and can go on to form full target 1 final product. In this example, there are 30 thermocycles comprising 5 minutes at a digestion temperature (where the temperature is 37 °C) followed by 5 minutes at the annealing temperature (where the temperature is 16 °C). It would however be understood that, whilst 30 cycles is a preferred option, 10 or more cycles, 20 or more cycles, 30 or more cycles, 40 or more cycles, 50 or more cycles would also be useful and the aim of the cycling is to increase the amount of full target 1 final product present and reduce the amount of cleaved nucleic acid fragment sequences of interest 2a-e that have re-ligated to a restriction recognition site 4 instead of forming the final target 1.

Methods of, and systems and apparatus for thermocycling are very well known e.g. from PCR systems and one skilled in the art would be able to carryout the temperature changes (as well as identifying appropriate digestion and ligation temperatures and times) without undue burden. Notably, unlike in the enzymatic slippage reaction described above, the annealing and ligation at this stage of the M-Gate assembly, does not require hybridisation conditions that permit mismatched duplex formation (and preferably occurs under conditions that do not permit mismatched duplex formation).

As the single-stranded overhangs have been designed to ensure directed assembly of the nucleic acid fragment sequences of interest 2a-e, this means that they form into the target nucleic acid 1. It is preferred that there is full overlap between complementary single-stranded overhangs.

Experimental work Experiments were carried out to confirm that digestion and ligation could be performed using Type IIB restriction enzymes. As it is not always possible to design to avoid hairpins and self-complementary sites (and other such features that can be considered undesirable in many DNA synthesis methods) a section of gene fragment was investigated which formed hairpins and secondary complementary sections. More particularly, MLH1 is a human gene for DNA repair, and a short fragment of this gene (69 bp) was taken to be the target of these experiments.

Type IIB recognition sites are removed during digestion with their associated restriction endonuclease and contain entirely customisable bases at their ends. This allows for scar free duplexes to be generated with non-pa lindromic sticky ends.

In this experimental work, three sequences were designed to incorporate the recognition site for BsaXl (which is 5'-(N)9AC(N)sCTCC(N)10-375'-(N)7GGAG(N)5GC(N)12-3'). Where "N" can be any nucleotide of choice allowing the user to select nucleotides to result in the desired sticky ends.

Oligo-20 (SEQ ID 1)/Oligo-21 (SEQ ID 2)(5'-TAT GAG CTT CGT GGC CGG CGT GAA AAA AAA 5 AAA CAA AAA CTC CAA AAA AAA AA-3'/5'-TCA CGC CGG CCA CGA AGC TCA TAT TTT TTT TTT GGA GTT TTT GTT ITT Oligo-22 (SEQ ID 3)/Oligo-23 (SEQ ID 4) (5'-TCA GGA GGC TGG ACG AGA CCG TGA AAA AAA AAA CAA AAA CTC CAA AAA AAT GA-375'-CAC GGT CTC GTC CAG CCT CCT GAT CAT TTT TTT 10 GGA GTT TTT GTT ITT ITT TT-3'), and Oligo-24 (SEQ ID 5)/Oligo-25 (SEQ ID 6) (5'-GTG AAC AGG ATC GCC GCC GGC GAA AAA AAA AAA CAA AAA CTC CAA AAA AAG TG-3'/5'-TCG CCG GCG GCG ATC CTG TTC ACC ACT TTT TTT GGA GTT TTT GTT ITT ITT TT-3').

These three sequences have overlapping sticky ends post digestion so that they can be ligated together in a specific order.

