Method for PCR

Abstract

The present invention relates to a method for producing large quantities of double stranded DNA material in an amount suitable for downstream applications, such as for in vitro transcription of RNA for therapeutic use. In particular, the present invention relates to a method of performing polymerase chain reaction (PCR) using a microwave synthesis reactor.

Description

[0001]METHOD FOR PCR FIELD OF THE INVENTIONThe present invention relates to a method for producing large quantities of double strandedDNA material in an amount suitable for downstream applications, such as for in vitrotranscription of RNA for therapeutic use. In particular, the present invention relates to a method of performing polymerase chain reaction (PCR) using a microwave synthesis reactor. BACKGROUND TO THE INVENTION Polymerase chain reaction (PCR) represents a standard technique to amplify DNA fragments of various lengths. These DNA templates can then be further used in downstream applicationssuch as RNA synthesis. Generally, PCR is conducted in standard PCR thermocyclersharbouring thermoblocks with e.g. 32, 96 or 384 wells, allowing different total PCR reactionvolumes. Depending on the device and block format, several mL of PCR reaction mixture can be prepared for one PCR run. The whole reaction mixture is then distributed in individual wells.In case large amounts of DNA templates are needed to be produced via PCR, many individualwells must be prepared. After the PCR is completed, pooling of several, and often more than100, individual reactions is required to obtain adequate amounts of PCR product (i.e. DNA)for further processing. Even though available PCR thermocyclers rely on many years of experience and perform to a high standard with good DNA yields, the current PCR cyclers are based on thermoblocks (e.g.32, 96 or 384 well plates) with limited capacities. For example, thermocyclers generallyproduce amounts of DNA corresponding to the average amount needed for smaller researchprojects but do not meet the DNA amounts required for larger projects, like production ofstarting material for therapeutic applications (e.g. therapeutic RNA) or for clinical trials suchas animal or clinical trial material (ATM / CTM). For these studies, often milligrams of DNA are required for subsequent RNA production. To produce these large quantities of DNA, many 96- well plates must be filled with the PCR master mix before PCR is conducted. After the PCR is completed, the DNA template must be pooled from all 96-well plates, which is particularly time consuming and requires a lot of personnel. In addition, handling of many 96-well plates is susceptible to errors during pipetting or pooling of the PCR product, along with a risk of contamination of the PCR product.Moreover, the manufacture of certain therapeutic RNA requires long RNA sequences to beproduced, e.g. RNA of around 100 base pairs or more in length, or around 1000 base pairs ormore in length. Existing DNA production methods which have only been tested in the contextof smaller lengths of DNA / RNA would not necessarily be suitable for the production of theselonger length therapeutic RNAs. This is because – prior to testing – it would not be at all certainthat such DNA production methods could successfully be used to reproduce the longertherapeutic RNAs, in sufficient quantities, while maintaining an acceptable level of RNA integrity.Orrling et al., Chem. Commun., 2004, 790-791 proposed a method of microwavethermocycling to produce short lengths of DNA for general applications in molecular biology.The proposed method was only realistically directed at very short lengths of DNA, with noevidence or expectation that the method could be used for the production of therapeutic RNA,particularly large-scale, longer length therapeutic RNA. Orrling (2004) also does not consideror determine whether the method would result in an acceptable level of RNA integrity.Taken together, the potential disadvantages indicate that an alternative method to theconventional PCR using a PCR thermocycler is needed in order to improve the productionprocess of large amounts of DNA, e.g. for the production of therapeutic RNA.SUMMARY OF THE INVENTION The present invention illustrates for the first time that a microwave synthesis reactor can be used to perform PCR cycles in a single reaction volume, leading to the successful production of large-scale, longer length therapeutic RNA molecules with excellent levels of RNA integrity. The present invention provides a method for producing double stranded DNA material, wherein the method comprises one or more polymerase chain reaction (PCR) cycle(s), wherein the, or each, PCR cycle comprises the steps of: (i) denaturing double stranded DNA molecule(s) into separate DNA strands; (ii) annealing primers to each DNA strand; and (iii) producing new double stranded DNA molecules by elongating DNA from the primers to produce a new DNA strand that is complementary to each of the DNA strands of step (ii); wherein the new double stranded DNA molecules form the double stranded DNA material; wherein the, or each, PCR cycle is performed using a microwave synthesis reactor in a single reaction volume. The present invention provides a method for producing double stranded DNA material for downstream synthesis of therapeutic RNA, wherein the method comprises one or more polymerase chain reaction (PCR) cycle(s), wherein the, or each, PCR cycle comprises the steps of: (i) denaturing double stranded DNA molecule(s) into separate DNA strands; (ii) annealing primers to each DNA strand; and (iii) producing new double stranded DNA molecules by elongating DNA from the primers to produce a new DNA strand that is complementary to each of the DNA strands of step (ii); wherein the new double stranded DNA molecules form the double stranded DNA material; wherein the, or each, PCR cycle is performed using a microwave synthesis reactor in a single reaction volume. The present invention provides a method for producing double stranded DNA material in a sufficient amount for downstream synthesis of therapeutic RNA, wherein the method comprises one or more polymerase chain reaction (PCR) cycle(s), wherein the, or each, PCR cycle comprises the steps of: (i) denaturing double stranded DNA molecule(s) into separate DNA strands; (ii) annealing primers to each DNA strand; and (iii) producing new double stranded DNA molecules by elongating DNA from the primers to produce a new DNA strand that is complementary to each of the DNA strands of step (ii); wherein the new double stranded DNA molecules form the double stranded DNA material; wherein the, or each, PCR cycle is performed using a microwave synthesis reactor in a single reaction volume.In some embodiments, the double stranded DNA material may be suitable for use as atemplate for in vitro transcription (IVT) to produce the therapeutic RNA.In some embodiments, the method according to the invention may comprise a further step ofin vitro transcription (IVT) to produce therapeutic RNA from the double stranded DNA material.In some embodiments, the RNA may be mRNA.In some embodiments, the double stranded DNA material may comprise 0.01-130.00 mg ofDNA. In some embodiments, the double stranded DNA material may comprise 0.01-0.10 mg of DNA.In some embodiments, the double stranded DNA material may comprise 0.01-0.09 mg, 0.01-0.08 mg, 0.01-0.07 mg, 0.01-0.06 mg, 0.01-0.05 mg, 0.01-0.04 mg, 0.01-0.03 mg, 0.01-0.02 mg, 0.02-0.10 mg, 0.03-0.10 mg, 0.04-0.10 mg, 0.05-0.10 mg, 0.06-0.10 mg, 0.07-0.10 mg, 0.08-0.10 mg, 0.09-0.10 mg of DNA.In some embodiments, the double stranded DNA material may comprise 0.1-1.0 mg of DNA.In some embodiments, the double stranded DNA material may comprise 0.1-0.9 mg, 0.1-0.8mg, 0.1-0.7 mg, 0.1-0.6 mg, 0.1-0.5 mg, 0.1-0.4 mg, 0.1-0.3 mg, 0.1-0.2 mg, 0.2-1.0 mg, 0.3- 1.0 mg, 0.4-1.0 mg, 0.5-1.0 mg, 0.6-1.0 mg, 0.7-1.0 mg, 0.8-1.0 mg, 0.9-1.0 mg of DNA. In some embodiments, the double stranded DNA material may comprise 1-130 mg of DNA.In some embodiments, the double stranded DNA material may comprise 1-120 mg, 1-110 mg,1-100 mg, 1-90 mg, 1-80 mg, 1-70 mg, 1-60 mg, 1-50 mg, 1-40 mg, 1-30 mg, 1-20 mg, 1-10 mg, 10-130 mg, 20-130 mg, 30-130 mg, 40-130 mg, 50-130 mg, 60-130 mg, 70-130 mg, 80- 130 mg, 90-130 mg, 100-130 mg, 110-130 mg, 120-130 mg of DNA. In some embodiments, the method may comprise repeating steps (i) to (iii), wherein the new double stranded DNA molecules formed in step (iii) are used as the double stranded DNA molecules in step (i) of the subsequent PCR cycle.In some embodiments, the PCR cycle may be performed for a total of at least 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 cycles.In some embodiments, the single reaction volume may be a volume of 0.5 to 1,000 mL.In some embodiments, the single reaction volume may be a volume of 0.5-10.0 mL.In some embodiments, the single reaction volume may be a volume of 0.5-9.5 mL, 0.5-9.0 mL,0.5-8.5 mL, 0.5-8.0 mL, 0.5-7.5 mL, 0.5-7.0 mL, 0.5-6.5 mL, 0.5-6.0 mL, 0.5-5.5 mL, 0.5-5.0 mL, 0.5-4.5 mL, 0.5-4.0 mL, 0.5-3.5 mL, 0.5-3.0 mL, 0.5-2.5 mL, 0.5-2.0 mL, 0.5-1.5 mL, 0.5- 1.0 mL, 1.0-10.0 mL, 1.5-10.0 mL, 2.0-10.0 mL, 2.5-10.0 mL, 3.0-10.0 mL, 3.5-10.0 mL, 4.0- 10.0 mL, 4.5-10.0 mL, 5.0-10.0 mL, 5.5-10.0 mL, 6.0-10.0 mL, 6.5-10.0 mL, 7.0-10.0 mL, 7.5- 10.0 mL, 8.0-10.0 mL, 8.5-10.0 mL, 9.0-10.0 mL, or 9.5-10.0 mL.In some embodiments, the single reaction volume may be a volume of 10-100 mL.In some embodiments, the single reaction volume may be a volume of 10-95 mL, 10-90 mL,10-85 mL, 10-80 mL, 10-75 mL, 10-70 mL, 10-65 mL, 10-60 mL, 10-55 mL, 10-50 mL, 10-45 mL, 10-40 mL, 10-35 mL, 10-30 mL, 10-25 mL, 10-20 mL, 10-15 mL, 15-100 mL, 20-100 mL, 25-100 mL, 30-100 mL, 35-100 mL, 40-100 mL, 45-100 mL, 50-100 mL, 55-100 mL, 60-100 mL, 65-100 mL, 70-100 mL, 75-100 mL, 80-100 mL, 85-100 mL, 90-100 mL, or 95-100 mL.In some embodiments, the single reaction volume may be a volume of 100-1000 mL.In some embodiments, the single reaction volume may be a volume of 100-950 mL, 100-900mL, 100-850 mL, 100-800 mL, 100-750 mL, 100-700 mL, 100-650 mL, 100-600 mL, 100-550 mL, 100-500 mL, 100-450 mL, 100-400 mL, 100-350 mL, 100-300 mL, 100-250 mL, 100-200 mL, 100-150 mL, 150-1,000 mL, 200-1,000 mL, 250-1,000 mL, 300-1,000 mL, 350-1,000 mL, 400-1,000 mL, 450-1,000 mL, 500-1,000 mL, 550-1,000 mL, 600-1,000 mL, 650-1,000 mL, 700-1,000 mL, 750-1,000 mL, 800-1,000 mL, 850-1,000 mL, 900-1,000 mL, or 950-1,000 mL.In some embodiments, the single reaction volume may be a volume of 3.0 mL.In some embodiments, the single reaction volume may be a volume of 20.0 mL.In some embodiments, each of steps (i), (ii) and (iii) of the method according to the inventionmay be performed separately. In some embodiments, step (ii) and step (iii) of the method according to the invention may be performed simultaneously.In some embodiments, the method according to the invention may comprise:(i) an initial denaturation step preceding the one or more PCR cycle(s); (ii) the one or more PCR cycle(s); and(iii) a final elongation step proceeding the one or more PCR cycle(s); wherein: (a) the temperature for the initial denaturation step is 88-105°C; (b) the temperature for step (i) of the PCR cycle is 88-105°C; (c) the temperature for step (ii) of the PCR cycle is 50-76°C; (d) the temperature for step (iii) of the PCR cycle is 68-76°C; and / or (e) the temperature for the final elongation step is 68-76°C. In some embodiments, the method according to the invention may comprise: (i) an initial denaturation step preceding the one or more PCR cycle(s); (ii) the one or more PCR cycle(s); and (iii) a final elongation step proceeding the one or more PCR cycle(s); wherein: (a) the temperature for the initial denaturation step is set as 94°C; (b) the temperature for step (i) of the PCR cycle is set as 94°C; (c) the temperature for step (ii) of the PCR cycle is set as 72°C;(d) the temperature for step (iii) of the PCR cycle is set as 72°C; and / or(e) the temperature for the final elongation step is set as 72°C. In some embodiments, the actual temperature inside the microwave synthesis reactor for step(i) of the PCR cycle may be 91-109°C.In some embodiments, the actual temperature inside the microwave synthesis reactor for step(i) of the PCR cycle may be 97-98°C. In some embodiments, the method according to the invention may comprise: (i) an initial denaturation step preceding the one or more PCR cycle(s); (ii) the one or more PCR cycle; and (iii) a final elongation step proceeding the one or more PCR cycle(s); wherein: (a) the temperature for the initial denaturation step is set as 98°C; (b) the temperature for step (i) of the PCR cycle is set as 98°C; (c) the temperature for step (ii) of the PCR cycle is set as 72°C;(d) the temperature for step (iii) of the PCR cycle is set as 72°C; and / or(e) the temperature for the final elongation step is set as 72°C.In some embodiments, the double stranded DNA molecules forming the double stranded DNAmaterial may be at least 54 base pairs in length.In some embodiments, the double stranded DNA molecules forming the double stranded DNAmaterial may be 54-10,000 base pairs in length.In some embodiments, the double stranded DNA molecules forming the double stranded DNAmaterial may be 100-1,000 base pairs in length.In some embodiments, the double stranded DNA molecules may be 100-950 base pairs inlength, 100-900 base pairs in length, 100-850 base pairs in length, 100-800 base pairs inlength, 100-750 base pairs in length, 100-700 base pairs in length, 100-650 base pairs in length, 100-600 base pairs in length, 100-550 base pairs in length, 100-500 base pairs in length, 100-450 base pairs in length, 100-400 base pairs in length, 100-350 base pairs in length, 100-300 base pairs in length, 100-250 base pairs in length, 100-200 base pairs in length, 100-150 base pairs in length, 150-1,000 base pairs in length, 200-1,000 base pairs in length, 250-1,000 base pairs in length, 300-1,000 base pairs in length, 350-1,000 base pairs in length, 400-1,000 base pairs in length, 450-1,000 base pairs in length, 500-1,000 base pairs in length, 550-1,000 base pairs in length, 600-1,000 base pairs in length, 650-1,000 base pairs in length, 700-1,000 base pairs in length, 750-1,000 base pairs in length, 800-1,000 base pairs in length, 850-1,000 base pairs in length, 900-1,000 base pairs in length, or 950-1,000 base pairs in length. In some embodiments, the double stranded DNA molecules forming the double stranded DNA material may be at least 100 base pairs in length. In some embodiments, the double stranded DNA molecules forming the double stranded DNA material may be at least 150 base pairs in length. In some embodiments, the double stranded DNA molecules forming the double stranded DNA material may be at least 500 base pairs in length. In some embodiments, the double stranded DNA molecules forming the double stranded DNA material may be at least 1,000 base pairs in length. In some embodiments, the double stranded DNA molecules forming the double stranded DNAmaterial may be 1,000-10,000 base pairs in length.In some embodiments, the double stranded DNA molecules may be 1,000-9,500 base pairsin length, 1,000-9,000 base pairs in length, 1,000-8,500 base pairs in length, 1,000-8,000 base pairs in length, 1,000-7,500 base pairs in length, 1,000-7,000 base pairs in length, 1,000-6,500 base pairs in length, 1,000-6,000 base pairs in length, 1,000-5,500 base pairs in length, 1,000- 5,000 base pairs in length, 1,000-4,500 base pairs in length, 1,000-4,000 base pairs in length,1,000-3,500 base pairs in length, 1,000-3,000 base pairs in length, 1,000-2,500 base pairs inlength, 1,000-2,000 base pairs in length, 1,000-1,500 base pairs in length, 1,500-10,000 base pairs in length, 2,000-10,000 base pairs in length, 2,500-10,000 base pairs in length, 3,000- 10,000 base pairs in length, 3,500-10,000 base pairs in length, 4,000-10,000 base pairs in length, 4,500-10,000 base pairs in length, 5,000-10,000 base pairs in length, 5,500-10,000 base pairs in length, 6,000-10,000 base pairs in length, 6,500-10,000 base pairs in length, 7,000-10,000 base pairs in length, 7,500-10,000 base pairs in length, 8,000-10,000 base pairs in length, 8,500-10,000 base pairs in length, 9,000-10,000 base pairs in length, or 9,500- 10,000 base pairs in length. In some embodiments, the double stranded DNA molecules forming the double stranded DNA material may each comprise a contiguous nucleotide sequence of about 1,000 to about 4,500 base pairs in length.In some embodiments, the reaction volume may not comprise bovine serum albumin (BSA).In some embodiments, the method according to the invention may comprise a further step ofpurifying the double stranded DNA material.In some embodiments, the double stranded DNA material may be purified with magneticbeads.The present invention also provides use of a microwave synthesis reactor for producing doublestranded DNA material in a single reaction volume in a sufficient amount for downstream synthesis of therapeutic RNA. BRIEF DESCRIPTION OF THE FIGURESFigure 1: Agarose gel electrophoresis of the purified DNA templates, which weresynthesized using the microwave synthesis reactor Monowave 400 and the standardPCR cycler. Four DNA templates (DNA template 1-4) were synthesized in a total PCRreaction volume of 3 mL and 20 mL using the microwave synthesis reactor Monowave 400.As a control, the DNA templates were produced in the standard PCR cycler. After synthesis,the DNA templates were purified. Following purification, 100 ng of the synthesized DNAtemplates were loaded and separated on a 1% agarose gel. The expected sizes for the DNA templates are: 4.4 kbp for DNA template 1, 1.1 kbp for DNA template 2, 1.8 kbp for DNA template 3 and 2 kbp for DNA template 4.Figure 2: Measurement of the RNA integrity after in vitro transcription. (A)-(D) RNA wassynthesized from the four DNA templates (DNA template 1-4) which were previously produced in the microwave synthesis reactors Monowave 400 and Discover 2.0 with a single reaction volume of 3 mL and 20 mL and as a control in the standard PCR cycler. For all manufactured RNAs, the integrity of the RNA was determined with the Fragment Analyzer. The measurement was performed in triplicates for each sample. Figure 3: Agarose gel electrophoresis of the purified DNA templates, which weresynthesized using the microwave synthesis reactor Discover 2.0. Four DNA templates(DNA template 1-4) were synthesized in a total PCR reaction volume of 3 mL and 20 mL usingthe microwave synthesis reactor Discover 2.0. After DNA purification, 100 ng of each samplewere loaded and separated on a 1% agarose gel. The expected sizes for the DNA templates are: 4.4 kbp for DNA template 1, 1.1 kbp for DNA template 2, 1.8 kbp for DNA template 3 and 2 kbp for DNA template 4. Figure 4: Agarose gel electrophoresis of further purified DNA templates, which weresynthesized using the microwave synthesis reactor Discover 2.0. DNA template 2 wassynthesized in a total PCR reaction volume of 50 mL, and a fifth DNA template (DNA template 5) was synthesized in a total PCR reaction volume of 50 mL and 70 mL using the microwave synthesis reactor Discover 2.0. After DNA purification, 100 ng of each sample were loaded and separated on a 1% agarose gel. The expected sizes for the DNA templates are: 1.1 kbpfor DNA template 2, and 1.4 kbp for DNA template 5.DETAILED DESCRIPTION OF THE INVENTION DOUBLE STRANDED DNA MATERIAL Whilst it is known that the production of DNA can be performed using the conventionalpolymerase chain reaction (PCR) device and protocol in multi well-plates in small individualvolumes, this is not suitable for the production of large quantities of DNA, in particular the largeamounts of DNA required as the starting material for the production of RNA that is to be used in therapeutic applications (e.g. RNA-based therapies). It will be understood that production of RNA for RNA-based therapies may be referred to as“large-scale” synthesis of RNA or “industrial-scale” synthesis of RNA. As used herein,“production” and “synthesis” in relation to RNA can be considered as interchangeable terms.In order to produce larger quantities of DNA without the need for filling and pooling multiplewells of multiple PCR plates, an alternative approach for producing DNA in a single, large