Nucleic acid synthesis system, device and method

Abstract

The present invention relates to a system for synthesising nucleic acids. In particular, to the cell-free synthesis of long nucleic acids such as whole gene sequences at high yield. The invention also relates to a method of synthesising nucleic acids and a device for the same.

Description

[0001] Nucleic Acid Synthesis System, Device and Method

[0002] Technical Field

[0003] The present invention relates to a systems, methods and devices for synthesising 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). 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 base pairs 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 ligated together.

[0008] Once the partial gene sequences are ligated together to form the whole gene sequence, 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 containing the 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.

[0009] 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 nucleic acid sequences such as gene sequences.

[0010] Summary of the Invention

[0011] A system for nucleic acid synthesis comprising: a nucleic acid synthesis device; at least one array comprising a plurality of surface-bound oligonucleotides; at least one partially complementary oligonucleotide configured to be at least partially complementary to one of the surface-bound oligonucleotides such that under appropriate hybridisation conditions it will at least partially hybridise to one of the array of surface-bound oligonucleotides to generate an overlapping oligonucleotide having at least one overhanging region; means for extending the overlapping oligonucleotide to provide an extended oligonucleotide; means for cleaving the extended oligonucleotide to provide at least one cleaved oligonucleotide; means for assembling the at least one cleaved oligonucleotide to provide a synthesised nucleic acid product.

[0012] Advantageously, the system of the present invention can produce high yields of nucleic acid sequences in a cell-free manner. Cell-free, high yield production of nucleic acids is important in fields such as genetic medicine where large quantities of highly specific sequences need to be produced for use in therapeutics. The system can also produce long sequences of nucleic acids with higher efficiency and accuracy than present methods.

[0013] The means for assembling can be any technique suitable for physically joining at least two nucleic acid sequences. Examples of such techniques include Gibson Assembly®, PCR assembly and enzymatic ligation with a DNA ligase.

[0014] Optionally, the system further comprises: means for extending the synthesised nucleic acid product to generate an extended synthesised nucleic acid product; means for cleaving the extended synthesised nucleic acid product to generate a plurality of synthesised nucleic acid products. Advantageously, where the synthesised nucleic acid product is extended and cleaved, this second extension can be conducted in solution, rather than on a surface, which can produce higher yields of the end product.

[0015] Alternatively, all of the extension can be carried out on the surface before cleaving to obtain a plurality of cleaved oligonucleotides which can be ligated to provide a plurality of synthesised nucleic acid products. This is particularly advantageous where modifications are to be incorporated into the sequence as it enables a higher level of control over whereabouts in the sequence the modifications are incorporated.

[0016] Optionally, in use, the plurality of partially complementary oligonucleotides are hybridised to the plurality of surface-bound oligonucleotides and are configured to be denatured from the plurality of surface-bound oligonucleotides and re-hybridised under conditions which promote the formation of an overlapping oligonucleotide.

[0017] Optionally, each of the plurality of surface-bound oligonucleotides comprises at least one repeating sequence and each of the plurality of partially complementary oligonucleotides comprises at least one sequence that is complementary to the repeating sequence. Further, at least one of the surface-bound oligonucleotide and / or the partially complementary oligonucleotide comprises at least two repeats of the repeating sequence and / or its complementary sequence.

[0018] The repeating sequence can be any sequence which will repeat when the surface-bound oligonucleotides is extended.

[0019] Advantageously, when one of the surface-bound oligonucleotides and partially complementary oligonucleotide comprises multiple repeats of a repeating sequence and the other oligonucleotide comprises at least one sequence that is complementary to the repeating sequence, the surface-bound oligonucleotide and partially complementary oligonucleotide can hybridise, under appropriate conditions, in such a way that an overhanging region is created, said overhanging region comprising at least a part of the repeating sequence or its complementary sequence. Therefore, during the extension further units of the repeating sequence and its complementary sequence are incorporated. Further denaturing, reannealing and extending steps can be conducted to continue to generate an extended double stranded oligonucleotide comprising multiple repeating sequences and complementary sequences. The extended oligonucleotide can be cleaved by restriction enzymes to generate multiple cleaved oligonucleotides, each containing one copy of the repeating sequence and complementary sequence. The multiple cleaved oligonucleotides can then be ligated to other cleaved oligonucleotides of different sequences to generate multiple copies of the same synthesised nucleic acid product.

[0020] Optionally, the plurality of surface-bound oligonucleotides comprise at least two copies of a repeating sequence.

[0021] Optionally, the repeating sequence is at least 8 base pairs in length.

[0022] Optionally, the repeating sequence is 8 base pairs in length.

[0023] An array comprising a range of different repeat sequences of 8 base pairs can allow for every possible variation of 8 base sequence to be present on a relatively small array footprint - thus providing an option for any combination of bases to be produced. However, it would be understood that in some instances the array can include or be made up of other lengths of repeat sequence which is particularly appropriate if large volumes of predetermined oligonucleotides, genes or gene fragments are desired.

[0024] Optionally, there is a restriction site provided at least one end of the repeating sequence.

[0025] Optionally, and preferably, the sequence of bases which make up the restriction site are separate and in addition to the bases which make up the repeat sequence. For example, a sequence of 8 bases may be flanked at the 3' end by an additional 3 bases making up a restriction site. The sequence may also be flanked at the 5' end by a further additional 3 bases making up a second restriction site.

[0026] Optionally, the restriction site may be a restriction site for a type 11 B restriction enzyme.

[0027] A type IIB restriction enzyme is a restriction endonuclease which cleaves both sides of its recognition sequence leaving 3 base 3' overhangs. Advantageously, type IIB restriction endonucleases digest at each end of the restriction site therefore resulting in the removal of the restriction site from the digested DNA. For example, BsaXI is a type IIB restriction endonuclease which cuts at the following site: 5'xKN)gAC(N)5 CTCC(N)io^3' (where N is any base). Advantageously, the sequences can be designed to create the desired 3 base overhang sequence for use in downstream processes such as assembly.

[0028] Alternatively, the sequence of bases which make up the restriction site are part of the bases which make up the repeat sequence. For example, a sequence of 8 bases may include one or more restriction sites within the 8 base pair sequence.

[0029] Optionally, the sequence of bases which make up the restriction site are partially formed from the bases which make up the repeat sequence and partially in addition to the repeat sequence.

[0030] Optionally, the array of surface-bound oligonucleotides is located on a solid substrate.

[0031] Optionally, the array of surface-bound oligonucleotides comprises a plurality of surfacebound oligonucleotides, wherein together the plurality of surface-bound oligonucleotides corresponds to all possible nucleic acid sequences.

[0032] Advantageously, when the plurality of surface-bound oligonucleotides together corresponds to every possible sequence, the system can produce any nucleic acid sequence. Alternatively, the array of surface-bound oligonucleotides comprises a plurality of surfacebound oligonucleotides, wherein together the plurality of surface bound oligonucleotides correspond to a set of nucleic acid sequences that can be used to produce one or more predetermined nucleic acid sequences.

[0033] Optionally, the surface bound oligonucleotides are positioned at least 2nm apart; or at least 5nm apart; or at least lOnm apart; or at least 15nm apart.

[0034] Reducing the surface density of the surface bound oligonucleotides by spacing them apart reduces steric hindrance.

[0035] Optionally duplicates of some or all of the surface bound oligonucleotides are provided on the array. Preferably duplicates of surface bound oligonucleotides are spaced at least 50nm apart from each other.

[0036] Optionally, the system may further comprise a means for incorporating modifications.

[0037] Optionally, the modifications may include modified nucleotides, artificial bases, loop structures. Optionally, the modified nucleotides are alkyne, azide or phosphorothioate modified nucleotides. Optionally, these may include: 5-Br-dUTP, 7-deaza-7-l-dATP, 6-S- dGTP,5-l-dCTP, 5-(octadiynyl)-dCTP.

[0038] Optionally, the at least one overhanging region is a 5' overhanging region.

[0039] Optionally, the synthesised nucleic acid product is a gene or gene fragment.

[0040] Optionally, the nucleic acid synthesis device further comprises a control module.

[0041] Advantageously, the control module can run computer programs to automate aspects of the system and therefore make the system faster and easier to use. Optionally, the nucleic acid synthesis device further comprises a temperature control means.

[0042] Advantageously, a temperature control means can vary the temperature of the nucleic acid synthesis device to provide optimal conditions for different reactions occurring within the device. Where reactions occur under optimal conditions, they are faster and more efficient.

[0043] Optionally, the nucleic acid synthesis device further comprises at least one assembly chamber.

[0044] Optionally, the means for assembling the at least one cleaved oligonucleotide is a surfacebound assembly means.

[0045] Optionally, the means for assembly comprises the Gibson assembly®.

[0046] Advantageously, the Gibson assembly® can be used to simultaneously assemble multiple sequences based on sequence identity.

[0047] Optionally, the means for assembly comprises PCR assembly.

[0048] Optionally, the double stranded sequences can be dehybridised and PCR assembly can be conducted on both the forward and reverse strands.

[0049] Advantageously, conducting PCR assembly on both the forward and reverse strands increases the yield of the assembled sequence compared to conducting PCR assembly on the forward strand alone.

[0050] Optionally, the product of PCR assembly is amplified by PCR amplification.

[0051] Optionally, the means for assembly comprises enzymatic ligation with a DNA ligase. Optional ly, the means for assembly comprises at least two means for assembly selected from the following list: the Gibson assembly®, PCR assembly enzymatic ligation with a DNA ligase.

[0052] Advantageously, PCR assembly and enzymatic ligation with a DNA ligase can be used to ligate small sequences into larger sequences and the Gibson assembly® can then be used to ligate the resulting larger sequences.

[0053] Optionally, the nucleic acid synthesis device further comprises a means for inputting a chosen sequence.

[0054] Optionally, the nucleic acid synthesis device further comprises a plurality of microfluidic channels.

[0055] Advantageously, the microfluidic channels can readily move fluid components such as nucleic acid products, oligonucleotides and reagents around the nucleic acid synthesis device, reducing the amount of manual input required from a user and reducing the risk of human error.

[0056] Optionally, the at least one array of surface-bound oligonucleotides comprises a plurality of beads.

[0057] Optionally, the beads are glass beads or magnetic beads.

[0058] Advantageously, beads can be easily transported between different reaction sites.

[0059] Optionally, the at least one array of surface-bound oligonucleotides comprises a rough surface.

[0060] Advantageously, a rough surface increases the surface area of the array thereby providing a larger area for containing a higher number of surface-bound oligonucleotides on a smaller area, allowing the overall footprint of the array to be reduced. Optionally, the at least one array of surface-bound oligonucleotides is located within a microreactor.