Extended DNA oligonucleotides were synthesised via an enzymatic slippage reaction (e.g. of the type described in EP3759253 (incorporated herein by reference) to give extended oligonucleotides containing two repeating units, the first unit being a nucleic acid fragment sequence of interest and the second unit being a recognition site for a Type IIB or Type IIB-like restriction enzyme. An example extended DNA oligonucleotide synthesis method includes the steps of: i) providing a single stranded primer oligonucleotide/polynucleotide comprising at least two tandem repeats of a sequence comprising the nucleic acid fragment sequence of interest followed by the recognition site (an 'oligoseed'), ii) contacting the primer polynucleotide with a single stranded template oligonucleotide /polynucleotide comprising at least two tandem repeats that are complementary to the sequence of the primer polynucleotide under hybridisation conditions that permit mismatched duplex formation between a sequence and its complement such that a 5' overhang of the template polynucleotide is generated, wherein the 5' overhang comprises at least one tandem repeat that is complementary to the sequence of the primer polynucleotide; and iii) contacting the mismatched duplexes with a thermostable 5' to 3' polymerase and nucleotides under extension conditions that permit polynucleotide extension in a 5' to 3' direction; and iv) denaturing the duplex of iii) under denaturing conditions to generate a single stranded immobilised polynucleotide; and v) repeating steps ii) to iii) at least once to increase the number of tandem repeats in the immobilised polynucleotide.

As used herein, "hybridisation conditions" refer to the reagents and reaction conditions (e.g. temperature, time etc) that are used. It describes conditions for hybridization and washing. Typically, hybridisation conditions may be stringent or moderate. The hybridisation conditions used in the context of the DNA oligonucleotides synthesis methods described above permit mismatched duplex formation. Preferably, the hybridisation between the sequence of the primer oligonucleotide /polynucleotide and the complementary sequence of the template oligonucleotide will form a stable duplex at 65C and below. It is preferred that a mismatched duplex may be formed at temperatures up to 65 °C, for example between 55C and 65 C, optionally for a time period of between 1 to 30 seconds. However, hybridisation conditions used in the later steps during M-Gate assembly where the complementary overhangs anneal and ligate do not need to permit mismatched duplex formation, and preferably do not permit a mismatched duplex to form.

As used herein, "extension conditions" refer to the reagents and reaction conditions (e.g. temperature, time etc) that are used. It describes conditions for extension of a polynucleotide e.g. a primer oligonucleotide. Appropriate extension conditions are well known in the art.

Preferably, extension is performed at a temperature of between about 65°C and 75°C, optionally for a time period of between 30 to 120 seconds. Appropriate conditions may be found, for example, in Whitfield CJ, Turley AT, Tuite EM, Connolly BA, Pike AR. Enzymatic Method for the Synthesis of Long DNA Sequences with Multiple Repeat Units.Angewandte Chemie International Edition 2015, 54(31), 8971-8974.

Appropriate denaturing conditions are well known in the art and include subjecting the extended duplex to a temperature of about 75-100 °C, preferably about 90 to 98 °C, optionally for about 15 to 30 seconds.

Moderate and stringent conditions are known to those skilled in the art and can be found in available references (e.g., Current Protocols in Molecular Biology, John Wiley & Sons, N.Y., 1989, 6.3.1-6.3.6). Aqueous and non-aqueous methods are described in that reference and either can be used. A preferred example of stringent hybridization conditions are hybridization in 6x sodium chloride/sodium citrate (SSC) at about 45°C, followed by one or more washes in 0.2x SSC, 0.1% (w/v) SDS at 50°C. Another example of stringent hybridization conditions are hybridization in 6x SSC at about 45°C, followed by one or more washes in 0.2x SSC, 0.1% (w/v) SDS at 55°C. A further example of stringent hybridization conditions are hybridization in 6x SSC at about 45°C, followed by one or more washes in 0.2x SSC, 0.1% (w/v) SDS at 60°C. Preferably, stringent hybridization conditions are hybridization in 6x SSC at about 45°C, followed by one or more washes in 0.2x SSC, 0.1% (w/v) SDS at 65°C. Particularly preferred stringency conditions (and the conditions that should be used if the practitioner is uncertain about what conditions should be applied to determine if a molecule is within a hybridization limitation of the invention) are 0.5 molar sodium phosphate, 7% (w/v) SDS at 65°C, followed by one or more washes at 0.2x SSC, 1% (w/v) SDS at 65°C.

