Method for enzymatic synthesis of oligonucleotides
First Claim
1. 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.
0 Assignments
0 Petitions
Accused Products
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.
161 Citations
18 Claims
-
1. 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. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 18)
-
- 14. 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.
Specification