reaction volume was sought, which would minimise the risk for contamination and also speed up the overall production process. As shown herein, the present inventors have surprisingly found that it is possible to use microwave synthesis reactors to perform PCR to produce large amounts of double strandedDNA of high quality, which are suitable for use as templates for downstream RNA productionby in vitro transcription (IVT). Indeed, prior to the present invention, it was not known thatmicrowave-based devices would be suitable for effecting the temperature changes needed toperform a full PCR cycle such that large amounts of double stranded DNA of high quality couldbe produced, particularly for therapeutic RNA applications requiring longer lengths of DNA, e.g.100 base pairs or more, 150 base pairs or more, 500 base pairs or more, 1,000 base pairs or more in length. The present invention provides a method for producing double stranded DNA material, wherein the method comprises one or more polymerase chain reaction (PCR) cycle(s), wherein the, or each, PCR cycle comprises the steps of: (i) denaturing double stranded DNA molecule(s) into separate DNA strands; (ii) annealing primers to each DNA strand; and (iii) producing new double stranded DNA molecules by elongating DNA from the primers to produce a new DNA strand that is complementary to each of the DNA strands of step (ii); wherein the new double stranded DNA molecules form the double stranded DNA material; wherein the, or each, PCR cycle is performed using a microwave synthesis reactor in a single reaction volume. In some embodiments of such a method, the double stranded DNA molecules forming the double stranded DNA material are at least about 100 base pairs in length.In some embodiments, the double stranded DNA molecules forming the double stranded DNAmaterial are at least 100 base pairs in length. In some embodiments, the double stranded DNA molecules forming the double stranded DNA material are at least about 150 base pairs in length. In some embodiments, the double stranded DNA molecules forming the double stranded DNA material are at least 150 base pairs in length. In some embodiments, the double stranded DNA molecules forming the double stranded DNAmaterial are at least about 500 base pairs in length.In some embodiments, the double stranded DNA molecules forming the double stranded DNA material are at least 500 base pairs in length. In some embodiments, the double stranded DNA molecules forming the double stranded DNA material are at least about 1,000 base pairs in length. In some embodiments, the double stranded DNA molecules forming the double stranded DNA material are at least 1,000 base pairs in length.In some embodiments, the method produces double stranded DNA material suitable for afurther step (iv) of in vitro transcription (IVT) to produce therapeutic RNA from the doublestranded DNA material produced in step (iii). For example, the double stranded DNA materialis produced in sufficient quantities and with an acceptable level of RNA integrity for a furtherstep (iv) of in vitro transcription (IVT) to produce therapeutic RNA.In some embodiments, the method comprises a further step (iv) of in vitro transcription (IVT)to produce therapeutic RNA from the double stranded DNA material produced in step (iii).The present invention also provides a method for producing double stranded DNA material for downstream synthesis of therapeutic RNA, wherein the method comprises one or more polymerase chain reaction (PCR) cycle(s), wherein the, or each, PCR cycle comprises the steps of: (i) denaturing double stranded DNA molecule(s) into separate DNA strands; (ii) annealing primers to each DNA strand; and (iii) producing new double stranded DNA molecules by elongating DNA from the primers to produce a new DNA strand that is complementary to each of the DNA strands of step (ii); wherein the new double stranded DNA molecules form the double stranded DNA material; wherein the, or each, PCR cycle is performed using a microwave synthesis reactor in a single reaction volume. In some embodiments of such a method, the double stranded DNA molecules forming the double stranded DNA material are at least about 100 base pairs in length. In some embodiments, the double stranded DNA molecules forming the double stranded DNA material are at least 100 base pairs in length. In some embodiments, the double stranded DNA molecules forming the double stranded DNA material are at least about 150 base pairs in length. In some embodiments, the double stranded DNA molecules forming the double stranded DNA material are at least 150 base pairs in length. In some embodiments, the double stranded DNA molecules forming the double stranded DNA material are at least about 500 base pairs in length. In some embodiments, the double stranded DNA molecules forming the double stranded DNA material are at least 500 base pairs in length. In some embodiments, the double stranded DNA molecules forming the double stranded DNA material are at least about 1,000 base pairs in length. In some embodiments, the double stranded DNA molecules forming the double stranded DNA material are at least 1,000 base pairs in length. In some embodiments of such a method, the method comprises a further step (iv) of in vitro transcription (IVT) to produce therapeutic RNA from the double stranded DNA material produced in step (iii).The present invention also provides a method for producing double stranded DNA material ina sufficient amount for downstream synthesis of therapeutic RNA, wherein the method comprises one or more polymerase chain reaction (PCR) cycle(s), wherein the, or each, PCR cycle comprises the steps of: (i) denaturing double stranded DNA molecule(s) into separate DNA strands; (ii) annealing primers to each DNA strand; and (iii) producing new double stranded DNA molecules by elongating DNA from the primers to produce a new DNA strand that is complementary to each of the DNA strands of step (ii); wherein the new double stranded DNA molecules form the double stranded DNA material;wherein the, or each, PCR cycle is performed using a microwave synthesis reactor in a singlereaction volume. In some embodiments of such a method, the double stranded DNA molecules forming the double stranded DNA material are at least about 100 base pairs in length. In some embodiments, the double stranded DNA molecules forming the double stranded DNA material are at least 100 base pairs in length. In some embodiments, the double stranded DNA molecules forming the double stranded DNA material are at least about 150 base pairs in length. In some embodiments, the double stranded DNA molecules forming the double stranded DNA material are at least 150 base pairs in length. In some embodiments, the double stranded DNA molecules forming the double stranded DNA material are at least about 500 base pairs in length. In some embodiments, the double stranded DNA molecules forming the double stranded DNA material are at least 500 base pairs in length. In some embodiments, the double stranded DNA molecules forming the double stranded DNA material are at least about 1,000 base pairs in length. In some embodiments, the double stranded DNA molecules forming the double stranded DNA material are at least 1,000 base pairs in length. In some embodiments of such a method, the method comprises a further step (iv) of in vitro transcription (IVT) to produce therapeutic RNA from the double stranded DNA material produced in step (iii). Further embodiments and aspects set forth herein are understood as applying to each / any of the three methods listed in this section above.The present invention also provides a method, e.g. for producing double stranded DNAmaterial for downstream synthesis of therapeutic RNA, wherein the method comprises one or more polymerase chain reaction (PCR) cycle(s), wherein the, or each, PCR cycle comprises the steps of: (i) denaturing double stranded DNA molecule(s) into separate DNA strands; (ii) annealing primers to each DNA strand; and (iii) producing new double stranded DNA molecules by elongating DNA from the primers to produce a new DNA strand that is complementary to each of the DNA strands of step (ii); wherein the new double stranded DNA molecules form the double stranded DNA material; wherein the, or each, PCR cycle is performed using a microwave synthesis reactor in a single reaction volume.The present invention also provides a method, e.g. for producing double stranded DNAmaterial in a sufficient amount for downstream synthesis of therapeutic RNA, wherein the method comprises one or more polymerase chain reaction (PCR) cycle(s), wherein the, or each, PCR cycle comprises the steps of: (i) denaturing double stranded DNA molecule(s) into separate DNA strands; (ii) annealing primers to each DNA strand; and (iii) producing new double stranded DNA molecules by elongating DNA from the primers to produce a new DNA strand that is complementary to each of the DNA strands of step (ii); wherein the new double stranded DNA molecules form the double stranded DNA material; wherein the, or each, PCR cycle is performed using a microwave synthesis reactor.The present invention also provides a method, e.g. for producing double stranded DNAmaterial for downstream synthesis of therapeutic RNA, wherein the method comprises one or more polymerase chain reaction (PCR) cycle(s), wherein the, or each, PCR cycle comprises the steps of: (i) denaturing double stranded DNA molecule(s) into separate DNA strands; (ii) annealing primers to each DNA strand; and (iii) producing new double stranded DNA molecules by elongating DNA from the primers to produce a new DNA strand that is complementary to each of the DNA strands of step (ii); wherein the new double stranded DNA molecules form the double stranded DNA material; wherein the, or each, PCR cycle is performed using a microwave synthesis reactor. It will be understood that the “double stranded DNA molecule(s)” may refer to the DNA that isused as the initial template for the PCR in step (i) of the method according to the invention.In some embodiments, the double stranded DNA molecule(s) used as the initial template for the PCR in step (i) may be genomic DNA. In some embodiments, the double stranded DNA molecule(s) used as the initial template for the PCR in step (i) may be recombinant DNA. In some embodiments, the double stranded DNA molecule(s) used as the initial template for the PCR in step (i) may be recombinant plasmid DNA.In some embodiments, the double stranded DNA molecule(s) used as the initial template forthe PCR in step (i) may be cDNA.In some embodiments, the double stranded DNA molecule(s) used as the initial template for the PCR in step (i) may be linear DNA.In some embodiments, the double stranded DNA molecule(s) used as the initial template forthe PCR in step (i) may comprise synthetic nucleotides.In some embodiments, the double stranded DNA molecule(s) used as the initial template forthe PCR in step (i) may comprise modified nucleotides.It will be understood that the double stranded DNA material may be considered as the DNA product that is produced at the end point of the PCR cycle. In some embodiments, the double stranded DNA material produced by the method may besuitable for use as a template for in vitro transcription (IVT) to produce the therapeutic RNA.In some embodiments, the double stranded DNA material may be, or may be derived from, genomic DNA. In some embodiments, the double stranded DNA material may be, or may be derived from, recombinant DNA. In some embodiments, the double stranded DNA material may be, or may be derived from, cDNA. In some embodiments, the double stranded DNA material may be, or may be derived from, linear DNA. In some embodiments, the double stranded DNA material may comprise synthetic nucleotides. In some embodiments, the double stranded DNA material may comprise modified nucleotides. It will be understood that the production of RNA for therapeutic applications by IVT requireslarge quantities of DNA as the starting template material. As such, it will be understood thatthe “sufficient amount” of double stranded DNA material will be a quantity of DNA that isappropriately large enough to be suitable for use as a template for IVT to produce therapeutic RNA. In some embodiments, the sufficient amount may comprise at least 0.01 mg of DNA. In some embodiments, the sufficient amount may comprise at least 0.1 mg of DNA. In some embodiments, the sufficient amount may comprise at least 1 mg of DNA. In some embodiments, the sufficient amount may comprise at least 10 mg of DNA. In some embodiments, the sufficient amount may comprise at least 100 mg of DNA. In some embodiments, the sufficient amount may comprise 0.01-130.00 mg of DNA. In some embodiments, the sufficient amount may comprise 0.01-0.10 mg of DNA. In some embodiments, the sufficient amount may comprise 0.01-0.09 mg, 0.01-0.08 mg, 0.01- 0.07 mg, 0.01-0.06 mg, 0.01-0.05 mg, 0.01-0.04 mg, 0.01-0.03 mg, or 0.01-0.02 mg of DNA. In some embodiments, the sufficient amount may comprise 0.02-0.10 mg, 0.03-0.10 mg, 0.04- 0.10 mg, 0.05-0.10 mg, 0.06-0.10 mg, 0.07-0.10 mg, 0.08-0.10 mg, or 0.09-0.10 mg of DNA. In some embodiments, the sufficient amount may comprise at least 0.01 mg, at least 0.02 mg, at least 0.03 mg, at least 0.04 mg, at least 0.05 mg, at least 0.06 mg, at least 0.07 mg, at least 0.08 mg, at least 0.09 mg, or at least 0.10 mg of DNA. In some embodiments, the sufficient amount may comprise 0.1-1.0 mg of DNA. In some embodiments, the sufficient amount may comprise 0.1-0.9 mg, 0.1-0.8 mg, 0.1-0.7 mg, 0.1-0.6 mg, 0.1-0.5 mg, 0.1-0.4 mg, 0.1-0.3 mg, or 0.1-0.2 mg of DNA. In some embodiments, the sufficient amount may comprise 0.2-1.0 mg, 0.3-1.0 mg, 0.4-1.0 mg, 0.5-1.0 mg, 0.6-1.0 mg, 0.7-1.0 mg, 0.8-1.0 mg, or 0.9-1.0 mg of DNA. In some embodiments, the sufficient amount may comprise at least 0.1 mg, at least 0.2 mg, at least 0.3 mg, at least 0.4 mg, at least 0.5 mg, at least 0.6 mg, at least 0.7 mg, at least 0.8 mg, at least 0.9 mg, or at least 1.0 mg of DNA. In some embodiments, the sufficient amount may comprise 1-130 mg of DNA. In some embodiments, the sufficient amount may comprise 1-125 mg, 1-120 mg, 1-115 mg, 1-110 mg, 1-105 mg, 1-100 mg, 1-95 mg, 1-90 mg, 1-85 mg, 1-80 mg, 1-75 mg, 1-70 mg, 1- 65 mg, 1-60 mg, 1-55 mg, 1-50 mg, 1-45 mg, 1-40 mg, 1-35 mg, 1-30 mg, 1-25 mg, 1-20 mg, 1-15 mg, 1-10 mg, or 1-5 mg of DNA. In some embodiments, the sufficient amount may comprise 5-130 mg, 10-130 mg, 15-130 mg, 20-130 mg, 25-130 mg, 30-130 mg, 35-130 mg, 40-130 mg, 45-130 mg, 50-130 mg, 55-130 mg, 60-130 mg, 65-130 mg, 70-130 mg, 75-130 mg, 80-130 mg, 85-130 mg, 90-130 mg, 95- 130 mg, 100-130 mg, 105-130 mg, 110-130 mg, 115-130 mg, 120-130 mg, or 125-130 mg of DNA. In some embodiments, the sufficient amount may comprise at least 1 mg, at least 5 mg, at least 10 mg, at least 15 mg, at least 20 mg, at least 25 mg, at least 30 mg, at least 35 mg, at least 40 mg, at least 45 mg, at least 50 mg, at least 55 mg, at least 60 mg, at least 65 mg, at least 70 mg, at least 75 mg, at least 80 mg, at least 85 mg, at least 90 mg, at least 95 mg, at least 100 mg, at least 105 mg, at least 110 mg, at least 115 mg, at least 120 mg, at least 125 mg, or at least 130 mg of DNA. In some embodiments, the sufficient amount may comprise more than 130 mg of DNA. In some embodiments, the double stranded DNA material may comprise at least 0.01 mg of DNA. In some embodiments, the double stranded DNA material may comprise at least 0.1 mg of DNA.In some embodiments, the double stranded DNA material may comprise at least 1 mg of DNA.In some embodiments, the double stranded DNA material may comprise at least 10 mg of DNA. In some embodiments, the double stranded DNA material may comprise at least 100 mg of DNA. In some embodiments, the double stranded DNA material may comprise 0.01-130.00 mg of DNA. In some embodiments, the double stranded DNA material may comprise 0.01-0.10 mg of DNA. In some embodiments, the double stranded DNA material may comprise 0.01-0.09 mg, 0.01- 0.08 mg, 0.01-0.07 mg, 0.01-0.06 mg, 0.01-0.05 mg, 0.01-0.04 mg, 0.01-0.03 mg, or 0.01-0.02 mg of DNA.In some embodiments, the double stranded DNA material may comprise 0.02-0.10 mg, 0.03- 0.10 mg, 0.04-0.10 mg, 0.05-0.10 mg, 0.06-0.10 mg, 0.07-0.10 mg, 0.08-0.10 mg, or 0.09- 0.10 mg of DNA. In some embodiments, the double stranded DNA material may comprise at least 0.01 mg, atleast 0.02 mg, at least 0.03 mg, at least 0.04 mg, at least 0.05 mg, at least 0.06 mg, at least0.07 mg, at least 0.08 mg, at least 0.09 mg, or at least 0.10 mg of DNA.In some embodiments, the double stranded DNA material may comprise 0.1-1.0 mg of DNA. In some embodiments, the double stranded DNA material may comprise 0.1-0.9 mg, 0.1-0.8 mg, 0.1-0.7 mg, 0.1-0.6 mg, 0.1-0.5 mg, 0.1-0.4 mg, 0.1-0.3 mg, or 0.1-0.2 mg of DNA.In some embodiments, the double stranded DNA material may comprise 0.2-1.0 mg, 0.3-1.0mg, 0.4-1.0 mg, 0.5-1.0 mg, 0.6-1.0 mg, 0.7-1.0 mg, 0.8-1.0 mg, or 0.9-1.0 mg of DNA. In some embodiments, the double stranded DNA material may comprise at least 0.1 mg, at least 0.2 mg, at least 0.3 mg, at least 0.4 mg, at least 0.5 mg, at least 0.6 mg, at least 0.7 mg, at least 0.8 mg, at least 0.9 mg, or at least 1.0 mg of DNA.In some embodiments, the double stranded DNA material may comprise 1-130 mg of DNA.In some embodiments, the double stranded DNA material may comprise 1-125 mg, 1-120 mg, 1-115 mg, 1-110 mg, 1-105 mg, 1-100 mg, 1-95 mg, 1-90 mg, 1-85 mg, 1-80 mg, 1-75 mg, 1- 70 mg, 1-65 mg, 1-60 mg, 1-55 mg, 1-50 mg, 1-45 mg, 1-40 mg, 1-35 mg, 1-30 mg, 1-25 mg,1-20 mg, 1-15 mg, 1-10 mg, or 1-5 mg of DNA.In some embodiments, the double stranded DNA material may comprise 5-130 mg, 10-130mg, 15-130 mg, 20-130 mg, 25-130 mg, 30-130 mg, 35-130 mg, 40-130 mg, 45-130 mg, 50-130 mg, 55-130 mg, 60-130 mg, 65-130 mg, 70-130 mg, 75-130 mg, 80-130 mg, 85-130 mg, 90-130 mg, 95-130 mg, 100-130 mg, 105-130 mg, 110-130 mg, 115-130 mg, 120-130 mg, or 125-130 mg of DNA. In some embodiments, the double stranded DNA material may comprise at least 1 mg, at least 5 mg, at least 10 mg, at least 15 mg, at least 20 mg, at least 25 mg, at least 30 mg, at least35 mg, at least 40 mg, at least 45 mg, at least 50 mg, at least 55 mg, at least 60 mg, at least65 mg, at least 70 mg, at least 75 mg, at least 80 mg, at least 85 mg, at least 90 mg, at least 95 mg, at least 100 mg, at least 105 mg, at least 110 mg, at least 115 mg, at least 120 mg, at least 125 mg, or at least 130 mg of DNA. In some embodiments, the double stranded DNA material may comprise more than 130 mg of DNA.As described herein, performing the method of the present invention in a single reactionvolume obviates the need to pool together multiple wells from multiple PCR plates in order toobtain a sufficient quantity of double stranded DNA material for downstream IVT. It will therefore be understood that the term “single reaction volume” means that the, or each,PCR cycle is performed in full in the same reaction volume (e.g. one vial, one tube, one flasketc.). It will be understood that the amount of double stranded DNA material that can be producedmay be associated with the reaction volume in which the, or each, PCR cycle occurs. It willtherefore be understood that a larger reaction volume may correlate with a larger quantity ofdouble stranded DNA material.In some embodiments, the single reaction volume may be referred to as a sample (e.g. asingle sample). In some embodiments, the single reaction volume may be referred to as aliquid sample (e.g. a single liquid sample).In some embodiments, the single reaction volume may be a volume of more than 0.5 mL.In some embodiments, the single reaction volume may be a volume of 0.5-1,000 mL.In some embodiments, the single reaction volume may be a volume of 0.5-10.0 mL. In some embodiments, the single reaction volume may be a volume of 0.5-9.5 mL, 0.5-9.0 mL, 0.5-8.5 mL, 0.5-8.0 mL, 0.5-7.5 mL, 0.5-7.0 mL, 0.5-6.5 mL, 0.5-6.0 mL, 0.5-5.5 mL, 0.5-5.0 mL, 0.5-4.5 mL, 0.5-4.0 mL, 0.5-3.5 mL, 0.5-3.0 mL, 0.5-2.5 mL, 0.5-2.0 mL, or 0.5-1.5 mL, 0.5-1.0 mL. In some embodiments, the single reaction volume may be a volume of 1.0-10.0 mL, 1.5-10.0 mL, 2.0-10.0 mL, 2.5-10.0 mL, 3.0-10.0 mL, 3.5-10.0 mL, 4.0-10.0 mL, 4.5-10.0 mL, 5.0-10.0 mL, 5.5-10.0 mL, 6.0-10.0 mL, 6.5-10.0 