DNA clean-up
Fragment isolation
Competent cells
Probe Labeling
Probe Detection


The enzyme DNA ligase can covalently join DNA fragments with compatible ends, e.g to insert a donor DNA fragment into a vector. The commonly used enzyme is isolated from bacteriophage T4 infected cells and is commercially available.
Ligases are efficient at sealing the broken phosphordiester bonds in an annealed pair of cohesive (or "sticky") ends, essentially the reverse of an restriction enzyme action. T4 ligase can even ligate (with low efficiency) one blunt end to another.

  See the Movie about
in vitro Ligation

Read the Movie Text


Compatible ends

Some restriction enzymes make strand incisions immediately opposite one another, producing "blunt end" DNA fragments. Most enzymes make slightly staggered incisions, resulting in "sticky ends", out of which one strand protrudes (3' or 5' overhangs)
(remember also the movie about some commonly used restriction enzymes that you have seen).
Because recognition sequences and cleavage sites differ between restriction enzymes, the length and the exact sequence of a sticky-end "overhang", as well as whether it is the 5' end or the 3' end strand that overhangs, depends on which enzyme produced it.
Base-pairing between overhangs with complementary sequences enables two fragments to be joined by DNA ligase.
A sticky-end fragment can be ligated not only to the fragment from which it was originally cleaved, but also to any other fragment with a compatible sticky end. Restriction enzymes having different recognition sites may have identical (compatible!) sticky overhangs.
 If a restriction enzyme has a non-degenerate palindromic cleavage site (or more precisely: when it generates non-degenerate palindromic overhangs), all ends that it produces are compatible. Ends produced by different enzymes may also be compatible.
The size of the fragments is not important: for example, in case of ligation of a donor fragment to a vector fragment, the insert may be much bigger than the vector..

Degeneracy: When more than one nucleotide is possible at a particular position in the recognition site of a restriction enzyme (ambiguity of recognition).