The present invention relates to novel nucleotide complexes which enhance the enzymatic production of DNA. The nucleotide complexes includes a divalent cation at a ratio of 0.2-1.5 divalent cations per nucleotide, whilst monovalent ions (other than hydrogen or hydronium ions from any solvent) are minimal. The nucleotide complexes have desirable properties for the synthesis of deoxyribonucleic acid (DNA), in particular cell-free enzymatic synthesis of DNA, preferably on a large or industrial scale. Further, the invention includes improved processes for preparing said nucleotide complexes. Further complexes are disclosed which possess unique properties in accelerating DNA synthesis at higher concentrations, these complexes also containing zwitterion molecules.
The present invention relates to a novel closed linear DNA vector, which is suitable for use in the production of lentiviral particles. Notably, the present invention relates to a new configuration of the vector including the transgene (often termed the "payload" vector), which enables a greater yield of infectious lentiviral particles, notably a greater yield of lentiviral particles carrying a transgene, to be prepared when compared to closed linear DNA vectors lacking this configuration. Further, the inventors have developed improvements in lentiviral production with closed linear DNA, through optimisation of vector input quantities and construct ratios. The invention furthermore relates to a method of generating infectious lentiviral particles using the construct, optionally in conjunction with improved production vectors and/or optimised methodology.
The present invention relates to an improved process for synthesis of deoxyribonucleic acid (DNA), in particular cell-free enzymatic synthesis of DNA, preferably on a large or industrial scale, with an improved yield and/or with an improved efficiency. The invention requires the use of nucleotide complexes wherein the nucleotide is associated with a mixture of divalent and monovalent cations. Preferably, the divalent cation may be magnesium or manganese.
The present invention relates to an improved process for synthesis of deoxyribonucleic acid (DNA), in particular cell-free enzymatic synthesis of DNA, preferably on a large scale, with an improved yield and/or with an improved efficiency. The species of cation present in the nucleotide salt as the counter-ion is critical to the yield, efficiency and fidelity of high yielding enzymatic DNA synthesis reaction. The processes herein use alternative cations as counter-ions for the ionic nucleotides, permitting the use of higher concentrations of nucleotides in DNA synthesis, and further allowing for more favourable reaction conditions to be used.
Reprogramming allows the "conversion" of any mature or somatic cell of the human or animal body into a pluripotent stem cell. Reprogramming can be performed through the introduction of exogenous factors, usually transcription factors, into the mature cell. This process allows the production of induced pluripotent stem cells without the use of embryos, with the advantage that they can be produced from an individual to return/re-implant to the same individual.The inventors have developed a method of transient expression of exogenous reprogramming factors using a transient vector, wherein the vector is a closed linear DNA. Surprisingly, pluripotent stem cells developed in this manner are stable and closer in phenotype to natural stem cells such as ESCs.
The present invention relates to improved processes for production of closed linear deoxyribonucleic acid (DNA), in particular cell-free enzymatic production of closed linear DNA molecules, preferably using a closed linear DNA as a template for DNA synthesis. The invention further relates to a novel closed linear DNA species, suitable for use as a template in the improved processes for production of closed linear DNA. Further, the invention pertains to the intermediate products of the processes, since this enables the production of larger quantities of closed linear DNA from the template than with methods known in the art.
The present invention relates to an in vitro cell-free process for production of deoxyribonucleotides (DNAs) comprising at least one hairpin, corresponding DNA products and uses thereof, and oligonucleotides and kits useful in the process of the invention.
The present invention relates to an improved process for synthesis of DNA, RNA, proteins and like molecules, in particular cell-free enzymatic synthesis of DNA, preferably in large scale. The present invention relates to the synthesis of DNA using strand-displacement replication and the addition of nucleotides to the reaction mixture is controlled, thus controlling yield. The reaction mixture contains a starting amount of nucleotides, polymerase and DNA template, to which further nucleotides are supplied in a controlled manner.