Templates and methods for nucleic acid synthesis

Abstract

The present invention relates to the use of templates with junction regions in the circularisation of linear nucleic acids; and to templates with junction regions for use in nucleic acid synthesis, methods of making said templates for use in nucleic acid synthesis, and methods of using said templates to synthesise nucleic acids.

Description

[0001] Templates and Methods for Nucleic Acid Synthesis

[0002] Technical Field

[0003] The present invention relates to templates with one or more junctions and their use in the circularisation of linear nucleic acids and in nucleic acid synthesis; as well as to methods of making templates for use in nucleic acid synthesis, and methods of using templates to synthesise nucleic acids.

[0004] Background

[0005] Nucleic acids, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), are synthesised naturally in vivo by enzymatic processes utilising polymerase enzymes. Advancements in molecular biology have led scientists to develop methods of artificially synthesising nucleic acids to be used as research tools and in therapeutics and commercial applications.

[0006] Currently, in order to obtain specific nucleic acid sequences (such as a particular gene sequence), specifically designed oligonucleotide primers are used to amplify the gene sequence or part of the gene sequence using the polymerase chain reaction (PCR). These oligonucleotide primers are short oligonucleotide sequences which provide a template in the PCR reaction. Oligonucleotide primers are typically chemically synthesised by a process known as the phosphoramidite method. The phosphoramidite method is limited to the synthesis of oligonucleotides which are less than 200 bases in length so is unsuitable for the synthesis of longer sequences such as whole gene sequences.

[0007] Where the gene sequence is too long to be amplified as a single sequence using PCR, multiple sets of overlapping primers are used to amplify multiple partial gene sequences, which can subsequently be assembled. It is often difficult to isolate and characterise the target gene sequence as there will often be several products of similar length present in the resulting ligation mixture and the yield of the target sequence within the mixture is often low. There are particular difficulties associated with complex sequences such as homopolymers. Once the target gene sequence has been isolated, the gene sequence is cloned into a plasmid vector. The plasmid vector will typically contain additional sequences such as a sequence conferring antibiotic resistance and restriction enzyme sites. A host organism (typically bacteria) is then transformed with the plasmid containing the gene sequence. The bacteria are then cultured to replicate the plasmid containingthe gene sequence of interest. Successfully transformed bacteria can be isolated by treatment with an antibiotic for which they contain a resistance gene. The isolated bacteria are subsequently harvested, the plasmids containing the gene sequence are isolated and the gene sequences excised from the plasmid and purified. This multi-step process is long and labour intensive. In addition, the use of living cells to replicate a sequence can result in issues such as the introduction of mutations into the gene sequence. There are additional concerns surroundingthe use of antibiotics in traditional cloning techniques and the potential for overuse of antibiotics to contribute to antimicrobial resistance. Also, when DNA has been synthesised or isolated from cells, it must be demonstrated that it is free from contaminants.

[0008] With recent advancements in emerging fields such as genetic medicine, there is a great need to move away from this traditional method of synthesising nucleic acids to a faster, more efficient means, which does not rely on cells and can reliably produce high yields of long and / or complex nucleic acid sequences, such as gene sequences.

[0009] Summary of the Invention

[0010] DNA junctions constitute a fundamental structural basis for DNA nanotechnology.

[0011] According to an aspect of the present invention, there is provided one or more junctions for use in nucleic acid synthesis.

[0012] There is provided a junction region comprising: a first nucleic acid strand comprising a target bindingor template sequence, and a 5’ junction sequence located substantially at the 5’ end of the first nucleic acid strand; an additional nucleic acid strand comprising a target bindingor template sequence, and a 3’ junction sequence located substantially at the 3’ end of the additional nucleic acid strand; wherein the 5’ junction sequence is at least partially complementary to the 3’ junction sequence. Under annealing conditions the 5’ junction sequence and 3’ junction sequence anneal to form said junction region.

[0013] There is also provided a bridged junction region consisting of a junction region (the first nucleic acid strand and the additional nucleic acid strand) and a bridging strand. The bridging strand includes a bridge sequence that is complementary to at least part of the template sequence (the part closest to the junction sequence) on the first strand and complementary to at least part of the template sequence (the part closest to the junction sequence) on the additionalstrand, such that under annealing conditions it binds to, and thus bridges, the junction formed by the first and additional strand.

[0014] There is also an initiation sequence which is the initiation site of the target that will be elongated and / or ligated - this may be present as part of the bridging strand.

[0015] The 3’ end of the bridging strand crosses the junction region.

[0016] Under annealing conditions, the three strands (the first nucleic acid strand, the additional nucleic acid strand and the bridging strand) anneal to form a 3-way junction or bridged junction. The initiation sequence (also referred to as a primer sequence) is typically different to the bridge sequences but could overlap with or be the bridge sequences in some embodiments.

[0017] Preferably the portion of the bridge sequence which is complementary to at least part of the template sequence on the first strand, and the portion of the bridge sequence which is complementary to at least part of the template sequence on the additional strand, have no additional bases between them.

[0018] The 5’ junction sequence is located upstream of the target binding or template sequence on the first nucleic acid strand. The 3’ junction sequence is downstream of the target binding or template sequence on the additional nucleic acid strand.

[0019] Optionally, the 5’ junction sequence is at least partially complementary to the 3’ junction sequence when read in the opposite direction.

[0020] The junction region has a 3’ single stranded overhang on the 5’>3’ strand and a 5’ single stranded overhang on the 3’>5’ strand, said 3’ single stranded overhang comprising the target binding or template sequence of the first nucleic acid strand, and said 5’ single stranded overhang comprising the target binding or template sequence of the additional nucleic acid strand. The ‘junction’ is formed where the two strands (the first nucleic acid strand and the additional nucleic acid strand) meet. The bridged junction also has the third bridging strand (which may also include the initiation site of the target), which must cross the junction with at least three bases.

[0021] The two junction strands i.e. the first nucleic acid strand and the additional nucleic acid strand create a complementary ssDNA region for binding the third bridging strand (initiation site of the target), which must cross the junction with at least three bases. Preferably, the target binding or template sequence of the first nucleic acid strand is not complementary to the target binding or template sequence of the additional nucleic acid strand.

[0022] Optionally, there are one or more optional bases located between the 5’ junction sequence and the target binding or template sequence and / or between the 3’ junction sequence and the target binding or template sequence. The one or more optional bases may be a sequence selected from the following: linker sequence, spacer sequence, secondary structure sequence, and / or restriction enzyme target sequence.

[0023] Advantageously, a linker or spacer sequence can provide flexibility and reduce steric hindrance between the junction and target binding sequences, enhancing binding efficiency. Including a secondary structure sequence increases the junction's stability, helps with proper folding, minimises misfolding, and controls the structure's angle and geometry. In addition, it may used as a functional site for binding or enzymatic reactions. Including a restriction enzyme target sequence allows the use of restriction enzymes to cut and manipulate the nucleic acid strands.

[0024] There is also provided a method of forming an oligonucleotide with at least one junction region according to the first aspect, comprising: annealing together the first nucleic acid strand and the additional nucleic acid strand. Then, these two strands can be ligated to create a reusable fixed template with close-ended DNA atthe said junction region (either in a circular or hairpin-like structure).

[0025] Advantageously, as the 5’ junction sequence is at least partially complementary to the 3’ junction sequence when read in the opposite direction, this results in a junction forming. The junction has a secondary structure at room temperature or body temperature (preferably at both room temperature and body temperature) which extends outwards and away from the template sequence and which is formed due to base pairing between the complementary regions forming a paired double helix (this is unlike a structure with sticky ends which overlap to give an in-line structure). The junction structure has similarities to a stem or stem loop but is formed from the joining of two single strand ends ratherthan within a single strand (and it may or may not contain an unpaired loop region). The two single stranded ends may be present on two separate strands, or in some specific cases may be the opposite ends of the same strand.

[0026] Optionally, the 5’ junction sequence is at least partially complementary to the 3’ junction sequence when read in the opposite direction.

[0027] There is also provided a multi-junction template comprising a plurality of junction regions, for use in synthesis of a target nucleic acid sequence, wherein when the first, additional strands and optionally the bridging strands are annealed, wherein the template sequences are the complement of the target nucleic acid sequence.

[0028] In an aspect the multi-junction template comprises: a first nucleic acid strand comprising a target binding or template sequence, and a 5’ junction sequence located substantially at the 5’ end of the first nucleic acid strand; a plurality of additional nucleic acid strands comprising a target binding or template sequence, and a 3’ junction sequence located substantially atthe3’ end of the additional nucleic acid strand; wherein the 5’ junction sequence is at least partially complementary to a 3’ junction sequence, such that under annealing conditions the 5’ junction sequence and said partially complementary 3’ junction sequence anneal to form said junction region; and wherein at least one of the plurality of additional nucleic acid strands is a middle nucleic acid strand which further comprises a 5’ junction sequence located substantially at the 5’ end of the middle nucleic acid strand; wherein the 5’ junction sequence of the at least one middle nucleic strand is at least partially complementary to the 3’ junction sequence of an additional strand, such that under annealing conditions said 5’ and 3’ junction sequences anneal to form at least a first junction region and a second junction region; when the first and additional strands are annealed, the template sequences are the complement of a target nucleic acid sequence. In other words, A first nucleic acid strand that includes a target binding or template sequence and a 5’ junction sequence located substantially at the 5’ end of the first nucleic acid strand; a plurality of additional nucleic acid strands, each comprising a target binding or template sequence and a 3’ junction sequence located substantially at the 3’ end of the additional nucleic acid strand; wherein the 5’ junction sequence is at least partially complementary to a 3’ junction sequence, such that under annealing conditions, the 5’ junction sequence and the partially complementary 3’ junction sequence anneal to form a junction region; wherein at least one of the plurality of additional nucleic acid strands is a middle nucleic acid strand that further comprises a 5’ junction sequence located substantially at the 5’ end of the middle nucleic acid strand; wherein the 5’ junction sequence of the at least one middle nucleic acid strand is at least partially complementary to the 3’ junction sequence of an additional strand, such that under annealing conditions, the 5’ and 3’ junction sequences anneal to form at least a first junction region and a second junction region; when the first and additional strands are annealed, the template sequences are complementary to a target nucleic acid sequence

[0029] The 5’ and 3’ junction sequences are selected or engineered to form complementary (or partially complementary) pairs which will anneal together under annealing conditions - effectively bringing the associated template sequence portions together. These ‘pairs’ of junction sequences may all differ from each other to ensure the template sequences are brought together in a specific combination to allow for long and very specific sequences to be made.

