A nucleic acid assembly method using Type IIB enzymes

Abstract

The present invention relates to a method for assembling nucleic acid, comprising providing an extended oligonucleotide comprising at least two repeating units, a fragment of the sequence of interest and a recognition site of a Type IIB or IIB-like restriction enzyme, digesting said oligonucleotide to generate cleaved sequences of interest with single-stranded overhangs and annealing two or more cleaved sequences where the overhangs are complementary. Preferably the overhangs are generated at each end, are three or more base in length and non-palindromic. The method is plasmid-free. The digestion and annealing steps are repeated. The annealed sequences are ligated to form a longer length linear DNA or assembled using polymerase cycling assembly. The method can be performed in a single pot reaction.