mL, 7.0-10.0 mL, 7.5-10.0 mL, 8.0-10.0 mL, 8.5-10.0 mL, 9.0-10.0 mL, or 9.5-10.0 mL.In some embodiments, the single reaction volume may be a volume of at least 0.5 mL, at least1 mL, at least 1.5 mL, at least 2 mL, at least 2.5 mL, at least 3 mL, at least 3.5 mL, at least 4 mL, at least 4.5 mL, at least 5 mL, at least 5.5 mL, at least 6 mL, at least 6.5 mL, at least 7 mL, at least 7.5 mL, at least 8 mL, at least 8.5 mL, at least 9 mL, at least 9.5 mL, or at least 10 mL. In some embodiments, the single reaction volume may be a volume of at least 3 mL.In some embodiments, the single reaction volume may be a volume of about 3 mL.In some embodiments, the single reaction volume may be a volume of 10-100 mL. In some embodiments, the single reaction volume may be a volume of 10-95 mL, 10-90 mL, 10-85 mL, 10-80 mL, 10-75 mL, 10-70 mL, 10-65 mL, 10-60 mL, 10-55 mL, 10-50 mL, 10-45 mL, 10-40 mL, 10-35 mL, 10-30 mL, 10-25 mL, 10-20 mL, or 10-15 mL. In some embodiments, the single reaction volume may be a volume of 15-100 mL, 20-100 mL, 25-100 mL, 30-100 mL, 35-100 mL, 40-100 mL, 45-100 mL, 50-100 mL, 55-100 mL, 60-100 mL, 65-100 mL, 70-100 mL, 75-100 mL, 80-100 mL, 85-100 mL, 90-100 mL, or 95-100 mL. In some embodiments, the single reaction volume may be a volume of at least 10 mL, at least 15 mL, at least 20 mL, at least 25 mL, at least 30 mL, at least 35 mL, at least 40 mL, at least 45 mL, at least 50 mL, at least 55 mL, at least 60 mL, at least 65 mL, at least 70 mL, at least 75 mL, at least 80 mL, at least 85 mL, at least 90 mL, at least 95 mL, or at least 100 mL. In some embodiments, the single reaction volume may be a volume of at least 20 mL. In some embodiments, the single reaction volume may be a volume of about 20 mL. In some embodiments, the single reaction volume may be a volume of 100-1000 mL. In some embodiments, the single reaction volume may be a volume of 100-950 mL, 100-900mL, 100-850 mL, 100-800 mL, 100-750 mL, 100-700 mL, 100-650 mL, 100-600 mL, 100-550mL, 100-500 mL, 100-450 mL, 100-400 mL, 100-350 mL, 100-300 mL, 100-250 mL, 100-200mL, or 100-150 mL.In some embodiments, the single reaction volume may be a volume of 150-1,000 mL, 200-1,000 mL, 250-1,000 mL, 300-1,000 mL, 350-1,000 mL, 400-1,000 mL, 450-1,000 mL, 500-1,000 mL, 550-1,000 mL, 600-1,000 mL, 650-1,000 mL, 700-1,000 mL, 750-1,000 mL, 800- 1,000 mL, 850-1,000 mL, 900-1,000 mL, or 950-1,000 mL. In some embodiments, the single reaction volume may be a volume of at least 100 mL, at least 150 mL, at least 200 mL, at least 250 mL, at least 300 mL, at least 350 mL, at least 400 mL, at least 450 mL, at least 500 mL, at least 550 mL, at least 600 mL, at least 650 mL, at least 700 mL, at least 750 mL, at least 800 mL, at least 850 mL, at least 900 mL, at least 950 mL, or at least 1000 mL. In some embodiments, the single reaction volume may be a volume of more than 1,000 mL. It will be understood that the double stranded DNA material may be defined according to the number of base pairs (i.e. the length in base pairs) forming the double stranded DNA molecules produced by the PCR method. It will be understood that the length in base pairs described herein refers to the typical, or average, length in number of base pairs of the double stranded DNA molecules forming the double stranded DNA material. The person skilled in theart will be aware that in some cases, due to incomplete reactions, the PCR may result inincomplete double stranded DNA molecules or double stranded DNA molecules with breakages. In some embodiments, the double stranded DNA molecules forming the double stranded DNA material may be at least about 54 base pairs in length. In some embodiments, the double stranded DNA molecules forming the double stranded DNAmaterial may be at least 54 base pairs in length.In some embodiments, the double stranded DNA molecules forming the double stranded DNA material may be at least about 58 base pairs in length. In some embodiments, the double stranded DNA molecules forming the double stranded DNA material may be at least 58 base pairs in length. In some embodiments, the double stranded DNA molecules forming the double stranded DNA material may be at least about 100 base pairs in length. In some embodiments, the double stranded DNA molecules forming the double stranded DNA material may be at least 100 base pairs in length. In some embodiments, the double stranded DNA molecules forming the double stranded DNA material may be at least about 150 base pairs in length. In some embodiments, the double stranded DNA molecules forming the double stranded DNA material may be at least 150 base pairs in length.In some embodiments, the double stranded DNA molecules forming the double stranded DNAmaterial may be at least about 154 base pairs in length. In some embodiments, the double stranded DNA molecules forming the double stranded DNA material may be at least 154 base pairs in length. In some embodiments, the double stranded DNA molecules forming the double stranded DNA material may be at least about 158 base pairs in length. In some embodiments, the double stranded DNA molecules forming the double stranded DNA material may be at least 158 base pairs in length. In some embodiments, the double stranded DNA molecules forming the double stranded DNA material may be 100-1,000 base pairs in length. In some embodiments, the double stranded DNA molecules forming the double stranded DNA material may be 100-950 base pairs in length, 100-900 base pairs in length, 100-850 base pairs in length, 100-800 base pairs in length, 100-750 base pairs in length, 100-700 base pairs in length, 100-650 base pairs in length, 100-600 base pairs in length, 100-550 base pairs in length, 100-500 base pairs in length, 100-450 base pairs in length, 100-400 base pairs in length, 100-350 base pairs in length, 100-300 base pairs in length, 100-250 base pairs in length, 100-200 base pairs in length, or 100-150 base pairs in length. In some embodiments, the double stranded DNA molecules forming the double stranded DNA material may be 150-1,000 base pairs in length, 200-1,000 base pairs in length, 250-1,000 base pairs in length, 300-1,000 base pairs in length, 350-1,000 base pairs in length, 400-1,000 base pairs in length, 450-1,000 base pairs in length, 500-1,000 base pairs in length, 550-1,000 base pairs in length, 600-1,000 base pairs in length, 650-1,000 base pairs in length, 700-1,000 base pairs in length, 750-1,000 base pairs in length, 800-1,000 base pairs in length, 850-1,000 base pairs in length, 900-1,000 base pairs in length, or 950-1,000 base pairs in length. In some embodiments, the double stranded DNA molecules forming the double stranded DNA material may be at least 100 base pairs in length, at least 150 base pairs in length, at least200 base pairs in length, at least 250 base pairs in length, at least 300 base pairs in length, atleast 350 base pairs in length, at least 400 base pairs in length, at least 450 base pairs in length, at least 500 base pairs in length, at least 550 base pairs in length, at least 600 base pairs in length, at least 650 base pairs in length, at least 700 base pairs in length, at least 750 base pairs in length, at least 800 base pairs in length, at least 850 base pairs in length, at least 900 base pairs in length, at least 950 base pairs in length, or at least 1,000 base pairs in length. In some embodiments, the double stranded DNA molecules forming the double stranded DNA material may be at least about 100 base pairs in length. In some embodiments, the double stranded DNA molecules forming the double stranded DNA material may be at least 100 base pairs in length. In some embodiments, the double stranded DNA molecules forming the double stranded DNA material may be at least about 150 base pairs in length. In some embodiments, the double stranded DNA molecules forming the double stranded DNA material may be at least 150 base pairs in length. In some embodiments, the double stranded DNA molecules forming the double stranded DNA material may be at least about 1,000 base pairs in length.In some embodiments, the double stranded DNA molecules forming the double stranded DNAmaterial may be at least 1,000 base pairs in length. In some embodiments, the double stranded DNA molecules forming the double stranded DNA material may be at least about 1,500 base pairs in length. In some embodiments, the double stranded DNA molecules forming the double stranded DNA material may be at least 1,500 base pairs in length. In some embodiments, the double stranded DNA molecules forming the double stranded DNA material may be at least about 3,00 base pairs in length. In some embodiments, the double stranded DNA molecules forming the double stranded DNA material may be at least 3,000 base pairs in length. In some embodiments, the double stranded DNA molecules forming the double stranded DNA material may be 1,000-10,000 base pairs in length. In some embodiments, the double stranded DNA molecules forming the double stranded DNA material may be 1,000-9,500 base pairs in length, 1,000-9,000 base pairs in length, 1,000- 8,500 base pairs in length, 1,000-8,000 base pairs in length, 1,000-7,500 base pairs in length, 1,000-7,000 base pairs in length, 1,000-6,500 base pairs in length, 1,000-6,000 base pairs in length, 1,000-5,500 base pairs in length, 1,000-5,000 base pairs in length, 1,000-4,500 base pairs in length, 1,000-4,000 base pairs in length, 1,000-3,500 base pairs in length, 1,000-3,000 base pairs in length, 1,000-2,500 base pairs in length, 1,000-2,000 base pairs in length, or 1,000-1,500 base pairs in length. In some embodiments, the double stranded DNA molecules forming the double stranded DNA material may be 1,500-10,000 base pairs in length, 2,000-10,000 base pairs in length, 2,500- 10,000 base pairs in length, 3,000-10,000 base pairs in length, 3,500-10,000 base pairs inlength, 4,000-10,000 base pairs in length, 4,500-10,000 base pairs in length, 5,000-10,000base pairs in length, 5,500-10,000 base pairs in length, 6,000-10,000 base pairs in length, 6,500-10,000 base pairs in length, 7,000-10,000 base pairs in length, 7,500-10,000 base pairs in length, 8,000-10,000 base pairs in length, 8,500-10,000 base pairs in length, 9,000-10,000 base pairs in length, or 9,500-10,000 base pairs in length. In some embodiments, the double stranded DNA molecules forming the double stranded DNAmaterial may be at least 1,000 base pairs in length, at least 1,500 base pairs in length, at least2,000 base pairs in length, at least 2,500 base pairs in length, at least 3,000 base pairs in length, at least 3,500 base pairs in length, at least 4,000 base pairs in length, at least 4,500 base pairs in length, at least 5,000 base pairs in length, at least 5,500 base pairs in length, at least 6,000 base pairs in length, at least 6,500 base pairs in length, at least 7,000 base pairs in length, at least 7,500 base pairs in length, at least 8,000 base pairs in length, at least 8,500 base pairs in length, at least 9,000 base pairs in length, at least 9,500 base pairs in length, or at least 10,000 base pairs in length. In some embodiments, the double stranded DNA molecules forming the double stranded DNAmaterial may be about 1,000 to about 4,500 base pairs in length.In some embodiments, the double stranded DNA molecules forming the double stranded DNA material may be at least about 500 base pairs in length. In some embodiments, the double stranded DNA molecules forming the double stranded DNA material may be at least about 1,000 base pairs in length. In some embodiments, the double stranded DNA molecules forming the double stranded DNAmaterial may be at least about 1,500 base pairs in length.In some embodiments, the double stranded DNA molecules forming the double stranded DNA material may be at least about 2,000 base pairs in length. In some embodiments, the double stranded DNA molecules forming the double stranded DNA material may be at least about 3,000 base pairs in length. In some embodiments, the double stranded DNA molecules forming the double stranded DNA material may be at least about 4,000 base pairs in length. In some embodiments, the double stranded DNA molecules forming the double stranded DNA material may be at least about 4,500 base pairs in length. POLYMERASE CHAIN REACTION (PCR) The polymerase chain reaction (PCR) is a technique well-known in the art that allows for the amplification of DNA from a starting DNA template using thermal cycling (i.e. different steps are performed at different temperatures). The general protocol for PCR is well-known to the person skilled in the art. In brief, the general PCR protocol comprises a cycle of three main steps. A first step of denaturing double stranded DNA into separate DNA strands; a second step to allow primers to anneal to each of the denatured DNA strands; and a third elongation step, where a DNA polymerase uses DNA nucleotides to form a new DNA strand that is complementary to each of the initial denatured DNA strands, ultimately forming new double stranded DNA molecules. This cycle can be repeated multiple times, with each cycle doubling the amount of DNA that was present at the start of that cycle. As such, a PCR cycle of the method according to the invention comprises the steps of: (i) denaturing double stranded DNA molecule(s) into separate DNA strands; (ii) annealing primers to each DNA strand; and (iii) producing new double stranded DNA molecules by elongating DNA from the primers to produce a new DNA strand that is complementary to each of the DNA strands of step (ii). In addition to the above, a one-time, denaturation step may be performed initially, preceding the one or more the PCR cycle(s) described above. It will be understood that this initial denaturation step is performed only once, independent of the number of PCR cycles. In addition to the above, a one-time elongation step may be performed, proceeding the one or more the PCR cycle(s) described above. It will be understood that this final elongation step is performed only once, independent of the number of PCR cycles. As described herein, the method of the present invention may be based on producing a sufficient amount of double stranded DNA material for downstream IVT to produce therapeutic RNA, wherein the amount of double stranded DNA material that is produced may be selected for by varying the number of PCR cycles. It will be understood that the new double stranded DNA molecules produced at the end of the PCR cycle form the sufficient amount of double stranded DNA material that may then be used as the DNA template for IVT to produce the therapeutic RNA, as described herein. It will also be understood that the double stranded DNA material may undergo a purification step prior to producing RNA using IVT. It will be understood that the PCR cycle may be repeated multiple times in order to increase the amount of double stranded DNA material produced. It will also be understood that once a sufficient amount of double stranded DNA material has been obtained, no further PCR cycles may be required and the double stranded DNA material can then be used as the DNA template material for producing RNA using IVT. In some embodiments, the double stranded DNA material formed of new double stranded DNA molecules produced at the end of one PCR cycle may be used as the starting material for IVT, or the double stranded DNA material may undergo further PCR cycles in order to amplify the amount of double stranded DNA material, before being used as the starting material for IVT. In some embodiments, the method according to the invention may comprise repeating steps (i) to (iii), wherein the new double stranded DNA molecules formed in step (iii) are used as the double stranded DNA molecules in step (i) of the subsequent PCR cycle. It will be understood that the skilled person will know how many PCR cycles to perform in total, in order to obtain a desired sufficient amount of double stranded DNA material. In some embodiments each of steps (i), (ii) and (iii) of the method according to the invention may be performed separately. In some embodiments, step (ii) and step (iii) of the method according to the invention may be performed simultaneously. In some embodiments, the method according to the invention may comprise the following sequential steps: (a) a one-time initial denaturation step preceding the one or more PCR cycle(s); (b) the one or more PCR cycle(s) according to the invention, comprising: (i) denaturing double stranded DNA molecule(s) into separate DNA strands; (ii) annealing primers to each DNA strand; and (iii) producing new double stranded DNA molecules by elongating DNA from the primers to produce a new DNA strand that is complementary to each of the DNA strands of step (ii); wherein step (b) is repeated for as many cycles as is required and the new double stranded DNA molecules formed in step (iii) are used as the double stranded DNA molecules in step (i) of the subsequent PCR cycle; and (c) a one-time final elongation step proceeding the one or more PCR cycle(s). In some embodiments, after the one-time final elongation step has been completed, there maybe a holding step wherein the reaction may be held at a holding temperature.In some embodiments, the PCR cycle may be performed for a total of at least 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, or 40 cycles. In some embodiments, the PCR cycle may be performed for a total of 2 or fewer, 3 or fewer, 4 or fewer, 5 or fewer, 6 or fewer, 7 or fewer, 8 or fewer, 9 or fewer, 10 or fewer, 11 or fewer, 12 or fewer, 13 or fewer, 14 or fewer, 15 or fewer, 16 or fewer, 17 or fewer, 18 or fewer, 19 or fewer, 20 or fewer, 21 or fewer, 22 or fewer, 23 or fewer, 24 or fewer, 25 or fewer, 26 or fewer, 27 or fewer, 28 or fewer, 29 or fewer, 30 or fewer, 31 or fewer, 32 or fewer, 33 or fewer, 34 or fewer, 35 or fewer, 36 or fewer, 37 or fewer, 38 or fewer, 39 or fewer, or 40 or fewer cycles. In some embodiments, the PCR cycle may be performed for a total of 1-40 cycles. In some embodiments, the PCR cycle may be performed for a total of 1-35 cycles. In some embodiments, the PCR cycle may be performed for a total of 1-30 cycles. In some embodiments, the PCR cycle may be performed for a total of 1-25 cycles. In some embodiments, the PCR cycle may be performed for a total of 1-20 cycles. In some embodiments, the PCR cycle may be performed for a total of 1-15 cycles. In some embodiments, the PCR cycle may be performed for a total of 1-10 cycles. In some embodiments, the PCR cycle may be performed for a total of 1-5 cycles.In some embodiments, the PCR cycle may be performed for a total of 22 cycles.In some embodiments, the PCR cycle may be performed for a total of 23 cycles.In some embodiments, the PCR cycle may be performed for a total of 28 cycles.In some embodiments, the PCR cycle may be performed for a total of more than 40 cycles.The person skilled in the art will be able to determine how many PCR cycles will be required, in order to obtain the desired amount of double stranded DNA material. As will be known in the art, the temperature of each step of the PCR method may vary depending on various factors, such as the DNA polymerase that is used, the sequence length of the starting DNA molecules, the % guanine-cytosine (GC) content of the starting DNA molecules, and / or the primers used during the annealing step. The person skilled in the art will know how to optimise the temperature of each step as appropriate. In some embodiments, the temperature for the initial denaturation step may be 88-105°C. In some embodiments, the temperature for the initial denaturation step may be 88-104°C, 88- 103°C, 88-102°C, 88-101°C, 88-100°C, 88-99°C, 88-98°C, 88-97°C, 88-96°C, 88-95°C, 88- 94°C, 88-93°C, 88-92°C, 88-91°C, 88-90°C, or 88-89°C. In some embodiments, the temperature for the initial denaturation step may be 89-105°C, 90- 105°C, 91-105°C, 92-105°C, 93-105°C, 94-105°C, 95-105°C, 96-105°C, 97-105°C, 98-105°C, 99-105°C, 100-105°C, 101-105°C, 102-105°C, 103-105°C, or 104-105°C. In some embodiments, the temperature for the initial denaturation step may be 88-99°C. In some embodiments, the temperature for the initial denaturation step may be 88-98°C, 88- 97°C, 88-96°C, 88-95°C, 88-94°C, 88-93°C, 88-92°C, 88-91°C, 88-90°C, or 88-89°C. In some embodiments, the temperature for the initial denaturation