[0030] The multi-junction template, is particularly useful in the synthesis of long target DNA sequences and DNA sequences which are difficult to synthesise using currently known technologies, such as DNA sequences comprising multiple repeating units or highly repetitive sequences (e.g. poly A sequences) with precisely controlled lengths that are not achievable by other methods.

[0031] The present invention overcomes issues with current in vitro nucleic acid synthesis technologies as it allows the synthesis of long nucleic acid sequences and / or difficult to synthesise sequences. Current commercial nucleic acid synthesis technologies are generally limited to producing single-stranded DNA sequences of approximately 200 nucleotides in length. Furthermore, no existing DNA assembly methods are known to generate homogeneous double-stranded DNA with high fidelity. The templates and methodologies described in the present invention enable the synthesis of longer and more complex nucleic acid sequences.

[0032] Optionally, the 5’ junction sequence is at least partially complementary to the 3’ junction sequence when read in the opposite direction.

[0033] Preferably, the multi-junction template comprises a plurality of additional nucleic acid strands and wherein at least one of the plurality of additional nucleic acid strands is a middle nucleic acid strand which further comprises a 5’ junction sequence located at the 5’ end of the middle nucleic acid strand; wherein the 5’ junction sequence of the at least one middle nucleic acid strand is at least partially complementary to the 3’ junction sequence of an additional strand, such that under annealing conditions said 5’ and 3’ junction sequences anneal to form at least a first junction region and a second junction region.

[0034] The plurality of additional nucleic acid strands can include one or more middle strands and / or a terminal strand.

[0035] Preferably there are a plurality of bridging strands, each bridging strand having a portion of the bridge sequence which is complementary to at least part of the template sequence on a first strand or additional, and a portion of the bridge sequence which is complementary to at least part of the template sequence on a different additional strand. Each of the plurality of bridging strands bridges a different junction on the template.

[0036] Optionally, the template is readable in a 3’ to 5’ direction.

[0037] The template can be either strand (3’>5’ or 5’>3’) depending on which strand the target nucleic acid is. If the template is 3’>5, the bridging nucleic acid strand which acts as the initiation site (primer or long oligonucleotide) sequence will be complementary to the 3’ end of the template sequence on the first nucleic acid strand. If the template is 5’>3’, the primer sequence will be complementary to the 5’ end of the template sequence on the additional nucleic acid strand.

[0038] Optionally, one of the first nucleic acid strand or plurality of additional nucleic acid strands is a terminating strand comprising a terminating sequence located substantially at the 5’ end or 3’ of the terminating strand.

[0039] Preferably, all the junctions in the multi-junction template are connected (bridged) by bridging strands. The bridging strands can act as initiation site sequences.

[0040] If the template is readable in a 3’>5’ direction, the terminating strand will be one of the additional nucleic acid strands and the terminating sequence will be at the 5’ end of the terminating strand.

[0041] If the template is readable in a 5’>3’ direction, the terminating strand will be the first nucleic acid strand and the terminating sequence will be at the 3’ end of the terminating strand.

[0042] Preferably, each 5’ junction sequence has a distinct nucleic acid sequence.

[0043] Advantageously, when each 5’ junction sequence has a distinct sequence, multiple junctions can simultaneously be assembled into the correct order. Due to complementary base pairing, the distinct 5’ sequences will only bind to their complementary 3’ junction sequence and therefore multiple junctions can be assembled into a large template in a single pot reaction.

[0044] Preferably, each 5’ junction sequence, 3’ junction sequence and target binding or template sequence has a distinct sequence.

[0045] Advantageously, when each sequence is distinct non-specific assembly of the junction can be avoided.

[0046] The template with one or more junction regions, preferably a multi-junction template, of the present invention can self-assemble under annealing conditions as the 5’ junction sequence of each nucleic acid strand has a distinct sequence which is at least partially complementary with the 3’ junction sequence of another nucleic acid strand which contains the template sequence which is the neighbouring part of the complementary sequence of the target nucleic acid sequence.

[0047] According to a related aspect of the present invention there is provided a method of manufacturing a template with one or more junction regions of the first aspect, preferably a multi-junction template, the method comprising: contacting at least the first nucleic acid strands and the additional nucleic acid strand under annealing conditions. The method may further comprise contacting the first nucleic acid strand and the additional nucleic acid strand with a ligase enzyme under ligation conditions. This can be used to create a fixed template with close-ended DNA at the said junction region, which is a hairpin-like structure.

[0048] Advantageously, when the template has undergone ligation, it will not disassemble under denaturing conditions. This allows the target DNA to be easily purified from the template using denaturising conditions and ensures that the ligated template can be readily reused in subsequent synthesis reactions. Optionally, the method of manufacturing can be adapted to manufacture multiple templates by including the steps of: providing a single stranded primer that is immobilised to a surface at one end and having the other end exposed to allow for primer extension, said primer being complementary to the 3’ end of the template or the 5’ end of the template; annealing the template to the primer under annealing conditions; extending the primer, using the template as a template, in the presence of nucleotides and enzymes that are able to open or linearise the one or more junction regions, to give a surface bound strand that is complementary to the template; denaturing the template from the surface bound strand under denaturing conditions; annealing a reverse primer to the surface bound strand; extending the reverse primer in the presence of a mixture of helicase and polymerase and bases; denaturing the strands so that they separate; and optionally repeating the annealing a reverse primer, extending the reverse primer and denaturing steps.

[0049] Optionally, the enzyme is a mixture of helicase and polymerase.

[0050] Optionally the single stranded primer that is immobilised to a surface is immobilised to a surface using succinimidyl ester surface chemistry

[0051] Optionally the surface is a chip, slide or bead.

[0052] Optionally the surface material is selected from mica, glass, silicon, magnetic silica beads, polypropylene. It is preferred that the surface is a material suitable for use with silane chemistry. Also, metals and metaloxide surfacesfor use with thioland phosphate.

[0053] Preferably the surface is pre-functionalized with N-hydroxysuccinimide (NHS) ester terminal groups.

[0054] Advantageously NHS ester terminal groups are used to covalently couple 5'-amino- modified primers. According to a related aspect of the present invention there is provided a method for synthesising a target nucleic acid comprising: contacting the template of an earlier aspect with at least one bridge sequence and a primer sequence under annealing conditions to form a template-primer-bridge complex; contacting the template-primer- bridge complex with a polymerase under extension conditions to synthesise the target nucleic acid; wherein said at least one bridge sequence is complementary to at least the 5’ end of one template sequence and the 3’ end of a neighbouring template sequence; wherein said primer sequence is complementary to at least the 3’ end of the template sequence of the first nucleic acid strand or wherein said primer sequence is complementary to at least the 5’ end of the template sequence of the additional nucleic acid strand.

[0055] The method for synthesising a target nucleic acid can also encompass the use of the template, particularly a multi-junction template of an earlier aspect in the in the synthesis of a target nucleic acid.

[0056] Optionally, the 5’ junction sequence is at least partially complementary to the 3’ junction sequence when read in the opposite direction.

[0057] Optionally, the bridging strand is at least 4 bases in length

[0058] Optionally, the bridge sequence is at least 4 bases in length.

[0059] Preferably, the method further comprises a plurality of bridging strands having bridge sequences, each of the plurality of bridge strands being at least partially complementary to the 5’ end of one template sequence and at least partially complementary to the 3’ end of the neighbouringtemplate sequence.

[0060] Optionally, the primer or the initiation site sequence is on the same nucleic acid strand (bridging strand) as the at least one bridge sequence. Optionally, the primer orthe initiation site sequence is on a separate nucleic acid strand (bridging strand) to the at least one bridge sequence.

[0061] Preferably, the synthesised target nucleic acid is denatured from the template.

[0062] Preferably, the target nucleic acid sequence is isolated from the template by a suitable isolation means.

[0063] Optionally, the suitable isolation means is a size selection method.

[0064] Optionally, the suitable isolation means is selected from: high-performance liquid chromatography; gel electrophoresis followed by extraction; ultracentrifugation; spin column purification; use of functionalised magnetic beads to bind target molecules.

[0065] Optionally, the template is a reuseable template.

[0066] Advantageously, the template can be denatured from the target sequence and reused as a template in subsequent reactions to generate more of the target sequence. As the template is not consumed in the reaction, it can be reused in subsequent reactions to generate more target DNA.

[0067] Preferably, the 3’ junction sequences and 5’ junction sequences are each at least 6 bases in length. Most preferably the 3’ junction sequences and 5’ junction sequences are each at least 6 bases in length, with at least 3 bases of each sequence being complementary to the other. In some cases at least one of the junction sequences are at least 6 bases in length.

[0068] In some cases at least one of the junction sequences are at least 10 bases in length.

