Method and kit for regenerating reusable initiators for nucleic acid synthesis

A method for nucleic acid synthesis and regeneration of a reusable initiator for such synthesis, comprising:

• exposing an initiator attached to a solid support for nucleic acid synthesis to a linking nucleotide in the presence of a polymerase so that the linking nucleotide is incorporated to the initiator, the linking nucleotide having a substrate base, a substrate sugar, and a 3′ hydroxyl group;
• exposing the initiator containing the linking nucleotide to nucleotide monomers in the presence of the polymerase, so that a nucleic acid is synthesized and is coupled to the initiator right after the linking nucleotide;
• providing a mono-functional DNA glycosylase, the linking nucleotide with the substrate base being recognizable and excisable by the mono-functional DNA glycosylase;
• subjecting the substrate base to an excision treatment with the mono-functional DNA glycosylase, so that the substrate base is excised by the mono-functional DNA glycosylase to generate an abasic site;
• providing an abasic site endonuclease, the resulting abasic site being recognizable and the substrate sugar being cleavable by the abasic site endonuclease;
• subjecting the abasic site to a cleavage treatment with the abasic site endonuclease, so that the substrate sugar and the backbone of the nucleic acid at the abasic site are both cleaved to release the nucleic acid from the initiator, so that a 3′-terminal nucleotide of the initiator has a 3′ phosphate group, and so that a 5′-terminal nucleotide of the synthesized nucleic acid has a 5′ phosphate group;
• providing a 3′ phosphatase activity-possessing enzyme;
• and
• subjecting the 3′-terminal nucleotide of the initiator to a dephosphorylation treatment with the 3′ phosphatase activity-possessing enzyme, so that the 3′ phosphate group of the 3′-terminal nucleotide of the initiator is converted back to the original 3′ hydroxyl group.