step may be 89-99°C, 90- 99°C, 91-99°C, 92-99°C, 93-99°C, 94-99°C, 95-99°C, 96-99°C, 97-99°C, or 98-99°C. In some embodiments, the temperature for the initial denaturation step may be selected from the list consisting of: 88°C, 89°C, 90°C, 91°C, 92°C, 93°C, 94°C, 95°C, 96°C, 97°C, 98°C, 99°C, 100°C, 101°C, 102°C, 103°C, 104°C, or 105°C. In some embodiments, the temperature for step (i) of the PCR cycle may be 88-105°C. In some embodiments, the temperature for step (i) of the PCR cycle may be 88-104°C, 88- 103°C, 88-102°C, 88-101°C, 88-100°C, 88-99°C, 88-98°C, 88-97°C, 88-96°C, 88-95°C, 88- 94°C, 88-93°C, 88-92°C, 88-91°C, 88-90°C, or 88-89°C. In some embodiments, the temperature for step (i) of the PCR cycle may be 89-105°C, 90- 105°C, 91-105°C, 92-105°C, 93-105°C, 94-105°C, 95-105°C, 96-105°C, 97-105°C, 98-105°C, 99-105°C, 100-105°C, 101-105°C, 102-105°C, 103-105°C, or 104-105°C. In some embodiments, the temperature for step (i) of the PCR cycle may be 88-99°C. In some embodiments, the temperature for step (i) of the PCR cycle may be 88-98°C, 88- 97°C, 88-96°C, 88-95°C, 88-94°C, 88-93°C, 88-92°C, 88-91°C, 88-90°C, or 88-89°C.In some embodiments, the temperature for step (i) of the PCR cycle may be 89-99°C, 90-99°C, 91-99°C, 92-99°C, 93-99°C, 94-99°C, 95-99°C, 96-99°C, 97-99°C, or 98-99°C. In some embodiments, the temperature for step (i) of the PCR cycle may be selected from the list consisting of: 88°C, 89°C, 90°C, 91°C, 92°C, 93°C, 94°C, 95°C, 96°C, 97°C, 98°C, 99°C, 100°C, 101°C, 102°C, 103°C, 104°C, or 105°C. In some embodiments, the temperature for step (ii) of the PCR cycle may be 50-76°C. In some embodiments, the temperature for step (ii) of the PCR cycle may be 50-75°C, 50- 74°C, 50-73°C, 50-72°C, 50-71°C, 50-70°C, 50-69°C, 50-68°C, 50-67°C, 50-66°C, 50-65°C, 50-64°C, 50-63°C, 50-62°C, 50-61°C, 50-60°C, 50-59°C, 50-58°C, 50-57°C, 50-56°C, 50- 55°C, 50-54°C, 50-53°C, 50-52°C, or 50-51°C. In some embodiments, the temperature for step (ii) of the PCR cycle may be 51-76°C, 52- 76°C, 53-76°C, 54-76°C, 55-76°C, 56-76°C, 57-76°C, 58-76°C, 59-76°C, 60-76°C, 61-76°C, 62-76°C, 63-76°C, 64-76°C, 65-76°C, 66-76°C, 67-76°C, 68-76°C, 69-76°C, 70-76°C, 71- 76°C, 72-76°C, 73-76°C, 74-76°C, or 75-76°C. In some embodiments, the temperature for step (ii) of the PCR cycle may be selected from the list consisting of: 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, or 76°C. In some embodiments, the temperature for step (iii) of the PCR cycle may be 68-76°C. In some embodiments, the temperature for step (iii) of the PCR cycle may be 68-75°C, 68- 74°C, 68-73°C, 68-72°C, 68-71°C, 68-70°C, or 68-69°C. In some embodiments, the temperature for step (iii) of the PCR cycle may be 69-76°C, 70- 76°C, 71-76°C, 72-76°C, 73-76°C, 74-76°C, or 75-76°C.In some embodiments, the temperature for step (iii) of the PCR cycle may be selected fromthe list consisting of: 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, or 76°C. In some embodiments, the temperature for the final elongation step may be 68-76°C. In some embodiments, the temperature for the final elongation step may be 68-75°C, 68-74°C, 68-73°C, 68-72°C, 68-71°C, 68-70°C, or 68-69°C. In some embodiments, the temperature for the final elongation step may be 69-76°C, 70-76°C, 71-76°C, 72-76°C, 73-76°C, 74-76°C, or 75-76°C. In some embodiments, the temperature for the final elongation step may be selected from the list consisting of: 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, or 76°C. In some embodiments, after the one-time final elongation step has been completed, the reaction may be held at a holding temperature of 20-40°C. In some embodiments, the holding temperature may be 20-39°C, 20-38°C, 20-37°C, 20-36°C, 20-35°C, 20-34°C, 20-33°C, 20-32°C, 20-31°C, 20-30°C, 20-29°C, 20-28°C, 20-27°C, 20-26°C, 20-25°C, 20-24°C, 20-23°C, 20-22°C, or 20-21°C.In some embodiments, the holding temperature may be 21-40°C, 22-40°C, 23-40°C, 24-40°C, 25-40°C, 26-40°C, 27-40°C, 28-40°C, 29-40°C, 30-40°C, 31-40°C, 32-40°C, 33-40°C, 34- 40°C, 35-40°C, 36-40°C, 37-40°C, 38-40°C, or 39-40°C. In some embodiments, the holding temperature may be selected from the list consisting of: 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, or 40°C. In some embodiments, the holding temperature may be less than 20°C. In some embodiments, the holding temperature may be 4-20°C. In some embodiments, the holding temperature may be 10-20°C. In some embodiments, the holding temperature may be 4-10°C.In some embodiments, the method according to the invention may comprise:(a) a one-time initial denaturation step preceding the one or more PCR cycle(s), wherein the temperature may be 88-105°C; (b) the one or more PCR cycle(s) according to the invention, comprising: (i) denaturing double stranded DNA molecule(s) into separate DNA strands wherein the temperature may be 88-105°C; (ii) annealing primers to each DNA strand wherein the temperature may be 50-76°C; and (iii) producing new double stranded DNA molecules by elongating DNA from theprimers to produce a new DNA strand that is complementary to each of the DNA strands of step (ii), wherein the temperature may be 68-76°C; wherein step (b) is repeated for as many cycles as is required and the new double stranded DNA molecules formed in step (iii) are used as the double stranded DNAmolecules in step (i) of the subsequent PCR cycle; and (c) a one-time final elongation step proceeding the one or more PCR cycle(s), wherein the temperature may be 68-76°C. In some embodiments, the method according to the invention may comprise: (a) a one-time initial denaturation step preceding the one or more PCR cycle(s), wherein the temperature may be 88-99°C; (b) the one or more PCR cycle(s) according to the invention, comprising: (i) denaturing double stranded DNA molecule(s) into separate DNA strands wherein the temperature may be 88-99°C; (ii) annealing primers to each DNA strand wherein the temperature may be 50-76°C; and (iii) producing new double stranded DNA molecules by elongating DNA from the primers to produce a new DNA strand that is complementary to each of the DNA strands of step (ii), wherein the temperature may be 68-76°C; wherein step (b) is repeated for as many cycles as is required and the new double stranded DNA molecules formed in step (iii) are used as the double stranded DNA molecules in step (i) of the subsequent PCR cycle; and (c) a one-time final elongation step proceeding the one or more PCR cycle(s), wherein the temperature may be 68-76°C.In some embodiments, after step (c) has been completed, the reaction may be held at aholding temperature of 20-40°C. It will be understood that, depending on the microwave synthesis reactor used, the temperature that may be programmed for a particular step of the method described herein may not equate to the actual temperature that is reached in the reaction volume. The personskilled in the art will know how to adjust the programmed temperature of each step of themethod described herein, depending on the microwave synthesis reactor used, so that each step is performed at the desired temperature. In some embodiments, the temperature for the initial denaturation step may be set as 88- 105°C. In some embodiments, the temperature for the initial denaturation step may be set as 88- 104°C, 88-103°C, 88-102°C, 88-101°C, 88-100°C, 88-99°C, 88-98°C, 88-97°C, 88-96°C, 88- 95°C, 88-94°C, 88-93°C, 88-92°C, 88-91°C, 88-90°C, or 88-89°C. In some embodiments, the temperature for the initial denaturation step may be set as 89- 105°C, 90-105°C, 91-105°C, 92-105°C, 93-105°C, 94-105°C, 95-105°C, 96-105°C, 97-105°C, 98-105°C, 99-105°C, 100-105°C, 101-105°C, 102-105°C, 103-105°C, or 104-105°C. In some embodiments, the temperature for the initial denaturation step may be set as 88- 99°C. In some embodiments, the temperature for the initial denaturation step may be set as 88- 98°C, 88-97°C, 88-96°C, 88-95°C, 88-94°C, 88-93°C, 88-92°C, 88-91°C, 88-90°C, or 88- 89°C. In some embodiments, the temperature for the initial denaturation step may be set as 89- 99°C, 90-99°C, 91-99°C, 92-99°C, 93-99°C, 94-99°C, 95-99°C, 96-99°C, 97-99°C, or 98- 99°C. In some embodiments, the temperature for the initial denaturation step may be set as a temperature selected from the list consisting of: 88°C, 89°C, 90°C, 91°C, 92°C, 93°C, 94°C, 95°C, 96°C, 97°C, 98°C, 99°C, 100°C, 101°C, 102°C, 103°C, 104°C, or 105°C. In some embodiments, the temperature for step (i) of the PCR cycle may be set as 88-105°C.In some embodiments, the temperature for step (i) of the PCR cycle may be set as 88-104°C,88-103°C, 88-102°C, 88-101°C, 88-100°C, 88-99°C, 88-98°C, 88-97°C, 88-96°C, 88-95°C, 88-94°C, 88-93°C, 88-92°C, 88-91°C, 88-90°C, or 88-89°C. In some embodiments, the temperature for step (i) of the PCR cycle may be set as 89-105°C, 90-105°C, 91-105°C, 92-105°C, 93-105°C, 94-105°C, 95-105°C, 96-105°C, 97-105°C, 98- 105°C, 99-105°C, 100-105°C, 101-105°C, 102-105°C, 103-105°C, or 104-105°C. In some embodiments, the temperature for step (i) of the PCR cycle may be set as 88-99°C. In some embodiments, the temperature for step (i) of the PCR cycle may be set as 88-98°C, 88-97°C, 88-96°C, 88-95°C, 88-94°C, 88-93°C, 88-92°C, 88-91°C, 88-90°C, or 88-89°C. In some embodiments, the temperature for step (i) of the PCR cycle may be set as 89-99°C, 90-99°C, 91-99°C, 92-99°C, 93-99°C, 94-99°C, 95-99°C, 96-99°C, 97-99°C, or 98-99°C. In some embodiments, the temperature for step (i) of the PCR cycle may be set as atemperature selected from the list consisting of: 88°C, 89°C, 90°C, 91°C, 92°C, 93°C, 94°C,95°C, 96°C, 97°C, 98°C, 99°C, 100°C, 101°C, 102°C, 103°C, 104°C, or 105°C. In some embodiments, the temperature for step (ii) of the PCR cycle may be set as 50-76°C.In some embodiments, the temperature for step (ii) of the PCR cycle may be set as 50-75°C,50-74°C, 50-73°C, 50-72°C, 50-71°C, 50-70°C, 50-69°C, 50-68°C, 50-67°C, 50-66°C, 50- 65°C, 50-64°C, 50-63°C, 50-62°C, 50-61°C, 50-60°C, 50-59°C, 50-58°C, 50-57°C, 50-56°C, 50-55°C, 50-54°C, 50-53°C, 50-52°C, or 50-51°C. In some embodiments, the temperature for step (ii) of the PCR cycle may be set as 51-76°C, 52-76°C, 53-76°C, 54-76°C, 55-76°C, 56-76°C, 57-76°C, 58-76°C, 59-76°C, 60-76°C, 61- 76°C, 62-76°C, 63-76°C, 64-76°C, 65-76°C, 66-76°C, 67-76°C, 68-76°C, 69-76°C, 70-76°C, 71-76°C, 72-76°C, 73-76°C, 74-76°C, or 75-76°C. In some embodiments, the temperature for step (ii) of the PCR cycle may be set as a temperature selected from the list consisting of: 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, or 76°C. In some embodiments, the temperature for step (iii) of the PCR cycle may be set as 68-76°C. In some embodiments, the temperature for step (iii) of the PCR cycle may be set as 68-75°C, 68-74°C, 68-73°C, 68-72°C, 68-71°C, 68-70°C, or 68-69°C. In some embodiments, the temperature for step (iii) of the PCR cycle may be set as 69-76°C, 70-76°C, 71-76°C, 72-76°C, 73-76°C, 74-76°C, or 75-76°C. In some embodiments, the temperature for step (iii) of the PCR cycle may be set as a temperature selected from the list consisting of: 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, or 76°C. In some embodiments, the temperature for the final elongation step may be set as 68-76°C. In some embodiments, the temperature for the final elongation step may be set as 68-75°C, 68-74°C, 68-73°C, 68-72°C, 68-71°C, 68-70°C, or 68-69°C. In some embodiments, the temperature for the final elongation step may be set as 69-76°C, 70-76°C, 71-76°C, 72-76°C, 73-76°C, 74-76°C, or 75-76°C. In some embodiments, the temperature for the final elongation step may be set as atemperature selected from the list consisting of: 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C,75°C, or 76°C. In some embodiments, after the one-time final elongation step has been completed, the reaction may be held at a holding temperature that may be set as 20-40°C. In some embodiments, the holding temperature may be set as 20-39°C, 20-38°C, 20-37°C, 20-36°C, 20-35°C, 20-34°C, 20-33°C, 20-32°C, 20-31°C, 20-30°C, 20-29°C, 20-28°C, 20- 27°C, 20-26°C, 20-25°C, 20-24°C, 20-23°C, 20-22°C, or 20-21°C. In some embodiments, the holding temperature may be set as 21-40°C, 22-40°C, 23-40°C, 24-40°C, 25-40°C, 26-40°C, 27-40°C, 28-40°C, 29-40°C, 30-40°C, 31-40°C, 32-40°C, 33- 40°C, 34-40°C, 35-40°C, 36-40°C, 37-40°C, 38-40°C, or 39-40°C. In some embodiments, the holding temperature may be set as a temperature selected from the list consisting of: 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, or 40°C. In some embodiments, the method according to the invention may comprise: (a) a one-time initial denaturation step preceding the one or more PCR cycle(s), wherein the temperature may be set as 88-105°C; (b) the one or more PCR cycle(s) according to the invention, comprising: (i) denaturing double stranded DNA molecule(s) into separate DNA strands wherein the temperature may be set as 88-105°C; (ii) annealing primers to each DNA strand wherein the temperature may be set as 50- 76°C; and (iii) producing new double stranded DNA molecules by elongating DNA from the primers to produce a new DNA strand that is complementary to each of the DNA strands of step (ii), wherein the temperature may be set as 68-76°C; wherein step (b) is repeated for as many cycles as is required and the new double stranded DNA molecules formed in step (iii) are used as the double stranded DNA molecules in step (i) of the subsequent PCR cycle; and (c) a one-time final elongation step proceeding the one or more PCR cycle(s), wherein the temperature may be set as 68-76°C.In some embodiments, the method according to the invention may comprise:(a) a one-time initial denaturation step preceding the one or more PCR cycle(s), wherein the temperature may be set as 88-99°C; (b) the one or more PCR cycle(s) according to the invention, comprising: (i) denaturing double stranded DNA molecule(s) into separate DNA strands wherein the temperature may be set as 88-99°C; (ii) annealing primers to each DNA strand wherein the temperature may be set as 50- 76°C; and (iii) producing new double stranded DNA molecules by elongating DNA from the primers to produce a new DNA strand that is complementary to each of the DNA strands of step (ii), wherein the temperature may be set as 68-76°C; wherein step (b) is repeated for as many cycles as is required and the new double stranded DNA molecules formed in step (iii) are used as the double stranded DNA molecules in step (i) of the subsequent PCR cycle; and (c) a one-time final elongation step proceeding the one or more PCR cycle(s), wherein the temperature may be set as 68-76°C. In some embodiments, after step (c) has been completed, the reaction may be held at a holding temperature that may be set as 20-40°C. In some embodiments, the temperature for the initial denaturation step may be set as 94°C. In some embodiments, the temperature for the initial denaturation step may be set as 98°C. In some embodiments, the temperature for step (i) of the PCR cycle may be set as 94°C. In some embodiments, the temperature for step (i) of the PCR cycle may be set as 98°C. In some embodiments, the temperature for step (ii) of the PCR cycle may be set as 72°C. In some embodiments, the temperature for step (iii) of the PCR cycle may be set as 72°C. In some embodiments, the temperature for the final elongation step may be set as 72°C. In some embodiments, after the one-time final elongation step has been completed, the reaction may be held at a holding temperature that is set as 30°C. In some embodiments, the method according to the invention may comprise:(a) a one-time initial denaturation step preceding the one or more PCR cycle(s), wherein thetemperature may be set as 94°C; (b) the one or more PCR cycle(s) according to the invention, comprising: (i) denaturing double stranded DNA molecule(s) into separate DNA strands wherein the temperature may be set as 94°C; (ii) annealing primers to each DNA strand wherein the temperature may be set as 72°C; and (iii) producing new double stranded DNA molecules by elongating DNA from the primers to produce a new DNA strand that is complementary to each of the DNA strands of step (ii), wherein the temperature may be set as 72°C; wherein step (b) is repeated for as many cycles as is required and the new double stranded DNA molecules formed in step (iii) are used as the double stranded DNA molecules in step (i) of the subsequent PCR cycle; and (c) a one-time final elongation step proceeding the one or more PCR cycle(s), wherein the temperature may be set as 72°C.In some embodiments, after step (c) has been completed, the reaction may be held at aholding temperature that is set as 30°C. As described herein, it will be understood that the temperature that may be programmed for a particular step of the method described herein may not equate to the actual temperature that is reached in the reaction volume. In some embodiments, the actual temperature inside the microwave synthesis reactor for step (i) of the PCR cycle may be 91-109°C. In some embodiments, the actual temperature inside the microwave synthesis reactor for step (i) of the PCR cycle may be 91-108°C, 91-107°C, 91-106°C, 91-105°C, 91-104°C, 91-103°C, 91-102°C, 91-101°C, 91-100°C, 91-99°C, 91-98°C, 91-97°C, 91-96°C, 91-95°C, 91-94°C, 91- 93°C, or 91-92°C. In some embodiments, the actual temperature inside the microwave synthesis reactor for step (i) of the PCR cycle may be 92-109°C, 93-109°C, 94-109°C, 95-109°C, 96-109°C, 97-109°C, 98-109°C, 99-109°C, 100-109°C, 101-109°C, 102-109°C, 103-109°C, 104-109°C, 105-109°C, 106-109°C, 107-109°C, or 108-109°C. In some embodiments, the actual temperature inside the microwave synthesis reactor for step (i) of the PCR cycle may be 97-98°C. In some embodiments, the actual temperature inside the microwave synthesis reactor for step (i) of the PCR cycle may be 97°C or 98°C. In some embodiments, the method according to the invention may comprise: (a) a one-time initial denaturation step preceding the one or more PCR cycle(s), wherein the temperature may be set as 98°C; (b) the one or more PCR cycle(s) according to the invention, comprising: (i) denaturing double stranded DNA molecule(s) into separate DNA strands wherein the temperature may be set as 98°C; (ii) annealing primers to each DNA strand wherein the temperature may be set as72°C; and (iii) producing new double stranded DNA molecules by elongating DNA from the primers to produce a new DNA strand that is complementary to each of the DNA strands of step (ii), wherein the temperature may be set as 72°C; wherein step (b) is repeated for as many cycles as is required and the new double stranded DNA molecules formed in step (iii) are used as the double stranded DNA molecules in step (i) of the subsequent PCR cycle; and (c) a one-time final elongation step proceeding the one or more PCR cycle(s), wherein the temperature may be set as 72°C. In some embodiments, after step (c) has been completed, the reaction may be held at a holding temperature that is set as 30°C. As will be known in the art, the duration of each step of the PCR method may vary depending on various factors, such as the DNA polymerase that is used, the sequence length of the starting DNA molecules, the % guanine-cytosine (GC) content of the starting DNA molecules, and / or the primers used during the annealing step. The person skilled in the art will know how to optimise the duration of each step as appropriate. In some embodiments, the duration for the initial denaturation step may be for 30 seconds to 300 seconds.In some embodiments, the duration for the initial denaturation step may be for 30 seconds to270 seconds, 30 seconds to 240 seconds, 30 seconds to 210 seconds, 30 seconds to 180 seconds, 30 seconds to 150 seconds, 30 seconds to 120 seconds, 30 seconds to 90 seconds, or 30 seconds to 60 seconds. In some embodiments, the duration for the initial denaturation step may be for 60 seconds to 300 seconds, 90 seconds to 300 seconds, 120 seconds to 300 seconds, 150 seconds to 300 seconds, 180 seconds to 300 seconds, 210 seconds to 300 seconds, 240 seconds to 300 seconds, or 270 seconds to 300 seconds. In some embodiments, the duration for step (i) of the PCR cycle may be for 10 seconds to 120 seconds. In some embodiments, the duration for step (i) of the PCR cycle may be for 10 seconds to 110 seconds, 