US 11,905,541 B2
Efficient product cleavage in template-free enzymatic synthesis of polynucleotides
Sandrine Creton, Le Kremlin-Bicêtre (FR)
Assigned to DNA Script SAS, Le Kremlin-Bicêtre (FR)
Filed by DNA Script SAS, Le Kremlin-Bicêtre (FR)
Filed on Jun. 10, 2022, as Appl. No. 17/837,660.
Application 17/837,660 is a continuation of application No. 16/981,595, granted, now 11,359,221, previously published as PCT/EP2020/053417, filed on Feb. 11, 2020.
Claims priority of application No. 19305174 (EP), filed on Feb. 12, 2019.
Prior Publication US 2023/0105977 A1, Apr. 6, 2023
This patent is subject to a terminal disclaimer.
Int. Cl. C12P 19/34 (2006.01); C12P 19/38 (2006.01); C12Q 1/68 (2018.01); C12N 9/12 (2006.01); C12Q 1/6806 (2018.01); C12Q 1/6876 (2018.01); C12N 15/10 (2006.01); C12P 19/14 (2006.01)
CPC C12P 19/14 (2013.01) [C12P 19/34 (2013.01)] 15 Claims
 
1. A method of synthesizing a polynucleotide having a predetermined sequence, the method comprising the steps of:
a) providing an initiator having a 3′-penultimate deoxyinosine and a 3′-terminal nucleotide having 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 the 3′-O-blocked nucleoside triphosphate to form 3′-O-blocked elongated fragments, and (ii) deblocking the elongated fragments to form elongated fragments having free 3′-hydroxyls, until the polynucleotide is formed, wherein said template-independent DNA polymerase is a terminal deoxynucleotidyl transferase; and
c) treating the polynucleotide with an endonuclease V activity to cleave the polynucleotide from the initiator, wherein said endonuclease V activity is provided by a prokaryotic endonuclease V.