Granted Patent B2

US 12,618,092 · App. 17/802,485 · Granted May 5, 2026

Inventors: Adrian Horgan (Le Kremlin-Bicêtre, FR); Xavier Godron (Le Kremlin-Bicêtre, FR)

Assignee: DNA Script

C12P19/34B01J19/0046C12N9/1264B01J2219/00423B01J2219/00452B01J2219/00695B01J2219/00722C12Y207/07031

Abstract

Claims (20)

1 . 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.

2 . The method of claim 1 , wherein said cycles and said short cycles have the same duration.

3 . The method of claim 1 , further including a step of cleaving said polynucleotides from said synthesis supports.

4 . The method of claim 1 , wherein said template-free polymerase is a terminal deoxynucleotidyl transferase (TdT).

5 . The method of claim 4 , wherein said TdT is a TdT variant having an amino acid sequence at least 90 percent identical to SEQ ID NO: 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30, or an amino acid sequence at least 90 percent identical to SEQ ID NO: 16, 17, 18, 19, or 20 and having a Q4E/S/D/N substitution,

wherein the TdT variant (i) is capable of synthesizing a nucleic acid fragment without a template, and (ii) is capable of incorporating a 3′-O-protected-nucleotide onto a free 3′-hydroxyl of a polynucleotide.

6 . The method of claim 1 , wherein protease treatment is performed either in each cycle or periodically during the synthesis process.

7 . The method of claim 1 , further comprising, after completing the synthesis of the polynucleotides,

(i) cleaving the completed polynucleotides from the solid supports, and

(ii) purifying the completed polynucleotides.

8 . The method of claim 7 , wherein the cleaving and the purifying are performed in the reaction chambers.

9 . The method of claim 7 , wherein the polynucleotides still attached to the solid supports are transferred to other reaction vessels for the cleaving and the purifying.

Assignments (1)

Priority Claims (1)

Continuity (1)

References Cited (57)