Abstract: In one embodiment of the invention, a method is provided for preparing plasmid from host cells which contain the plasmid, comprising: (a) providing a plasmid solution comprised of unligatable open circular plasmid; (b) reacting the unligatable open circular plasmid with one or more enzymes and appropriate nucleotide cofactor(s), such that unligatable open circular plasmid is converted to 3?-hydroxyl, 5?-phosphate nicked plasmid; (c) reacting the 3?-hydroxyl, 5?-phosphate nicked plasmid with a DNA ligase and DNA ligase nucleotide cofactor, such that 3?-hydroxyl, 5?-phosphate nicked plasmid is converted to relaxed covalently closed circular plasmid; and (d) reacting the relaxed covalently closed circular plasmid with a DNA gyrase and DNA gyrase nucleotide cofactor, such that relaxed covalently closed circular plasmid is converted to negatively supercoiled plasmid. In other embodiments, DNA gyrase is replaced with reverse DNA gyrase or reaction (d) is not performed.

Abstract: An oligonucleotide is synthesized by adding a 3'-phosphate blocked nucleotide to a primer, removing the blocking group from the primer-blocked nucleotide product using a thermostable 3'-phosphatase enzyme, and repeating these steps until the desired nucleotides have been added to the primer. A suitable phosphatase enzyme for use in this method is a thermostable phosphatase derived from the hyperthermophilic archaebacterium Pyrococcus furiosus.

Abstract: Enzymatic synthesis of oligonucleotides is performed by the steps of: (a) combining a primer and a blocked nucleotide in the presence of a chain extending enzyme to form a primer-blocked nucleotide product containing the blocked nucleotide coupled to the primer at its 3'-end; (b) removing the blocking group from the 3' end of the primer-blocked nucleotide product; and (c) repeating the cycle of steps (a) and (b), using the primer-nucleotide product of step (b) as the primer for step (a) in the next cycle, for sufficient cycles to form the oligonucleotide product. Cycles may optionally include the step of converting any unreacted blocked nucleotide to an unreactive form which is substantially less active as a substrate for the chain extending enzyme. Cycles may also include the step of removing the blocking group from unreacted blocked nucleotide. This step is unnecessary, however, when the same nucleotide is added in two or more successive cycles.

Abstract: Enzymatic synthesis of a repeat region of an oligonucleotide may be performed by the steps of: (a) combining a primer and a blocked nucleotide in the presence of a chain extending enzyme whereby a primer-blocked nucleotide product is formed containing the blocked nucleotide coupled to the primer at its 3'-end; (b) removing the blocking group from the 3'-end of the primer-blocked nucleotide product using a 3'-phosphatase enzyme substantially without removing the 3'-phosphate blocking group from unreacted 3'-phosphate-blocked nucleotide; and (c) repeating the cycle of steps (a) and (b), using the primer-nucleotide product of step (b) as the primer for step (a) in the next cycle, for sufficient cycles to form the oligonucleotide product. These cycles are performed preferably in a single vessel without intermediate purification of oligonucleotide product.

Abstract: Enzymatic synthesis of a portion of an oligonucleotide is performed by a cycle of synthetic steps: (a) combining an oligonucleotide primer and a blocked nucleotide in a reaction mixture in the presence of a chain extending enzyme, such that a primer-blocked nucleotide product is formed, wherein the blocked nucleotide substrate comprises (i) a nucleotide to be added to form part of the defined sequence and (ii) a blocking group attached to the nucleotide effective to prevent the addition of more than one blocked nucleotide to the primer; and (b) removing the blocking group from the 3' end of the primer-blocked nucleotide product to form a primer-nucleotide product. Phosphate is generated in at least one synthetic step. A precipitate is formed in the cycle comprising phosphate and at least one precipitation cation. The precipitation of phosphate reduces its unfavorable effect on the method. Preferably, cycles of the method are repeated without intermediate purification of primer-nucleotide product or precursor.

Abstract: Enzymatic synthesis of oligonucleotides may be performed in a single vessel without intermediate purification, by the steps of:(a) combining a nucleotide primer sequence and a blocked nucleotide in the presence of a chain extending enzyme whereby a reaction mixture is formed containing the blocked nucleotide coupled to the nucleotide primer sequence at its 3' end;(b) inactivating the chain extending enzyme;(c) removing the blocking group from the primer-blocked nucleotide to form a primer-nucleotide product; and converting any unreacted blocked nucleotide to an unreactive form which is substantially less active as a substrate for the chain extending enzyme than the blocked nucleotide.