[0061] Advantageously, a microreactor provides a convenient vessel in which reactions can take place involving small volumes of reagents. Use of small volumes of reagents, as is possible in a microreactor, saves cost and is energy efficient and has faster reaction speeds.

[0062] Optionally, the at least one partially complementary oligonucleotide is insertable into the nucleic acid synthesis device.

[0063] Optionally, the at least one partially complementary oligonucleotide is located in a partially complementary oligonucleotide cartridge configured to be insertable into the nucleic acid synthesis device.

[0064] Advantageously, when the partially complementary oligonucleotide cartridge is a separate component which is insertable into the nucleic acid synthesis device, the consumable partially complementary oligonucleotides can be readily replaced without the need to replace the entire device or replenish an internal store. This reduces the cost associated with using the system.

[0065] Optionally, the means for extending the surface-bound oligonucleotide and partially complementary oligonucleotide comprise at least a polymerase enzyme and a plurality of nucleotides.

[0066] Typically, the means for extending the surface-bound oligonucleotide will be using PCR. Standard PCR reagents include a polymerase, nucleotides, DNA polymerase, a polymerasespecific buffer, sterile water, template DNA and primers. In the present invention, the surfacebound oligonucleotide and complementary oligonucleotide provide the template DNA and primers. These reagents may be contained integrally within the system or alternatively, supplied to the system via removeable cassettes, cartridges or any other suitable means. Typically, the means for extending the surface-bound oligonucleotide comprises hybridising the surface-bound and complementary oligonucleotides under conditions which promote partial annealing of the complementary oligonucleotide to the surface-bound oligonucleotide to generate an overhanging region on each oligonucleotide. The means for extending the surface-bound oligonucleotide also comprises the reagents and conditions necessary for PCR such that the overhanging region of the complementary oligonucleotide acts as a template for the PCR-mediated extension of the surface-bound oligonucleotide, and that the overhanging region of the surface-bound oligonucleotide acts as a template for the PCR- mediated extension of the complementary oligonucleotide.

[0067] Optionally, the means for extending the overlapping oligonucleotide are insertable into the nucleic acid synthesis device and / or are located in a store on the nucleic acid synthesis device.

[0068] Optionally, the means for cleaving the extended oligonucleotide to provide at least one cleaved oligonucleotide comprises at least one restriction enzyme.

[0069] Optionally, the means for cleaving the extended oligonucleotide to provide at least one cleaved oligonucleotide comprises a plurality of types of restriction enzymes.

[0070] Optionally, the restriction enzyme is a programmable restriction enzyme.

[0071] Advantageously, a programmable restriction enzyme can be guided to target its activity to a specific sequence such that one single species of restriction enzyme can be used to digest a plurality of different sequences. For example, the programmable restriction enzyme may be Tth Argonaute (TtAgo).

[0072] Optionally, the at least one restriction enzyme is located in a store on the nucleic acid synthesis device. Optionally, the at least one restriction enzyme is insertable into the nucleic acid synthesis device.

[0073] Optionally, the at least one restriction enzyme is located in a restriction enzyme cartridge configured to be insertable into the nucleic acid synthesis device.

[0074] Advantageously, when the at least one restriction enzyme is located in a cartridge which is insertable into the device, the consumable restriction enzymes can be readily replaced without the need to replace the entire device or replenish an internal store. This reduces the cost associated with using the system.

[0075] Optionally, the plurality of restriction enzymes is stored such that each type of restriction enzyme is separated from other types of restriction enzymes.

[0076] Optionally, the means for extending the surface-bound oligonucleotides is located in an extension cartridge which is insertable into the nucleic acid synthesis device.

[0077] Advantageously, when the means for extending the surface-bound oligonucleotides is located in a cartridge which is insertable into the device, the consumable extension reagents can be readily replaced without the need to replace the entire device or replenish an internal store. This reduces the cost associated with using the system.

[0078] Optionally, the system further comprises a means for purifying the synthesised nucleic acid product.

[0079] Advantageously, when the system comprises a purification means the output is a ready-to- use synthesised nucleic acid product and therefore further steps are not needed before use of the product.

[0080] Optionally, the system further comprises at least one waste outlet. Optionally, the system further comprises a quality control means.

[0081] Optionally, the quality control means quantifies the concentration of the synthesised nucleic acid product.

[0082] Optionally, the quality control means confirms the sequence of the synthesised nucleic acid product.

[0083] A method for synthesising nucleic acids comprising the steps:

[0084] (i) selecting a chosen nucleic acid sequence;

[0085] (ii) obtaining a plurality of partially complementary oligonucleotides;

[0086] (iii) bringing said plurality of partially complementary oligonucleotides into contact with an array of surface-bound oligonucleotides under hybridising conditions; wherein each of the plurality of partially complementary oligonucleotides is at least partially complementary to at least one surface-bound oligonucleotide on the array of surface-bound oligonucleotides; such that each of the plurality of partially complementary oligonucleotides hybridises to one surface-bound oligonucleotide on the array of surface bound oligonucleotides creating an overlapping oligonucleotide having least one overhanging region;

[0087] (iv) extending the overlapping oligonucleotide to provide a plurality of extended oligonucleotides;

[0088] (v) cleaving the plurality of extended oligonucleotides to provide a plurality of cleaved oligonucleotides;

[0089] (vi) ligating the plurality of cleaved oligonucleotides to provide a synthesised nucleic acid product corresponding to the chosen nucleic acid sequence.

[0090] Optionally, step (ii) further comprises the steps:

[0091] (ii.a) generating a complementary sequence which is complementary to the chosen nucleic acid sequence;

[0092] (ii.b) converting the complementary sequence into a plurality of complementary overlapping sequences; wherein the plurality of partially complementary oligonucleotides correspond to the plurality of complementary overlapping sequences.

[0093] Optionally, the each of the plurality of extended oligonucleotides comprise a plurality of repeating sequences.

[0094] Optionally, the cleaved oligonucleotides correspond to an individual repeating sequence.

[0095] Optionally, the method is conducted on a device.

[0096] Optionally, the device is a benchtop device.

[0097] Optionally, the bringing said plurality of partially complementary oligonucleotides into contact with an array of surface-bound oligonucleotides is carried out in a temporally separated manner.

[0098] A nucleic acid synthesis device comprising: an input module for inputting a chosen nucleic acid sequence; at least one array comprising a plurality of surface-bound oligonucleotides; a means for extending at least two of the plurality of surface-bound oligonucleotides on the array of surface-bound oligonucleotides to provide a plurality of extended oligonucleotides; a means for cleaving the plurality of extended oligonucleotides to provide a plurality of cleaved oligonucleotides; a assembly chamber; a means for assembling the plurality of cleaved oligonucleotides within the assembly chamber to provide the synthesised nucleic acid product, wherein the synthesised nucleic acid product corresponds to the chosen nucleic acid sequence; an outlet Brief Description of the Drawings

[0099] 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:

[0100] Figure 1 shows a schematic of extension of a surface-bound oligonucleotide according to certain embodiments of the invention.

[0101] Figure 2 shows an example of an array according to certain embodiments of the present invention.

[0102] Figure 3 shows the mean fluorescence intensity of surface-bound oligonucleotides before extension and after extension by 5 PCR cycles (fig 3A) by 10 PCR cycles (fig 3B) and by 15 cycles.

[0103] Figure 4 shows gel electrophoresis images and corresponding intensity graphs of extended and digested DNA products.

[0104] Figure 5 is a gel electrophoresis image showing extended DNA product after digestion with different quantities of restriction enzyme

[0105] Figure 6 shows a gel electrophoresis image of ligation products.

[0106] Figure 7 shows the fluorescence intensity of the surface-bound oligonucleotide before and after extension by 15 PCR cycles and dehybridisation.

[0107] Figure 8 shows the fluorescence intensity of the surface-bound oligonucleotide before after extension by 15 PCR cycles, dehybridisation from the partially complementary oligonucleotide and rehybridization with the partially complementary oligonucleotide. Figure 9 shows an overlapping oligonucleotide as in example 3.

[0108] Figure 10 shows the UV-Vis spectra of an extended product of example 3.

[0109] Figure 11 shows a visualisation and fluorescence intensity of the gel electrophoresis of the extended oligonucleotide oligonucleotide as in example 3.

[0110] Figure 12 shows the binding efficiency of DNA to Dynabeads® before extension.

[0111] Figure 13 shows the binding efficiency of extended DNA to Dynabeads®.

[0112] Figure 14 shows an agarose gel image of TP53 exon 1-3 extended DNA product.

[0113] Figure 15 shows an agarose gel image of TP53 exon 1-3 BsaXI digest products.

[0114] Figure 16 shows an agarose gel image of partial TP53 exon 1 extended DNA product.

[0115] Figure 17 shows an agarose gel image of partial TP53 exon 1 Bpll digest product.

[0116] Figure 18 shows Tapestation™ electrophoresis of Gibson assembly® products.

[0117] Figure 19 is a simplified schematic diagram showing a graphical representation of PCR assembly.

[0118] Figure 20 shows Tapestation™ electrophoresis of PCR assembly products.

[0119] Figure 21 shows Tapestation™ electrophoresis of extended biotinylated products.

[0120] Figure 22 shows Tapestation™ electrophoresis of Bael digest products on beads. Figure 23 shows Tapestation™ electrophoresis of TP53 exon 1 extension products.

[0121] Figure 24 shows Tapestation™ electrophoresis of TP53 exon 1 Bael digest products.

[0122] 5

[0123] Detailed Description

[0124] Example 1 - Nucleic acid synthesis system for synthesising pre-determined nucleic acid products

[0125] According to one example of the present invention, a synthetic nucleic acid synthesis system is provided for synthesising multiple pre-determined synthesised nucleic acid products. In this example, the synthesised nucleic acid products are genes. For example, the genes may be genes involved in the DNA repair pathway such as LIG3, XPC and RAD51.

[0126] The system comprises a nucleic acid synthesis device with multiple arrays of surface-bound oligonucleotides. Each array is configured to produce one gene product.

[0127] On each array, there is a plurality of surface-bound oligonucleotides. One of these surfacebound oligonucleotides 100 is shown in figure 1. The surface 101 is a solid glass surface. The surface bound oligonucleotides 100 are covalently bound to the surface 101 by an APEGDMES (acetalpolyethyleneglycoldimethylethoxysilane) linker. Each of the surface-bound oligonucleotides 100 comprises two repeating copies of sequence X corresponding to part of the gene sequence. Hybridised to each of the surface-bound oligonucleotides 100 is a partially complementary oligonucleotide 102 which is exactly complementary to the surface-bound oligonucleotide to which it is hybridised and therefore contains two copies of a sequence complementary to sequence X - sequence Y.