Figure 1 illustrates how the DNA extends via an enzymatic slippage reaction to give a series of repeating units, namely repeating fragment sequence of interest (U1) and recognition sites for Type IIB restriction endonuclease BsaXl (U2) e.g. U1 U2 U1 U2 U1 etc. Here, after annealing duplexes in an AB conformation, they were subjected to 20 heat-cool cycles in order to enzymatically extend them into repeating units. Gel electrophoresis was used to characterise the lengths of the extended DNA and, as shown in Figure 2, the three different extended oligonucleotides comprising repeating units extended over 500 by and exceeded the maximum length (20k bp) of the ladder. The synthesis of the extended oligonucleotides was followed by a restriction digest with BsaXl to give separated fragment sequence of interest portions and the restriction site portions -the fragment sequences of interest having single stranded overlaps i.e. 'sticky ends'.

All three extended oligonucleotides were digested, and as they all contained the recognition site for BsaXl they were all able to digest together under the same conditions. (Whilst it is preferred that all of the extended oligonucleotides contain the same recognition sites, in some embodiments of the present invention different extended oligonucleotides could contain different recognition sites and multiple restriction enzymes could be used). In this experiment, digestion was carried out in T4 Ligase buffer (although buffers such as rCutSmart could also be used). Five different reactions were made up which contained various concentrations of each extended product; 20 ng/RL, 15 ng/kL, 10 ng/RL, 5 ng/mL, ing/mL. These reactions were incubated at 37 °C for 1 hour. Gel electrophoresis was used to analyse the digest products of each of these reactions. The recognition site is 33 by long and each fragment sequence of interest is 23 by (the target sequence being the 69bp fragment of MLH1 which is made up of the three fragment sequences of interest). Figure 3 (i) shows the gel electrophoresis image which has the lowest intensity band at just over 50 by which would correspond to a recognition site and a target sequence of interest combined (56 bp). Although further analysis in the ImageJ plot in Figure 3 (ii) shows that two peaks are faintly present between approximately 25 and 35 bp. These faint peaks show the presence of both the fragment sequence of interest and the recognition site.

The decreasing concentrations of DNA digested in Figure 3 prevent accurate visualisation of the digest products. To improve this a large scale digest reaction (1.25 mL) was made up with a concentration of DNA at 0.8 ng/RL. This is 25 times more dilute than the initial reaction (20 ng/RL) but with the same 1 kg of DNA. After digestion, the reaction was concentrated using a speed vac"' vacuum concentrator before purification on with the oligo cleanup method on a Monarch'"' DNA cleanup column. The purified product was then ran on a 4% MetaPhorTm aga rose gel to show the lengths of DNA formed.

Figure 4 shows a more significant band at the fragment sequence of interest length of 23 by and at the BsaXl restriction site length of 33 bp. The band for the combination of these two is still visible at 56 by but is less significant than seen previously in comparison to the fragment sequence of interest band. One unit of a restriction enzyme is defined as digesting 1 kg of lambda DNA in an hour. Lambda DNA contains 19 BsaXl restriction sites per strand which equates to 3.6 x1011 per kg. whereas sequences Oligo-20, Oligo-21, Oligo-22, Oligo-23, Oligo- 24 and Oligo-25, contain 1.8 x1013 sites per Lig on average, which is SO times more sites.

The digest reaction could be adjusted to yield an increase in the target products. For example it could be 50 times more dilute but would require much more enzyme.

Confirmation of appropriate yield and ligation to give target sequence The expected products of the target sequences of interest were made to confirm that the yield was sufficient at this scale to carry out ligation (unlike with prior art size exclusion separation methods which have been shown previously to not yield enough DNA to carry out a ligation post digestion at this scale). Oligonucleotides ('oligos') were made using a DNA Script Syntax with TiEOS method. This method already gives phosphorylated ends to the DNA therefore the kinase step prior to ligation is not required. These were printed with the target concentration of 4 LIM. 23mer-24 failed to reach this and only made 40 ttL of 2.2 p.M. This was still used but was in a significantly lower concentration to the other oligonucleotides.

