Enzymatic synthesis of repeat regions of oligonucleotides

Details Enzymatic synthesis of repeat regions of oligonucleotides Enzymatic synthesis of repeat regions of oligonucleotides

1993-12-02

1996-05-14

Naff, David M.

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Preparing compound containing saccharide radical

435 911, 435 913, 435 915, 435 9153, 536 253, 536 2531, 935 17, C12P 1934, C07H 2100

Patent

active

055166644

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.
Also disclosed is a process for synthesizing an oligonucleotide having a defined sequence including at least one repeat region and one non-repeating region, wherein at least one non-repeating region is synthesized by reaction cycles using the steps of extending a primer with a 3'-blocked nucleotide, inactivating unreacted 3'-blocked nucleotide, and removing the blocking group from the extended primer. The disclosed processes may be used to synthesize repeat regions of oligoribonucleotides.

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