[0128] When in use, using a user interface present on the device, the user selects which of the predetermined genes they wish to synthesise and inputs a desired concentration. The control module then selects the array which corresponds to the users selected gene.

[0129] The control module then activates the temperature control module to raise the temperature in the selected array to enable denaturing 101 of the surface-bound 100 and partially complementary oligonucleotides 102. The temperature is then adjusted to provide conditions that promote partial annealing 103 of the surface-bound 100 and partially complementary oligonucleotides 102 to generate an overlapping oligonucleotide 110 with two 5' overhanging regions 104a, 104b.

[0130] A cartridge containing a means for extending the oligonucleotides is inserted into the device. The cartridge contains extension reagents, such as polymerase enzyme, buffers and nucleotides to enable the polymerase-mediated extension of the surface-bound 100 and partially complementary oligonucleotides 102. The control module controls the addition of the extension reagents into the array. The temperature control means raises and lowers the temperature of the array to facilitate the PCR reaction to extend 105 the surface-bound 100 and partially complementary oligonucleotides 102 to provide a first extended surface-bound oligonucleotide 100a and a first extended partially complementary oligonucleotide 102a.

[0131] The first extended surface-bound oligonucleotide 100a and a first extended partially complementary oligonucleotide 102a are denatured 106 from one another by raising the temperature of the array. The temperature is once more adjusted to provide conditions that promote partial annealing 107 of the first extended surface-bound oligonucleotide 100a and a first extended partially complementary oligonucleotide 102a. The partial annealing of the first extended surface-bound oligonucleotide 100a and a first extended partially complementary oligonucleotide 102a generates an overhanging region on each oligonucleotide 108a, 108b.

[0132] Extension reagents are added into the array and the temperature of the array is adjusted to facilitate a second extension reaction to provide a second extended surface-bound oligonucleotide 100b and a second extended partially complementary oligonucleotide 102b.

[0133] These steps (denaturing, partial annealing and extending) can be repeated any number of times as is necessary to synthesise enough repeating sequence units so as to achieve the concentration of gene product as selected by the user. This can be calculated by an algorithm running on the control module of the device. Following the extension reaction, the extended oligonucleotides are cleaved from the surface-bound and partially complementary oligonucleotides using an appropriate restriction enzyme. A cartridge containing restriction enzymes and associated reagents is inserted into the device and the enzymes and reagents are brought into contact with the extended oligonucleotides on the oligonucleotide array.

[0134] In certain embodiments, the surface-bound oligonucleotide may comprise a restriction site at its 3' end such that the extended product can readily be cleaved from the oligonucleotide whilst leaving the original surface-bound oligonucleotide intact. Alternatively, or in addition the partially complementary oligonucleotide may comprise a restriction site at it 5' end.

[0135] Once the extended products are cleaved from the surface-bound and partially complementary oligonucleotides, they are further cleaved by restriction enzymes into their constituent sequences to provide cleaved double-stranded oligonucleotides, i.e. a plurality of individual units comprising sequence X and sequence Y. These cleaved oligonucleotides are then transported to an assembly chamber. In certain embodiments, the restriction enzyme is the same enzyme as is used to cleave the extended product from the surface-bound oligonucleotide such that the cleavage from the surface and into cleaved oligonucleotides can occur as a single digest step. In alternative embodiments, the restriction enzyme used to generate the cleaved oligonucleotide is different from the restriction enzyme used to cleave the extended product from the surface-bound oligonucleotide.

[0136] The steps described above are also conducted on other surface-bound and partially complementary oligonucleotides having different sequences on the array. For example, a surface-bound oligonucleotide having repeating units of sequence A and a partially complementary oligonucleotide having repeating units of sequence B and a surface-bound oligonucleotide having repeating units of sequence C and a partially complementary oligonucleotide having repeating units of sequence D. Once cleaved, the double-stranded cleaved oligonucleotides comprising individual units of sequence A and B, C and D are also transported into the assembly chamber with the double-stranded oligonucleotides comprising sequences X and Y.

[0137] In this example, the means for assembly is enzymatic ligation. A cartridge containing ligation reagents including a ligase enzyme and buffer is inserted into the device. The ligation reagents are transported to the assembly chamber. The sequences are configured such that they can be ligated to form the pre-determined gene which the array is configured to synthesise. Each surface-bound oligonucleotide is configured to generate one part of the gene sequence which will ligate to at least one neighbouring part of the gene sequence, said neighbouring part having been generated by a different surface-bound oligonucleotide on the array. For example, the chosen gene may be gene X which comprises sequences A, C and X. In this scenario, a plurality of cleaved oligonucleotides coding sequences A, C and X will be ligated in the assembly chamber to generate a plurality of double-stranded gene X products as required to reach the concentration selected by the user. In some embodiments, the complementary sequences (sequences B, D and Y) are not required for the gene product and the gene product is supplied as a single-stranded product. Where a single-stranded product is required, the double-stranded sequence can be denatured using heat and the desired single-stranded product can be supplied to the user. Alternatively, the unwanted strand can be tagged with a phosphate group and a specific enzyme can be used to digest the unwanted phosphate- tagged strand.

[0138] The extension and assembly steps may be carried out in stages or simultaneously depending on the complexity of the gene.

[0139] Following the ligation, the gene X products are purified and quantified. The sequence of the gene X product may also be checked to ensure that it is correct. Purification of the gene product can be conducted using any suitable method. Suitable methods of purification would be known by the skilled person and may include purification by spin / gravity column or by ethanol precipitation. Similarly, suitable methods of quantification would be known to the skilled person, these may include UV-Vis spectrophotometry or fluorescence-based quantification techniques.

[0140] Example 2 - Nucleic acid synthesis system for synthesising any nucleic acid product

[0141] In another embodiment of the present invention, a synthetic nucleic acid synthesis system is provided to synthesise any nucleic acid sequence. In this embodiment, the nucleic acid sequence may be a gene sequence or partial gene sequence.

[0142] The system comprises a nucleic acid synthesis device with an array of surface-bound oligonucleotides. Each surface-bound oligonucleotide comprises a different sequence of 8 nucleotides such that every possible 8 nucleotide sequence (8-mer) is present in one of the surface-bound oligonucleotides.

[0143] When in use, using a user interface present on the device, the user inputs the gene they wish to synthesise and inputs a desired concentration. The control module then computes the complementary sequence and divides the complementary sequence into 8 nucleotide lengths. The applicable partially complementary oligonucleotides are selected and one or more cartridges containing partially complementary oligonucleotides are inserted into the device. The selected partially complementary oligonucleotides are brought into contact with the surface-bound oligonucleotides on the oligonucleotide array.

[0144] The annealing, extension and denaturing steps are performed in the same manner as described in example 1. Once the final extension step has occurred, the unbound partially complementary oligonucleotides are washed away from the array and the extended oligonucleotide is cleaved from the surface-bound oligonucleotide leaving the original surface-bound oligonucleotide. The extended oligonucleotide comprises multiple 8-mer repeats, each 8-mer repeat in the extended oligonucleotide corresponds to the same 8-mer segment of the chosen gene sequence. The extended oligonucleotide is cleaved into a plurality of cleaved oligonucleotides, each cleaved oligonucleotide comprising one 8-mer. The resulting cleaved oligonucleotides are transported to the assembly chamber alongside other cleaved oligonucleotides corresponding to other 8-mer segments of the chosen gene sequence. The extension and assembly steps may take place in a number of stages as appropriate for the complexity of the chosen gene sequence. For example, if the chosen gene sequence requires the assembly of 10 different 8-mer cleaved oligonucleotides, the 10 different 8-mers may be produced and ligated simultaneously by introducing all 10 partially complementary oligonucleotides to the array simultaneously. Alternatively, the extension and assembly steps may be repeated a number of times. For example in a first stage, 4 of the 10 partially complementary oligonucleotides which correspond to neighbouring sequences of the chosen gene sequence may be introduced into the array in a first extension and cleavage reaction resulting in the production of 4 different 8-mer cleaved oligonucleotides. The resulting 8-mers may then be transported to the assembly chamber and ligated to provide a partial gene sequence. In a second stage of extension and cleavage reactions, the remaining 6 partially complementary oligonucleotides may be introduced into the array to produce the remaining 6 cleaved oligonucleotide 8-mers. These 8-mers may then be transported into the assembly chamber to be ligated to the partial gene sequence produced in stage one to provide a full gene sequence of the chosen gene or gene fragment.

[0145] In certain embodiments, spatial addressability may be utilised to determine which of the surface-bound oligonucleotides are extended. Spatial addressability is a term describing the ability to switch extension on and off at different locations on a solid substrate. For example, the surface-bound oligonucleotides may be bound to a solid substrate and each area of that solid substrate may hold a distinct surface-bound oligonucleotide. Using spatial addressability, different areas of the solid substrate can be activated, only permitting extension of those surface-bound oligonucleotides within activated areas. Extension does not occur in areas which have not been activated. Activation may be achieved by activating polymerases in selected areas, such as by the addition of a necessary co-factor or alternatively, activation could be achieved by the removal of blocking factors on the oligonucleotides which would otherwise prevent extension occurring.

[0146] Alternatively, spatial addressability can be achieved by selectively supplying the complementary oligonucleotides to the array of surface-bound oligonucleotides such that the activation occurs by partial hybridisation between the surface-bound and complementary oligonucleotides which allows extension to take place. The surface-bound oligonucleotides which have not been supplied with a complementary oligonucleotide remain inactive, preventing extension from occurring.

[0147] Each cartridge of partially complementary oligonucleotides may comprise multiple copies of the same partially complementary oligonucleotide. Alternatively, each cartridge may comprise multiple copies of a number of different partially complementary oligonucleotides which are stored separately from one another within the cartridge.

[0148] The control module may be configured to run a computer program. The computer program may be configured to compute a sequence complementary to a user's chosen sequence and determine which partially complementary oligonucleotides are needed to generate the chosen sequence. The computer program may also determine which restriction enzyme(s) are suitable for cleaving the extended product. The computer program may also be configured to determine how many stages of extension and assembly are required to efficiently produce the chosen gene product. The computer program may determine the optimal temperatures for different stages of the process according to the specific enzymes, sequences and optimal outcomes. The computer program may also be configured to control microfluidic movement of products and reagents in the device. The computer program may control conditions within the device such as temperature and pressure.

