Enzymatic synthesis of repeat regions of oligonucleotides

A method for synthesizing an oligonucleotide having a defined sequence including at least one repeat region in which a repeated nucleotide appears more than once in succession and at least one non-repeating region, wherein at least one non-repeating region of the oligonucleotide is synthesized by reaction cycles comprising the steps of

• extending a primer with a 3'-blocked nucleotide substrate to form an extended primer having a blocking group at the 3'-end;
• inactivating unreacted 3'-blocked nucleotide substrate to render it less reactive than blocked nucleotide as a substrate for the primer extension reaction of step (a);
• and
• removing the blocking group from the extended primer to render it available for subsequent reaction cycles;
• and at least one repeat region is synthesized by reaction cycles comprising the steps of
• enzymatically coupling an oligonucleotide primer with a 3'-phosphateblocked repeated nucleotide to form a 3'-phosphate blocked primer;
• and
• removing the 3'-blocking group from the 3'-phosphate-blocked primer using a 3'-phosphatase enzyme substantially without removing the 3'-phosphate blocking group from unreacted 3'-phosphate-blocked repeated nucleotide;
• wherein unfeacted 3'-phosphate blocked repeated nucleotide from a first cycle is utilized as substrate in at least one successive cycle without prior separation from the primer from which the 3'-phosphate blocking group has been removed.

A method for synthesizing a repeat region of an oligoribonucleotide having a defined sequence, said repeat region including a repeated ribonucleotide that appears more than once in succession, comprising the steps of:

• (a) enzymatically coupling an oligonucleotide primer with a 3'-phosphate-blocked repeated ribonucleotide to form a 3'-phosphate blocked primer;
• (b) removing the 3'-phosphate blocking group from the 3'-phosphate-blocked primer using a 3'-phosphatase enzyme substantially without removing the 3'-phosphate blocking group from unreacted 3'-phosphate-blocked repeated ribonucleotide;
• and
• (c) repeating steps (a) and (b) using unreacted 3'-phosphate-blocked repeated ribonucleotide from step (b) as the 3'-phosphate-blocked repeated ribonucleotide of step (a) and the deblocked primer product of step (b) as the oligonucleotide primer of step (a) without prior separation of the unreacted 3'-phosphate-blocked repeated ribonucleotide from the deblocked primer product.