Method for the enzymatic synthesis of oligonucleotides using thermostable 3'-phosphatase

A method for synthesizing a portion of an oligonucleotide of defined sequence, comprising the steps of:

• (a) combining an oligonucleotide primer and a blocked nucleotide, or a blocked nucleotide precursor that forms a blocked nucleotide substrate in situ, in a reaction mixture in the presence of RNA Ligase, such that a primer-blocked nucleotide product is formed, wherein the blocked substrate comprises a nucleotide to be added to form part of the defined sequence and a 3'-phosphate blocking group attached to the nucleotide effective to prevent the addition of more than one blocked substrate to the oligonucleotide primer;
• (b) removing the blocking group from the 3' end of the primer-blocked nucleotide product using a thermostable 3'-Phosphatase to form a primer-nucleotide product;
• and
• (c) repeating a cycle of steps (a) and (b) using the primer-nucleotide product from step (b) as the oligonucleotide primer of step (a) of the next cycle, without prior purification of the primer-nucleotide product from the reaction mixture.