[0149] In certain embodiments of the present invention, extension of the surface-bound oligonucleotide and complementary oligonucleotide is conducted such that 2 units of the repeating sequence are incorporated into the surface-bound oligonucleotide to create an extended product having the surface-bound oligonucleotide plus additional repeating sequences and corresponding extended complementary oligonucleotide. The additional repeating sequences can then be cleaved from the surface-bound oligonucleotide and each other into the constituent repeating sequences. The repeating sequences can then be ligated with other sequences to generate a template of the desired end product. The template can then be extended in solution to obtain the desired number of repeating sequence units before being cleaved into the individual desired end products. This embodiment is particularly useful where no modifications are being included in the extended oligonucleotide as the majority of the extension takes place in solution, which is faster and generates a higher yield than extension on a surface.

[0150] Alternatively, the extension may take place entirely on the surface. In this embodiment, the extension of the surface-bound and complementary oligonucleotides takes place on the surface in the manner described above in examples 1 and 2 (i.e. extension, denaturation and annealing). Once the desired number of repeating sequence units have been incorporated into the extended oligonucleotide, the extended oligonucleotide can be cleaved from the surface-bound and complementary oligonucleotides and further cleaved into its constituent repeating units before being ligated with other sequences. During the assembly, modifications can be incorporated into the sequence in a controlled manner. This embodiment is particularly useful where modifications are being incorporated as it allows more control over where the modifications are included into the sequence.

[0151] In certain embodiments, the ends of the sequence may be closed. The closing of sequences can be carried out during the cleavage of the extended oligonucleotide into cleaved oligonucleotides. Experimental Data

[0152] Figure 2 shows an example of an array according to certain embodiments of the present invention. The array is provided by a plurality of glass beads onto which fluorescent-tagged DNA has been immobilised via a linker module.

[0153] Two different sizes of beads, 0.5 mm and 1 mm, were treated with Oz plasma for 10 minutes, they were then transferred to a vial with APEGDMES (acetalpolyethyleneglycoldimethylethoxysilane) linker in toluene and heated at 65 °C for 60 hrs before being washed with toluene, ethanol and water through a glass frit. These were then heated in a vacuum oven at 120 °C for 40 minutes to completely dry. Beads were then placed into 5 mL syringes with caps on and cotton wool in the outlet. DNA (200 pL, 5 pM in 10% acetic acid) was added to the beads for 1 hr, NaCNBHs (200 pL, 16 pM in 50% methanol) was then added for 60 hrs. The beads were then washed through with 0.5X PBS 3 times and water 3 times by pushing the plunger down the syringe. Nitrogen was used to push the beads and cotton wool out of the syringe into a glass vial.

[0154] The beads were then placed onto microscope slides and viewed under a 10X lens on the fluorescent microscope on the fluorescein setting.

[0155] The sequences used are shown in the table below:

[0156] Figure 3 shows the mean fluorescence intensity of surface-bound oligonucleotides before extension and after extension by 5 PCR cycles (fig 3A) by 10 PCR cycles (fig 3B) and by 15 cycles. The graphs shows fluorescence intensity of the linker molecule alone stained with PicoGreen (Linker), the surface-bound oligonucleotide stained with PicoGreen ([AaGh) and the extended product stained with PicoGreen (extended DNA). Each graph demonstrates an increase in fluorescence of the extended product when compared to the linker and the surface-bound oligonucleotide. This demonstrates that the extension reaction is successful in extending the surface-bound oligonucleotide.

[0157] Figure 4 shows gel electrophoresis images and corresponding intensity graphs of extended and digested DNA products. Figure 4A shows the digested DNA product in lane 1 eluted in water and in lane 2 eluted in elution buffer. Lane 3 shows the undigested extended DNA product. Figure 4B shows the DNA product after different lengths of digestion. Lane 1 shows the product after a 1 hour digest, lane 2 shows the product after a 2 hour digest and lane 3 shows the product after a 3 hour digest. After a one hour digest, the strongest band is over 700 bp in length and comprises undigested extended DNA product. Some faint bands can be seen at shorter lengths (e.g. 30 and 45 bp). This is also demonstrated in the corresponding graph showing no strong peaks in the 1 hour digest product. After the 2 hour digest, there is significantly less of the undigested extended product. On the graph, peaks can be seen around 15, 30, 45 and 60 base pairs indicating partial digestion of the extended DNA product. After 3 hours of digestion, one band can be seen at 15 bp indicating complete digestion of the extended DNA product. This can also be seen in the corresponding graph whereby there is one large peak around 15 bp and a lack of peaks at other lengths.

[0158] Figure 5 is a gel electrophoresis image showing extended DNA product after digestion with different quantities of restriction enzyme. No difference in digestion efficiency was observed with higher quantities of the enzyme.

[0159] Figure 6 shows a gel electrophoresis image of ligation products. Stepwise ligation reactions were prepared by ligation of sequence pairs (1+2, 3+4, 5+6, 7+8, 9+10, 11+12). Sequence pairs were then ligated together (1+2+3+4, 5+6+7+8, 9+10+11+12) the resulting sequences were subsequently ligated in a final step to provide a final ligated sequence comprising sequences 1 to 12.

[0160] Reactions were incubated at room temperature for 2 hours.

[0161] Lane one shows the ligation product of sequences 1 and 2 of the gene sequence which is shown at the expected length of 40bp. Lane 2 shows the ligation product of sequences 1 to 4 of the gene sequence which is shown at the expected length of 80 base pairs. Lane 3 contains the ligation product of sequences 1 to 8 of the gene sequence, shown at the expected length of 160 bp. Lane 4 contains the full gene product comprising sequences 1-12, the ligation product can be seen at the expected size of 240 bp.

[0162] Figure 7 shows the fluorescence intensity of the linker stained with PicoGreen (Linker), surface-bound oligonucleotide hybridised with a complementary oligonucleotide and stained with PicoGreen ([AsGJs) and extended surface-bound and complementary oligonucleotide stained with PicoGreen after extension by 15 PCR cycles.

[0163] Figure 8 shows the fluorescence intensity of the linker stained with PicoGreen (Linker), surface-bound oligonucleotide hybridised to a complementary oligonucleotide and stained with PicoGreen ([A3G]5) and extended surface-bound oligonucleotide stained with PicoGreen after extension by 15 PCR cycles, dehybridisation from the partially complementary oligonucleotide and rehybridization with the partially complementary oligonucleotide.

[0164] Example 3 - extension of an ABCD repeating sequence

[0165] In this example, a double stranded sequence containing 4 repeating sequences was extended and digested according to the present invention. 3.1 Preparation of overlapping oligonucleotides

[0166] The surface-bound oligonucleotide comprises sequences A, B and C is a 5' to 3' direction and the complementary oligonucleotide comprises sequences D, B and E in a 3' to 5' direction.

[0167] Sequence A is complementary to sequence E, sequence C is complementary to sequence D and sequence B is self-complementary. Hybridisation between the surface-bound and complementary oligonucleotide results in an overlapping oligonucleotide as shown in figure 9.

[0168] The overlapping oligonucleotides (sequences shown below) were prepared by adding the single stranded surface-bound and complementary oligonucleotides (10 pL of each SS oligonucleotide (100 pM)) to HEPES and potassium acetate DNA annealing buffer (480 pL, IX) in an Eppendorf. HEPES and potassium acetate buffer is made from 10 mM HEPES, 100 mM KCI and 1 mM EDTA. The solution was vortexed for a few seconds to ensure thorough mixing, and then heated to 95 °C for 10 minutes before being cooled slowly to room temperature (roughly 25 °C). When not in use, overlapping oligonucleotides were stored at -20 °C in the freezer.

[0169] Surface-bound oligonucleotide (SEQ ID 3): 5’TGG ACT CTC TCA GAT ATC ATC GAC T3‘ Complementary oligonucleotide (SEQ ID 4): 5'TGA GAG AGT CCA GAT ATC AGT CGA T3'

[0170] EcoRV restriction sites are shown in bold.

[0171] 3.2 Extension of overlapping oligonucleotides

[0172] Nanopure-H2O (60 pL) was added to 10 pL each of dATP, dTTP, dCTP, dGTP (all 100 mM) to provide a 100 pL dNTP mix of 10 mM concentration.

[0173] The extension reaction mixture was prepared in a thin-walled 200 pL Eppendorf by adding nanopure-H2O (36 pL) to ThermoPol buffer solution (5 pL, 10X), overlapping r oligonucleotide (5 pL, 2 pM), primer-specific dNTP mix (2.5 pL, 10 mM), and MgSO4 (1 pL, 100 mM). The mixture was then vortexed for a few seconds before adding DeepVent® DNA polymerase enzyme (0.5 pL, 1U). At 75 °C, one unit of DeepVent® (exo-) DNA polymerase enzyme can incorporate 10 nmol of dNTPs. To not destroy the polymerase enzyme, the mixture was mixed gently with a pipette tip instead of using a vortex. After 10 cycles, more primer-specific dNTP mix (2.5 |_ L, lOmM) was added, along with polymerase enzyme (0.5 pL, 1U). The Eppendorf was then placed back in the thermocycler on the same setting for a further 10 cycles (20 cycles in total). When not in use, reagents and products were stored at -20 °C.

[0174] Each cycle consisted of:

[0175] 1) Denaturing at 95 °C for 30 seconds.

[0176] 2) Annealing at 55 °C for 30 seconds.

[0177] 3) Elongation at 72 °C for 120 seconds.

[0178] After 20 cycles, the thermocycler cooled the mixture to 4 °C and held the mixture at this temperature for as long as required.

[0179] 3.3 Purification of extended oligonucleotides

[0180] For purification, the Monarch® PCR and DNA Cleanup Kit was used. DNA binding buffer (100 pL) was added to the extended oligonucleotide solution and mixed with a pipette before transferring the whole sample to a thick-walled tube and column. The column was centrifuged at 13000 RPM for 1 minute, then the discarded buffer was disposed of in the aqueous waste. DNA wash buffer (200 pL) was added to the column, which was then centrifuged at 13000 RPM for 1 minute. This step was repeated, then all the wash solution was disposed of in the aqueous waste. The column was centrifuged again at 13000 RPM for 1 minute to ensure all excess liquid had been removed and the column was fully dry - any waste was disposed of in the aqueous waste. A 1.5 mL Eppendorf tube was heated to 60 °C in the heating block, into which the column (with DNA attached to the silica) was placed. This heating step was to increase yield. Elution buffer (20 pL) was added to the column in the heating block and was kept at 65 °C for 5 minutes. The sample was centrifuged for a final time at 13000 RPM for 1 minute. The purified extended oligonucleotide product was collected in the 1.5 mL Eppendorf tube. All binding / wash / elution buffers were stored at room temperature.

