Method for synthesizing nucleic acids, in particular long nucleic acids, use of said method and kit for implementing said method
Inventors
Ybert, Thomas • Gariel, Sylvain
Assignees
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Publication Number
US-11685941-B2
Publication Date
2023-06-27
Expiration Date
Abstract
The invention relates to a method for synthesising long nucleic acids, including at least one cycle of elongating initial fragments of nucleic acids, including a) a phase comprising the enzymatic addition of nucleotides to said fragments, b) a phase comprising the purification of the fragments having a correct sequence, c) an optional phase of enzymatic amplification, each cycle being performed in a reaction medium which is compatible with enzymatic addition and amplification, such as an aqueous medium, the synthesis method also comprising, at the end of all the elongation cycles, a last step of final amplification. The invention also relates to the use of the method for the production of genes, or sequences of synthetic nucleic acids, DNA or RNA. The invention further relates to a kit for implementing said method.
Core Innovation
The invention relates to a cyclic enzymatic method for the synthesis of a nucleic acid sequence, including genes, by repeated elongation cycles. Initial nucleic acid fragments or elongated fragments are attached to a first support and are elongated by contacting with a modified nucleoside triphosphate and a template-independent DNA polymerase. The modified nucleotide comprises a protective group that prevents multiple additions of nucleotides.
The method is performed by repeating elongation and deprotecting steps until a nucleic acid sequence is obtained. Elongated fragments are then contacted again with a modified nucleoside triphosphate and a template-independent DNA polymerase to elongate elongated fragments, followed by detaching the elongated fragments from the first support. The invention includes purifying elongated fragments and deprotecting the remaining protection groups.
The document further describes selective retention and wide removal of incorrect-sequence fragments through attachment to a first and then a second support, often magnetic. Supports are configured so that fragments are noncovalently or orthogonally attached and detached by physicochemical condition changes, enabling removal of incorrect-sequence fragments and purification of the correct-sequence fragments. The invention also describes suitable template-independent polymerization enzymes, including terminal deoxynucleotidyl transferase (TdT), and protected 3′-end nucleotides to limit uncontrolled additions.
Claims Coverage
The partial content provides one independent claim (independent_claim_id clm-00001). Across the independent claim and its dependent refinements, the claim coverage includes five inventive features centered on cyclic elongation, protective-group chemistry, and post-elongation handling.
Cyclic synthesis with support-attached elongation fragments
Attaching initial nucleic acid fragments or elongated fragments to a first support; contacting the attached fragments with a modified nucleoside triphosphate and a template-independent DNA polymerase for elongation; deprotecting the elongated fragments; and repeating the elongation and deprotection steps until a nucleic acid sequence is obtained.
Protective-group controlled template-independent nucleotide addition
Elongating the initial and subsequent fragments by contacting with a modified nucleoside triphosphate and a template-independent DNA polymerase, wherein the modified nucleotide comprises a protective group that prevents multiple additions of nucleotides.
Deprotection, detachment from the first support, purification, and final deprotection
After the sequence is obtained, contacting the elongated fragments again for elongation, detaching the elongated fragments from the first support, purifying elongated fragments, and deprotecting the protection groups.
Protective-group chemistry prevents multiple additions
Using modified nucleoside triphosphate comprising a protective group that prevents multiple additions of nucleotides, including embodiments where the modified nucleoside triphosphate comprises a 3′-O-protected nucleoside triphosphate and/or includes a protected nitrogenous base.
Optional amplification of released elongated fragments
Including an embodiment where the method further amplifies released elongated fragments.
Overall, the claims coverage focuses on cyclic elongation with support-attached fragments, using template-independent DNA polymerase and protective-group modified nucleosides that prevent multiple additions, followed by deprotection, detachment from the first support, purification, and final deprotection.
Stated Advantages
Not explicitly described in patent.
Documented Applications
Not explicitly described in patent.
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