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.
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