[0181] 3.4 UV / Vis Spectroscopy

[0182] UV / Vis spectra, overlapping oligonucleotide concentrations, and purity ratios were recorded using a Thermo Scientific™ NanoDrop™ One Microvolume-UV / Vis Spectrophotometer allowing analysis of 1 pL samples, using surface tension to hold the small volume of sample in place between two pedestals. Most measurements of samples and blanks were recorded using the dsDNA setting. Blanks were obtained using Monarch® DNA Elution Buffer each time before performing measurements of each DNA sample. Between each measurement, a lint free wipe was used to clean the pedestals and limit any contamination between samples. An absorption band at 260 nm would confirm the presence of DNA.

[0183] Figure 10 shows the UV-Vis spectra of the purified extended oligonucleotide of example 3. The absorption band at 260nm confirms the presence of DNA in the sample.

[0184] 3.5 Digestion of extended oligonucleotides

[0185] Reaction mixture was made up using the extended oligonucleotide product, rCutSmart Buffer and EcoRV HF in the concentrations and volumes in the table below. This reaction mixture was heated to 37 °C for 3 hours before purple loading dye was added to deactivate the enzyme.

[0186] 3.6 Visualisation of cleaved oligonucleotide products via Agarose Gel Electrophoresis

[0187] A 4% MetaPhor agarose gel was used to run the cleaved oligonucleotide product alongside a low range ladder and the gel electrophoresis was analysed using Imaged. Gels were electrophoresed at 100 V (400 mA) for 60 to 90 minutes. All gels were imaged using UviProMWl software and a Uvitec Chemiluminescence fluorescence imaging box. All gels were analysed using ImageJ software, converting the data into graphs using Microsoft Excel.

[0188] Figure 11 shows a visualisation and fluorescence intensity of the gel electrophoresis of the extended oligonucleotide and cleaved oligonucleotide. Figure 11A shows the visualisation and fluorescence intensity of the gel electrophoresis of the extended oligonucleotide. Figure 11B shows the visualisation and fluorescence intensity of the gel electrophoresis of the cleaved oligonucleotide. Figure 11A shows a band at the expected length of 190bp. As can be seen in figure 11B, after digestion, DNA bands of 31, 18 and 13 base pairs can be detected. The bands at 18 and 13 bp correspond to the two products expected after digestion (either side of the restriction site). The 31 bp band represents extended oligonucleotides which have been cleaved only at one restriction site.

[0189] Example 4 - Extension and amplification of a sequence bound to a surface

[0190] 4.1 Preparation of duplexed DNA attached to beads

[0191] 650 pl beads (Dynabeads® MyOne™ Streptavidin Cl) were washed three times in 2x binding and washing (B&W) buffer (10 nM Tris-HCI (pH 7.5), ImM EDTA, 2 M NaCI) buffer before being resuspended in 1350 pl 2x B&W buffer (5 pg beads / pl). Separately, 10 pM of duplex DNA (sequences shown below) was prepared in 2x B&W buffer. 50 pl of prepared duplex DNA was added to 50 pl beads (50:50 ratio) and incubated at room temperature for approximately 15 mins to allow the DNA to bind to the beads. Following the incubation, a magnet was applied to separate the duplex-bound beads and the supernatant was aspirated. Three washing steps were conducted to remove any unbound duplex DNA. Washing steps comprised mixing the duplex-bound beads with 2x B&W buffer before applying the magnet and aspirating the supernatant. The resulting duplex-bound beads were then resuspended to give 0.5 pg DNA in 5pL water.

[0192] Biotin (SEQ ID 5) - TGG ACT CTC TCA ATG TGG ACT CTC TCA ATG

[0193] Complement (SEQ ID 6) - CAT TGA GAG AGT CCA CAT TGA GAG AGT CCA Figure 12 demonstrates the binding efficiency of the duplex DNA to the beads. The duplex DNA was attached to the beads as described above but the DNA was diluted to the desired concentrations before incubation with beads. To determine the binding efficiency at each of the different concentrations, the concentration of unbound DNA in the buffer was measured after incubation with the beads using the Qubit™ IX dsDNA HS Assay Kit and following the manufacturers guidance. As can be seen from the figure, approximately 100% of the DNA applied to the Dynabeads® was found to be bound to the beads until the saturation point was reached at around 1600ng DNA.

[0194] 4.2 Extension and amplification of duplex DNA bound to beads

[0195] A PCR reaction mix was prepared in accordance with Table 1 below. The reaction was run for

[0196] 20 cycles of the following: 30 seconds at 95 °C, 30 seconds at 55 °C and 120 seconds at 55 °C.

[0197] Table 1. PCR components

[0198] It was observed that the DNA products became detached from the beads during the extension process. This is likely due to the high temperatures used during extension and amplification. The DNA was rebound to the beads under the original binding conditions (described in 4.1). Figure 13 demonstrates that extended DNA could be rebound to the beads. 4.3 Removal of duplex DNA from beads

[0199] Following the extension and amplification, the beads were removed from the reaction mixture using a magnet and the supernatant aspirated. The beads were then subjected to three wash steps as outlined above in section 4.1. To remove the duplex DNA from the bead, the bond between the biotin on the duplex DNA and streptavidin on the beads was broken by incubating the beads with of 50 pl of 10 mM EDTA, pH 8.2, 95% formamide for 5 minutes at 65 °C or for 2 minutes at 90 °C. The beads were removed using a magnet and supernatant containing the extended DNA duplex was retained. The beads were washed in 2x B&W buffer before the magnet was applied again and supernatant retained.

[0200] The amount of DNA bound to beads was measured before and after extension using a Qubit™ fluorometer. Up to a 50-fold increase in DNA was observed after extension indicating successful extension of the duplex DNA.

[0201] Example 5 - Extension of TP53 exon 1 to exon 3 and digestion of the extended products with BsaXI

[0202] 5.1 Extension of TP53 exon 1 to exon 3

[0203] Single stranded oligonucleotides were formed into duplexes by mixing equal parts of complementary oligonucleotides and heating to 95°C for 10 minutes. The solution was then cooled slowly to room temperature. Duplexes of sequences TP53 1.1 to 3.1 and their complementary sequences were formed. The restriction site (highlighted in bold) of each sequence binds to the restriction site in the complementary sequence (highlighted in bold) to provide a duplex having an overhanging region on each strand. The overhanging regions on duplexes TP53 1.1 to 1.3 are 41bp; duplex TP53 2.1 has overhanging regions of 37bp, and; TP53 3.1 has overhanging regions of 25bp. The sequences are shown in the below table and the BsaXI site is shown in bold in each of the sequences.

[0204] The extension reaction mix was prepared in accordance with the below table.

[0205] The extension reaction mixture was placed in a thermocycler and subject to the following conditions:

[0206] 5.2 Purification and quantification of extended TP53 exon 1 to exon 3

[0207] The PCR products containing extended DNA were purified using the Monarch® PCR and DNA Clean-up kit and following the manufacturers guidance. The concentration of DNA in the purified PCR product was quantified using a Nanodrop™ microvolume spectrophotometer. The samples were eluted in 20 pL NP-water and 1 pL of each sample was analysed on the NanoDrop™. The results are shown below.

[0208] A 1% agarose gel was prepared to visualise the size of the extended DNA products. Agarose was dissolved in 0.5x TBE buffer, Sybr™ Safe DNA gel stain was added to allow visualisation of DNA. The gel was loaded with GeneRuler™ lkb Plus DNA ladder and samples were made up by mixing 5 pL each purified PCR product with 5 pL Nanopure (NP)-water and 3 pL purple loading dye. The samples were loaded into the gel and run at 100V for 60 minutes.

[0209] The gel was imaged under UV light. The results are shown in Figure 14. All samples exceeded 20,000bp demonstrating that the extension reaction had been successful.

[0210] 5.3 Digestion of extended products with BsaXI

[0211] Extended DNA from section 5.2 was used to set up a series of digest reactions with BsaXI restriction enzyme. Digestion reactions were prepared for each sample in accordance with the below table. Reactions were heated to 37 °C for 60 minutes and then the enzyme was denatured by heating the reaction to 65 °C for 15 minutes. Samples were held at 4 °C.

[0212] In order to determine whether the digest had been successful, the digest product was run on a 4% agarose gel, prepared as outlined in section 5.2. An Ultra-low range ladder was used. The result is shown in Figure 15. In each lane, two bands are expected, one band between 27 and 33 base pairs which is the restriction site. The expected size of the second band varies depending on the sequence as follows: TP53 1.1 expected at 38-44 bp; TP53 1.2 expected at 34-40 bp; TP53 1.3 expected at 38-44 bp; TP53 2.1 expected at 37-43 bp; TP53 2.2 expected at 37-43 bp; TP53 2.3 expected at 37-43 bp; TP53 3.1 expected at 25-31 bp. Example 6 - Extension of part of TP53 exon 1 and digestion of the extended product with Bpll

[0213] 6.1 Extension of part of TP53 exon 1 The duplex TP53 1.1 (corresponding to the first part of exon 1 of TP53 gene) was prepared as outlined in section 5.1 above. The extension was conducted as outlined in section 5.1. The sequence is shown in the table below, with the target site for Bpll is shown in bold in each of the sequences.

[0214] 6.2 Purification and quantification of extended partial TP53 exon 1

[0215] Purification and measurement of the extended product was conducted as outlined in section 5.2. A total of 1582 ng DNA was measured on the NanoDrop® microvolume spectrophotometer.

[0216] A 1% agarose gel was prepared and run in accordance with section 5.2. The resulting gel is shown in Figure 16. Smears on the gel at 1500 bp and higher are consistent with a successful extension reaction.

[0217] 6.3 Digestion of extended partial TP53 exon 1 product with BPII

[0218] Extended DNA product from section 6.2 was used in digest reactions with Bpll restriction enzyme.

[0219] Digest reactions were prepared in accordance with the below table. Reactions were heated to 37 °C for 30 minutes and then the enzyme was denatured by heating the reaction to 65 °C for 5 minutes. Samples were held at 4 °C.

[0220] In order to determine whether the digest had been successful, the digest product was run on a 4% agarose gel, prepared as outlined in section 5.2. An Ultra-low range ladder was used. The result is shown in Figure 17.

[0221] Example 7 - Gibson Assembly®

[0222] 7.1 Assembly by Gibson Assembly®

[0223] Five overlapping fragments were ligated using the Gibson Assembly® methodology. The sequences of each fragment are shown below. The fragments overlap by 20 base pairs, overlapping regions are highlighted in bold.

