Method for enzymatic synthesis of oligonucleotides
OwnerEdward D. Hymaninventor
Life of this patent
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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.
Claims18
A method for synthesizing an oligonucleotide of defined sequence, comprising the steps of:
- (a) combining (1) an oligonucleotide primer and (2) a blocked nucleotide or a blocked nucleotide precursor that forms a blocked nucleotide in situ, in a reaction mixture in the presence of a chain extending enzyme effective to couple the blocked nucleotide to the 3'-end of the oligonucleotide primer such that a primer-blocked nucleotide product is formed, said blocked nucleotide comprising a nucleotide to be added to form part of the defined sequence and a blocking group attached to the nucleotide effective to prevent the addition of more than one blocked nucleotide to the primer;
- (b) inactivating the chain extending enzyme;
- (c) removing the blocking group from the 3'-end of the primer-blocked nucleotide product to form a primer-nucleotide product, whereby the reaction mixture contains unreacted starting materials, primer-nucleotide product and reaction by-products;
- (d) converting any unreacted blocked nucleotide in the reaction mixture to an unreactive form which is less active as a substrate for the chain extending enzyme than the blocked nucleotide;
- (e) repeating a cycle of steps (a) through (d) using the primer-nucleotide product of step (c) as the primer of step (a) for sufficient cycles to obtain a synthesized oligonucleotide of the defined sequence;
- and
- (f) cleaving the initial primer used in the first cycle from synthesized oligonucleotide.
A method according to claim 14, further comprising the step of digesting the initial primer with an enzyme, substantially without digestion of the synthesized oligonucleotide.
Dependencies are inferred from the claim text.