Method for synthesizing nucleic acids, in particular long nucleic acids, use of said method and kit for implementing said method
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.
1. A method for the synthesis of a nucleic acid sequence, comprising:
a) attaching initial nucleic acid fragments or elongated fragments to a first support;
b) contacting the attached initial nucleic acid fragments or elongated fragments with a modified nucleoside triphosphate and a template-independent DNA polymerase so that initial nucleic acid fragments or elongated fragments are elongated by a modified nucleotide, wherein the modified nucleotide comprises a protective group that prevents multiple additions of nucleotides;
c) deprotecting the elongated fragments;
d) repeating steps b) and c) until a nucleic acid sequence is obtained;
e) contacting the elongated fragments with a modified nucleoside triphosphate and a template-independent DNA polymerase so that elongated fragments are elongated by a modified nucleotide, wherein the modified nucleotide comprises a protective group;
f) detaching the elongated fragments from the first support;
g) purifying elongated fragments; and
h) deprotecting the protection groups.
2. The method of claim 1 , further comprising amplifying said released elongated fragments.
3. The method of claim 1 , wherein said template-independent DNA polymerase is a terminal deoxynucleotidyl transferase.
4. The method of claim 1 , wherein said modified nucleoside triphosphate is a 3′-O-protected nucleoside triphosphate.
5. The method of claim 1 , wherein said step of purifying comprises specifically binding a 3′-O-protection group to said second support.
6. The method of claim 1 , wherein said modified nucleoside triphosphate has a 3′-O-protection group and a protected nitrogenous base.
7. The method of claim 1 , wherein said protective group in step e specifically binds to a second support and prevents multiple additions of nucleotides.
8. The method of claim 1 , wherein said protective group in step e is a first protective group that specifically binds to a second support and said modified nucleotide further comprises a second protective group that prevents multiple additions of nucleotides.
9. The method of claim 1 , wherein said protective group in step e is a first protective group that prevents multiple additions of nucleotides and said modified nucleotide further comprises a second protective group that specifically binds to a second support.