Increasing long-sequence yields in template-free enzymatic synthesis of polynucleotides

A method of synthesizing a polynucleotide having a predetermined sequence, the method comprising:

• (a) providing an initiator attached by a 5′ end to a solid support and having a 3′-terminal nucleotide with a free 3′-hydroxyl;
• (b) repeating cycles of
• (i) contacting under elongation conditions the initiator or elongated fragments having free 3′-O-hydroxyls with a 3′-O-blocked nucleoside triphosphate and a template-independent DNA polymerase so that the initiator or elongated fragments are elongated by incorporation of a 3′-O-blocked nucleoside triphosphate to form 3′-O-blocked elongated fragments, and
• (ii) deblocking the 3′-O-blocked elongated fragments to form elongated fragments having free 3′-hydroxyls, until polynucleotides having the predetermined sequences are formed;
• (c) generating double stranded polynucleotides by annealing primers to the 3′-ends of the polynucleotides formed in (b) and extending the primers with a template-dependent polymerase to create reverse complements of the polynucleotides;
• (d) providing reaction conditions with a hybridization stringency that dissociate failure sequences among the double stranded polynucleotides;
• and
• (e) digesting strands of the dissociated double stranded polynucleotides.