chapter  9
4 Pages

Molecular Cloning

The exogenous DNA of the donor organism is digested by the same restriction enzyme as that used for the linearization of the vector. The different fragments obtained thus also have at their ends part of the DNA sequence recognized by the restriction enzyme. When the open, linearized plasmid and the digested fragments of the donor DNA meet each other, there is hybridization (formation of hydrogen bonds) of complementary sticky ends, following the laws of complementarity of bases (A/T, C/G). An enzyme, ligase, allowing the formation of a covalent bond between these hybridized DNA fragments is then added to ligate the foreign DNA fragment to the plasmid. The effect of the ligase is to create phosphodiester bonds between the 5'-P and 3'-OH ends rendered adjacent by the hybridization of sticky ends of the fragment and the plasmid. The plasmid obtained, which is again circular, is called recombinant if it has integrated an insert.