Ligation of Nonmatching DNA Molecule Ends

Abstract

T4 DNA ligase can promote the in vitro ligation of blunt DNA ends to ends bearing a 2-nucleotide single-stranded protrusion. This was shown by digestion of plasmids pBR322 and pSP71 with the appropriate restriction enzymes followed by recircularization of the plasmids and transformation of Escherichia coli . It could be ruled out that such nonmatching ligations are due to the presence of contaminating nucleases. The efficiency of ligation is of the same order of magnitude as that obtained with blunt end ligations. The interaction of a number of different combinations of blunt and sticky ends, the latter bearing both 3′ and 5′ protrusions, was investigated. Ligation of nonmatching ends was shown to take place in all cases. Several ligation junctions were sequenced, showing that during the ligation process the 2-nucleotide protrusion is trimmed away. In two instances the ligation event was accompanied by the specific loss of either 3 or 15 nucleotide pairs as well as the protrusion. An intermolecular ligation involving nonmatching ends was also performed, demonstrating that this form of ligation can be usefully employed in molecular cloning experiments.