[0224] A Gibson Assembly® mastermix was created containing three enzymes which perform in a single buffer. An exonuclease enzyme creates single-stranded 3' overhanging regions to facilitate the annealing of fragments. A polymerase enzyme facilitates the addition of bases to fill the gaps in the annealed duplex fragments. Finally, a DNA ligase enzyme facilitates the joining of the fragments by catalysing the formation of phosphodiester bonds.

[0225] Fragment Bll / 18 was ligated with B19 / 26 in one reaction (rection 1). In a second reaction fragments B27 / 34, B35 / 40 and B41 / 42 were ligated (reaction 2).

[0226] The reactions were set up on ice as follows:

[0227] The reactions were mixed by gently pipetting before being incubated in a thermocycler at 50 °C for one hour.

[0228] 7.2 Tapestation™ electrophoresis of assembly products

[0229] Assembly product 1 (obtained from reaction 1) and assembly product 2 (obtained from reaction 2) were used in Tapestation™ electrophoresis to determine the size of each product. The ladder was prepared by mixing 1 pl D5000 ladder with 10 pl D5000 sample buffer. Samples were prepared by mixing 1 pl of DNA with 10 pl D5000 sample buffer. The samples and ladder were mixed at 2000 rpm for 1 minute before being loaded into a D5000 screentape on a TapeStation™ (Agilent). The results are shown in Figure 18. Panel A shows the gel image with the ladder in lane Al assembly product 1 (Bll / 18 and B19 / 26) in lane Cl and assembly product 2 (B27 / 34, B35 / 40 and B41 / 42) in in lane DI. Panels B and C show the quantification of the band intensities of lanes Cl (assembly product 1) and DI (assembly product 2). Panel B shows a peak at 961 bp, the expected length of assembly product 1 was 980 bp. Panel C shows a peak at 898 bp and the expected length of assembly product 2 was 899 bp. The difference between the expected length and the actual length (peak) can be accounted for by the margin of error of the Tapestation™ system.

[0230] Example 8 - Assembly of a Sequence by PCR Assembly

[0231] Figure 19 shows a graphical representation of an PCR assembly after extension and digestion of duplex sequences. Panel A shows three overlapping duplex oligonucleotides. Each duplex comprises a forward strand having (in the 5' to 3' direction) a target sequence, sticky end sequence and a restriction sequence. The reverse stand comprises (in the 5' to 3' direction) a complementary restriction site, a complementary target site and a complementary sticky end site. The sticky end sequence is configured to be complementary to the next part of the target sequence. The duplex overlapping oligonucleotides are extended to give extended duplex oligonucleotides, shown in panel B. The extension results in the multiplication of the target sequence, sticky end sequence and restriction sequence on both the forward and reverse strand. The extended sequences are then digested with a type 11 B restriction enzyme which removes the restriction sequence as shown in panel C. Following digestion, the duplexes are denatured into single strand sequences. The sticky end sequences are designed to overlap with the subsequent part of the target sequence, these therefore align in the desired order (panel D) and can be ligated via PCR assembly resulting in the double stranded target sequence (panel E).

[0232] An example of assembly by PCR assembly is described below. Oligonucleotides sequences having overlap regions of 20bp. The sequences of the oligonucleotides used are shown in the below table with overlapping regions highlighted in bold.

[0233] As the melting temperature (Tm) between each overlapping pair differed, a temperature gradient PCR was set up. Sequence IF was used as the forward primer and sequence 10R was used as the reverse primer.

[0234] The PCR product was analysed by Tapestation™ electrophoresis to determine whether the PCR assembly had been successful. The Tapestation™ was conducted as per section 7.2 above, using a D1000 screentape with 3 pL of D1000 sample buffer and 1 pL DNA. The results are shown in Figure 20. Panel A shows the gel image with the ladder in lane Al. Lanes Bl to Fl show the assembly products with melting temperatures of 56 °C, 58 °C, 60 °C, 62 °C and 66 °C respectively. Panels B to G show the quantification of the band intensities of lanes Bl to Fl respectively. The presence of bands at the expected length (408 bp) indicates that the PCR assembly was successful and appears to be more successful at lower annealing temperatures.

[0235] Example 9 - Immobilisation onto magnetic beads

[0236] 9.1 Immobilisation onto magnetic beads

[0237] 5' biotin TP53 1.1 BsaXI (sequence shown below) was immobilised onto Dynabeads™ MyOne™ Streptavidin Cl beads. 20 pL of beads were washed three times in B&W buffer and the supernatant removed. The beads were then resuspended in 40pL B&W buffer containing 2.5 pM DNA (5' biotin TP53 1.1 BsaXI). The beads were incubated with the DNA for 30 minutes at room temperature. The supernatant was removed, and beads were washed three times in B&W buffer.

[0238] The total amount of DNA in the DNA solution was measured before and after incubation with beads. DNA amount was measured using a Qubit™ according to the manufacturers protocol. The total amount of DNA in the solution after incubation with the beads reduced by 67%, indicating that 67% of the DNA was successfully immobilised to the beads.

[0239] 9.2 Hybridisation of complement

[0240] The DNA complement (TP53 1.1 BsaXI comp (sequence shown above), 0.45 pM in 40 pL B&W buffer) was added to immobilised beads as produced in section 9.1 (0.46 pM in 40 pL B&W buffer). The mixture was incubated at room temperature for 30 minutes. The supernatant was removed and beads washed three times in B&W buffer (100 pL).

[0241] The total amount of DNA was measured before and after incubation with beads using a Qubit™ according to the manufacturers protocol. Only very small amounts of DNA were detected in the supernatant after incubation with the beads, indicating that almost all the complement DNA had bound to the immobilised DNA.

[0242] Example 10 - Extension of biotinyated DNA, immobilisation onto beads and digestion with BsaXI

[0243] 10.1 - Generation of biotinylated duplex DNA

[0244] Duplex DNA was formed by adding biotinylated DNA (TP53 BsaXI 1.2, 100 pM) with its complement (TP53 BsaXI 1.2 comp, 100 pM). Sequences shown below. The mixture was heated to 95°C for 10 minutes and subsequently cooled to room temperature.

[0245] 10.2 - Extension of biotinylated DNA

[0246] A PCR reaction mix was prepared in accordance with the below table. The reaction was run for 20 cycles of the following: 30 seconds at 95 °C, 30 seconds at 55 °C and 120 seconds at 72 °C.

[0247] The extended product was used in Tapestation™ electrophoresis to determine its size. The ladder was prepared by mixing 1 pl genomic sample ladder with 10 pl genomic sample buffer. Samples were prepared by mixing 1 pl of DNA with 10 pl genomic sample buffer. The samples and ladder were mixed at 2000 rpm for 1 minute before being loaded into a screentape on a TapeStation™ (Agilent). The results are shown in Figure 21. Panel A shows the gel image with the ladder in lane Al extension product in lane Bl. Panel B shows the quantification of the band intensity of lane Bl (extension product). Panel B shows a peak at over 60,000 bp, indicating that the DNA has been successfully extended.

[0248] 10.3 - Purification and quantification of extended biotinylated DNA

[0249] The PCR products containing extended DNA were purified using the Monarch® PCR and DNA Clean-up kit and following the manufacturers guidance.

[0250] The concentration of DNA in the purified PCR product was quantified using a Nanodrop™ microvolume spectrophotometer. The sample was eluted in 20 pL NP-water and 1 pL of each sample was analysed on the NanoDrop™. A total of 180 ng DNA was measured in the sample.

[0251] 10.4 - Immobilisation of extended biotinylated DNA onto magnetic beads

[0252] Solutions of biotinylated extended DNA (as obtained in section 10.3) were made up to 10 ng / pL in 40 pL of B&W buffer. Beads (Dynabeads™ MyOne™ Streptavidin Cl beads) were washed three times in 100 pL B&W buffer, the supernatant was discarded, and the DNA solution was incubated with the beads for 30 minutes at room temperature. Beads were then washed three times in 100 pL B&W buffer. The total amount of DNA was measured before and after incubation with beads using a Qubit™ according to the manufacturers protocol. There was a significant reduction in the amount of DNA present after incubation with the beads, suggesting that the extended biotinylated DNA was successfully immobilised onto the beads.

[0253] 10.5 - Digestion of extended DNA on magnetic beads with BsaXI A digestion reaction mix was prepared in accordance with the below table. The beads with extended DNA produced in section 10.4 were added. The reaction mix was heated to 37 °C for 1 hour.

[0254] The digest product was used in Tapestation™ electrophoresis to determine its size.

[0255] The ladder was prepared by mixing 1 pl D1000 ladder with 3 pl D1000 sample buffer. Samples were prepared by mixing 1 pl of DNA with 3 pl D1000 sample buffer. The samples and ladder were mixed at 2000 rpm for 1 minute before being loaded into a D1000 screentape on a TapeStation™ (Agilent). The results are shown in Figure 22. Panel A shows the gel image with the ladder in lane ELI triplicate digest product in lanes Al, Bl and Cl. Panels B to D show the quantification of the band intensity of lanes Al to Cl (triplicate digest products). The target band size was 43 bp and bands were observed at 35 bp in each of the triplicate wells.

[0256] Example 11 - Extension and digestion with Bael

[0257] 11.1 - Generation of duplex DNA

[0258] 5 pM of oligonucleotides (TP53 Bael 1.1, TP53 Bael 1.2 and TP53 Bael 1.3) were combined with 5 pM of their complement oligonucleotides (TP53 Bael 1.1 comp, TP53 Bael 1.2 comp and TP53 Bael 1.3 comp). Sequences are shown in the table below. The mixtures were heated to 95 °C for 10 minutes. The duplexed oligonucleotides were left to cool to room temperature.

[0259] 11.2 - Extension of DNA

[0260] PCR reaction mixtures were prepared as follows: The mixtures were vortexed and placed into a thermocycler. The following PCR program was run for 20 cycles: 30 seconds at 95 °C, 30 seconds at 55 °C and 120 seconds at 72 °C.

[0261] The extended product was used in Tapestation™ electrophoresis to determine its size. The ladder was prepared by mixing 1 pl genomic sample ladder with 10 pl genomic sample buffer. Samples were prepared by mixing 1 pl of DNA with 10 pl genomic sample buffer. The samples and ladder were mixed at 2000 rpm for 1 minute before being loaded into a screentape on a TapeStation™ (Agilent). The results are shown in Figure 23. Panel A shows the gel image with the ladder in lane Al, extension product TP53 Bael 1.1 in lane Bl, extension product TP53 Bael 1.2 in lane Cl, and extension product TP53 Bael 1.3 in lane Cl. Panel B shows the quantification of the band intensity of lane Bl (TP53 Bael 1.1). Panel C shows the quantification of the band intensity of lane Bl (TP53 Bael 1.2). and Panel D shows the quantification of the band intensity of lane Bl (TP53 Bael 1.3). The resulting smear on the gel is consistent with successful extension.