[0069] Preferably, the template sequence is at least 6 bases. In some cases the template sequence is at least 6 bases. Optionally, the template is attached to a surface.

[0070] Advantageously, surface attachment of the template would allow for readily incorporatin the present method into devices to enable automation of the method.

[0071] According to another aspect of the present invention there is provided a method of circularising a linear single strand nucleic acid using a junction region according to the first aspect, wherein the target binding sequence of the first nucleic acid strand is at least partially complementary to a first end of the linear single strand nucleic acid; annealing the additional nucleic acid strand to a second end of the linear single strand nucleic acid, the second end being different to the first end, wherein the target binding sequence is at least partially complementary to a second end of the linear single strand nucleic acid, and the middle of this strand is partially complementary to last bases of the first end of the linear single strand; annealing the 5’ junction sequence to the 3’ junction sequence to form the junction region and circularising the single strand nucleic acid. The annealing process typically occurs in a single step for all strands.

[0072] Preferably the method occurs as a ‘single-pot’ reaction.

[0073] Advantageously the circularised single strands will serve as templates for new plasmids by annealing a primer or primers that can then be elongated and ligated e.g. by including the steps of: annealing a primer to the circularised nucleic acid strand; extending the primer in the presence of a polymerase and bases; and optionally denaturing the strands so that they separate. The newly created strands can then be circularised using the same method.

[0074] Optionally multiple plasmids can be manufactured by repeating the steps of annealing a primer, and extendingthe primerand optionally denaturingto produce further templates.

[0075] Preferably the junction region is ligated by contacting it with a ligase enzyme under ligation conditions. Preferably the method of circularising a linear single strand DNA can be used to circularise a DNA duplex by, prior to annealing the first nucleic acid strand, there is a step of: denaturing the DNA duplex to give a first linear single strand DNA and a second linear single strand DNA.

[0076] Optionally, the 5’ junction sequence is at least partially complementary to the 3’ junction sequence when read in the opposite direction.

[0077] Optionally, the 3’ junction sequences and 5’ junction sequences are at least 6 base pairs in length.

[0078] Optionally, wherein the template sequence is at least 20 base pairs. A further option, wherein the template sequence is at least 40 base pairs.

[0079] Various further features and aspects of the invention are defined in the claims.

[0080] 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.

[0081] Brief Description of the Drawings

[0082] 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:

[0083] Figure 1 is a simplified schematic diagram showing a method of synthesising nucleic acids using a single junction template in accordance with certain aspects of the present invention; Figure 2 is a simplified schematic diagram showing a ligated single junction template in accordance with certain embodiments of the present invention;

[0084] Figure 3 is a simplified schematic diagram showing a method of synthesising DNA using a three-junction template in accordance with certain embodiments of the present invention;

[0085] Figure 4a shows a template with bridging sequences and figures 4b and 4c show the characterisation of DNA synthesised usinga singlejunction template in accordance with the present invention;

[0086] Figure 5 shows the characterisation of DNA synthesised using a short single junction template in accordance with the present invention;

[0087] Figure 6 shows the characterisation of DNA synthesised using a long single junction template in accordance with the present invention;

[0088] Figure 7a shows a template with bridging sequences and figures 7b and 7c show the characterisation of DNA synthesised using a two-junction template in accordance with the present invention;

[0089] Figure 8 shows the characterisation of DNA synthesised using a two-junction template with different lengths in accordance with the present invention;

[0090] Figure 9a shows a template with bridging sequences and figures 9b and 9c show the characterisation of DNA synthesised using a three-junction template in accordance with the present invention;

[0091] Figure 10 shows the characterisation of poly A DNA sequence synthesised using a singlejunction template in accordance with the present invention; Figure 11 is a schematic diagram showing how multi-junction templates can be replicated in accordance with the present invention;

[0092] Figure 12 is a schematic diagram showing how the junction regions of the present invention can be used to circularise DNA e.g. to provide templates for plasmids;

[0093] Figure 13 shows the characterisation of DNA synthesised using a single junction template in accordance with the present invention; Figure 14 shows the characterisation of poly A DNA sequence synthesised using a three- junction template in accordance with the present invention.

[0094] Detailed Description

[0095] Figure 1 a is a simplified schematic diagram showing a method of synthesising nucleic acids using a single junction template. A template-primer-bridge complex 100 comprising a first strand 101, which is readable in a 3’ to 5’ direction. The first strand comprises a template sequence 102 which is flanked at its 5’ end by a 5’ junction sequence 103.

[0096] The single-junction template 100 also comprises a second strand 104 which is readable in a 3’ to 5’ direction. The second strand 104 comprises a 3’ junction sequence 105 at its 3’ end and a template sequence 106 at its 5’ end.

[0097] The first and second strands 101 , 104 may be synthetic DNA such as oligonucleotides.

[0098] An oligonucleotide is a relatively short sequence of nucleotides, typically ranging from about 2 to 100, sometimes 2-60, sometimes 2 to 25 bases in length, which can be either DNA or RNA. These molecules are synthetically produced and are widely used in genetic research, diagnostics, and molecular biology techniques such as PCR, sequencing, and as primers or probes due to their ability to bind specifically to complementary nucleic acid sequences.

[0099] It should be understood that, throughout this description, the term "nucleic acids" is intended to encompass not only naturally occurring DNA and RNA, but also their various analogues and derivatives. This includes, for example, modified DNA such as methylated DNA, phosphorothioate DNA, and locked nucleic acids (LNA), as well as RNA variants like 2'-O-methyl RNA and small interfering RNA (siRNA). Both naturally occurring and synthetic forms are included within the scope of the present invention.

[0100] The 5’ junction sequence 103 and 3’ junction sequence 105 are complementary, such that under annealing conditions, they together form a double stranded junction region 107. Each of the template sequences 102 and 106 is configured such that they are complementary to part of the target DNA. For example, if the target sequence is ACAACA, the complementary sequence is TGTTGT. Each template sequence corresponds to part of the complementary sequence of the target sequence. The 3’ junction sequence 105 of the second strand 104 is configured to be complementary to the 5’ junction sequence 103 of the first strand 101 , such when complementary base pairing occurs, the strands assemble in the correct order to give the complementary sequence of the target sequence.

[0101] The template sequences 102 and 106 are complementary to the target sequence which is being synthesised.

[0102] To synthesise the target DNA, the first strand 101 , the second strand 104, and the bridging strand 108 forming the initiation site (or primer sequence) and bridge are annealed together under appropriate annealing conditions. The 5’ end of the bridging strand is the primer sequence 109 and is complementary to the 3’ end of the template sequence 102 of the first strand 101 . The bridge sequence 110 is complementary to the remainder of the template sequence 102 of the first strand 101 .

[0103] The bridge sequence 110 is also complementary to at least the 3’ end of the template sequence 106 of the second strand 104. Under annealing conditions, the 5’ junction sequence 103 anneals to the 3’ junction sequence 105 by complementary base pairing, the primer sequence 109 anneals to the 3’ end of the template sequence 102 of the first strand 101 by complementary base pairing. The bridge sequence 110 anneals to the remainder of the template sequence 102 of the first strand 101 and the 3’ end of the template sequence 106 of the second strand 104 by complementary base pairing. The result is a template-primer-bridge complex 100 in which the bridge sequence 110 bridges the double stranded junction region 107 by annealing to both the 5’ end of the first strand 101 and the 3’ end of the second strand 104. In this particular embodiment, the primer / initiation sequence 109 and bridge sequence 110 are provided on a single oligonucleotide 112 (as shown in Fig. 1 b). The primer sequence 109 and bridge sequence 110 do not need to be present on the same strand and could therefore be provided as separate nucleic acid strands 113, or oligonucleotides (as shown in Fig. 1 c).

[0104] The template-primer-bridge complex 100 is then brought into contact with a polymerase and nucleotides under extension conditions. The polymerase extends the double stranded region that has been formed by the primer sequence 109 and bridge sequence 110 annealingto the template sequences 102, 106 using the remainder of the template sequence 106 as a template. The resulting DNA sequence is the target DNA 111 and is the complement sequence to the template sequence 102 of the first strand 101 and the template sequence 106 of the second strand 104. The target DNA 111 can be isolated by denaturingthe duplex and separating the target DNA 111 from the template sequences 102, 106. The template sequences 102, 106 can then be reused to generate more target DNA.

[0105] Reference to template-primer-bridge complexes can be understood to refer to template- ‘initiation sequence’-bridge complexes.

[0106] Figure 2 is a simplified schematic diagram showing a ligated single junction template in accordance with certain embodiments of the present invention. The template comprises a first strand 201 and second strand 202. The first strand 201 comprises a template sequence 203 and a 5’ junction sequence 204 located at the 5’ end of the template sequence. The second strand 202 comprises a template sequence 206 and a 3’ junction sequence 205 which is located at the 3’ end of the template sequence.

[0107] The 3’ junction sequence 205 of the second strand 202 is in part complementary to the 5’ junction sequence 204 of the first strand 201 and partially self-complementary such that under annealing conditions a partial hairpin loop 207 is formed in the 3’ junction sequence 205. The self-complementary region is located at the 3’ end of the 3’ junction sequence 205 such that hairpin loop is formed at the 3’ terminus of the 3’ junction sequence 205. Under annealing conditions, the 5’ junction sequence 204 of the first strand 201 anneals to the part of the 3’ junction sequence 205 of the second strand 202 to which it is complementary.

[0108] The annealed first and second strands 201 , 202 are then brought into contact with a DNA ligase enzyme under ligation conditions to generate the formation of a phosphodiester bond resulting in a ligated template 208.