10 seconds to 100 seconds, 10 seconds to 90 seconds, 10 seconds to 80 seconds, 10 seconds to 70 seconds, 10 seconds to 60 seconds, 10 seconds to 50 seconds, 10 seconds to 40 seconds, 10 seconds to 30 seconds, or 10 seconds to 20 seconds. In some embodiments, the duration for step (i) of the PCR cycle may be for 20 seconds to 120 seconds, 30 seconds to 120 seconds, 40 seconds to 120 seconds, 50 seconds to 120 seconds, 60 seconds to 120 seconds, 70 seconds to 120 seconds, 80 seconds to 120 seconds, 90 seconds to 120 seconds, 100 seconds to 120 seconds, or 110 seconds to 120 seconds. In some embodiments, the duration for step (ii) of the PCR cycle may be for 30 seconds to 300 seconds. In some embodiments, the duration for step (ii) of the PCR cycle may be for 30 seconds to 270 seconds, 30 seconds to 240 seconds, 30 seconds to 210 seconds, 30 seconds to 180 seconds, 30 seconds to 150 seconds, 30 seconds to 120 seconds, 30 seconds to 90 seconds, or 30 seconds to 60 seconds. In some embodiments, the duration for step (ii) of the PCR cycle may be for 60 seconds to 300 seconds, 90 seconds to 300 seconds, 120 seconds to 300 seconds, 150 seconds to 300 seconds, 180 seconds to 300 seconds, 210 seconds to 300 seconds, 240 seconds to 300 seconds, or 270 seconds to 300 seconds. In some embodiments, the duration for step (iii) of the PCR cycle may be for 30 seconds to 1200 seconds. In some embodiments, the duration for step (iii) of the PCR cycle may be for 30 seconds to 1170 seconds, 30 seconds to 1140 seconds, 30 seconds to 1110 seconds, 30 seconds to 1080 seconds, 30 seconds to 1050 seconds, 30 seconds to 1020 seconds, 30 seconds to 990 seconds, 30 seconds to 960 seconds, 30 seconds to 960 seconds, 30 seconds to 930 seconds, 30 seconds to 900 seconds, 30 seconds to 870 seconds, 30 seconds to 840 seconds, 30 seconds to 810 seconds, 30 seconds to 780 seconds, 30 seconds to 750 seconds, 30 seconds to 720 seconds, 30 seconds to 690 seconds, 30 seconds to 660 seconds, 30 seconds to 630 seconds, 30 seconds to 600 seconds, 30 seconds to 570 seconds, 30 seconds to 540 seconds, 30 seconds to 510 seconds, 30 seconds to 480seconds, 30 seconds to 450 seconds, 30 seconds to 420 seconds, 30 seconds to 390seconds, 30 seconds to 360 seconds, 30 seconds to 330 seconds, 30 seconds to 300 seconds, 30 seconds to 270 seconds, 30 seconds to 240 seconds, 30 seconds to 210 seconds, 30 seconds to 180 seconds, 30 seconds to 150 seconds, 30 seconds to 120 seconds, 30 seconds to 90 seconds, or 30 seconds to 60 seconds. In some embodiments, the duration for step (iii) of the PCR cycle may be for 60 seconds to 1200 seconds, 90 seconds to 1200 seconds, 120 seconds to 1200 seconds, 150 seconds to 1200 seconds, 180 seconds to 1200 seconds, 210 seconds to 1200 seconds, 240 seconds to 1200 seconds, 270 seconds to 1200 seconds, 300 seconds to 1200 seconds, 330 seconds to1200 seconds, 360 seconds to 1200 seconds, 390 seconds to 1200 seconds, 420 seconds to1200 seconds, 450 seconds to 1200 seconds, 480 seconds to 1200 seconds, 510 seconds to 1200 seconds, 540 seconds to 1200 seconds, 570 seconds to 1200 seconds, 600 seconds to 1200 seconds, 630 seconds to 1200 seconds, 660 seconds to 1200 seconds, 690 seconds to 1200 seconds, 720 seconds to 1200 seconds, 750 seconds to 1200 seconds, 780 seconds to 1200 seconds, 810 seconds to 1200 seconds, 840 seconds to 1200 seconds, 870 seconds to 1200 seconds, 900 seconds to 1200 seconds, 930 seconds to 1200 seconds, 960 seconds to 1200 seconds, 990 seconds to 1200 seconds, 1020 seconds to 1200 seconds, 1050 seconds to 1200 seconds, 1080 seconds to 1200 seconds, 1110 seconds to 1200 seconds, 1140 seconds to 1200 seconds, or 1170 seconds to 1200 seconds. In some embodiments, the duration for step (iii) of the PCR cycle may be for 30 seconds to 300 seconds. In some embodiments, the duration for step (iii) of the PCR cycle may be for 30 seconds to 270 seconds, 30 seconds to 240 seconds, 30 seconds to 210 seconds, 30 seconds to 180 seconds, 30 seconds to 150 seconds, 30 seconds to 120 seconds, 30 seconds to 90 seconds, or 30 seconds to 60 seconds. In some embodiments, the duration for step (iii) of the PCR cycle may be for 60 seconds to 300 seconds, 90 seconds to 300 seconds, 120 seconds to 300 seconds, 150 seconds to 300 seconds, 180 seconds to 300 seconds, 210 seconds to 300 seconds, 240 seconds to 300 seconds, or 270 seconds to 300 seconds. In some embodiments, the combined duration for step (ii) and step (iii) of the PCR cycle may be for 30 seconds to 1200 seconds. In some embodiments, the combined duration for step (ii) and step (iii) of the PCR cycle maybe for 30 seconds to 1170 seconds, 30 seconds to 1140 seconds, 30 seconds to 1110seconds, 30 seconds to 1080 seconds, 30 seconds to 1050 seconds, 30 seconds to 1020 seconds, 30 seconds to 990 seconds, 30 seconds to 960 seconds, 30 seconds to 960 seconds, 30 seconds to 930 seconds, 30 seconds to 900 seconds, 30 seconds to 870 seconds, 30 seconds to 840 seconds, 30 seconds to 810 seconds, 30 seconds to 780 seconds, 30 seconds to 750 seconds, 30 seconds to 720 seconds, 30 seconds to 690 seconds, 30 seconds to 660 seconds, 30 seconds to 630 seconds, 30 seconds to 600 seconds, 30 seconds to 570 seconds, 30 seconds to 540 seconds, 30 seconds to 510 seconds, 30 seconds to 480 seconds, 30 seconds to 450 seconds, 30 seconds to 420 seconds, 30 seconds to 390 seconds, 30 seconds to 360 seconds, 30 seconds to 330 seconds, 30 seconds to 300 seconds, 30 seconds to 270 seconds, 30 seconds to 240 seconds, 30 seconds to 210 seconds, 30 seconds to 180 seconds, 30 seconds to 150 seconds, 30 seconds to 120 seconds, 30 seconds to 90 seconds, or 30 seconds to 60 seconds. In some embodiments, the combined duration for step (ii) and step (iii) of the PCR cycle may be for 60 seconds to 1200 seconds, 90 seconds to 1200 seconds, 120 seconds to 1200 seconds, 150 seconds to 1200 seconds, 180 seconds to 1200 seconds, 210 seconds to 1200 seconds, 240 seconds to 1200 seconds, 270 seconds to 1200 seconds, 300 seconds to 1200 seconds, 330 seconds to 1200 seconds, 360 seconds to 1200 seconds, 390 seconds to 1200 seconds, 420 seconds to 1200 seconds, 450 seconds to 1200 seconds, 480 seconds to 1200 seconds, 510 seconds to 1200 seconds, 540 seconds to 1200 seconds, 570 seconds to 1200 seconds, 600 seconds to 1200 seconds, 630 seconds to 1200 seconds, 660 seconds to 1200 seconds, 690 seconds to 1200 seconds, 720 seconds to 1200 seconds, 750 seconds to 1200 seconds, 780 seconds to 1200 seconds, 810 seconds to 1200 seconds, 840 seconds to 1200 seconds, 870 seconds to 1200 seconds, 900 seconds to 1200 seconds, 930 seconds to 1200 seconds, 960 seconds to 1200 seconds, 990 seconds to 1200 seconds, 1020 seconds to 1200 seconds, 1050 seconds to 1200 seconds, 1080 seconds to 1200 seconds, 1110 seconds to 1200 seconds, 1140 seconds to 1200 seconds, or 1170 seconds to 1200 seconds. In some embodiments, the combined duration for step (ii) and step (iii) of the PCR cycle may be for 30 seconds to 300 seconds. In some embodiments, the combined duration for step (ii) and step (iii) of the PCR cycle may be for 30 seconds to 270 seconds, 30 seconds to 240 seconds, 30 seconds to 210 seconds, 30 seconds to 180 seconds, 30 seconds to 150 seconds, 30 seconds to 120 seconds, 30 seconds to 90 seconds, or 30 seconds to 60 seconds. In some embodiments, the combined duration for step (ii) and step (iii) of the PCR cycle may be for 60 seconds to 300 seconds, 90 seconds to 300 seconds, 120 seconds to 300 seconds, 150 seconds to 300 seconds, 180 seconds to 300 seconds, 210 seconds to 300 seconds, 240 seconds to 300 seconds, or 270 seconds to 300 seconds. In some embodiments, the duration for the final elongation step may be for 30 seconds to 1200 seconds. In some embodiments, the duration for the final elongation step may be for 30 seconds to1170 seconds, 30 seconds to 1140 seconds, 30 seconds to 1110 seconds, 30 seconds to1080 seconds, 30 seconds to 1050 seconds, 30 seconds to 1020 seconds, 30 seconds to 990 seconds, 30 seconds to 960 seconds, 30 seconds to 960 seconds, 30 seconds to 930 seconds, 30 seconds to 900 seconds, 30 seconds to 870 seconds, 30 seconds to 840 seconds, 30 seconds to 810 seconds, 30 seconds to 780 seconds, 30 seconds to 750 seconds, 30 seconds to 720 seconds, 30 seconds to 690 seconds, 30 seconds to 660 seconds, 30 seconds to 630 seconds, 30 seconds to 600 seconds, 30 seconds to 570 seconds, 30 seconds to 540 seconds, 30 seconds to 510 seconds, 30 seconds to 480 seconds, 30 seconds to 450 seconds, 30 seconds to 420 seconds, 30 seconds to 390 seconds, 30 seconds to 360 seconds, 30 seconds to 330 seconds, 30 seconds to 300 seconds, 30 seconds to 270 seconds, 30 seconds to 240 seconds, 30 seconds to 210 seconds, 30 seconds to 180 seconds, 30 seconds to 150 seconds, 30 seconds to 120 seconds, 30 seconds to 90 seconds, or 30 seconds to 60 seconds. In some embodiments, the duration for the final elongation step may be for 60 seconds to 1200 seconds, 90 seconds to 1200 seconds, 120 seconds to 1200 seconds, 150 seconds to 1200 seconds, 180 seconds to 1200 seconds, 210 seconds to 1200 seconds, 240 seconds to1200 seconds, 270 seconds to 1200 seconds, 300 seconds to 1200 seconds, 330 seconds to1200 seconds, 360 seconds to 1200 seconds, 390 seconds to 1200 seconds, 420 seconds to 1200 seconds, 450 seconds to 1200 seconds, 480 seconds to 1200 seconds, 510 seconds to 1200 seconds, 540 seconds to 1200 seconds, 570 seconds to 1200 seconds, 600 seconds to 1200 seconds, 630 seconds to 1200 seconds, 660 seconds to 1200 seconds, 690 seconds to 1200 seconds, 720 seconds to 1200 seconds, 750 seconds to 1200 seconds, 780 seconds to 1200 seconds, 810 seconds to 1200 seconds, 840 seconds to 1200 seconds, 870 seconds to 1200 seconds, 900 seconds to 1200 seconds, 930 seconds to 1200 seconds, 960 seconds to 1200 seconds, 990 seconds to 1200 seconds, 1020 seconds to 1200 seconds, 1050 seconds to 1200 seconds, 1080 seconds to 1200 seconds, 1110 seconds to 1200 seconds, 1140 seconds to 1200 seconds, or 1170 seconds to 1200 seconds. In some embodiments, the duration for the final elongation step may be for 30 seconds to 300 seconds. In some embodiments, the duration for the final elongation step may be for 30 seconds to 270 seconds, 30 seconds to 240 seconds, 30 seconds to 210 seconds, 30 seconds to 180 seconds, 30 seconds to 150 seconds, 30 seconds to 120 seconds, 30 seconds to 90 seconds, or 30 seconds to 60 seconds. In some embodiments, the duration for the final elongation step may be for 60 seconds to 300 seconds, 90 seconds to 300 seconds, 120 seconds to 300 seconds, 150 seconds to 300seconds, 180 seconds to 300 seconds, 210 seconds to 300 seconds, 240 seconds to 300seconds, or 270 seconds to 300 seconds. In some embodiments, the reaction may be held at the holding temperature for a duration of 30 seconds to 600 seconds. In some embodiments, the reaction may be held for more than 600 seconds. In some embodiments, the reaction may be held for a duration of 30 seconds to 540 seconds, 30 seconds to 480 seconds, 30 seconds to 420 seconds, 30 seconds to 360 seconds, 30 seconds to 300 seconds, 30 seconds to 240 seconds, 30 seconds to 180 seconds, 30 seconds to 120 seconds, or 30 seconds to 60 seconds. In some embodiments, the reaction may be held for a duration of 60 seconds to 600 seconds, 120 seconds to 600 seconds, 180 seconds to 600 seconds, 240 seconds to 600 seconds, 300seconds to 600 seconds, 360 seconds to 600 seconds, 420 seconds to 600 seconds, 480seconds to 600 seconds, or 540 seconds to 600 seconds. In some embodiments, the duration for the initial denaturation step may be for 50 seconds.In some embodiments, the duration for step (i) of the PCR cycle may be for 20 seconds.In some embodiments, the combined duration for step (ii) and step (iii) of the PCR cycle may be for 200 seconds. In some embodiments, the duration for the final elongation step may be for 180 seconds. In some embodiments, the reaction may be held for a duration of 380 seconds. In some embodiments: (a) the duration for the initial denaturation step may be for 50 seconds; (b) the duration for step (i) of the PCR cycle may be for 20 seconds; (c) the combined duration for step (ii) and step (iii) of the PCR cycle may be for 200 seconds; and (d) the duration for the final elongation step may be for 180 seconds. Suitable reagents for performing PCR will be known in the art, including: water, buffers, cations(e.g. magnesium cations and / or potassium cations), dimethylsulfoxide (DMSO), detergents,DNA polymerases, primers (i.e. forward primers and reverse primers), plasmid DNA, templateDNA, cDNA, genomic DNA, and nucleotide mix (e.g. deoxynucleotides (dNTPs)).The person skilled in the art will be able to select the most appropriate reagents for performing the PCR. The person skilled in the art will also be able to select an appropriate amount of the reagent(s) in order to optimise the PCR. In some embodiments, the reaction volume may comprise one or more of the reagentsselected from the list consisting of: water, buffer, cations (e.g. magnesium cations and / orpotassium cations), DMSO, detergents, DNA polymerase, forward primer, reverse primer,plasmid DNA, template DNA, cDNA, genomic DNA, and nucleotide mix. In some embodiments, the reaction volume may comprise all of the reagents selected fromthe list consisting of: water, buffer, DNA polymerase, forward primer, reverse primer, templateDNA and nucleotide mix.It will be understood that the “double stranded DNA molecule(s)” in step (i) of the method according to the invention may be the template DNA referred to herein, which is used as the initial template for the PCR.In some embodiments, the nucleotide mix (e.g. dNTP mix) may comprise dATP, dCTP, dGTPand dTTP. In some embodiments, the nucleotide mix (e.g. dNTP mix) may comprise dATP, dCTP, dGTPand dTTP in any suitable ratio or at any suitable concentration.In some embodiments, dUTP may be used instead of dTTP. It will be understood that the person skilled in the art will be able to select suitable primers (forward primer and reverse primer) depending on the template DNA that is to be amplified by PCR. It will be understood that the DNA polymerase may be any suitable DNA polymerase known in the art. In some embodiments, the DNA polymerase may be selected from the list consisting of: aprokaryotic DNA polymerase, a eukaryotic DNA polymerase, a bacteriophage DNApolymerase, a high fidelity polymerase, and a heat-stable DNA polymerase. In some embodiments, the reaction volume may not comprise bovine serum albumin (BSA). THERAPEUTIC RNATherapeutic RNA is a class of therapeutics based on RNA molecules, which include, forexample, messenger RNA (mRNA) therapies and antisense RNA.In some embodiments, the therapeutic RNA is at least about 100 base pairs in length. In some embodiments, the therapeutic RNA is at least 100 base pairs in length. In some embodiments, the therapeutic RNA is at least about 150 base pairs in length. In some embodiments, the therapeutic RNA is at least 150 base pairs in length. In some embodiments, the therapeutic RNA is at least about 1,000 base pairs in length. In some embodiments, the therapeutic RNA is at least 1,000 base pairs in length.mRNA therapies involve administering mRNA molecules encoding proteins or peptides ofinterest. The mRNA is then translated within cells to synthesise the encoded protein(s) orpeptide(s), which can then exert functional (e.g. therapeutic) effect(s) in vivo.As used herein, the terms “RNA therapy”, RNA-based therapy”, “RNA therapeutics” and “therapeutic RNA” can be considered as interchangeable. In some embodiments, the double stranded DNA material may be suitable for use as atemplate for in vitro transcription (IVT) to produce the therapeutic RNA.In some embodiments, the method according to the invention may comprise a further step ofin vitro transcription (IVT) to produce therapeutic RNA from the double stranded DNA material.IVT is a process that is well-known in the art and involves transcribing DNA sequences into corresponding RNA sequences. During IVT, a template DNA sequence is bound by an RNA polymerase, which uses RNA nucleotides to produce a sequence of RNA that is complementary to the template DNA sequence. It will be understood that the double stranded DNA material produced by the method of the invention may be used as the DNA template for IVT to produce the therapeutic RNA. It will also be understood that the IVT step occurs after the sufficient amount of double stranded DNA material has been produced. In other words, in some embodiments, once a sufficient amount of the double stranded DNAmaterial has been produced, there may be a further step of in vitro transcription (IVT) toproduce therapeutic RNA from the double stranded DNA material. In some embodiments, the therapeutic RNA is mRNA or an antisense RNA. In some embodiments, “therapeutic RNA” used herein does not include aptamers. The present invention also provides a method for producing therapeutic RNA, wherein the method comprises the steps of: (a) producing double stranded DNA material, wherein the method comprises one or more polymerase chain reaction (PCR) cycle(s), wherein the, or each, PCR cycle comprises the steps of: (i) denaturing double stranded DNA molecule(s) into separate DNA strands; (ii) annealing primers to each DNA strand; and (iii) producing new double stranded DNA molecules by elongating DNA from the primers to produce a new DNA strand that is complementary to each of the DNA strands of step (ii); wherein the new double stranded DNA molecules form the double stranded DNA material; wherein steps (i)-(iii) are repeated for as many cycles as is required and the new double stranded DNA molecules formed in step (iii) are used as the double stranded DNA molecules in step (i) of the subsequent PCR cycle; wherein the, or each, PCR cycle is performed using a microwave synthesis reactor in a single reaction volume; and (b) in vitro transcription (IVT) to produce therapeutic RNA from the double stranded DNAmaterial. In some embodiments, the method for producing therapeutic RNA may comprise the steps of: (a) producing double stranded DNA material, wherein the method comprises one or more polymerase chain reaction (PCR) cycle(s), a one-time initial denaturation step preceding the one or more PCR cycle(s), and a one-time final elongation step proceeding the one or more PCR cycle(s), wherein the, or each, PCR cycle comprises the following sequential steps: (i) denaturing double stranded DNA molecule(s) into separate DNA strands; (ii) annealing primers to each DNA strand; and (iii) producing new double stranded DNA molecules by elongating DNA from the primers to produce a new DNA strand that is complementary to each of theDNA strands of step (ii); wherein the new double stranded DNA molecules form the double stranded DNA material; wherein steps (i)-(iii) are repeated for as many cycles as is required and the new double stranded DNA molecules formed in step (iii) are used as the double stranded DNA molecules in step (i) of the subsequent PCR cycle; wherein the, or each, PCR cycle is performed using a microwave synthesis reactor in a single reaction volume; and (b) in vitro transcription (IVT) to produce therapeutic RNA from the double stranded DNAmaterial. The present invention also provides a method for producing therapeutic RNA, wherein the method comprises the steps of: (a) producing double stranded DNA material for downstream synthesis of therapeutic RNA, wherein the method comprises one or more polymerase chain reaction (PCR) cycle(s), wherein the, or each, PCR cycle comprises the steps of: (i) denaturing double stranded DNA molecule(s) into separate DNA strands; (ii) annealing primers to each DNA strand; and (iii) producing new double stranded DNA molecules by elongating DNA from the primers to produce a new DNA strand that is complementary to each of the DNA strands of step (ii); wherein the new double stranded DNA molecules form the double stranded DNA material; wherein steps (i)-(iii) are repeated for as many cycles as is required and the new double stranded DNA molecules formed in step (iii) are used as the double stranded DNA molecules in step (i) of the subsequent PCR cycle; wherein the, or each, PCR cycle is performed using a microwave synthesis reactor in a single reaction