Abstract

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, preferable 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.

IPC Classes  ?

• C12Q 1/6844 - Nucleic acid amplification reactions

Abstract

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.

IPC Classes  ?

• C12P 19/34 - Polynucleotides, e.g. nucleic acids, oligoribonucleotides

Abstract

An in vitro process for the production of closed linear deoxyribonucleic acid (DNA) comprises (a) contacting a DNA template comprising at least one protelomerase target sequence with at least one DNA polymerase in the presence of one or more primers under conditions promoting amplification of the template; and (b) contacting amplified DNA produced in (a) with at least one protelomerase under conditions promoting production of closed linear DNA. A kit provides components necessary in the process.

IPC Classes  ?

• C12Q 1/6853 - Nucleic acid amplification reactions using modified primers or templates
• C12N 15/11 - DNA or RNA fragments; Modified forms thereof
• A61K 39/00 - Medicinal preparations containing antigens or antibodies
• A61K 48/00 - Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• C12N 9/02 - Oxidoreductases (1.), e.g. luciferase
• C12Q 1/6844 - Nucleic acid amplification reactions
• A61P 37/08 - Antiallergic agents
• A61P 39/02 - Antidotes
• A61P 37/04 - Immunostimulants
• A61P 35/00 - Antineoplastic agents
• A61P 33/00 - Antiparasitic agents
• A61P 37/00 - Drugs for immunological or allergic disorders

Abstract

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.

IPC Classes  ?

• C12Q 1/6844 - Nucleic acid amplification reactions

Abstract

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.

IPC Classes  ?

• C12P 19/34 - Polynucleotides, e.g. nucleic acids, oligoribonucleotides

Abstract

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.

IPC Classes  ?

• C12N 15/85 - Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
• C12N 5/074 - Adult stem cells

Abstract

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.

IPC Classes  ?

• C12P 19/34 - Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• C12Q 1/6844 - Nucleic acid amplification reactions

Abstract

An in vitro process for the production of closed linear deoxyribonucleic acid (DNA) comprises (a) contacting a DNA template comprising at least one protelomerase target sequence with at least one DNA polymerase in the presence of one or more primers under conditions promoting amplification of the template; and (b) contacting amplified DNA produced in (a) with at least one protelomerase under conditions promoting production of closed linear DNA. A kit provides components necessary in the process.

IPC Classes  ?

• C12Q 1/68 - Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
• C12Q 1/6853 - Nucleic acid amplification reactions using modified primers or templates
• C12N 15/11 - DNA or RNA fragments; Modified forms thereof
• A61K 39/00 - Medicinal preparations containing antigens or antibodies
• A61K 48/00 - Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• C12N 9/02 - Oxidoreductases (1.), e.g. luciferase
• C12Q 1/6844 - Nucleic acid amplification reactions
• A61P 37/08 - Antiallergic agents
• A61P 39/02 - Antidotes
• A61P 37/04 - Immunostimulants
• A61P 35/00 - Antineoplastic agents
• A61P 33/00 - Antiparasitic agents
• A61P 37/00 - Drugs for immunological or allergic disorders

Abstract

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.

IPC Classes  ?

• C07H 21/00 - Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• C12Q 1/6844 - Nucleic acid amplification reactions

Abstract

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.

IPC Classes  ?

• C12Q 1/6844 - Nucleic acid amplification reactions

Abstract

Apparatus for biochemical synthesis having a reaction vessel, a temperature control device for the reaction vessel, a plurality of reservoirs for reaction components, and supply/withdrawal systems, e.g. reciprocating syringe pumps and switchable valves. Agitation of a reaction mixture is effected by withdrawing a part of the mixture from the reaction vessel and returning it thereto.

IPC Classes  ?

• B01F 15/00 - Accessories for mixers
• B01L 7/00 - Heating or cooling apparatus; Heat insulating devices
• B01F 5/10 - Circulation mixers
• B01J 19/00 - Chemical, physical or physico-chemical processes in general; Their relevant apparatus
• B01F 13/10 - Mixing plant, including combinations of dissimilar mixers
• B01F 15/06 - Heating or cooling systems
• B01F 11/00 - Mixers with shaking, oscillating, or vibrating mechanisms
• B01F 13/08 - Magnetic mixers
• C12P 19/34 - Polynucleotides, e.g. nucleic acids, oligoribonucleotides

Abstract

A primer for the amplification of a DNA template comprising a protelomerase target sequence, particularly for production of closed linear DNA, which primer is capable of specifically binding to a palindromic sequence within a protelomerase target sequence and priming amplification in both directions.

IPC Classes  ?

• C12Q 1/68 - Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
• C12P 19/34 - Polynucleotides, e.g. nucleic acids, oligoribonucleotides