Duplexes of fragment sequences of interest 23mer-20/23mer-21, 23mer-22/23mer-23 and 23mer-24/23mer-25 were first made by heating to 95 °C for 10 minutes before being left to cool slowly in a heating block. The three duplexes were then combined in equal ratios with T4 ligase and corresponding buffer at room temperature for 1 hour. The starting duplexes and the ligation reaction were ran on a 4% Metaphor'"' agarose gel and visualised to give Figure 5. The starting duplex lengths (23 bp) were seen in all wells (bottom box shown on Fig 5(i)) with a weaker band seen in the ligation well. A weaker band was also seen in Lane 4, this is due to 23mer-24 having a lower concentration than the other oligonucleotides. Two higher products were seen in the ligation well (shown in the middle and upper boxes shown on Figure 5(i)). The middle box shows a band at around SO by which correlates with the ligation of two duplexes together (46 bp). The strongest band is shown in the upper box at around 70 by which is the target length of all three sections ligating together (69 bp) to give the target sequence (i.e. in this case the 69bp fragment of MLH1). Importantly, no longer products are seen in this reaction as there are no palindromic sticky ends.

This method of using Type IIB restriction sites allows for complete customisability on replicated oligonucleotides sequences and selective ligation of sections together post digestion. Here there is a complete methodology, including the pre-step of synthesising extended oligonucleotides from short DNA into long repeating lengths, followed by the digestion by a restriction endonuclease into short sections which are then able to ligate together to give long lengths of target custom DNA. Use of a Type IIB restriction endonuclease removes the limitation found with Type IIP sites which influence the end bases of a sequence.

One-pot methodology As shown above, the extension and digestion of DNA that contains Type IIB restriction sites has been shown to produce custom sticky-ended sequences suitable for gene fragment assembly. The inventors have developed and confirmed the viability of cycling between a Type IIB restriction enzyme digest and sticky-ended ligation in a single reaction pot that produces long linear DNA strand.

As descried above, three sequences of extended-DNA (Oligo-20/01igo-21, Oligo-22/Oligo-23, Oligo-24/Oligo-25), were digested and the ligation of the equivalent TiEOS fragments into a longer 69 by sequence, was demonstrated.

In a preferred one pot embodiment of the method of the invention each of the three extended oligonucleotide DNA samples are initially digested into two portions; one portion containing the cleaved restriction sites and the other the respective cleaved fragment sequences of interest. The three fragment sequences of interest contain overhangs that match and are able to ligate together to give the desired 69 by sequence. Clearly however, the target fragment overhangs also match the restriction site overhangs to which they were initially attached to prior to digestion. A ligation step re-assembles the DNA fragments but it can be seen that various different products can be formed as the restriction sites can either ligate back to where they were prior to digestion or the targets can ligate into the desired 69 by conformation. This range of ligation possibilities leads to a number of different products being formed.

In order to direct the method to maximise the amount of target being formed (the target here being the 69bp fragment of MLH1). Following the initial ligation, the DNA is once again heated to the activation temperature of the restriction enzyme to initiate a second digest. The target fragments that have ligated to one another cannot be digested as they no longer contain the restriction site for BsaXl. However, the DNA that has ligated back to the restriction sites is once again able to be re-digested. Further ligations and digestions are cycled in this manner, to maximise the yield of the 69 by sequence. Figure 6 illustrates this methodology.