[0109] Whilst the examples above are useful, there is a particular benefit when a multi-junction template is used. This allows long target nucleic acid sequences to be synthesised and / or the synthesis of target sequences which are often difficult to synthesise using traditional synthesis methods. Furthermore, when a multi-junction template is ligated and attached to a surface, it facilitates the integration of the current method into devices for automating nucleic acid synthesis.

[0110] Figure 3 is a simplified schematic diagram showing a method of synthesising a poly A sequence using a three-junction template in accordance with certain embodiments of the present invention.

[0111] Manufacture of multi-junction template

[0112] The template 301 comprises 4 nucleic acid strands: a first nucleic acid strand 302, two middle nucleic acid strands 303, 304 and a terminal nucleic acid strand 305. The first nucleic acid strand 301 comprises a first template sequence 302a (75 bases) and a first 5’ junction sequence 302b which is located at the 5’ end of the first nucleic acid strand 302, upstream of the first template sequence. The first of the two middle nucleic acid strands 303 comprises a second template sequence 303a (75 bases), a first 3’ junction sequence 303b and second 5’ junction sequence 303c located at the 3’ and 5’ ends of the second template sequence 303a, respectively. The second of the two middle nucleic acid strands 304 comprises a third template sequence 304a (75 bases), a second 3’ junction sequence 304c and third 5’ junction sequence 304b located at the 3’ and 5’ ends of the third template sequence 304a, respectively. The terminal nucleic acid strand 305 comprises a fourth template sequence 305a (75 bases), a fourth 3’ junction sequence 305b located at the 3’ end of the terminal nucleic acid strand, downstream of the fourth template sequence, and a terminating sequence 305c located at the 5’ end of the fourth template sequence 305a. The terminating sequence 305c may include a restriction site to allow the terminating sequence to be cleaved from the synthesised DNA product.

[0113] The middle nucleic acid strands 303, 304 and terminal nucleic acid strand 305 are additional strands. The first nucleic acid strand 302 and terminal nucleic acid strands 305 may include additional sequences at their ends. For example, the terminal nucleic acid strand 305 may include a poly T sequence on its 5’ end. Alternatively, the terminal nucleic acid sequence may contain a sequence which is partially complementary to another sequence to allow for assembly into a longer sequence.

[0114] The first 5’ junction sequence 302b (located on the first nucleic acid strand 302) is complementary to the first 3’ junction sequence 303b (located on the second nucleic acid strand 303). The second 5’ junction sequence 303c (located on the second nucleic acid strand 303) is complementary to the second 3’ junction sequence 304c (located on the third nucleic acid strand 304). The third 5’ junction sequence 304b (located on the third nucleic acid strand 304) is complementary to the third 3’ junction sequence 305b (located on the fourth nucleic acid strand 305).

[0115] The initiation or primer sequence 309 and the first bridge sequence 310 are on a single nucleic acid bridging strand (100 bases). The initiation primer sequence 309 includes a restriction site to allow the primer sequence to be removed from the synthesised DNA product. The primer sequence 309 is complementary to the 3’ end of the first template sequence 302a. The first bridge sequence 310 is complementary to the remainder of the first template sequence 302a and the neighbouring 25 nucleotides of the second template sequence 303a such that the first bridge sequence forms a bridge over the first junction 306. The second bridge sequence 311 (80 bases) is complementary to the 30 to 50 5’ most nucleotides of the second template sequence 303a and the 30 to 503’ most nucleotides of the third template sequence 304a, such that under annealing conditions, the second bridge sequence anneals to the 5’ end of the second template sequence 303a and the 3’ end of the third template sequence 304a so as to form a bridge over the second junction 307. If the sequence is a homogenous sequence, such as a poly A sequence, the bridge sequence will fit between the second and third template sequence randomly and therefore the number of nucleotides bound on each sequence will vary.

[0116] The third bridge sequence 312 (120 bases) is complementary to the 25 bases 5’ most nucleotides of the third template sequence 304a and the 75 bases 3’ most nucleotides of the fourth template sequence 305a, and 20 bases to terminal primer 305c; such that under annealing conditions, the third bridge sequence 312 anneals to the 5’ end of the third template sequence 304a and the 3’ end of the fourth template sequence 305a to form a bridge over the third junction 308.

[0117] The template is designed such that, when assembled, the first, second, third and fourth template sequences 302a, 303a, 304a, 305a are together complementary to the target sequence. Each of the junction sequences, the primer and the terminating sequences has a distinct sequence such that the strands will anneal specifically to their complementary sequence under appropriate conditions and thus will assemble in a predetermined order. This template assembly occurs in a single one-pot reaction. Under annealing conditions, the first 5’ and 3’ junction sequences 302b and 303b anneal to form a first junction region 306. The second 5’ and second 3’ junction sequences 303c and 304c anneal to form a second junction region 307. The third 5’ and 3’ junction sequences 304b and 305b anneal to form a third junction region 308. All these junctions with the bridges 310, 311 and 312 anneal to form bridged junctions in a template-primer- bridge complex.

[0118] The template-primer-bridge complex is brought into contact with a polymerase and nucleotides under extension conditions. The extension reaction adds nucleotides between the bridge sequences resulting in the synthesised DNA product 313 comprising the target sequence 314, the primer sequence 309 and a terminating sequence 315. The synthesised DNA product is brought into contact with a ligase enzyme to ligate the synthesised DNA product.

[0119] Both the primer sequence 309 and terminating sequence 315 may comprise restriction endonuclease sites such that these sites can be cleaved from the synthesised DNA product to result in the target DNA sequence. In order to do so, the synthesised DNA product is brought into contact with a suitable restriction endonuclease (e.g. BsaXI) under conditions which allow the enzyme to cleave the DNA.

[0120] In order to isolate the target DNA sequence, the mixture including the template can be subjected to High-performance liquid chromatography, or any other suitable means of isolating target DNA. Once the target DNA sequence has been isolated, the template can be reused to synthesise more target DNA.

[0121] Use of templates for ligation of target sequences

[0122] In certain embodiments, templates can be used to bring together nucleic acid sequences to facilitate ligation of these sequences (Figures 4-7).

[0123] Self-replication of templates

[0124] Figure 11 is a schematic diagram showing a template with a plurality of junction regions 601a, 601b, 601c, 601d, which can self-replicate by annealing to primers attached to a surface using succinimidyl ester surface chemistry. The surface can be mica, glass slides, silicon chips, or magnetic slica beads. These surfaces are pre-functionalized with N-hydroxysuccinimide (NHS) ester terminal groups, which are used to covalently couple 5'-amino-modified primers.

[0125] The plurality of junction regions 601 a, 601 b, 601 c, 601 d are annealed and ligated to form a multi-junction template 602 as described above. The multi-junction template is brought into contact with a surface-bound primer 603, which is complementarily to the 3’ end of the template 602. The primer is elongated using a mixture of helicase and polymerase to open the junction regions and produce a single strand 604 complementary to the template 602 and which is immobilised on the surface. The template 602 can be released from the surface by denaturation and recycled again. The surface-attached complementary single strand 604 will act as a template to replicate the template by adding a reverse primer 605 and undergoing an elongation and denaturation cycle.

[0126] It is preferred in this case that the initial template is ligated as it will remain ligated when released by denaturation, and will also be available for in solution extension if appropriate primers are provided.

[0127] Circularisation of linear nucleic acid usi

[0128] Circularising linear plasmids is a well-known challenge, specifically if the singlestranded overhangs are short or if the plasmid is long. The junction regions of the present invention can be used to address this issue by circularising a linear plasmid without requiring sticky ends.

[0129] In Figure 12, the process of circularising a linear plasmid is shown schematically. To achieve this, a linear DNA duplex 701 is denatured into a first linear single strand 701a and second strand 701b.

[0130] Nucleic acid strands 702a, 702b, 702c, 702d are designed to partially complement the ends of each of the first and second linear single strands 701 a, 701 b. More particularly, there is provided a first nucleic acid 702a comprising both a target binding sequence 703 that is at least partially complementary to a first end of the first linear single strand 701 a, and a 5’ junction sequence 704 located substantially at the 5’ end of the first linear strand 701 a; and an additional nucleic acid strand 702b comprising three parts; a target binding sequence 705 that is at least partially complementary to a second end of the first linear single strand nucleic acid, and a 3’ junction sequence 706 located substantially at the 3’ end of the second linear strand 702b. In the centre of 702b, there is a short sequence 707 (preferably at least four bases) complementary to the first end of the first linear single strand 701 a.

[0131] The 5’ junction sequence 704 is at least partially complementary to the 3’ junction sequence 706 when read in the opposite direction.

[0132] The 5’ junction sequence 704 is then annealed to the 3’ junction sequence 706 to form a junction region 708 thus circularisingthe single strand nucleic acid.

[0133] Essentially the same happens for the second strand to give two circularised nucleic acid templates. It is generally preferred that the templates are brought into contact with a ligase under ligation conditions before multiple plasmids are produced by annealing primers 709a, 709b to the newly circularised templates and extending the primers under extension conditions in the presence of the usual bases and a polymerase to give a double stranded plasmid 710a, 710b. Optionally the plasmids can be denatured and the primer addition and extension steps repeated to produce more plasmids.

[0134] Subsequently, the circulated single strands will serve as templates for new plasmids by annealing primers to be elongated and ligated.

[0135] Experimental data

[0136] 1.0 Template with one junction

[0137] 1 .1 Template assembly

[0138] The junction in this experiment is 45 base pairs, and the target is 150 base pairs long(SEQ ID 68). In this example, the target has been synthesised by ligation of two sequences. The first strand, second strand, bridge and terminating sequences are synthetic oligonucleotides, synthesised using a SYNTAX DNA printing system. The sequences of these oligonucleotides are shown below.

