Nonenzymatic plasmid ligation mediated by minor groove-binding molecules.
DNA ligation is the weak link in the chain of gene cloning. We have developed a straightforward nonenzymatic alternative to this reaction that employs easily available commercial reagents. The method uses the affinity of distamycin for the minor groove to join DNA ends together. Phosphodiester bonds are formed after cyanoimidazole-promoted phosphate activation in the presence of manganese(II) cations. When transfected into eukaryotic cells, the chemically ligated plasmid is transcribed even more efficiently than after enzymatic ligation. At present this technique compares favorably with T4 ligation for AT-rich cohesive termini, but in principle it could be extended to any restriction site.
Laboratoire de Chimie Génétique, CNRS URA 1386, Faculté de Pharmacie de Strasbourg, Illkirch, France.
G
G
Zuber
J P
JP
Behr
D003001:Cloning, Molecular / Q000379:methods*; D011088:DNA Ligases; D004269:DNA, Bacterial / Q000737:chemistry*; D004214:Distamycins / Q000737:chemistry*; D008024:Ligands; D010957:Plasmids / Q000737:chemistry*; D014158:Transcription, Genetic; D014162:Transfection
D004269:DNA, Bacterial; D004214:Distamycins; D008024:Ligands; D011088:DNA Ligases