Three reactions containing all three extended sequences at 20 ng/p.L were monitored; one (1) contained both ligase and restriction enzyme, one (2) with just the restriction enzyme and lastly one (3) with only the ligase. These were subjected to 5 minutes at 37 °C and 5 minutes at 16 °C cycled 30 times. A 4% MetaPhor agarose gel was used to visualise the products of these reactions, shown in Figure 7. Lanes 1 and 2 showed multiple bands as expected from the restriction digest of the long DNA into smaller fragments. Lane 3 showed only the long extended DNA and appeared to be stuck in the well at the top of the gel. In Lane 2 the extended DNA has been clearly digested into shorter fragments and are more intense than the equivalent bands observed in Lane 1. In Lane 1 the DNA has clearly been digested, although somewhat less efficiently than in Lane 2. In addition, it does not appear as though the fragments in Lane 1 have been ligated due to the absence of bands that are not already present in Lane 2.

In order to increase the amount of product at the target length (69 bp) the heat-cool conditions were optimised. Incubation times were increased to 30 minutes for digestion and 15 minutes for ligation, the same reaction mixtures were then made up and subjected to 30 cycles. Once again gel electrophoresis was used to characterise the reactions, shown in Figure 8. Lane 2 shows strong digest bands of expected lengths (23 by target fragment, 33 by restriction site, 56 by target plus restriction site etc.), and some faint bands that appear in Lane 1 that match these lengths.

It was noted that if there is too much DNA in a reaction compared to the amount of restriction enzyme, in this case BsaXI, full digestion is not realised. One skilled in the art would understand how to optimise the method to encourage full digestion, optionally the restriction enzyme would be used in excess or an optimisation assay could be carried out. To encourage full digestion, and therefore ligation, the same concentration of DNA (0.8 ng/p.L) as previously optimised was investigated. Twelve identical 100 jtl_ reactions were made up and then combined after reaction for purification to yield 20 pL. Once again the control experiments of only restriction enzyme and only ligase were run alongside the reaction with both enzymes.

Gel electrophoresis shows clear digest bands of the target lengths in Lanes 1 and 2 of Figure 9. The bands of the targets were slightly more intense when only the restriction enzyme is present (Lane 2) compared to where both ligase and restriction enzymes were used (Lane 1), which may be due to some ligation occurring. The higher intensity could also be due to the ligase inhibiting the digest or just a slight reduction in the activity of the restriction enzyme in Lane 1.

The target strand is close in length to that of the side products from incomplete digestion, so it is challenging to clearly differentiate using an agarose gel. Sequence 23mer-22/23mer-23 sits between 23mer-20/23mer-21 and 23mer-24/23mer-25 in the final target product.

Therefore, without 23mer-22/23mer-23 the ligation product will not be visible, as it is central position links the outer two sequences. To determine if ligation is occurring within the cycling reaction a control was run without including the extended Oligo-22/01igo-23.1nstead of using a standard agarose gel to visualise the product a TapestationTM (Agilent Technologies Inc.) was instead used as this provides a higher resolution of the bands.

Figure 10 shows the D1000 screentapeTM, interpretation of the intermediate ladder lengths by the software is an approximation and so does not give exact values. Lane 1 shows a wide band, just below the 100 by ladder band, which looks to be made up of two bands, one of which is not present in the other lanes. This length would correspond to the target product of 69 by and indicates that described methodology (termed M-Gate' by the inventors) is a feasible approach.

To further confirm that the target was made PCR was used. PCR is able to amplify sections of DNA that correspond with primer sequences. Amplification of the three reactions shown previously in Figure 10 was carried out with primer sequences Primer-20 (5'-TCG CCG GCG GCG ATC CTG-3') (SEQ ID 7) and Primer-25 (5'-TCG CCG GCG GCG ATC-3') (SEQ ID 8). These were analysed on the Tapestation TM with D1000 screentapeTM. results showed that the M-Gate reaction has produced the target product and is a viable one-pot method.

PCA Alternative Whilst the preferred embodiment of the invention uses ligation to direct assembly, there is an alternative embodiment that can use PCA.

Again, the extended oligonucleotides 3a-e from the previously described 'production of extended oligonucleotides' method can be provided -preferably in a single reaction pot, container or vessel. This may be the same pot, container or vessel that was used to produce the extended oligonucleotides or may be a different pot, container or vessel.