[0139] The template-primer-bridge complex is assembled by annealing the four oligonucleotides. 2 pL of the oligos V2 NR 150 T1 , V2 NR 150 T2, V2 NR 150 T3 and V2 NR 150 T4 have mixed and annealed in the thermocycler by heating to 98 °C for 10 min and cold down to 4 °C, at rate of 2 °C every 10 min.

[0140] 1 .2 Synthesis of DNA using a one junction template A ligation reaction mix was prepared in accordance with the table below:

[0141] The synthesis reaction mix was incubated at 16 °C for 2 hours. Then it was heated to 65 °C for 10 minutes. After the ligation, the ligated sequence was amplified using PCR.

[0142] PCR reaction was set up as follows: Primers:

[0143] Mix all the reagents and oligos before adding the enzyme usinga vortex for 2 to 3 seconds. Add the Q5 polymerase enzyme and mix gently by pipetting. Place the tube in the thermocycler and run the following program: 1 .3 Characterisation of synthesised DNA

[0144] The reaction product was analysed usingTapeStation D1000 Screen Tape Assay and the Oxford Nanopore GridlON, and the results are shown in Figure 4.

[0145] 1.3.1 - Tapestation D1000 Screen Tape Protocol

[0146] The D1000 Reagents were left to equilibrate at Room Temperature for 30 minutes. The D1000 ScreenTape device was inserted into the ScreenTape nest of the TapeStation instrument. Reagents and samples were vortexed before used. The ladder was prepared as follows: 3 pl D1000 Sample Buffer and 1 pl D1000 Ladder and added to a tube strip. 1 pl of the sample was added to 3 pl D1000 Sample Buffer and loaded into the tube strip. Liquids were mixed at 2000 rpm for 1 minute and subsequently spun down for 1 minute. Samples were loaded into and run on the Tapestation.

[0147] 1.3.2 Results

[0148] Results are shown in figure 4. Lane EL1 shows the ladder, and lane A1 shows the synthesised nucleic acid product after ligation and amplification by PCR.

[0149] The band at 157 bp in lane A1 shows the nucleic acid product after the ligation and the PCR. This band is close to the length of the target sequence, which is 150 bp. The bands over 260 bp may be due to aggregation or random secondary structures from the templates. The sequencing results confirm that the NR 150 sequence is correct (SEQ ID 68).

[0150] 2.0 Template with a short one junction

[0151] 2.1 Template assembly

[0152] The junction in this experiment is 11 base pairs, and the target is 178 base pairs long. In this example, the target has been synthesised by elongation of one sequence. The first strand, second strand and bridge sequences are synthetic oligonucleotides, synthesised using a SYNTAX DNA printing system. The sequences of these oligonucleotides are shown below.

[0153] The template-primer-bridge complex is assembled by annealing the three oligonucleotides. 5 pL of the oligos Junction Template 1 , Junction Template 2 and Target Template have mixed and annealed in the thermocycler by heating to 98 °C for 10 min and cold down to 4 °C, at rate of 2 °C every 10 min.

[0154] 2.2 Synthesis of DNA using a one junction template Elongation of the Target fragment using DNA Polymerase 1 Large Klenow Fragment

[0155] An elongation reaction mix was prepared in accordance with the table below:

[0156] 1 . Setup each reaction in a 0.2ml PCR tube.

[0157] 2. Reaction reagents are setup as follows. 3. Combine the reagents and mix by pipetting. 4. Heat the reaction mix to 37 °C for 30 minutes in the Thermal Cycler.

[0158] 5. Success of the reaction assessed using a D1000 Screentape on the Tapestation.

[0159] After the elongation, the sequence was amplified using PCR. PCR reaction was set up as follows:

[0160] Primers:

[0161] Mix all the reagents and oligos before adding the enzyme usinga vortex for 2 to 3 seconds. Add the Q5 polymerase enzyme and mix gently by pipetting. Place the tube in the thermocycler and run the following program: 2.3 Characterisation of synthesised DNA

[0162] The reaction product was analysed using TapeStation D1000 Screen Tape Assay, and the results are shown in Figure 5.

[0163] 2.3.1 - Tapestation D1000 Screen Tape Protocol

[0164] The D1000 Reagents were left to equilibrate at Room Temperature for 30 minutes. The D1000 ScreenTape device was inserted into the ScreenTape nest of the TapeStation instrument. Reagents and samples were vortexed before used. The ladder was prepared as follows: 3 pl D1000 Sample Buffer and 1 pl D1000 Ladder and added to a tube strip. 1 pl of the sample was added to 3 pl D1000 Sample Buffer and loaded into the tube strip. Liquids were mixed at 2000 rpm for 1 minute and subsequently spun down for 1 minute. Samples were loaded into and run on the Tapestation.

[0165] 2.3.2 Results

[0166] Results are shown in figure 5. In the first gel, lane EL1 shows the ladder, and lane B1 shows the synthesised nucleic acid product after elongation. In the second gel, lane EL1 shows the ladder, lane C1 shows the prudact after amplification by PCR.

[0167] The band at 172 bp in lane C1 in the second gel shows the nucleic acid product after the elongation and the PCR. This band is close to the length of the target sequence, which is 178 bp. The bands over 300 bp may be due to aggregation or random secondary structures from the templates.

[0168] 3.0 Template with a long one junction

[0169] 3.1 Template assembly

[0170] The junction in this experiment is 55 base pairs, and the target is 90 base pairs long. In this example, the target has been synthesised by elongation of one sequence. The first strand, second strand and bridge sequences are synthetic oligonucleotides, supplied by Invitrogen. The sequences of these oligonucleotides are shown below. The template-primer-bridge complex is assembled by annealing the three oligonucleotides.

[0171] 1 . Setup each reaction in a 0.2ml PCR tube. 2. Reaction reagents are setup as follows.

[0172] 3. Combine the reagents and mix by pipetting.

[0173] 4. Heat the reaction mix to 98 °C for 10 minutes, then cool by 2 °C every 10 minutes in the thermal cycler. 3.2 Synthesis of DNA using a one junction template Elongation of the Target fragment using DNA Polymerase 1 Large Klenow Fragment

[0174] An elongation reaction mix was prepared in accordance with the table below:

[0175] 1 . Setup each reaction in a 0.2ml PCR tube. 2. Reaction reagents are setup as follows.

[0176] 3. Combine the reagents and mix by pipetting.

[0177] 4. Heat the reaction mix to 37 °C for 30 minutes in the Thermal Cycler.

[0178] 5. Success of the reaction assessed using a D1000 Screentape on the Tapestation. After the elongation, the sequence was amplified using PCR.

[0179] PCR reaction was set up as follows:

[0180] Primers:

[0181] Mix all the reagents and oligos before adding the enzyme usinga vortex for 2 to 3 seconds. Add the Q5 polymerase enzyme and mix gently by pipetting. Place the tube in the thermocycler and run the following program:

[0182] 3.3 Characterisation of synthesised DNA

[0183] The reaction product was analysed usingTapeStation D1000 Screen Tape Assay, and the results are shown in Figure 6.

[0184] 3.3.7 - Tapestation D1000 Screen Tape Protocol

[0185] The D1000 Reagents were left to equilibrate at Room Temperature for 30 minutes. The D1000 ScreenTape device was inserted into the ScreenTape nest of the TapeStation instrument. Reagents and samples were vortexed before used. The ladder was prepared as follows: 3 pl D1000 Sample Buffer and 1 pl D1000 Ladder and added to a tube strip. 1 pl of the sample was added to 3 pl D1000 Sample Buffer and loaded into the tube strip. Liquids were mixed at 2000 rpm for 1 minute and subsequently spun down for 1 minute. Samples were loaded into and run on the Tapestation.

[0186] 3.3.2 Results Results are shown in figure 6. In the first gel, lane EL1 shows the ladder, lane C1 shows the synthesised nucleic acid product after elongation. In the second gel, lane EL1 shows the ladder, and lane B1 shows the product after amplification by PCR. The band at 96 bp in lane B1 in the second gel shows the nucleic acid product after the elongation and the PCR. This band is close to the length of the target sequence, which is 90 bp.

[0187] 4.0 Template with two junctions 4.1 Template assembly

[0188] The junction in this experiment is 40 base pairs, and the target is 230 base pairs long (SEQ ID 69). In this example, the target has been synthesised by ligation of two sequences. The first, second, third strand, bridge and terminating sequences are synthetic oligonucleotides, synthesised using a SYNTAX DNA printing system. The sequences of these oligonucleotides are shown below. The template-primer-bridge complex (fig 7a) is assembled by annealing the five oligonucleotides. 2 pL of the oligos V2 NR 230 T1 , V2 NR 230 T2, V2 NR 230 T3, V2 NR 230 T4 and V2 NR 230 T5 have mixed and annealed in the thermocycler by heating to 98 °C for 10 min and cold down to 4 °C, at rate of 2 °C every 10 min.

[0189] 4.2 Synthesis of DNA using a two-junction template

[0190] A ligation reaction mix was prepared in accordance with the table below: The synthesis reaction mix was incubated at 16 °C for 2 hours. Then it was heated to 65

[0191] °C for 10 minutes.

[0192] After the ligation, the ligated sequence was amplified using PCR.

[0193] PCR reaction was set up as follows:

[0194] Primers:

[0195] Mix all the reagents and oligos before adding the enzyme usinga vortex for 2 to 3 seconds. Add the Q5 polymerase enzyme and mix gently by pipetting. Place the tube in the thermocycler and run the following program:

[0196] 4.3 Characterisation of synthesised DNA

[0197] The reaction product was analysed usingTapeStation D1000 Screen Tape Assay and the

[0198] Oxford Nanopore GridlON, and the results are shown in Figure 7.

