Ligation of oligonucleotides

Abstract

A method to produce a target double-stranded (ds) DNA polynucleotide by annealing single-stranded oligonucleotides (ss oligos) and ligating a ds assembly, comprising the following steps: a) annealing at least 2 ss oligos comprising matching sequences for hybridization to produce a starter ds oligo with two overhangs, and hybridizing one of said overhangs with a nucleotide (NA) building block, preferably any one of a ss oligo, ds oligo or ds polynucleotide, thereby obtaining a ds assembly with two overhangs of at least 2 nucleotides (nt), which ds assembly comprises at least one nick in the ds part of the ds assembly; and b) hybridizing to the ds assembly at least one extender ss oligo (ExtON), which comprises a sticky terminal sequence of at least 2 nt on one side, and an identifiable tail on the other side, which sticky sequence is matching for hybridization with an overhang of the ds assembly, thereby obtaining an extended ds assembly; and c) ligating the nucleotides within the extended ds assembly by a ligation reaction, thereby resolving said at least one nick; and d) removing said at least one ExtON from the extended ds assembly upon said ligation reaction, thereby producing the target ds polynucleotide.

Dependencies are inferred from the claim text.