C - Chemistry – Metallurgy – 12 – P
Patent
C - Chemistry, Metallurgy
12
P
C12P 19/34 (2006.01) B01J 19/00 (2006.01) C07H 21/00 (2006.01) C12M 1/40 (2006.01) C12Q 1/68 (2006.01)
Patent
CA 2150670
Enzymatic synthesis of oligonucleotides may be performed by the steps of: (a) combining a primer and a blocked nucleotide in the presence of a chain attending 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; 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 be performed either with or without inactivation of unreacted blocked nucleotide substrate, i.e., converting any unreacted blocked nucleotide to an unreactive form which is substantially less active as a substrate for the chain extending enzyme. Cycles may be performed with or without removal of the blocking group from unreacted blocked nucleotide. When the blocked nucleotide used in a given cycle is not to be reused in the next cycle, then the given cycle is performed with inactivation of unreacted blocked nucleotide and favorably with removal of tbe blocking group from unreacted blocked nucleotide. When the blocked nucleotide used in a given cycle is to be reused in the next cycle, then the given cycle is performed without removal of the blocking group from unreacted blocked nucleotide and favorably without inactivation of unreacted blocked nucleotide. These cycles are performed perferably in a single vessel witbout intermediate purification of oligonucleotide product. The removal of the blocking group is preferably performed using an enzyme. The inactivation of unreacted blocked nucleotide is preferably performed using an enzyme or enzyme conbination.
Borden Ladner Gervais Llp
Hyman Edward David
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