volume; and (b) in vitro transcription (IVT) to produce therapeutic RNA from the double stranded DNAmaterial. In some embodiments, the method for producing therapeutic RNA may comprise the steps of: (a) producing double stranded DNA material for downstream synthesis of therapeutic RNA, wherein the method comprises one or more polymerase chain reaction (PCR) cycle(s), a one-time initial denaturation step preceding the one or more PCR cycle(s), and a one-time final elongation step proceeding the one or more PCR cycle(s), wherein the, or each, PCR cycle comprises the following sequential steps: (i) denaturing double stranded DNA molecule(s) into separate DNA strands; (ii) annealing primers to each DNA strand; and (iii) producing new double stranded DNA molecules by elongating DNA from the primers to produce a new DNA strand that is complementary to each of the DNA strands of step (ii); wherein the new double stranded DNA molecules form the double stranded DNA material; wherein steps (i)-(iii) are repeated for as many cycles as is required and the new double stranded DNA molecules formed in step (iii) are used as the double stranded DNA molecules in step (i) of the subsequent PCR cycle; wherein the, or each, PCR cycle is performed using a microwave synthesis reactor in a single reaction volume; and (b) in vitro transcription (IVT) to produce therapeutic RNA from the double stranded DNAmaterial. The present invention also provides a method for producing therapeutic RNA, wherein the method comprises the steps of: (a) producing double stranded DNA material in a sufficient amount for downstream synthesis of therapeutic RNA, wherein the method comprises one or more polymerase chain reaction (PCR) cycle(s), wherein the, or each, PCR cycle comprises the steps of: (i) denaturing double stranded DNA molecule(s) into separate DNA strands; (ii) annealing primers to each DNA strand; and (iii) producing new double stranded DNA molecules by elongating DNA from the primers to produce a new DNA strand that is complementary to each of the DNA strands of step (ii); wherein the new double stranded DNA molecules form the double stranded DNA material; wherein steps (i)-(iii) are repeated for as many cycles as is required and the new double stranded DNA molecules formed in step (iii) are used as the doublestranded DNA molecules in step (i) of the subsequent PCR cycle; wherein the, or each, PCR cycle is performed using a microwave synthesis reactor in a single reaction volume; and (b) in vitro transcription (IVT) to produce therapeutic RNA from the double stranded DNAmaterial. In some embodiments, the method for producing therapeutic RNA may comprise the steps of: (a) producing double stranded DNA material in a sufficient amount for downstream synthesis of therapeutic RNA, wherein the method comprises one or more polymerasechain reaction (PCR) cycle(s), a one-time initial denaturation step preceding the one or more PCR cycle(s), and a one-time final elongation step proceeding the one or more PCR cycle(s), wherein the, or each, PCR cycle comprises the following sequentialsteps: (i) denaturing double stranded DNA molecule(s) into separate DNA strands; (ii) annealing primers to each DNA strand; and (iii) producing new double stranded DNA molecules by elongating DNA from the primers to produce a new DNA strand that is complementary to each of theDNA strands of step (ii); wherein the new double stranded DNA molecules form the double stranded DNA material; wherein steps (i)-(iii) are repeated for as many cycles as is required and the new double stranded DNA molecules formed in step (iii) are used as the double stranded DNA molecules in step (i) of the subsequent PCR cycle; wherein the, or each, PCR cycle is performed using a microwave synthesis reactor in a single reaction volume; and (b) in vitro transcription (IVT) to produce therapeutic RNA from the double stranded DNAmaterial. In some embodiments, the therapeutic RNA may be single stranded. In some embodiments, the therapeutic RNA may be double stranded. In some embodiments, the therapeutic RNA may undergo further processing such that it becomes double stranded. In some embodiments, the therapeutic RNA may undergo further processing such that it comprises synthetic nucleotides. In some embodiments, the therapeutic RNA may undergo further processing such that itcomprises modified nucleotides.In some embodiments, the therapeutic RNA may be mRNA. In some embodiments, the therapeutic RNA may be therapeutic mRNA.In some embodiments, the therapeutic RNA may be siRNA.In some embodiments, the therapeutic RNA may be antisense oligonucleotide(s). In some embodiments, the therapeutic RNA may be immune stimulating RNA. In some embodiments, the therapeutic RNA may not be an aptamer. MICROWAVE SYNTHESIS REACTOR The present invention is based on using a large-scale PCR using a microwave synthesis reactor. Microwave synthesis reactors are known in the art and have been used previously for chemicalsynthesis reactions such as solvent extractions. These devices have the capabilities toproduce fast and uniform heating for all types of reactions, typically up to 300°C.Microwave synthesis reactors can typically be configured for one reaction at a time, meaningthat one reaction vessel of varying size can be used.The present inventors have surprisingly found that microwave synthesis reactors can be usedto successfully perform large-scale PCR, using the reaction volumes described herein.Indeed, until the present invention, it was not known that microwave synthesis reactors couldbe used for large-scale PCR, in particular to perform full PCR cycles in a single reactionvolume, utilising different temperatures at different stages wherein the temperatures changerapidly and homogeneously within the reaction volume, in order to produce double strandedDNA material in the sufficient amount described herein for downstream therapeutic RNAsynthesis. In addition, it was also not known that microwave synthesis reactors could be used to producedouble stranded DNA molecules of the length (in base pairs) as described herein. It wastherefore surprising that microwave synthesis reactors can be used for generating doublestranded DNA material that is suitable as templates for in vitro transcription reactions usingreaction volumes in the millilitre scale described herein.As described herein, examples of microwave synthesis reactors that are suitable for use inthe present invention include the Monowave 400 (Anton Paar GmbH) and the Discover 2.0(CEM GmbH). Other known providers of microwave synthesis reactors include Milestone andLabX. Other suitable microwave synthesis reactors will be known in the art. PURIFICATION In some embodiments, the method according to the invention may comprise a further step of purifying the double stranded DNA material. In some embodiments, the step of purifying the double stranded DNA material may occur after the sufficient amount of double stranded DNA material has been produced.In other words, in some embodiments, once (a sufficient amount of) the double stranded DNAmaterial has been produced, there may be a further step of purifying the double stranded DNA material. In some embodiments, the step of purifying the double stranded DNA material may occur afterthe (sufficient amount of) double stranded DNA material has been produced, and before thein vitro transcription (IVT) step described herein is performed.In other words, in some embodiments, once (a sufficient amount of) the double stranded DNAmaterial has been produced, there may be a further step of purifying the double stranded DNAmaterial, followed by a further step of in vitro transcription (IVT) to produce therapeutic RNAfrom the purified double stranded DNA material.It will be understood that any purification step may be used when performing the method of the present invention. The person skilled in the art will be able to perform purification step(s) as appropriate. In some embodiments, the double stranded DNA material may be purified with magnetic beads. In some embodiments, the double stranded DNA material may be purified by silica column purification. In some embodiments, the double stranded DNA material may be purified by chromatographic purification. In some embodiments, the double stranded DNA material may be purified by tangential flow filtration (TFF). USE The present invention also provides use of a microwave synthesis reactor for producing double stranded DNA material in a single reaction volume in a sufficient amount for downstream synthesis of therapeutic RNA. The present invention also provides use of a microwave synthesis reactor for producing double stranded DNA material in a single reaction volume for downstream synthesis of therapeutic RNA. The present invention also provides use of a microwave synthesis reactor for producing double stranded DNA material in a single reaction volume. The present invention also provides use of a microwave synthesis reactor for producing double stranded DNA material in a sufficient amount for downstream synthesis of therapeutic RNA. The present invention also provides use of a microwave synthesis reactor for producing double stranded DNA material for downstream synthesis of therapeutic RNA. The present invention also provides use of a microwave synthesis reactor for producing double stranded DNA material.It will be understood that the embodiments of the microwave synthesis reactor, the doublestranded DNA material, the sufficient amount, the single reaction volume, and the therapeuticRNA described above with regards to the methods according to the invention also applies tothe uses according to the invention. OTHER EMBODIMENTS The present invention provides a method for producing double stranded DNA material in a sufficient amount for downstream synthesis of therapeutic RNA, wherein the method comprises one or more polymerase chain reaction (PCR) cycle(s), wherein the, or each, PCR cycle comprises the steps of: (i) denaturing double stranded DNA molecule(s) into separate DNA strands; (ii) annealing primers to each DNA strand; and (iii) producing new double stranded DNA molecules by elongating DNA from the primers to produce a new DNA strand that is complementary to each of the DNA strands of step (ii); wherein the new double stranded DNA molecules form the double stranded DNA material; wherein the, or each, PCR cycle is performed in a single reaction volume; wherein the PCRcycle is performed for a total of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 cycles.The present invention provides a method for producing double stranded DNA material in a sufficient amount for downstream synthesis of therapeutic RNA, wherein the method comprises one or more polymerase chain reaction (PCR) cycle(s), wherein the, or each, PCR cycle comprises the steps of: (i) denaturing double stranded DNA molecule(s) into separate DNA strands; (ii) annealing primers to each DNA strand; and (iii) producing new double stranded DNA molecules by elongating DNA from the primers to produce a new DNA strand that is complementary to each of the DNA strands of step (ii); wherein the new double stranded DNA molecules form the double stranded DNA material;wherein the PCR cycle is performed for a total of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 cycles. The present invention provides a method for producing double stranded DNA material fordownstream synthesis of therapeutic RNA, wherein the method comprises one or morepolymerase chain reaction (PCR) cycle(s), wherein the, or each, PCR cycle comprises the steps of: (i) denaturing double stranded DNA molecule(s) into separate DNA strands; (ii) annealing primers to each DNA strand; and (iii) producing new double stranded DNA molecules by elongating DNA from the primers to produce a new DNA strand that is complementary to each of the DNA strands of step (ii); wherein the new double stranded DNA molecules form the double stranded DNA material; wherein the, or each, PCR cycle is performed in a single reaction volume; wherein the PCRcycle is performed for a total of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 cycles. The present invention provides a method for producing double stranded DNA material for downstream synthesis of therapeutic RNA, wherein the method comprises one or morepolymerase chain reaction (PCR) cycle(s), wherein the, or each, PCR cycle comprises thesteps of: (i) denaturing double stranded DNA molecule(s) into separate DNA strands; (ii) annealing primers to each DNA strand; and (iii) producing new double stranded DNA molecules by elongating DNA from the primers to produce a new DNA strand that is complementary to each of the DNA strands of step (ii); wherein the new double stranded DNA molecules form the double stranded DNA material;wherein the PCR cycle is performed for a total of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 cycles. The present invention provides a method for producing double stranded DNA material, wherein the method comprises one or more polymerase chain reaction (PCR) cycle(s), wherein the, or each, PCR cycle comprises the steps of: (i) denaturing double stranded DNA molecule(s) into separate DNA strands; (ii) annealing primers to each DNA strand; and (iii) producing new double stranded DNA molecules by elongating DNA from the primers to produce a new DNA strand that is complementary to each of the DNA strands of step (ii); wherein the new double stranded DNA molecules form the double stranded DNA material;wherein the, or each, PCR cycle is performed in a single reaction volume; wherein the PCRcycle is performed for a total of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 cycles. The present invention provides a method for producing double stranded DNA material, wherein the method comprises one or more polymerase chain reaction (PCR) cycle(s), wherein the, or each, PCR cycle comprises the steps of: (i) denaturing double stranded DNA molecule(s) into separate DNA strands; (ii) annealing primers to each DNA strand; and (iii) producing new double stranded DNA molecules by elongating DNA from the primers to produce a new DNA strand that is complementary to each of the DNA strands of step (ii); wherein the new double stranded DNA molecules form the double stranded DNA material;wherein the PCR cycle is performed for a total of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 cycles. In some embodiments, the PCR cycle may be performed for a total of 2 or fewer, 3 or fewer,4 or fewer, 5 or fewer, 6 or fewer, 7 or fewer, 8 or fewer, 9 or fewer, 10 or fewer, 11 or fewer,12 or fewer, 13 or fewer, 14 or fewer, 15 or fewer, 16 or fewer, 17 or fewer, 18 or fewer, 19 or fewer, 20 or fewer, 21 or fewer, 22 or fewer, 23 or fewer, 24 or fewer, 25 or fewer, 26 or fewer,27 or fewer, 28 or fewer, 29 or fewer, 30 or fewer, 31 or fewer, 32 or fewer, 33 or fewer, 34 orfewer, 35 or fewer, 36 or fewer, 37 or fewer, 38 or fewer, 39 or fewer, or 40 or fewer cycles. In some embodiments, the PCR cycle may be performed for a total of 1-40 cycles. In some embodiments, the PCR cycle may be performed for a total of 1-35 cycles. In some embodiments, the PCR cycle may be performed for a total of 1-30 cycles. In some embodiments, the PCR cycle may be performed for a total of 1-25 cycles. In some embodiments, the PCR cycle may be performed for a total of 1-20 cycles. In some embodiments, the PCR cycle may be performed for a total of 1-15 cycles. In some embodiments, the PCR cycle may be performed for a total of 1-10 cycles. In some embodiments, the PCR cycle may be performed for a total of 1-5 cycles.In some embodiments, the PCR cycle may be performed for a total of 22 cycles.In some embodiments, the PCR cycle may be performed for a total of 23 cycles.In some embodiments, the PCR cycle may be performed for a total of 28 cycles.In some embodiments, the PCR cycle may be performed for a total of more than 40 cycles.The person skilled in the art will be able to determine how many PCR cycles will be required,in order to obtain the desired amount of double stranded DNA material. GENERAL TERMS AND DEFINITIONS This disclosure is not limited by the exemplary methods and materials disclosed herein, and any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of this disclosure. Numeric ranges are inclusive of the numbers defining the range. Unless otherwise indicated, any nucleic acid sequences are written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively. Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upperand lower limits of that range is also specifically disclosed. Each smaller range between anystated value or intervening value in a stated range and any other stated or intervening valuein that stated range is encompassed within this disclosure. The upper and lower limits of thesesmaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassedwithin this disclosure, subject to any specifically excluded limit in the stated range. Where thestated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in this disclosure. It must be noted that as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. The terms "comprising", "comprises" and "comprised of' as used herein are synonymous with "including", "includes" or "containing", "contains", and are inclusive or open-ended and do not exclude additional, non-recited members, elements or method steps. The terms "comprising", "comprises" and "comprised of' also include the term "consisting of'. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that such publications constitute prior art to the claims appended hereto. NUMBERED PARAGRAPHS The present invention may be described by way of the following numbered paragraphs: 1. A method for producing double stranded DNA material, wherein the method comprises one or more polymerase chain reaction (PCR) cycle(s), wherein the, or each, PCR cycle comprises the steps of: (i) denaturing double stranded DNA molecule(s) into separate DNA strands; (ii) annealing primers to each DNA strand; and (iii) producing new double stranded DNA molecules by elongating DNA from the primers to produce a new DNA strand that is complementary to each of the DNA strands of step (ii); wherein the new double stranded DNA molecules form the double stranded DNA material; wherein the, or each, PCR cycle is performed using a microwave synthesis reactor in a single reaction volume. 2. A method for producing double stranded DNA material for downstream synthesis of therapeutic RNA, wherein the method comprises one or more polymerase chain reaction (PCR) cycle(s), wherein the, or each, PCR cycle comprises the steps of: (i) denaturing double stranded DNA molecule(s) into separate DNA strands; (ii) annealing primers to each DNA strand; and (iii) producing new double stranded DNA molecules by elongating DNA from the primers to produce a new DNA strand that is complementary to each of the DNA strands of step (ii); wherein the new double stranded DNA molecules form the double stranded DNA material; wherein the, or each, PCR cycle is performed using a microwave synthesis reactor in a single reaction volume. 