[0199] 4.3.1 - Tapestation D1000 Screen Tape Protocol

[0200] The D1000 Reagents were left to equilibrate at Room Temperature for 30 minutes. The D1000 ScreenTape device was inserted into the ScreenTape nest of the TapeStation instrument. Reagents and samples were vortexed before used. The ladder was prepared as follows: 3 pl D1000 Sample Buffer and 1 pl D1000 Ladder and added to a tube strip. 1 pl of the sample was added to 3 pl D1000 Sample Buffer and loaded into the tube strip. Liquids were mixed at 2000 rpm for 1 minute and subsequently spun down for 1 minute. Samples were loaded into and run on the Tapestation.

[0201] 4.3.2 Results Results are shown in figure 7. Lane EL1 shows the ladder, and lane A1 shows the synthesised nucleic acid product after ligation and amplification by PCR.

[0202] The band at 209 bp in lane A1 shows the nucleic acid product after the ligation and the PCR. This band is close to the length of the target sequence, which is 230 bp. The bands over 300 bp may be due to aggregation or random secondary structures from the templates. The sequencing results confirm that the NR 230 sequence is correct.

[0203] 5.0 Templates with two junctions at different lengths 5.1 Templates assembly

[0204] The junctions in this experiment are 11 , 22 and 33 base pairs, and the targets are 316, 272 and 228 base pairs long. In this example, the target has been synthesised by elongation of one sequence. The first strand, second strand, third strand and two bridge sequences are synthetic oligonucleotides, synthesised using a SYNTAX DNA printing system. The sequences of these oligonucleotides are shown below.

[0205] The template-primer-bridge complex is assembled by annealing the three oligonucleotides.

[0206] 1 . Setup each reaction in a 0.2ml PCR tube.

[0207] 2. Reaction reagents are setup as follows.

[0208] 3. Combine the reagents and mix by pipetting.

[0209] 4. Heat the reaction mix to 98 °C for 10 minutes then cool by 2 °C every 10 minutes in the thermal cycler.

[0210] 5.2 Synthesis of DNA using a two-junction template

[0211] Elongation of the Target fragment using DNA Polymerase 1 Large Klenow Fragment

[0212] An elongation reaction mix was prepared in accordance with the table below:

[0213] 1 . Setup each reaction in a 0.2 ml PCR tube.

[0214] 2. Reaction reagents are setup as follows.

[0215] 3. Combine the reagents and mix by pipetting.

[0216] 4. Heat the reaction mix to 37 °C for 30 minutes in the Thermal Cycler.

[0217] 5. Success of the reaction assessed using a D1000 Screentape on the Tapestation.

[0218] Ligation Procedure

[0219] 1 . Prepare the following reaction mixture on ice for each pair of kinased duplexes.

[0220] 2. Add the reagents (ligase buffer, duplex solution and water) to a thin walled 500 pL tube (volumes and concentrations shown in the table below)

[0221] 3. Vortex for 10 seconds 4. Add ligase enzyme and mix gently with pipette - N.B. do not vortex once enzyme has been added

[0222] 5. Incubate at 16 °C for 2 hours

[0223] 6. Repeat this as many times as necessary to build up longer fragments until the full sequence has been assembled.

[0224] 7. Heat inactivate for 10 minutes at 65 °C.

[0225] Success of the reaction assessed using a D1000 Screentape on the Tapestation.

[0226] After the elongation, the sequence was amplified using PCR.

[0227] PCR reaction was set up as follows:

[0228] Primers:

[0229] Mix all the reagents and oligos before adding the enzyme usinga vortex for 2 to 3 seconds. Add the Q5 polymerase enzyme and mix gently by pipetting. Place the tube in the thermocycler and run the following program:

[0230] 5.3 Characterisation of synthesised DNA

[0231] The reaction product was analysed usingTapeStation D1000 Screen Tape Assay, and the results are shown in Figure 8.

[0232] 5.3.7 - Tapestation D1000 Screen Tape Protocol

[0233] The D1000 Reagents were left to equilibrate at Room Temperature for 30 minutes. The D1000 ScreenTape device was inserted into the ScreenTape nest of the TapeStation instrument. Reagents and samples were vortexed before used. The ladder was prepared as follows: 3 pl D1000 Sample Buffer and 1 pl D1000 Ladder and added to a tube strip. 1 pl of the sample was added to 3 pl D1000 Sample Buffer and loaded into the tube strip. Liquids were mixed at 2000 rpm for 1 minute and subsequently spun down for 1 minute. Samples were loaded into and run on the Tapestation.

[0234] 5.3.2 Results Results are shown in figure 8. Lane EL1 shows the ladder, and lanes A1 , B1 and C1 show the synthesised nucleic acid product after elongation, ligation and amplification for the target 316, 272 and 228 base pairs, respectively. The bands at 359, 291 and 254 bp are close to the length of the target sequences 316, 272 and 228 bp, respectively.

[0235] 6.0 Template with three junctions

[0236] 6.1 Template assembly The four strands, two bridges and terminating sequence are synthetic oligonucleotides, synthesised using a SYNTAX DNA printing system. The sequences of these oligonucleotides are shown below.

[0237] The template-primer-bridge complex is assembled by annealing the five oligonucleotides. 2 pL of the oligos NR 300 T1 , NR 300 T2, NR 300 T3, NR 300 T4, NR 300 T5, NR 300 T6 and NR 300 T7 have mixed and annealed in the thermocycler by heating to 98 °C for 10 min and cold down to 4 °C, at rate of 2 °C every 10 min.

[0238] 6.2 Synthesis of DNA using a three-junction template

[0239] A ligation reaction mix was prepared in accordance with the table below: The synthesis reaction mix was incubated at 16 °C for 2 hours. Then it was heated to 65 °C for 10 minutes.

[0240] After the ligation, the ligated sequence was amplified using PCR.

[0241] PCR reaction was set up as follows:

[0242] Primers:

[0243] Mix all the reagents and oligos before adding the enzyme usinga vortex for 2 to 3 seconds. Add the Q5 polymerase enzyme and mix gently by pipetting. Place the tube in the thermocycler and run the following program: 6.3 Characterisation of synthesised DNA

[0244] The reaction product was analysed usingTapeStation D1000 Screen Tape Assay and the

[0245] Oxford Nanopore GridlON, and the results are shown in Figure 9.

[0246] 6.3.7 - Tapestation D1000 Screen Tape Protocol The D1000 Reagents were left to equilibrate at Room Temperature for 30 minutes. The D1000 ScreenTape device was inserted into the ScreenTape nest of the TapeStation instrument. Reagents and samples were vortexed before used. The ladder was prepared as follows: 3 pl D1000 Sample Buffer and 1 pl D1000 Ladder and added to a tube strip. 1 l of the sample was added to 3 pl D1000 Sample Buffer and loaded into the tube strip. Liquids were mixed at 2000 rpm for 1 minute and subsequently spun down for 1 minute. Samples were loaded into and run on the Tapestation.

[0247] 6.3.2 Results

[0248] Results are shown in figure 9. Lane EL1 shows the ladder, and lane A1 shows the synthesised nucleic acid product after ligation and amplification by PCR. The band at 266 bp in lane A1 shows the nucleic acid product after the ligation and the PCR. This band is close to the length of the target sequence (SEQ ID 70), which is 300 bp. The sequencing results confirm that the NR 300 sequence is correct.

[0249] 7.0 Synthesis of a Poly A sequence 7.1 Template assembly or manufacture

[0250] The first strand, second strand, bridge and terminating sequences are synthetic oligonucleotides, synthesised using a SYNTAX DNA printing system. The sequences of these oligonucleotides are shown below. The template-primer-bridge complex is assembled by annealing the four oligonucleotides. 5 pL of the oligos V4 Poly A140 T1 , V4 Poly A O T2, V4 Poly A O T3 and V4 Poly AMO T4 have mixed and annealed in the thermocycler by heating to 98 °C for 10 min and cold down to 4 °C, at rate of 2 °C every 10 min.

[0251] 7.2 Synthesis of DNA using a one junction template

[0252] A ligation reaction mix was prepared in accordance with the table below: The synthesis reaction mix was incubated at 16 °C for 30 minutes. Then it was heated to 65 °C for 10 minutes.

[0253] After the ligation, the ligated sequence was amplified using 2-step PCR in 10 % DMSO. 2-step PCR reaction was set up as follows:

[0254] Primers:

[0255] Mix all the reagents and oligos before adding the enzyme usinga vortex for 2 to 3 seconds. Add the Q5 polymerase enzyme and mix gently by pipetting. Place the tube in the thermocycler and run the following program:

[0256] 7.3 Characterisation of synthesised DNA

[0257] The reaction product was analysed usingTapeStation D1000 Screen Tape Assay, and the results are shown in Figure 10.

[0258] 7.3.1 - Tapestation D1000 Screen Tape Protocol

[0259] The D1000 Reagents were left to equilibrate at Room Temperature for 30 minutes. The D1000 ScreenTape device was inserted into the ScreenTape nest of the TapeStation instrument. Reagents and samples were vortexed before used. The ladder was prepared as follows: 3 pl D1000 Sample Buffer and 1 pl D1000 Ladder and added to a tube strip. 1 pl of the sample was added to 3 pl D1000 Sample Buffer and loaded into the tube strip. Liquids were mixed at 2000 rpm for 1 minute and subsequently spun down for 1 minute. Samples were loaded into and run on the Tapestation.

