DNA ligation by selection.

Abstract

resultant fragments is further destroyed at its unique restriction-purification site. The crude digestion products are ligated in situ to generate a single plasmid containing the two fragments that can be selected for directly. Two of the two-fragment plasmids are then ligated to make a plasmid with a four-fragment insert, and the process is continued recursively to lengthen the size of the insert in each cycle. Although the method has been developed to stitch together synthetic genes, a modified version could as well be used to ligate multiple DNA fragments from other sources (e.g., PCR). All enzymes were obtained from New England Biolabs (Beverly, MA, USA) and used as recommended. Molecular biological techniques were performed using standard protocols (2). Synthetic DNA fragments were designed and prepared by a modification of reported methods (3–5). A linker duplex for uracil-DNA glycosylase-based ligation-independent cloning (UDG-LIC) (6,7) comprised of oligonucleotides UDG-L1 and UDG-L2, which contained a 5′ overhang [nucleotides (nt) 1–4] compatible with HindIII and a 3′ EcoRI overhang (nt 66–69), was introduced into HindIII/EcoRI-digested pUC18 to give pKOS293-118-72. The linker contained a SacI site (nt 36–41), a NotI site (nt 6–13), and two N. BbvCIA sites (nt 14–20 and 58–64), and upon introduction destroyed the HindIII site. The chloramphenicol resistance gene (CmR) was amplified from pACYC184 with the primers Cm-forward and CmDNA ligation by selection