A Type IIB restriction enzyme 5 that recognises the Type IIB restriction recognition site 4 is again provided, however a DNA polymerase, dNTPs and appropriate buffers are provided in this version.

The temperature is taken to the activation temperature ('digestion temperature') of the Type IIB restriction enzyme to initiate a digest of extended oligonucleotides 3a-e. The extended oligonucleotides 3a-e are digested with the Type IIB restriction enzyme to provide multiple cleaved nucleic acid fragment sequences of interest 2a-e with single-stranded overhangs, as well as separate Type IIB restriction recognition sites 4. The temperature is then lowered to an annealing temperature such that cleaved nucleic acid sequences of interest 2a-e which have complementary single-stranded overhangs then anneal. In this case the overlapping single-stranded overhangs do not completely overlap and there are some single stranded portions left between annealed sections. DNA polymerase and dNTPs are provided and the temperature is raised to the extension temperature required for DNA polymerase activation such that the single stranded areas between annealed portions are filled in using known PCA methodology. The temperature is then cycled (thermocycled) between the digestion temperature, the annealing temperature (and in this embodiment preferably also the PCA extension temperature) multiple times.

The patent, scientific and technical literature referred to herein establish knowledge that was available to those skilled in the art at the time of filing. The entire disclosures of the issued patents, published and pending patent applications, and other publications that are cited herein are hereby incorporated by reference to the same extent as if each was specifically and individually indicated to be incorporated by reference. In the case of any inconsistencies, the present disclosure will prevail.

Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. For example, Singleton and Sainsbury, Dictionary of Microbiology and Molecular Biology, 2d Ed., John Wiley and Sons, NY (1994); and Hale and Marham, The Harper Collins Dictionary of Biology, Harper Perennial, NY (1991) provide those of skill in the art with a general dictionary of many of the terms used in the invention. Although any methods and materials similar or equivalent to those described herein find use in the practice of the present invention, the preferred methods and materials are described herein.

All of the features disclosed in this specification (including any accompanying claims, abstract and drawings), and/or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and/or steps are mutually exclusive. Each feature disclosed in this specification (including any accompanying claims, abstract and drawings) may be replaced by alternative features serving the same, equivalent or similar purpose, unless expressly stated otherwise. Thus, unless expressly stated otherwise, each feature disclosed is one example only of a generic series of equivalent or similar features. The invention is not restricted to the details of the foregoing embodiment(s). The invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed.

With respect to the use of substantially any plural and/or singular terms herein, those having skill in the art can translate from the plural to the singular and/or from the singular to the plural as is appropriate to the context and/or application. The various singular/plural permutations may be expressly set forth herein for sake of clarity.

It will be understood by those within the art that, in general, terms used herein, and especially in the appended claims are generally intended as "open" terms (e.g., the term "including" or "comprising" should be interpreted as "including but not limited to," the term "having" should be interpreted as "having at least," the term "includes" should be interpreted as "includes but is not limited to," etc.). It will be further understood by those within the art that if a specific number of an introduced claim recitation is intended, such an intent will be explicitly recited in the claim, and in the absence of such recitation no such intent is present. For example, as an aid to understanding, the following appended claims may contain usage of the introductory phrases "at least one" and "one or more" to introduce claim recitations. However, the use of such phrases should not be construed to imply that the introduction of a claim recitation by the indefinite articles "a" or "an" limits any particular claim containing such introduced claim recitation to embodiments containing only one such recitation, even when the same claim includes the introductory phrases "one or more" or "at least one" and indefinite articles such as "a" or "an" (e.g., "a" and/or "an" should be interpreted to mean "at least one" or "one or more"); the same holds true for the use of definite articles used to introduce claim recitations. In addition, even if a specific number of an introduced claim recitation is explicitly recited, those skilled in the art will recognize that such recitation should be interpreted to mean at least the recited number (e.g., the bare recitation of "two recitations," without other modifiers, means at least two recitations, or two or more recitations).

