IP Library › Granted Patent US 12,421,628
Granted Patent B2
US 12,421,628 · App. 17/259,839 · Granted Sep 23, 2025

Massively parallel enzymatic synthesis of nucleic acid strands

Inventors: Adrian Horgan (Le Kremlin-Bicêtre, FR); Xavier Godron (Le Kremlin-Bicêtre, FR); Thomas Ybert (Paris, FR); Robert Nicol (Le Kremlin-Bicêtre, FR)
Assignee: DNA Script
C40B50/06C12N15/1068G16B50/00
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Quick Facts
Patent No.
US 12,421,628
App. No.
17/259,839
Filed
Jan 12, 2021
Granted
Sep 23, 2025
Kind
B2
Art Unit
1684
USPC
506/26
Abstract

The invention is directed to methods for massively parallel template-free enzymatic synthesis of a plurality of different polynucleotides of predetermined sequences. In one aspect, methods of the invention employ large scale arrays of reaction sites each associated with at least one working electrode for controlling deprotection and deblocking steps at predetermined user selected sites. In another aspect, the invention provides template-free enzymatic synthesis with proofreading, wherein completed polynucleotides at predetermined reaction sites are sequenced using a sequencing by synthesis technique, particularly employing electrochemically labile blocking groups.

Claims (6)

1. A method of synthesizing a plurality of polynucleotides having predetermined sequences, the method comprising the steps of: (a) providing a spatially addressable array of reaction sites, wherein each reaction site is operationally associated with at least one working electrode and has disposed thereon initiators attached by their 5′-ends and having a 3′-O-electrochemically labile protecting group; (b) performing for each kind of nucleotide a cycle of (i) deprotecting initiators or elongated fragments at electrodes at predetermined addresses by generating a voltage difference between each of the electrodes at the predetermined addresses and a reference electrode so that the electrochemically labile protecting group is cleaved, thereby generating free 3′-hydroxyls on the initiators or elongated fragments at the electrodes of the predetermined addresses, (ii) contacting under elongation conditions the electrodes with a 3′-O-electrochemically labile-protected nucleoside triphosphate and a template-independent DNA polymerase so that the initiators or elongated fragments at the predetermined addresses are elongated by the incorporation of a 3′-electrochemically labile-protected nucleoside triphosphate to form 3′-O-electrochemically labile-protected elongated fragments; and (c) repeating step (b) until the array of polynucleotides of predetermined sequences is completed, wherein said electrochemically labile protecting group is an amino group and wherein said spatially addressable array of reaction sites is integrated in a semiconductor device.

2. The method of claim 1 , wherein said voltage difference between said electrodes at said predetermined addresses and a reference electrode activates an electroactive agent at said predetermined addresses which changes the pH at said predetermined addresses, thereby cleaving said electrochemically labile protecting group.

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

4. The method of claim 2 , wherein said template-independent DNA polymerase is a terminal deoxynucleotidyl transferase (TdT).

5. The method of claim 1 , wherein said semiconductor device is a Complementary Metal Oxide Semiconductor (CMOS) device.

6. The method of claim 5 , wherein said template-independent DNA polymerase is a terminal deoxynucleotidyl transferase (TdT).

Assignments (1)
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Mar 1, 2022
From: HORGAN, ADRIAN; GODRON, XAVIER; YBERT, THOMAS; NICOL, ROBERT
To: DNA SCRIPT
Reel/Frame 059135/0080 →
Priority Claims (1)
EP 18306000 · Jul 23, 2018 · regional
Continuity (1)
Related Publication 20210332351A1 · Oct 28, 2021
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