[0260] 7.3.2 Results Results are shown in figure 10. Lane EL1 shows the ladder, and lane A1 shows the control sample. The control sample includes all the oligonucleotides and reagents except for the Q5 polymerase enzyme in the PCR reaction. This control sample has been subjected to the same reaction conditions as the Poly A sample. Lane G1 shows the synthesised nucleic acid product after ligation and amplification by PCR.

[0261] The band at 216 bp in lane G1 shows the nucleic acid product after the ligation and the PCR. This band is close to the length of the target sequence, which is 183 bp. The bands over 300 bp may be due to aggregation or random secondary structures from the templates.

[0262] Eady experimental data 8.0 Template with one junction

[0263] 8.1 Template assembly

[0264] The first strand, second strand and bridge sequences are synthetic oligonucleotides, synthesised using a SYNTAX DNA printing system. The sequences of these oligonucleotides are shown below.

[0265] The template-primer-bridge complex is assembled by annealing the three oligonucleotides. 10 pL of each oligonucleotide (5 pM) is mixed in an Eppendorf tube with 4 pL of NEBuffer 2™ and 6 pL of H2O. The mixture is then heated to 95 °C and cooled to room temperature overnight.

[0266] 8.2 Synthesis of DNA using a one junction template

[0267] A synthesis reaction mix was prepared in accordance with the below table:

[0268] The synthesis reaction mix was incubated at 37 °C for 1 hour.

[0269] 8.3 Characterisation of synthesised DNA

[0270] The reaction products were analysed using TapeStation D1000 Screen Tape Assay and the results are shown in Figure 13.

[0271] 8.3.1 - Tapestation D1000 Screen Tape Protocol

[0272] The D1000 Reagents were left to equilibrate at Room Temperature for 30 minutes. The D1000 ScreenTape device was inserted into the ScreenTape nest of the TapeStation instrument. Reagents and samples were vortexed before used. The ladder was prepared as follows: 3 pl D1000 Sample Buffer and 1 pl D1000 Ladder and added to a tube strip. 1 pl of each sample was added to 3 pl D1000 Sample Buffer and loaded into the tube strip. Liquids were mixed at 2000rpm for 1 minute and subsequently spun down for 1 minute. Samples were loaded into and run in the Tapestation.

[0273] 8.3.2 Results Results are shown in figure 13. Lane EL1 shows the ladder and lane A1 is empty. Lane A2 shows the mixture of the first strand, second strand and bridge sequence. Lane B2 is the annealed template-primer-bridge complex. Lane C2 shows the synthesised nucleic acid product after elongation. The band at 196 bp in lane C2 showingthe nucleic acid product after elongation is in the expected range of 200-220 bp. The band at 140 bp is due to the oligonucleotides used to construct the template. The bands over 300 bp may be due to aggregation or random secondary structures from the templates. 9.0 Synthesis of a Poly A sequence

[0274] 9.1 Template assembly or manufacture

[0275] The first, second, third and fourth strands of the template, three bridge sequences and two primer sequences are synthetic oligonucleotides, synthesised using a SYNTAX DNA printing system. The sequences of these oligonucleotides are shown below.

[0276] The template is assembled by annealing the four template strand oligonucleotides (NR.T4.1 to NR.T4.4) and three bridge oligonucleotides (NR.P4.1 to NR.P4.3). 1 pL of each oligonucleotide (5 pM) is mixed in an Eppendorf tube with 4 pL of NEBuffer 2™ and 29 pL of H2O. The mixture is then heated to 95 °C and cooled to room temperature overnight.

[0277] 9.2 Synthesis of a Poly A sequence

[0278] A synthesis reaction mix was prepared in accordance with the below table:

[0279] The synthesis reaction mix was incubated at 37 °C for 30 minutes.

[0280] 9.3 Characterisation of the Poly A sequence

[0281] The synthesis reaction products were analysed using a TapeStation. The results are shown in figure 14.

[0282] 9.3.7 - Tapestation D5000 Screen tape Assay

[0283] The D5000 Reagents were left to equilibrate at Room Temperature for 30 minutes. The D5000 ScreenTape device was inserted into the ScreenTape nest of the TapeStation instrument. Reagents and samples were vortexed before use. The ladder was prepared as follows: 10 pl D5000 Sample Buffer and 1 pl D5000 Ladder and added to a tube strip. 1 pl of each sample was added to 10 pl D5000 Sample Buffer and loaded into the tube strip. Liquids were mixed at 2000rpm for 1 minute and subsequently spun down for 1 minute. Samples were loaded into and run in the Tapestation.

[0284] Figure 14 shows the results. Lane A1 contains the ladder, lane D2 contains the template and bridge sequences after annealing. Lane E2 contains the sample after elongation and lane F2 contains the sample after PCR amplification. Lane E2 shows a band at 328 bp, which corresponds to the expected size of 344 bp. However, after amplification with PCR (lane F2) a wide band can be seen containing the target band size. This wide band also contains smaller fragments which are portions of the templates (with 1 or 2 junctions) have also been amplified after PCR. The early-stage experiments show that the templates can be manufactured, and said templates can be used to synthesise DNA. Including a ligation step can further improve the method as shown in later work.

[0285] The term ‘complementary’ in the context of nucleotide sequences refers to when the sequence of one can bind to the sequence of the other in an anti-parallel sense wherein the 3'-end of one sequence binds to the 5'-end of the other sequence and each A, T(U), G, and C of one sequence is then aligned with a T(U), A, C, and G, respectively, of the other sequence.

[0286] The term ‘partially complementary’ in the context of the present invention means that at least some of the nucleotides of a first sequence are complementary to some of the nucleotides in another sequence. In some cases, the degree of complementarity between nucleotide sequences is at least 99% of nucleotides are complementary. Alternatively, at least 98% of nucleotides are complementary. Alternatively, at least 95% of nucleotides are complementary. Alternatively, at least 90% of nucleotides are complementary. Alternatively, at least 85% of nucleotides are complementary. Alternatively, at least 80% of nucleotides are complementary. Alternatively, at least 75% of nucleotides are complementary. Alternatively, at least 70% of nucleotides are complementary. Alternatively, at least 60% of nucleotides are complementary. Alternatively, at least 50% of nucleotides are complementary.

[0287] The term ‘amplification’, as applied to nucleic acids refers to any method that results in the formation of one or more copies of a nucleic acid, where preferably the amplification is exponential. One such method for enzymatic amplification of specific sequences of DNA is known as the polymerase chain reaction (PCR), as described by Saiki et al., 1986, Science 230:1350-1354.

[0288] The terms ‘nucleic acid’ and ‘polynucleotide’ are interchangeable and refer to any nucleic acid, whether DNA, RNA, cDNA, DNA-RNA, peptide nucleic acid (PNA), a hybrid or any mixture of the same. The terms ‘nucleic acid’, ‘polynucleotide’ and ‘nucleotide’ also specifically include nucleic acids composed of synthetic bases (i.e. bases other than the five biologically occurring bases - adenine, guanine, thymine, cytosine and uracil), modified bases or any combination of biologically occurring bases, synthetic bases and modified bases.

[0289] The polynucleotides of the present invention can be from a human or non-human mammal, or any other organism, derived from any recombinant source, synthesized in vitro or by chemical synthesis.

[0290] Nucleic acid sequences may be of genomic, synthetic or recombinant origin. The term ’nucleotide sequence', ’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

[0291] The terms “annealing’, ‘hybridisation’ and ‘binding’ (or ‘anneal’, ‘hybridise’ and ‘bind’) in the context of nucleic acid sequences, are used interchangeably herein. The ability of two nucleic acid sequences to anneal with each other is based on the degree of complementarity of the two nucleic acid sequences, which in turn is based on the fraction of matched complementary nucleotide pairs.

[0292] Theterms ‘sequence' and ’strand' referto a single continuous molecule of nucleic acids. The terms ‘substantially at the 3’ end’ and ‘substantially at the 5’ end’ refer to a sequence which is at the immediate 3’ or 5’ end of a nucleic acid strand or sequence or close to the 3’ or 5’ end of a nucleic acid strand or sequence.

[0293] The term ’annealing conditions' refers to the reagents and reaction conditions (e.g. temperature, time etc) that are used for annealing. For example, heating the sequences to be annealed to 95 °C for 10 minutes and then cooling the mixture to room temperature overnight. One skilled in the art would understand that such conditions are known and will be selected and refined based on the sequences. The conditions should be sufficient to allow for the junction sequences to anneal when they are complementary, or in some cases partially complementary, but will limit non-specific binding.

[0294] The terms ‘extension’ or ‘elongation’ in the context of nucleotide sequences, are used interchangeably herein. They refer to the extension of a 3’-end and / or a 5’ end of a polynucleotide by the addition of nucleotides or bases. Chain extension relevant to the present invention is generally template dependent, that is, the appended nucleotides are determined by the sequence of a template nucleic acid to which the extending chain is hybridised.

[0295] The term ‘enzymatic extension’ refers to the extension of a nucleic acid which is catalysed by an enzyme, such as a polymerase enzyme. It is preferred that the polymerase enzyme lacks the exonuclease activities of DNA Polymerase I, such as Klenow Fragment (exo-), to avoid removing nucleotides.

[0296] The term ’extension conditions’ refers to the reagents and reaction conditions (e.g. temperature, time etc) that are used. It describes conditions for extension of the primer polynucleotide. 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 C J, Turley A T, Tuite E M, Connolly B A, Pike A R. Enzymatic Method for the Synthesis of Long DNA Sequences with Multiple Repeat Units. Angewandte Chemie International Edition 2015, 54(31 ), 8971 -8974.

[0297] The term ‘circularise’ or ‘circularising’ refers to the structure of a nucleic acid having it’s ends are joined together to form a circular structure.