It will be appreciated that various embodiments of the present disclosure have been described herein for purposes of illustration, and that various modifications may be made without departing from the scope of the present disclosure. Accordingly, the various embodiments disclosed herein are not intended to be limiting, with the true scope being indicated by the following claims.

Claims (27)

1. CLAIMS1. A nucleic acid assembly method comprising: providing an extended oligonucleotide comprising at least two repeating units, the first unit being a nucleic acid fragment sequence of interest and the second unit being a recognition site for a Type IIB or Type IIB-like restriction enzyme; digesting said oligonucleotide with a Type IIB or Type IIB-like restriction enzyme to provide cleaved nucleic acid fragment sequences of interest with single-stranded overhangs; annealing two or more cleaved sequence portions where the single-stranded overhangs are complementary.
2. 2. A nucleic acid assembly method as in Claim 1 wherein the step of digesting said oligonucleotide with a Type IIB or Type IIB-like restriction enzyme provides cleaved nucleic acid fragment sequences of interest with single-stranded overhangs on each end.
3. 3. A nucleic acid assembly method as in any of the previous claims further comprising providing a plurality or set of extended oligonucleotides comprising at least two repeating units.
4. 4. A nucleic acid assembly method as in claim 3 wherein different extended oligonucleotides contain different nucleic acid fragment sequences of interest.
5. 5. A nucleic acid assembly method as in any of the previous claims wherein the repeating first units within an extended nucleotide may comprise different nucleic acid fragment sequences of interest.
6. 6. A nucleic acid assembly method as in any of the previous claims wherein the single-stranded overhangs are 2 or more bases in length and preferably the single-stranded overhangs are 3 or more bases in length.
7. 7. A nucleic acid assembly method as in any of the previous claims wherein the single-stranded overhangs are non-palindromic.
8. 8. A nucleic acid assembly method as in any of the previous claims wherein the method is plasmid-free.
9. 9. A nucleic acid assembly method as in any of the previous claims which is for assembling a target polynucleotide and which comprises the step of determining, for the target polynucleotide, an optimized overhang sequence for each nucleic acid fragment sequence of interest for joining into an ordered set to assemble the target polynucleotide.
10. 10. A nucleic acid assembly method as in any of the previous claims wherein the digestion, and annealing steps are repeated or cycled.
11. 11. A nucleic acid assembly method as in claim 10 wherein the digestion and annealing steps are repeated or cycled 10 or more times.
12. 12. A nucleic acid assembly method as in claim 10 wherein the digestion, and annealing steps are repeated or cycled 30 or more times.
13. 13. A nucleic acid assembly method as in any of the previous claims wherein the Type IIB or Type IIB-like restriction enzymes are selected from the following list: Name Recognition Sequence CspCl (10-11/12-13)CAANNNNNGTGG(12-13/10-11) Alol (7/12-13)GAACNNNNNNTCC(12-13/7) Ppil (7/12)GAACNNNNNCTC(13/8) Psrl (7/12)GAACNNNNNNTAC(12/7) BO (8/13)GAGNNNNNCTC(13/8) Fall (8/13)AAGNNNNNCTT(13/8) Bsp241 (8/13)GACNNNNNNTGG(12/7) BsaXl (9/12)ACNNNNNCTCC(10/7) HaehV (7/13)GAYNNNNNRTC(14/9) Cjel (8/14)CCANNNNNNGT(15/9) CjePI (7/13)CCANNNNNNNTC(14/8) Hin4l (8/13)GAYNNNNNVTC(13/8) Bael (10/15) ACNNNNGTAYC(12/7) Alfl (10/12)GCANNNNNNTGC(12/10) Bcgl (10/12)CGANNNNNNTGC(12/10) BsIFI (6/10)GGGAC(10/14)
14. 14. A nucleic acid assembly method as in any of the previous claims wherein the step of providing the extended oligonucleotide comprising at least two repeating units includes synthesising the extended oligonucleotide comprising at least two repeating units.
15. 15. A nucleic acid assembly method as in claim 14 wherein synthesis of the extended oligonucleotide comprising at least two repeating units occurs in solution or on a solid support.
16. 16. A nucleic acid assembly method as in either of claims 14 or 15 wherein the extended oligonucleotide is synthesised by an enzymatic method.
