Method and apparatus for enzymatic synthesis of polynucleotides

A method for synthesizing with a template-free polymerase a plurality of polynucleotides each with a predetermined sequence, wherein the template-free polymerase has reduced coupling efficiency at one or more inefficiency motifs, the method comprising the steps of:

• (a) providing a reaction chamber for each polynucleotide of the plurality of polynucleotides, each reaction chamber having disposed therein a synthesis support with initiators attached, wherein each initiator has a free 3′-hydroxyl, and wherein each reaction chamber has an inlet and an outlet and a filter that retains the synthesis support and that is operationally associated with the outlet so that reaction solutions exiting the reaction chamber pass through the filter;
• (b) providing a waste manifold operationally associated with the outlets of the reaction chambers so that whenever a positive pressure differential is created between the reaction chambers and the waste manifold, reaction solutions are removed from the reaction chambers;
• (c) repeating for each reaction chamber, until a polynucleotide of such reaction chamber is complete, cycles of the following reaction steps:
• (i) contacting in a coupling solution the initiator or a deprotected elongated fragment with a 3′-protected nucleoside triphosphate and a template-free polymerase so that the initiator or the deprotected elongated fragment is elongated by the 3′-protected nucleoside triphosphate to form a 3′-protected elongated fragment,
• (ii) deprotecting the 3′-protected elongated fragment with a deprotection solution, and
• (iii) applying a pressure differential between the reaction chambers and the waste manifold to remove reaction solution(s) from the reaction chambers;
• wherein the kind of 3′-protected nucleoside triphosphate contacted in step (i) in a reaction chamber is determined by the predetermined sequence of the reaction chamber, and
• wherein, prior to each cycle, one or more short cycles of step (i) is carried out in a reaction chamber whenever an inefficiency motif is present at a 3′ end of the deprotected elongated fragment of such reaction chamber, wherein the inefficiency motif is CCA, CTA, GCA, GTA or CCT.