[0298] The term ‘upstream’ refers to the relative position of a nucleic acid base or sequence being located towards the 5’ end of a nucleic acid.

[0299] The term ‘downstream’ refers to the relative position of a nucleic acid base or sequence being located towards the 3’ end of a nucleic acid.

[0300] The term ‘oligo’ or ‘oligonucleotide’ refers to a short sequence of nucleotides, typically comprising between about 10 and 100 nucleotides in length. Oligonucleotides may be single-stranded or double-stranded and are commonly synthesised chemically for use in various molecular biology applications, such as primers, probes, or template strands. The precise length and composition of an oligonucleotide can be tailored according to its intended use within nucleic acid synthesis or detection assays.

[0301] 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.

[0302] 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).

[0303] 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

CLAIMS1 . A multi-junction template comprising: a first nucleic acid strand comprising a target binding or template sequence, and a 5’ junction sequence located substantially at the 5’ end of the first nucleic acid strand; a plurality of additional nucleic acid strands comprising a target binding or template sequence, and a 3’ junction sequence located substantially at the 3’ end of each additional nucleic acid strand; wherein the 5’ junction sequence on the first nucleic acid strand is at least partially complementary to a 3’ junction sequence on an additional nucleic acid strand, such that under annealing conditions the 5’ junction sequence and the 3’ junction sequence anneal to form said junction region; and wherein at least one of the plurality of additional nucleic acid strands is a middle nucleic acid strand which further comprises a 5’ junction sequence located substantially at the 5’ end of the middle nucleic acid strand; wherein the 5’ junction sequence of the at least one middle nucleic strand is at least partially complementary to the 3’ junction sequence of another additional strand, such that under annealing conditions said 5’ and 3’ junction sequences anneal to form at least a first junction region between the first stand and a first additional strand which is a middle strand, and a second junction region between the first additional strand and a further additional strand, wherein when the first and additional strands are annealed, the template sequences are the complement of a target nucleic acid sequence.

2. A multi-junction template as in claim 1 , wherein: the 3’ junction sequence of an additional strand is in part complementary to the 5’ junction sequence of the first strand and partially self-complementary such that under annealing conditions a partial hairpin loop is formed in the 3’ junction sequence. The annealed first and second strands are then brought into contact with a DNA ligase enzyme under ligation conditions to create a ligated template.

3. A multi junction template as in claims 1 or 2 further comprising one or more bridging strands, said bridging strand comprising a bridge sequence that is complementary to at least part of the template sequence on the first strand and complementary to at least part of the template sequence on the additional strand, such that under annealing conditions it binds to, and thus bridges, the junction formed by the first and additional strand.

4. A multi-junction template according to any of the previous claims, further comprising an initiation sequence which is the initiation site of the target that will be elongated and / or ligated.

5. A multi-junction template as in claim 4 where the initiation sequence is present on a bridging strand.

6. A multi-junction template accordingto any of the previous claims, wherein one of the first nucleic acid strand or plurality of additional nucleic acid strands is a terminating strand comprising a terminating sequence located substantially at the 5’ end or 3’ end of the template sequence.

7. A multi-junction template for use in synthesis of a target nucleic acid sequence as in any of the previous claims, wherein each 5’ junction sequence has a distinct nucleic acid sequence from the other 5’ junction sequences.

8. A method of manufacturing the multi-junction template of any of the previous claims, the method comprising: contacting at least the first nucleic acid strand and the additional nucleic acid strands under annealing conditions.

9. A method of manufacturing the multi-junction template of any of claims 3 to 7 the method comprising: contacting at least the first nucleic acid strand, the additional nucleic acid strands and the initiation sequence and / or one or more bridging strands under annealing conditions.10.A method of manufacturing a template as in claim 8 or 9, wherein the method further comprises contacting at least the first nucleic acid strand and the additional nucleic acid strand with a ligase enzyme under ligation conditions.11.A method of manufacturing a template as in claims 8 to 10, adapted to manufacture multiple templates by includingthe steps of: providing a single stranded primer that is immobilised to the surface at one end, having the other end exposed to allow for primer extension, said primer being complementary to the 3’ end of the template or to the 5’ end of the template; annealingthe template to the primer under annealing conditions; extendingthe primer, usingthe template as a template, in the presence of nucleotides and enzymes that are able to open or linearise the one or more junction regions, to give a surface-bound strand that is complementary to the template; denaturingthe template from the surface-bound strand under denaturing conditions; annealing a reverse primer to the surface-bound strand; extending the reverse primer in the presence of a polymerase and bases; denaturingthe strands so they separate; optionally, repeating the steps of annealing a reverse primer, extending the reverse primer and denaturing the strands.

12. A method of manufacturing multiple templates as in claim 11 , wherein the enzymes are a mixture of helicase and polymerase.

13. A method of manufacturing multiple templates as any of claims 1 1 to 12, wherein the single stranded primer that is immobilised to a surface is immobilised to a surface using succinimidyl ester surface chemistry.

14. A method of manufacturing multiple templates as in claim 13, wherein the surface material is selected from mica, glass, silicon, magnetic silica beads, polypropylene, metals and metal oxides.

15. A method of manufacturing multiple templates as any of claims 1 1 to 14, wherein the surface is pre-functionalized with N-hydroxysuccinimide (NHS) ester terminal groups or any other chemical group that can immobilise primers on the surfaces.

16. A method for synthesising a target nucleic acid comprising: contacting the template of any of claims 1 to 7 with at least one bridge sequence and an initiation or primer sequence under annealing conditions to form a template-primer-bridge complex; contacting the template-primer-bridge complex with a polymerase under extension conditions to synthesise the target nucleic acid; wherein said at least one bridge sequence is complementary to at least the 5’ end of one template sequence and the 3’ end of a neighbouringtemplate sequence; wherein said primer sequence is complementary to at least the 3’ end of the template sequence of the first nucleic acid strand or wherein said primer sequence is complementary to at least the 5’ end of the template sequence of the additional nucleic acid strand.

17. A method as in claim 16, comprising a plurality of bridge sequences, each of the plurality of bridge sequences being at least partially complementary to the 5’ end of one template sequence and at least partially complementary to the 3’ end of the neighbouring template sequence.

18. A method as in any of claims 16 to 17, wherein the initiation or primer sequence is on the same nucleic acid strand as the at least one bridge sequence.

19. A method as in any of claims 16 to 17, wherein the primer sequence is on a separate nucleic acid strand to the at least one bridge sequence.

20. A method as in any of claims 16 to 19, wherein the synthesised target nucleic acid is denatured from the template.21 .A method as in claim 20, wherein the target DNA sequence is isolated from the template by a suitable isolation means.

22. A method as in claim 21 , wherein the suitable isolation means is selected from the following list: high-performance liquid chromatography; gel electrophoresis followed by extraction; ultracentrifugation; spin column purification; use of functionalised magnetic beads to bind target or template molecules; wash with an appropriate solution after denaturing the target or template molecules from the complementary molecules attached to a surface.23.A method as in any of claims 11 and 16 to 22, wherein the template is a reuseable template.

24. A method of circularising a linear single strand nucleic acid using a junction region, said junction region having a first nucleic acid strand comprising a target binding or template sequence, and a 5’ junction sequence located substantially at the 5’ end of the first nucleic acid strand; and an additional nucleic acid strand comprising a target binding or template sequence, and a 3’ junction sequence located substantially at the 3’ end of the additional nucleic acid strand; wherein the 5’ junction sequence is at least partially complementary to the 3’ junction sequence, such that under annealing conditions the 5’ junction sequence and the 3’ junction sequence anneal to form said junction region comprising: annealingthe first nucleic acid strand to a first end of the linear single strand nucleic acid, wherein the target binding sequence of the first nucleic acid strand is at least partially complementary to a first end of the linear single strand nucleic acid;annealingthe additional nucleic acid strand to a second end of the linear single strand nucleic acid, the second end being different to the first end, wherein the target binding sequence of the additional nucleic acid strand is at least partially complementary to a second end of the linear single strand nucleic acid; annealingthe 5’ junction sequence to the 3’ junction sequence to form a junction region and circularisingthe single strand nucleic acid.

25. A method of circularising a linear single strand nucleic acid as in claim 24 wherein the annealing process occurs substantially simultaneously in a single pot reaction.

26. A method of circularising linear single strand nucleic acids as in claims 24 or 25, wherein, multiple plasmids can be manufactured by repeating the steps of annealing a primer, and extending the primer and optionally denaturingto produce further templates.

27. A method of circularising linear single strand nucleic acids as in any of claims 24 to 26, wherein the junction region is ligated by contacting it with a ligase enzyme under ligation conditions.

28. A method of circularising linear single strand nucleic acids as in any of claims 24 to 27 wherein the method of circularising a linear single strand DNA can be used to circularise a DNA duplex by, prior to annealingthe first nucleic acid strand, there is a step of: denaturingthe DNA duplexto give a first linear single strand DNA and a second linear single strand DNA.

29. A multi-junction template as in any of claims 1 to 7 or methods as in any of claims 8 to 28 wherein the 3’ junction sequences and 5’ junction sequences are at least 6 base pairs in length.

29. A template as in any of claims 1 to 7 or 29 or a method as in any of claims 8 to 29 wherein the target binding or template sequence is at least 20 base pairs.

30. A template as in any of claims 1 to 7 or 28 to 29 or a method as in any of claims 8 to 29 wherein the template is attached to a surface. 31 . A template as in any of claims 1 to 7 or 28 to 30, or a method as in any of claims 8 to 30, wherein the 5’ junction sequence is at least partially complementary to the 3’ junction sequence when read in the opposite direction.

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