17. 17. A nucleic acid assembly method as in any of the previous claims wherein the step of digesting said oligonucleotide with a Type IIB or Type IIB-like restriction enzyme to provide cleaved nucleic acid fragment sequences of interest with single-stranded overhangs occurs at a digesting temperature, the digesting temperature being the activation temperature of the Type IIB or Type IIB-like restriction enzyme.
18. 18. A nucleic acid assembly method as claim 17 wherein the step of annealing two or more cleaved sequence portions where the single-stranded overhangs are complementary occurs at an annealing temperature which is different to the digesting temperature.
19. 19. A nucleic acid assembly method as in any of the previous Claims wherein the annealed sequence portions are ligated, after annealing, to form a longer length linear DNA.
20. 20. A nucleic acid assembly method as in claim 19 wherein the digestion, annealing and ligation occurs in a 'single pot reaction'.
21. 21. A nucleic acid assembly method as in any of claims 19 or 20 wherein the DNA ligase is a Type 2 ligase, or variant thereof such as a thermostable variant or high salt tolerant ligase.
22. 22. A nucleic acid assembly method as in any of claims 1 to 18 wherein the annealed sequence portions are assembled using polymerase cycling assembly (PCA).
23. 23. A nucleic acid assembly method as in claim 22 wherein the single stranded overhangs that are produced by restriction with a type 115 restriction endonuclease recognising the recognition sites of the cleavage sites, partially overlap such that portions of single stranded DNA remain between annealed sections.
24. 24. A nucleic acid assembly method as in any of claims 22 to 23 wherein the annealed sequence portions are extended by DNA polymerase such that single stranded DNA remaining between annealed sections is flied in.
25. 25. A nucleic acid assembly method as in any of claims 22 to 24 wherein the digestion, annealing and PCA occurs in a 'single pot reaction'.
26. 26. A nucleic acid assembly method as in any of the previous claims wherein, after cycling of the digestion and annealing steps, and when dependent on claims 19 to 25 after the subsequent ligation and/or PCA steps, the DNA is separated e.g. by HPLC.
27. 27. A method for producing a target nucleic acid as in any of the previous claims comprising: providing a set of extended oligonucleotides, each extended oligonucleotide comprising: (i) a recognition site for a Type IIB or Type IIB-like restriction enzyme, the cleavage sites for said restriction enzyme being provided upstream and downstream of said recognition site; (ii) a fragment sequence of interest, positioned between the upstream cleavage site of the recognition site of (i) and the downstream cleavage site of a further recognition site of Type IIB or Type IIB-like restriction enzyme; (iii) said further recognition site for a Type IIB or Type IIB-like restriction enzyme, the cleavage sites for said restriction enzyme being provided upstream and downstream of said further recognition site, wherein in a first extended oligonucleotide, a Type IIB or Type IIB-like restriction enzyme can produce at least one first cleaved fragment sequence of interest (ii) with single-stranded overhangs on each end by cleaving both the upstream cleavage site of item (i) and the downstream cleavage site of item (iii); and in a second extended oligonucleotide, a Type IIB or Type IIB-like restriction enzyme can produce at least one second cleaved fragment sequence of interest (ii) with single- 1 0 stranded overhangs on each end by cleaving both the upstream cleavage site of item (i) and the downstream cleavage site of item (iii); a single-stranded overhang of the first cleaved fragment sequence of interest from the first extended oligonucleotide being complementary to a single-stranded overhang of the second cleaved fragment sequence of interest from the second extended oligonucleotide; such that, under appropriate conditions, the first cleaved fragment sequence of interest and second cleaved fragment sequence of interest will anneal, and be optionally ligated after annealing, to form a linear DNA.