3. A method for producing double stranded DNA material in a sufficient amount for downstream synthesis of therapeutic RNA, wherein the method comprises one or morepolymerase chain reaction (PCR) cycle(s), wherein the, or each, PCR cycle comprises thesteps of: (i) denaturing double stranded DNA molecule(s) into separate DNA strands; (ii) annealing primers to each DNA strand; and (iii) producing new double stranded DNA molecules by elongating DNA from the primers to produce a new DNA strand that is complementary to each of the DNA strands of step (ii); wherein the new double stranded DNA molecules form the double stranded DNA material; wherein the, or each, PCR cycle is performed using a microwave synthesis reactor in a single reaction volume.4. The method of any one of paragraphs 1 to 3, wherein the double stranded DNAmaterial is suitable for use as a template for in vitro transcription (IVT) to produce thetherapeutic RNA.5. The method according to any one of paragraphs 1 to 4, comprising a further step of invitro transcription (IVT) to produce therapeutic RNA from the double stranded DNA material.6. The method according to any one of paragraphs 1-5, wherein the RNA is mRNA.7. The method according to any one of paragraphs 1-6, wherein the double stranded DNA material comprises 0.01-130.00 mg of DNA. 8. The method according to paragraph 7, wherein the double stranded DNA material comprises 0.01-0.10 mg of DNA, optionally wherein the double stranded DNA material comprises 0.01-0.09 mg, 0.01-0.08 mg, 0.01-0.07 mg, 0.01-0.06 mg, 0.01-0.05 mg, 0.01-0.04 mg, 0.01-0.03 mg, 0.01-0.02 mg, 0.02-0.10 mg, 0.03-0.10 mg, 0.04-0.10 mg, 0.05-0.10 mg, 0.06-0.10 mg, 0.07-0.10 mg, 0.08-0.10 mg, 0.09-0.10 mg of DNA. 9. The method according to paragraph 7, wherein the double stranded DNA material comprises 0.1-1.0 mg of DNA, optionally wherein the double stranded DNA material comprises 0.1-0.9 mg, 0.1-0.8 mg, 0.1-0.7 mg, 0.1-0.6 mg, 0.1-0.5 mg, 0.1-0.4 mg, 0.1-0.3 mg, 0.1-0.2 mg, 0.2-1.0 mg, 0.3-1.0 mg, 0.4-1.0 mg, 0.5-1.0 mg, 0.6-1.0 mg, 0.7-1.0 mg, 0.8-1.0 mg, 0.9- 1.0 mg of DNA. 10. The method according to paragraph 7, wherein the double stranded DNA material comprises 1-130 mg of DNA, optionally wherein the double stranded DNA material comprises 1-120 mg, 1-110 mg, 1-100 mg, 1-90 mg, 1-80 mg, 1-70 mg, 1-60 mg, 1-50 mg, 1-40 mg, 1- 30 mg, 1-20 mg, 1-10 mg, 10-130 mg, 20-130 mg, 30-130 mg, 40-130 mg, 50-130 mg, 60-130 mg, 70-130 mg, 80-130 mg, 90-130 mg, 100-130 mg, 110-130 mg, 120-130 mg of DNA.11. The method according to any one of paragraphs 1-10, wherein the method comprisesrepeating steps (i) to (iii), wherein the new double stranded DNA molecules formed in step (iii) are used as the double stranded DNA molecules in step (i) of the subsequent PCR cycle. 12. The method according to any one of paragraphs 1-11, wherein the PCR cycle isperformed for a total of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 cycles.13. The method according to any one of paragraphs 1-12, wherein the single reactionvolume is a volume of 0.5 to 1,000 mL.14. The method according to paragraph 13, wherein the single reaction volume is a volumeof 0.5-10.0 mL, optionally wherein the single reaction volume is a volume of 0.5-9.5 mL, 0.5- 9.0 mL, 0.5-8.5 mL, 0.5-8.0 mL, 0.5-7.5 mL, 0.5-7.0 mL, 0.5-6.5 mL, 0.5-6.0 mL, 0.5-5.5 mL, 0.5-5.0 mL, 0.5-4.5 mL, 0.5-4.0 mL, 0.5-3.5 mL, 0.5-3.0 mL, 0.5-2.5 mL, 0.5-2.0 mL, 0.5-1.5 mL, 0.5-1.0 mL, 1.0-10.0 mL, 1.5-10.0 mL, 2.0-10.0 mL, 2.5-10.0 mL, 3.0-10.0 mL, 3.5-10.0 mL, 4.0-10.0 mL, 4.5-10.0 mL, 5.0-10.0 mL, 5.5-10.0 mL, 6.0-10.0 mL, 6.5-10.0 mL, 7.0-10.0 mL, 7.5-10.0 mL, 8.0-10.0 mL, 8.5-10.0 mL, 9.0-10.0 mL, or 9.5-10.0 mL.15. The method according to paragraph 13, wherein the single reaction volume is a volumeof 10-100 mL, optionally wherein the single reaction volume is a volume of 10-95 mL, 10-90 mL, 10-85 mL, 10-80 mL, 10-75 mL, 10-70 mL, 10-65 mL, 10-60 mL, 10-55 mL, 10-50 mL, 10-45 mL, 10-40 mL, 10-35 mL, 10-30 mL, 10-25 mL, 10-20 mL, 10-15 mL, 15-100 mL, 20- 100 mL, 25-100 mL, 30-100 mL, 35-100 mL, 40-100 mL, 45-100 mL, 50-100 mL, 55-100 mL, 60-100 mL, 65-100 mL, 70-100 mL, 75-100 mL, 80-100 mL, 85-100 mL, 90-100 mL, or 95- 100 mL.16. The method according to paragraph 13, wherein the single reaction volume is a volumeof 100-1000 mL, optionally wherein the single reaction volume is a volume of 100-950 mL, 100-900 mL, 100-850 mL, 100-800 mL, 100-750 mL, 100-700 mL, 100-650 mL, 100-600 mL, 100-550 mL, 100-500 mL, 100-450 mL, 100-400 mL, 100-350 mL, 100-300 mL, 100-250 mL, 100-200 mL, 100-150 mL, 150-1,000 mL, 200-1,000 mL, 250-1,000 mL, 300-1,000 mL, 350- 1,000 mL, 400-1,000 mL, 450-1,000 mL, 500-1,000 mL, 550-1,000 mL, 600-1,000 mL, 650- 1,000 mL, 700-1,000 mL, 750-1,000 mL, 800-1,000 mL, 850-1,000 mL, 900-1,000 mL, or 950- 1,000 mL.17. The method according to paragraph 13 or paragraph 14, wherein the single reactionvolume is a volume of 3.0 mL.18. The method according to paragraph 13 or paragraph 15, wherein the single reactionvolume is a volume of 20.0 mL.19. The method according to any one of paragraphs 1-18, wherein step (ii) and step (iii)are performed simultaneously.20. The method according to any one of paragraphs 1-19, wherein the method comprises:(i) an initial denaturation step preceding the one or more PCR cycle(s); (ii) the one or more PCR cycle(s); and (iii) a final elongation step proceeding the one or more PCR cycle(s); wherein: (a) the temperature for the initial denaturation step is 88-105°C; (b) the temperature for step (i) of the PCR cycle is 88-105°C; (c) the temperature for step (ii) of the PCR cycle is 50-76°C; (d) the temperature for step (iii) of the PCR cycle is 68-76°C; and / or (e) the temperature for the final elongation step is 68-76°C.21. The method according to paragraph 20, wherein the method comprises:(i) an initial denaturation step preceding the one or more PCR cycle(s); (ii) the one or more PCR cycle(s); and (iii) a final elongation step proceeding the one or more PCR cycle(s); wherein: (a) the temperature for the initial denaturation step is set as 94°C; (b) the temperature for step (i) of the PCR cycle is set as 94°C; (c) the temperature for step (ii) of the PCR cycle is set as 72°C;(d) the temperature for step (iii) of the PCR cycle is set as 72°C; and / or(e) the temperature for the final elongation step is set as 72°C.22. The method according to paragraph 21, wherein the actual temperature inside themicrowave synthesis reactor for step (i) of the PCR cycle is 91-109°C, optionally wherein the actual temperature is 97-98°C.23. The method according to paragraph 20, wherein the method comprises:(i) an initial denaturation step preceding the one or more PCR cycle(s); (ii) the one or more PCR cycle; and (iii) a final elongation step proceeding the one or more PCR cycle(s); wherein: (a) the temperature for the initial denaturation step is set as 98°C; (b) the temperature for step (i) of the PCR cycle is set as 98°C; (c) the temperature for step (ii) of the PCR cycle is set as 72°C;(d) the temperature for step (iii) of the PCR cycle is set as 72°C; and / or(e) the temperature for the final elongation step is set as 72°C.24. The method according to any one of paragraphs 1-23, wherein the double strandedDNA molecules forming the double stranded DNA material are at least 54 base pairs in length.25. The method according to any one of paragraphs 1-24, wherein the double strandedDNA molecules forming the double stranded DNA material are at least 1,000 base pairs in length.26. The method according to paragraph 24 or 25, wherein the double stranded DNAmolecules forming the double stranded DNA material are 54-10,000 base pairs in length.27. The method according to any one of paragraphs 24 to 26, wherein the double strandedDNA molecules forming the double stranded DNA material are 100-1,000 base pairs in length, optionally wherein the double stranded DNA molecules are 100-950 base pairs in length, 100- 900 base pairs in length, 100-850 base pairs in length, 100-800 base pairs in length, 100-750 base pairs in length, 100-700 base pairs in length, 100-650 base pairs in length, 100-600 base pairs in length, 100-550 base pairs in length, 100-500 base pairs in length, 100-450 base pairs in length, 100-400 base pairs in length, 100-350 base pairs in length, 100-300 base pairs in length, 100-250 base pairs in length, 100-200 base pairs in length, 100-150 base pairs in length, 150-1,000 base pairs in length, 200-1,000 base pairs in length, 250-1,000 base pairs in length, 300-1,000 base pairs in length, 350-1,000 base pairs in length, 400-1,000 base pairs in length, 450-1,000 base pairs in length, 500-1,000 base pairs in length, 550-1,000 base pairs in length, 600-1,000 base pairs in length, 650-1,000 base pairs in length, 700-1,000 base pairs in length, 750-1,000 base pairs in length, 800-1,000 base pairs in length, 850-1,000 base pairs in length, 900-1,000 base pairs in length, or 950-1,000 base pairs in length.28. The method according to any one of paragraphs 24 to 26, wherein the double strandedDNA molecules forming the double stranded DNA material are 1,000-10,000 base pairs in length, optionally wherein the double stranded DNA molecules are 1,000-9,500 base pairs in length, 1,000-9,000 base pairs in length, 1,000-8,500 base pairs in length, 1,000-8,000 base pairs in length, 1,000-7,500 base pairs in length, 1,000-7,000 base pairs in length, 1,000-6,500 base pairs in length, 1,000-6,000 base pairs in length, 1,000-5,500 base pairs in length, 1,000- 5,000 base pairs in length, 1,000-4,500 base pairs in length, 1,000-4,000 base pairs in length, 1,000-3,500 base pairs in length, 1,000-3,000 base pairs in length, 1,000-2,500 base pairs in length, 1,000-2,000 base pairs in length, 1,000-1,500 base pairs in length, 1,500-10,000 basepairs in length, 2,000-10,000 base pairs in length, 2,500-10,000 base pairs in length, 3,000-10,000 base pairs in length, 3,500-10,000 base pairs in length, 4,000-10,000 base pairs in length, 4,500-10,000 base pairs in length, 5,000-10,000 base pairs in length, 5,500-10,000 base pairs in length, 6,000-10,000 base pairs in length, 6,500-10,000 base pairs in length, 7,000-10,000 base pairs in length, 7,500-10,000 base pairs in length, 8,000-10,000 base pairs in length, 8,500-10,000 base pairs in length, 9,000-10,000 base pairs in length, or 9,500- 10,000 base pairs in length.29. The method according to any one of paragraphs 24, 25, 26 or28, wherein the doublestranded DNA molecules forming the double stranded DNA material each comprise a contiguous nucleotide sequence of about 1,000 to about 4,500 base pairs in length.30. The method according to any one of paragraphs 1-29, wherein the reaction volumedoes not comprise bovine serum albumin (BSA).31. The method according to any one of paragraphs 1-30, comprising a further step ofpurifying the double stranded DNA material.32. The method according to paragraph 31, wherein the double stranded DNA material ispurified with magnetic beads.33. Use of a microwave synthesis reactor for producing double stranded DNA material ina single reaction volume in a sufficient amount for downstream synthesis of therapeutic RNA. The invention will now be further described by way of Examples, which are meant to serve to assist one of ordinary skill in the art in carrying out the invention and are not intended in any way to limit the scope of the invention. EXAMPLES EXAMPLE 1 The aim of this study was to generate PCR-based linear DNA templates based on PCR cycling protocols in a single reaction setup with a 3 mL and 20 mL PCR reaction volume. A PCR protocol was established that could be used for DNA production in a reaction volumeof 3 mL and 20 mL using the microwave synthesis reactor Monowave 400 from Anton PaarGmbH (Table 4).During PCR, the temperature of the sample was continuously measured with an integratedruby thermometer. With a set temperature of 94°C for the denaturation step it was possible to achieve an actual temperature of 97-98°C inside the reaction vessel. A uniform thermal cycling was achieved between ~98°C and ~72°C for the specified numberof cycles in the PCR cycling protocol, similar to the standard PCR cycler.Using the established PCR cycling protocol, various DNA templates of different lengths (from 1 kbp to more than 4 kbp) and GC content (from 50% to more than 57%) were able to be successfully synthesized in the microwave synthesis reactor. Synthesized DNA templates were purified and subsequent agarose gel electrophoresis showed a band of the correct size for each sample (Figure 1). As a control, DNA templates were produced in the standard PCR cycler (Figure 1).To further verify successful production, the identity of the synthesized DNA templates wasverified by DNA sequencing. Measurement of the DNA concentration of the purified DNA template showed similar values between samples that were manufactured in the Monowave 400 compared to the standard PCR cycler indicating that production of DNA template in a single reaction setup and an increased reaction volume does not impact DNA yield negatively (Table 1). Of note, for better comparison, only 600 µL of PCR product were purified for each sample / condition, irrespective of the total PCR reaction volume. To assess whether the DNA templates were suitable for subsequent RNA production, RNAwas synthesized via in vitro transcription and the quality of the resulting RNA was verified withthe Fragment Analyzer. The results showed that the synthesized RNAs had a good integrity (Figure 2). The RNA integrity was similar, irrespective of the device (Monowave 400 or standard PCR cycler) used for DNA template manufacturing via PCR. In addition to the Monowave 400 from Anton Paar GmbH, a second microwave synthesis reactor (Discover 2.0 from CEM GmbH) was tested for DNA template synthesis in a PCRreaction volume of 3 mL and 20 mL. Here, a second PCR cycling protocol was establishedwhere the temperature during the denaturation step was set to 98°C (Table 5). Measurement of the temperature inside the reaction vessel confirmed that a temperature around 98°C was reached during PCR. The data show that all DNA templates were manufactured successfully using the microwave synthesis reactor Discover 2.0. For each sample, a band of the correct size was visible on theagarose gel (Figures 3 and 4) and the identity of the DNA templates could be verified by DNAsequencing. All DNA template samples displayed the expected lengths within acceptabletolerance of agarose gel electrophoresis, indicating that the synthesis was successful. Anyapparent slight variations in the positioning of bands, whether from the DNA ladder or the manufactured DNA template, can be attributed to the intrinsic variability in DNA migration through the gel. In addition, the DNA concentration was similar for DNA templates produced in the device Discover 2.0 compared to the standard PCR cycler (Table 1). Furthermore, the resulting RNAs after in vitro transcription had a good quality irrespective of the reaction volume used for the single reaction PCR approach (Figure 2).In summary, the data show that microwave synthesis reactors are suitable for the large-scaleamplification of DNA templates (of different lengths and different GC content) for the subsequent manufacturing of therapeutic RNA. Table 1: Measurement of DNA concentration for purified DNA templates produced in the microwave synthesis reactor Monowave 400 and Discover 2.0 as well as thestandard PCR cycler. Five DNA templates were manufactured in the microwave synthesisreactor Monowave 400 and Discover 2.0 in a single reaction setup and a reaction volume as shown below. As a control, DNA templates were produced in 0.075 mL per reaction using the standard PCR cycler with eight reactions running in parallel leading to a total volume of 0.6 mL. After synthesis, DNA templates were purified. For each sample, 600 µL of the synthesized DNA template were used for purification (irrespective of the total PCR reaction volume) and after purification, the DNA was eluted in 88 µL of water.DNA concentration of purified DNA (elutedSingle PCR Total PCR in a total volume of 88 µL) [ng / µL] DNA reaction reaction Template PCR volume [mL] volume [mL] Monowave Discover 2.0 cycler 400 control 33 348.9 469.6120 20 571.3 364.40.075 0.6 579.93 3 535.6 342.120 20 503.3 538.52 50 50 340.20.075 0.6 703.93 3 703.7 826.4320 20 501.6 496.70.075 0.6 554.93 3 342.4 247.1420 20 250.3 190.80.075 0.6 419.550 50 346.5570 70 516.60.075 0.6 744.5Material and Methods: DNA templates were produced via PCR and subsequently purified. After DNA purification, the identity of the DNA template was confirmed by Sanger sequencing. Furthermore, all DNAtemplates were used for RNA synthesis via in vitro transcription. The integrity of the resultingRNA was determined using the Fragment Analyzer. PCR DNA templates were synthesized by PCR. Properties and details regarding the PCR template are listed in Table 2. The details of the reaction mixture are listed in Table 3.The PCR cycling protocols for the microwave synthesis reactor and standard PCR cyclers arelisted in Table 4-8. Table 2: Properties of the DNA templates which were produced during PCR using themicrowave synthesis reactor Monowave 400 and Discover 2.0 as well as the standardPCR cyclerDNA template ID DNA TemplateGC content length (bp) (%)1 4,380 55.12 1,140 50.33 1,815 53.44 2,045 57.65 1,377 55bp = base pairs; DNA = deoxyribonucleic acid; GC = guanine-cytosine; ID = identification;PCR = polymerase chain reaction.Table 3: PCR reaction mixture Reagent C (final reaction)Buffer 1.00 xDNA polymerase 0.04 U / µlWater -Primer fwd 0.5 - 0.8 µMPrimer rev 0.5 - 0.8 µMdNTP mix 0.2 mM eachPlasmid 15-30 ngC = concentration; fwd = forward; rev = reverse; U = Unit. Table 4: PCR cycling protocol for the synthesis of all DNA templates using the microwave synthesis reactor Monowave 400 Step Temperature (°C) Time (min:sec) CyclesInitial denaturation 94 00:50 1Denaturation 94 00:20Annealing & 22 72 03:20Elongation Final Elongation 72 03:00 1Hold 30 06:20 1DNA = deoxyribonucleic acid; min = minute(s); PCR = polymerase chain reaction; sec = second(s). Table 5: PCR cycling protocol for the synthesis of all DNA templates manufactured ina PCR volume of 3 mL and 20 mL using the microwave synthesis reactor Discover 2.0Step Temperature (°C) Time (min:sec) CyclesInitial denaturation 98 00:50 1Denaturation 98 00:20Annealing & 23 72 03:20Elongation Final Elongation 72 03:00 1DNA = deoxyribonucleic acid; min = minute(s); PCR = polymerase chain reaction; sec = second(s).Table 6: PCR cycling protocol for the synthesis of the DNA templates manufactured ina PCR volume of 50 mL using the microwave synthesis reactor Discover 2.0Step Temperature (°C) Time (min:sec) CyclesInitial denaturation 98 00:50 1Denaturation 98 00:20Annealing & 76 01:40Elongation 22 Annealing & 74 01:40Elongation Final Elongation 74 03:00 1DNA = deoxyribonucleic acid; min = minute(s); PCR = polymerase chain reaction; sec = second(s).Table 7: PCR cycling protocol for the synthesis of the DNA template 5 manufacturedin a PCR volume of 70 mL using the microwave synthesis reactor Discover 2.0Step Temperature (°C) Time (min:sec) CyclesInitial denaturation 98 00:50 1Denaturation 98 00:20Annealing & 76 01:40Elongation 22 Annealing & 75 01:40Elongation Final Elongation 74 03:00 1DNA = deoxyribonucleic acid; min = minute(s); PCR = polymerase chain reaction; sec =second(s).Table 8: PCR cycling protocol to amplify the DNA template 1-5 using the standardPCR cycler Step Temperature (°C) Time (min:sec) CyclesInitial denaturation 98 00:50 1Denaturation 98 00:20Annealing 69.8-71.7 00:42-01:00 22-28Elongation 71.7-72 00:35-02:20Final Elongation 72 03:00 1Hold 4 ∞ 1DNA = deoxyribonucleic acid; min = minute(s); PCR = polymerase chain reaction; sec = second(s). Agarose gel electrophoresis Agarose gel electrophoresis was performed to verify the successful synthesis of DNAtemplates. The purified DNA template (100 ng) was mixed with 6x loading dye and TE bufferand separated on a 1 % agarose gel. To visualize DNA using a gel documentation imaging system, 10000x GelRed was added to the agarose. DNA purification by magnetic beadsFollowing PCR, the DNA template was purified with magnetic beads. For each sample, 600 µlof the DNA template were used for magnetic bead purification. The DNA was eluted in 88 µl of water. DNA sequencing In order to check the identity of the synthesized DNA templates, samples were Sanger sequenced.In vitro transcriptionFor RNA generation, a small scale (100 µL) non-fedbatch T7 transcription (IVT) with following magnetic bead purification was performed.Analysis of the RNA integrityThe RNA integrity was determined by capillary electrophoresis using the Fragment Analyzer. For this, 90 ng / µL of RNA were used and each sample was analyzed in triplicate. Data analysis was performed using Agilent ProSize software. Here, the ProSize “smear” function was selected in order to determine the RNA integrity and the boundaries for the peak to be integrated were selected manually. Boundaries of the main peak were set at the start and end of the peak in the “smear analysis” settings. The baseline was set manually. RNA integrity values were exported and mean values were calculated for each sample. RNA and DNA measurements The DNA and RNA concentration was measured using the NanoDrop UV spectrophotometer. For each sample, the DNA concentration was measured in triplicates. All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in molecular biology or related fields are intended to be within the scope of the following claims.