[0262] 11.3 - Purification and quantification of extended DNA

[0263] The PCR products containing extended DNA were purified using the Monarch® PCR and DNA Clean-up kit and following the manufacturers guidance.

[0264] The concentration of DNA in the purified PCR product was quantified using a Nanodrop™ microvolume spectrophotometer. There was 20 pL of each purified PCR product. 1 pL of each sample was analysed on the NanoDrop™. DNA concentrations were as follows: TP53 Bael 1.1 - 115 ng / pL, TP53 Bael 1.2 - 99 ng / pL, TP53 Bael 1.3 - 119 ng / pL.

[0265] 11.4 - Digestion of extended DNA with Bael

[0266] Digestion reaction mixes were prepared as follows:

[0267] Reaction mixtures were heated to 37 °C for 1 hour. The digest products were used in Tapestation™ electrophoresis to determine their size.

[0268] The ladder was prepared by mixing 1 pl D1000 ladder with 3 pl D1000 sample buffer. Samples were prepared by mixing 1 pl of DNA with 3 pl D1000 sample buffer. The samples and ladder were mixed at 2000 rpm for 1 minute before being loaded into a D1000 screentape on a TapeStation™ (Agilent). The results are shown in Figure 24. Panel A shows the gel image with the ladder in lanes ELI and EL2 and TP53 Bael 1.1, 1.2 and 1.3 digest products in lanes Bl, Cl and DI, respectively. Panel B shows the quantification of the band intensity of lane Bl (TP53 Bael 1.1 digest product). Panel C shows the quantification of the band intensity of lane Cl (TP53 Bael 1.2 digest product). Panel D shows the quantification of the band intensity of lane DI (TP53 Bael 1.3 digest product). Target band size was 43 bp. TP53 Bael 1.1 gave a band of 43 bp, TP53 Bael 1.2 gave a band of 39 bp and 1.3 gave a band of 40 bp.

[0269] Example 12 - Collection of extended DNA using HPLC

[0270] 12,1 - Generation of duplex DNA 5 |1M of oligonucleotides (IF, 2R, 3F, 4R, 5F, 6R - sequences shown below) were combined with 5 |1M of their complement oligonucleotides (IF comp, 2R comp, 3F comp, 4R comp, 5F comp, 6R comp - sequences shown below) and heated to 95 °C for 10 minutes. The duplexed oligonucleotides were left to cool to room temperature. 12,2 Extension of DNA

[0271] PCR reaction mixtures were prepared as follows:

[0272] The mixtures were vortexed and placed into a thermocycler. The following PCR program was run for 20 cycles: 30 seconds at 95 °C, 30 seconds at 55 °C and 120 seconds at 72 °C.

[0273] 12.3 - Purification of extended DNA

[0274] The PCR products containing extended DNA were purified using the Monarch® PCR and DNA Clean-up kit and following the manufacturers guidance.

[0275] 12.4 - Digestion of extended DNA with BsaXI

[0276] Digestion reaction mixes were prepared as follows:

[0277] 12.5 - Separation by HPLC

[0278] High performance liquid chromatography (HPLC) can be used to isolate target sequence from other nucleic acids in a mixture. Before HPLC collection, samples were concentrated using SpeedVac vacuum concentrators. Samples were run through syringe filters and placed in the SpeedVac at 45 °C until only the glycerol component remained. Samples were made up to 100 pL with water and added to HPLC vials. Buffer A: Water with TEA A (80 mM) pH 7, Buffer B: Acetonitrile with TEA A (80 mM), buffers were run on a gradient of increasing buffer B percentage. 0 min 1% B. 30 min 20% B, 40 min 90% B. HPLC was performed using a flow rate of 0.4 mL / min, column heater at 50 °C, DAD set to 260 nm. 25 iL of sample was injected and below fraction collectors used:

[0279] Samples were freeze dried and re-dissolved in 10 pl water.

[0280] The collected fragment sizes were confirmed via gel electrophoresis.

[0281] Example 13 - System automation

[0282] 13.1 - Automation of system

[0283] The system of the invention has been automated and shown to produce up to 225 pg in 1ml output of DNA in 90 minutes using 6% of its capacity. Use of full capacity is estimated to generate up to 3.7 mg of DNA in a day per caddy in a 90-minute run.

[0284] The automated system utilises digital heating blocks, temperature probes, chillers and a controller encased within a single housing. The caddy is made from carbon fibre and holds up to 47 Eppendorf tubes plus a temperature probe. Caddies are held in place using electrically opposed permanent magnets.

[0285] The automated system comprises 6 heating blocks, 2 of which are held at 120 °C and four held at 72 °C. The system also comprises a chiller block capable of holding 2 caddies and a loading block which can also hold 2 caddies. A robotic arm is provided in the system for automated relocation of the samples between different blocks.

[0286] Each caddy has a microcontroller which is used to read the temperature and can communicate directly with the controller.

[0287] 13.2 - Extension of repeating sequencing using automated system

[0288] The sequence was duplexed with its complement by mixing 40 pL of each and heating to 95 °C for 10 minutes then colling to room temperature.

[0289] PCR reagents were prepared as follows:

[0290] The PCR reaction mix was vortexed and 1 ml was added to 4 Eppendorf tubes. Each Eppendorf was placed in one of the four reaction caddies. The System was programmed to perform 20 cycles of the following 30 seconds at 95 °C, 30 seconds at 55 °C and 120 seconds at 72 °C.

[0291] The total amount of DNA after extension was measured using a Qubit™ according to the manufacturers protocol. The total DNA produced ranged from 317 to 389 ng / pL across the four Eppendorf tubes giving a total yield of 341 pg DNA across the four reactions.

[0292] Example 14 - Continuous flow system

[0293] The system of the invention has been implemented in a continuous flow system. The continuous flow system comprises PTFE and tygon tubing through which fluid is driven via a peristaltic pump. Different areas of the tubing are maintained at different temperatures by heaters and chillers. The system also includes fill and empty valves to allow fluid to enter and leave the system.

[0294] Various further features and aspects of the invention are defined in the claims.

[0295] 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.

[0296] The term 'enzymatic extension' refers to the extension of a nucleic acid which is catalysed by an enzyme, such as a polymerase enzyme.

[0297] 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.

[0298] 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.

[0299] 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.

[0300] 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.

[0301] 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.

[0302] The term "array" refers to a solid support upon which a plurality of nucleic acids have been fixed. The solid support may be a glass surface, alternatively the solid support may be a plurality of glass or silicon beads or any other suitable surface. Where the solid support is a plurality of beads, each bead may hold multiple copies of the same sequence or multiple copies of different sequences.

[0303] The terms 'hybridisation' 'binding' and "annealing' (or 'hybridise', 'bind' and 'anneal') in the context of nucleotide sequences, are used interchangeably herein. The ability of two nucleotide sequences to hybridize with each other is based on the degree of complementarity of the two nucleotide sequences, which in turn is based on the fraction of matched complementary nucleotide pairs. The more nucleotides in a given sequence that are complementary to another sequence, the more stringent the conditions can be for hybridization and the more specific the binding of the two sequences will be. Increased stringency is typically achieved by elevating the temperature, increasing the ratio of cosolvents, lowering the salt concentration, and other such methods well known in the field.

[0304] The term "hybridisation conditions" refers to the reagents and reaction conditions (e.g. temperature, time etc) that are used for hybridisation. Typically, hybridisation conditions may be stringent or moderate. The hybridisation conditions used in the context of the methods described herein permit mismatched duplex formation and therefore may be either moderate or stringent. Preferably, the hybridisation between the unit sequence of the first single-stranded oligonucleotide and a second single-stranded oligonucleotide will form a stable duplex at 65° C. and below. It is preferred that a mismatched duplex may be formed at temperatures up to 65° C., for example between 55° C. and 65° C., optionally for a time period of between 1 to 30 seconds.

[0305] Moderate and stringent conditions are known to those skilled in the art and can be found in available references (e.g., Current Protocols in Molecular Biology, John Wiley & Sons, N.Y., 1989, 6.3.1-6.3.6). Aqueous and non-aqueous methods are described in that reference and either can be used. A preferred example of stringent hybridization conditions are hybridization in 6x sodium chloride / sodium citrate (SSC) at about 45° C., followed by one or more washes in 0.2xSSC, 0.1% (w / v) SDS at 50° C. Another example of stringent hybridization conditions are hybridization in 6xSSC at about 45° C, followed by one or more washes in 0.2xSSC, 0.1% (w / v) SDS at 55° C. A further example of stringent hybridization conditions are hybridization in 6xSSC at about 45° C., followed by one or more washes in 0.2xSSC, 0.1% (w / v) SDS at 60° C. Preferably, stringent hybridization conditions are hybridization in 6xSSC at about 45° C., followed by one or more washes in 0.2xSSC, 0.1% (w / v) SDS at 65° C. Particularly preferred stringency conditions (and the conditions that should be used if the practitioner is uncertain about what conditions should be applied to determine if a molecule is within a hybridization limitation of the invention) are 0.5 molar sodium phosphate, 7% (w / v) SDS at 65° C., followed by one or more washes at 0.2xSSC, 1% (w / v) SDS at 65° C.

[0306] 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.

[0307] 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 can be template dependent, such as Deep Vent® polymerase. 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. In the present invention, contact between the mismatched duplex, polymerase and nucleotides is under extension conditions that permit polynucleotide extension in a 5' to 3' direction. 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.

