Scarless template-free enzymatic synthesis of polynucleotides

A method of enzymatically synthesizing a plurality of polynucleotides, the method comprising:

• (a) providing a plurality of reaction sites on a solid support;
• (b) immobilizing a plurality of initiators on a surface of the solid support, wherein each initiator comprises a free 3′-hydroxyl group, at least one cleavable group, and at least one enzymatic cleavage site, wherein said at least one enzymatic cleavage site is positioned between the free 3′-hydroxyl group and said at least one cleavable group;
• (c) contacting the surface of the solid support with a 3′-O-protected nucleoside triphosphate and a template-free polymerase under suitable conditions for enzymatic extension of the initiators, wherein incorporation of the 3′-O-protected nucleoside triphosphate results in production of a 3′-O-protected extension product at each of the reaction sites;
• (d) performing one or more cycles of i) deprotecting the 3′-O-protected extension products at the reaction sites, wherein extension products having free 3′-hydroxyl groups are formed at the reaction sites, and ii) contacting the surface of the solid support with another 3′-O-protected nucleoside triphosphate and a template-free polymerase under suitable conditions for enzymatic extension of the extension products having free 3′-hydroxyl groups;
• (e) repeating step (d) until synthesis of the plurality of polynucleotides is completed;
• (f) cleaving said at least one cleavable group, thereby releasing the plurality of polynucleotides from the surface of the solid support;
• and
• (g) enzymatically cleaving said at least one enzymatic cleavage site, thereby removing polynucleotide scars from the plurality of polynucleotides to produce a plurality of scarless polynucleotides, wherein said enzymatically cleaving the at least one enzymatic cleavage site is performed simultaneously with or after said cleaving the at least one cleavable group.