Claims

CLAIMS 1. A method for producing double stranded DNA material for downstream synthesis of therapeutic RNA, wherein the method comprises one or more polymerase chain reaction(PCR) cycle(s), wherein the, or each, PCR cycle comprises the steps of:(i) denaturing double stranded DNA molecule(s) into separate DNA strands; (ii) annealing primers to each DNA strand; and (iii) producing new double stranded DNA molecules by elongating DNA from the primers to produce a new DNA strand that is complementary to each of the DNAstrands of step (ii); wherein the new double stranded DNA molecules form the double stranded DNA material;wherein the, or each, PCR cycle is performed using a microwave synthesis reactor in a singlereaction volume.

2. The method of claim 1, wherein the method is for producing double stranded DNAmaterial in a sufficient amount for downstream synthesis of therapeutic RNA.

3. The method of claim 1 or 2, wherein the double stranded DNA material is suitable foruse as a template for in vitro transcription (IVT) to produce the therapeutic RNA.

4. The method according to any one of claims 1-3, comprising a further step of in vitrotranscription (IVT) to produce therapeutic RNA from the double stranded DNA material.

5. The method according to any one of claims 1-4, wherein the RNA is mRNA.

6. The method according to any one of claims 1-5, wherein the double stranded DNA material comprises 0.01-130.00 mg of DNA.

7. The method according to any one of claims 1-6, wherein the method comprisesrepeating steps (i) to (iii), wherein the new double stranded DNA molecules formed in step (iii) are used as the double stranded DNA molecules in step (i) of the subsequent PCR cycle.

8. The method according to any one of claims 1-7, wherein the PCR cycle is performed for a total of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 cycles.

9. The method according to any one of claims 1-8, wherein the single reaction volume isa volume of 0.5 to 1,000 mL, optionally wherein the single reaction volume is a volume of 3.0mL or a volume of 20.0 mL.

10. The method according to any one of claims 1-9, wherein step (ii) and step (iii) areperformed simultaneously.

11. The method according to any one of claims 1-10, wherein the method comprises:(i) an initial denaturation step preceding the one or more PCR cycle(s); (ii) the one or more PCR cycle(s); and (iii) a final elongation step proceeding the one or more PCR cycle(s); wherein: (a) the temperature for the initial denaturation step is 88-105°C; (b) the temperature for step (i) of the PCR cycle is 88-105°C; (c) the temperature for step (ii) of the PCR cycle is 50-76°C; (d) the temperature for step (iii) of the PCR cycle is 68-76°C; and / or (e) the temperature for the final elongation step is 68-76°C; optionally wherein the method comprises: (a) (i) an initial denaturation step preceding the one or more PCR cycle(s); (ii) the one or more PCR cycle(s); and (iii) a final elongation step proceeding the one or more PCR cycle(s); wherein: (a) the temperature for the initial denaturation step is set as 94°C; (b) the temperature for step (i) of the PCR cycle is set as 94°C; (c) the temperature for step (ii) of the PCR cycle is set as 72°C;(d) the temperature for step (iii) of the PCR cycle is set as 72°C; and / or(e) the temperature for the final elongation step is set as 72°C; or (b) (i) an initial denaturation step preceding the one or more PCR cycle(s); (ii) the one or more PCR cycle; and (iii) a final elongation step proceeding the one or more PCR cycle(s); wherein: (a) the temperature for the initial denaturation step is set as 98°C; (b) the temperature for step (i) of the PCR cycle is set as 98°C;(c) the temperature for step (ii) of the PCR cycle is set as 72°C;(d) the temperature for step (iii) of the PCR cycle is set as 72°C; and / or(e) the temperature for the final elongation step is set as 72°C.

12. The method according to any one of claims 1-11, wherein the double stranded DNAmolecules forming the double stranded DNA material are at least 54 base pairs in length; optionally wherein the double stranded DNA molecules forming the double stranded DNA material are 54-10,000 base pairs in length.

13. The method according to any one of claims 1-11, wherein the double stranded DNAmolecules forming the double stranded DNA material are at least 1,000 base pairs in length, optionally wherein the double stranded DNA molecules forming the double stranded DNA material are 1,000-10,000 base pairs in length14. The method according to any one of claims 1-13, comprising a further step of purifyingthe double stranded DNA material; optionally wherein the double stranded DNA material is purified with magnetic beads.

15. Use of a microwave synthesis reactor for producing double stranded DNA material ina single reaction volume in a sufficient amount for downstream synthesis of therapeutic RNA.

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