[0308] The term "Gibson assembly®" is a method of isothermal in vitro recombination known to one skilled in the art and described e.g., in Gibson et al., (2009), Enzymatic assembly of DNA molecules up to several hundred kilobases, Nature Methods, 6(5): 343-348 and U.S. Pat. No. 8,968,999. The method generally comprises joining (assembling) nucleic acids via the steps of exonuclease digestion to create at least partially cohesive ends, annealing of ends, polymerase fill and ligation of ends. Exonuclease may be used in a reaction together with ligase to assemble multiple components. The reaction may occur at a fixed temperature or cycle between multiple temperatures, each ideal for the ligase or the exonuclease, respectively. Polymerase may be included in an assembly reaction with ligase and a 5'-to-3‘ exonuclease. The components in such a reaction may be designed such that components intended to assemble adjacent to each other share homologous sequences on their edges. For example, a component X to be assembled with component Y may have a 3' edge sequence of the form 5'-z-3', and the component Y may have a 5' edge sequence of the form 5'-z-3', where z is any nucleic acid sequence. Such homologous edge sequences may be referred to as 'gibson overlaps'. As the 5' exonuclease chews back the 5' end of dsDNA components with gibson overlaps it creates compatible 3' overhangs that hybridize to each other. The hybridized 3' ends may then be extended by the action of polymerase to the end of the template component, or to the point where the extended 3' overhang of one component meets the 5' cavity of the adjacent component, thereby forming a nick that may be sealed by a ligase. Such an assembly reaction where polymerase, ligase, and exonuclease are used together is often referred to as "Gibson assembly®". Gibson assembly® may be performed by using T5 exonuclease, Phusion polymerase, and Taq ligase, and incubating the reaction at 50 degrees Celsius. In said instance, the use of the thermophilic ligase, Taq, enables the reaction to proceed at 50 degrees Celsius, a temperature suitable for all three types of enzymes in the reaction. The term "Gibson assembly®" may generally refer to any assembly reaction involving polymerase, ligase, and exonuclease. Gibson assembly® may be used to assemble at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more components. Gibson assembly® may occur as a one- step, isothermal reaction or as a multi-step reaction with one or more temperature incubations. For example, Gibson assembly® may occur at temperatures of at least 30, 40, 50, 60, or 70 degrees, or less. The incubation time for a Gibson assembly® may be at least 1, 5, 10, 20, 40, or 80 minutes.

[0309] Gibson assembly® reactions may occur optimally when gibson overlaps between intended adjacent components are a certain length and have sequence features, such as sequences that avoid undesirable hybridization events such as hairpins, homodimers, or unwanted heterodimers. Generally, gibson overlaps of at least 20 bases are recommended. But Gibson overlaps may be at least 1, 2, 3, 5, 10, 20, 30, 40, 50, 60, 100, or more bases in length. The GC content of a gibson overlap may be anywhere from 0% to 100%. Though Gibson assembly® is commonly described with a 5' exonuclease, the reaction may also occur with a 3' exonuclease. As the 3' exonuclease chews back the 3' end of dsDNA components, the polymerase counteracts the action by extending the 3' end. This dynamic process may continue until the 5' overhang (created by the exonuclease) of two components (that share a gibson overlap) hybridize and the polymerase extends the 3' end of one component far enough to meet the 5' end of its adjacent component, thus leaving a nick that may be sealed by a ligase. The Gibson assembly® may be performed in one reaction using a single master mix comprising the required enzymes. Alternatively, the Gibson assembly® can be performed in two steps: the first comprising the exonuclease and annealing reaction and; the second comprising the DNA polymerase and assembly reaction. The term "PCR assembly" (also referred to as polymerase cycling assembly, polymerase cycling assembly (PCA) or assembly PCR) refers to a method of assembling long DNA sequences from shorter sequences. PCR assembly is a method which hybridises multiple overlapping oligonucleotides to generate a double stranded template. The double stranded template has gap regions where the oligonucleotides do not overlap. A polymerase enzyme is used to add bases to the double stranded template in the gap regions resulting in an extended double stranded sequence. Following PCR assembly, standard PCR techniques are often used to amplify the extended double stranded sequence.

[0310] 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.

[0311] 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.

[0312] 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).

[0313] 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 system for nucleic acid synthesis comprising: a nucleic acid synthesis device; at least one array of surface-bound oligonucleotides, said array comprising a plurality of surface-bound oligonucleotides; at least one partially complementary oligonucleotide configured to be at least partially complementary to one of the surface-bound oligonucleotides and such that under appropriate hybridisation conditions it will at least partially hybridise to one of the array of surface-bound oligonucleotides to generate an overlapping oligonucleotide having at least one overhanging region; means for extending the overlapping oligonucleotide to provide an extended oligonucleotide; means for cleaving the extended oligonucleotide to provide at least one cleaved oligonucleotide; means for assembling the at least one cleaved oligonucleotide to provide a synthesised nucleic acid product.

2. A system according to claim 1, wherein the system further comprises: means for extending the synthesised nucleic acid product to generate an extended synthesised nucleic acid product; means for cleaving the extended synthesised nucleic acid product to generate a plurality of synthesised nucleic acid products.

3. A system according to any previous claim, wherein the at least one partially complementary oligonucleotide is hybridised to at least one of the plurality of surfacebound oligonucleotides and is configured to be denatured from the at least one of the plurality of surface-bound oligonucleotides and re-hybridised under conditions which promote the formation of an overlapping oligonucleotide.

4. A system according to any previous claim, wherein each of the plurality of surfacebound oligonucleotides comprises at least one repeating sequence and each partially complementary oligonucleotide comprises at least one sequence that is complementary to the repeating sequence and at least one of the surface-bound oligonucleotide and the partially complementary oligonucleotide comprises at least two repeats of the repeating sequence or its complementary sequence.

5. A system according to claim 4, wherein the repeating sequence is 8 base pairs in length.

6. A system according to any previous claim, wherein the array of surface-bound oligonucleotides is located on a solid substrate.

7. A system according to any previous claim, wherein the array of surface-bound oligonucleotides comprises a plurality of surface-bound oligonucleotides, wherein together the plurality of surface-bound oligonucleotides corresponds to all possible nucleic acid sequences.

8. A system according to any of claims 1 to 6, wherein the array of surface-bound oligonucleotides comprises a plurality of surface-bound oligonucleotides, wherein together the plurality of surface-bound oligonucleotides correspond to a set of nucleic acid sequences that can be used to produce one or more predetermined nucleic acid sequences.

9. A system according to any previous claim, wherein the at least one overhanging region is a 5' overhanging region.

10. A system according to any previous claim, wherein the synthesised nucleic acid product is a gene or gene fragment.

11. A system according to any previous claim, wherein the nucleic acid synthesis device further comprises at least one feature selected from the following: a control module; a temperature control means; at least one assembly chamber; means for inputting a chosen sequence; a plurality of microfluidic channels; at least one waste outlet; a quality control means; means for purifying the synthesised nucleic acid product.

12. A system according to any previous claim, wherein the means for assembling the at least one cleaved oligonucleotide is a surface-bound assembly means.

13. A system according to any previous claim, wherein the at least one array of surfacebound oligonucleotides is located within a microreactor.

14. A system according to any previous claim, wherein the at least one partially complementary oligonucleotide is located in a partially complementary oligonucleotide cartridge configured to be insertable into the nucleic acid synthesis device.

15. A system according to any previous claims, wherein the means for extending the overlapping oligonucleotide comprises at least a polymerase enzyme and a plurality of nucleotides.

16. A system according to any previous claim, wherein the means for extending the overlapping oligonucleotide is located in a store on the nucleic acid synthesis device.

17. A system according to any previous claim, wherein the means for cleaving the extended oligonucleotide to provide at least one cleaved oligonucleotide comprises at least one restriction enzyme.

18. A system according to any previous claim, wherein the means for cleaving the extended oligonucleotide to provide at least one cleaved oligonucleotide comprises aplurality of types of restriction enzymes; and wherein the plurality of restriction enzymes is stored such that each type of restriction enzyme is separated from other types of restriction enzymes.

19. A system according to claim 17 or 18, wherein the restriction enzyme is located in a store on the nucleic acid synthesis device.

20. A system according to claim 17 or 18, wherein the at least one restriction enzyme is located in a restriction enzyme cartridge configured to be insertable into the nucleic acid synthesis device.

21. A system according to any previous claim, wherein the means for extending the overlapping oligonucleotide is located in an extension cartridge which is insertable into the nucleic acid synthesis device.

22. A system according to any of claims 11 to 21, wherein the quality control means quantifies the concentration of the synthesised nucleic acid product and / or wherein the quality control means confirms the sequence of the synthesised nucleic acid product.

23. A method for synthesising nucleic acids comprising the steps:(i) selecting a chosen nucleic acid sequence;(ii) obtaining a plurality of partially complementary oligonucleotides;(iii) bringing said plurality of partially complementary oligonucleotides into contact with an array of surface-bound oligonucleotides under hybridising conditions; wherein each of the plurality of partially complementary oligonucleotides is at least partially complementary to at least one surface-bound oligonucleotide on the array of surfacebound oligonucleotides; such that each of the plurality of partially complementary oligonucleotides hybridises to one surface-bound oligonucleotide on the array ofsurface bound oligonucleotides creating a plurality of overlapping oligonucleotides, each overlapping oligonucleotide having least one overhanging region;(iv) extending the plurality of overlapping oligonucleotides to provide a plurality of extended oligonucleotides;(v) cleaving the plurality of extended oligonucleotides to provide a plurality of cleaved oligonucleotides;(vi) ligating the plurality of cleaved oligonucleotides to provide a synthesised nucleic acid product corresponding to the chosen nucleic acid sequence.

24. A method according to claim 23, wherein step (ii) further comprises the steps:(ii.a) generating a complementary sequence which is complementary to the chosen nucleic acid sequence;(ii.b) converting the complementary sequence into a plurality of complementary overlapping sequences; wherein the plurality of partially complementary oligonucleotides correspond to the plurality of complementary overlapping sequences.

25. A method according to any of claims 23 to 24, wherein the method further comprises the following steps:(vii) extending the synthesised nucleic acid product to generate an extended synthesised nucleic acid product;(viii) cleaving the extended synthesised nucleic acid product to generate a plurality of synthesised nucleic acid products.

26. A method according to any of claims 23 to 25, wherein the each of the plurality of extended oligonucleotides comprise a plurality of repeating sequences.

27. A method according to any of claims 23 to 26, wherein the cleaved oligonucleotides correspond to an individual repeating sequence.

28. A method according to any of claims 23 to 27, wherein the method is conducted on a device, preferably the device is a benchtop device.

29. A method according to any of claims 23 to 28, wherein the bringing said plurality of partially complementary oligonucleotides into contact with an array of surface-bound oligonucleotides is carried out in a temporally separated manner.

30. A nucleic acid synthesis device comprising: an input for inputting a chosen nucleic acid sequence; at least one array of surface-bound oligonucleotides comprising a plurality of surfacebound oligonucleotides; a means for extending at least two of the plurality of surface-bound oligonucleotides on the array of surface-bound oligonucleotides to provide a plurality of extended oligonucleotides; a means for cleaving the plurality of extended oligonucleotides to provide a plurality of cleaved oligonucleotides; an assembly chamber; a means for assembling the plurality of cleaved oligonucleotides within the assembly chamber to provide the synthesised nucleic acid product, wherein the synthesised nucleic acid product corresponds to the chosen nucleic acid sequence; an output for receiving the synthesised nucleic acid product.

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