Systems, apparatus and kits for enzymatic polynucleotide synthesis

A method for synthesizing with a template-free polymerase a plurality of polynucleotides each with a predetermined sequence, the method comprising:

• (1) providing an apparatus comprising
• (a) a plurality of reaction chambers, each reaction chamber having 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 connected to the outlet so that reaction solutions exiting the reaction chamber pass through the filter;
• (b) a waste manifold configured to operatively connect to the outlets of the plurality of reaction chambers such that reaction solutions are removed from the reaction chambers and enter the waste manifold whenever a positive pressure differential is established between the plurality of reaction chambers and the waste manifold;
• (c) a fluid delivery system for delivering reaction solutions to the plurality of reaction chambers, the reaction solutions comprising 3′-O-protected nucleoside triphosphates, a deprotection solution, and a template-free polymerase;
• (d) a user interface for accepting nucleotide sequences of polynucleotides to be synthesized;
• and
• (e) a control system operationally connected to the user interface, the plurality of reaction chambers, the fluid delivery system and the waste manifold, wherein the control system assigns the predetermined sequence of each polynucleotide to a reaction chamber of the plurality of reaction chambers for synthesis;
• (2) repeating for each reaction chamber of the plurality of reactions chambers, under direction of the control system:
• (i) delivering under coupling conditions to the initiators or deprotected elongated fragments a 3′-O-protected nucleoside triphosphate and a template-free polymerase, wherein the coupling conditions include a predetermined coupling incubation time and incubation temperature to allow initiator oligonucleotides or deprotected elongated fragments to be elongated by the 3′-O-protected nucleoside triphosphate to form 3′-O-protected elongated fragments;
• (ii) delivering the deprotection solution to the plurality of reaction chambers so that the 3′-O-protected elongated fragments are deprotected;
• and
• (iii) producing a pressure differential between the plurality of reaction chambers and the waste manifold to remove deprotection solution from the plurality of reaction chambers at a predetermined rate.