Oct 1, 2009
-This process of creating a restriction map is based on the idea that there are
different recognition sites for the various other types of endonucleases.
-Some of the restriction enzymes will leave “sticky” or “staggered” ends that
allow for the creation of recombinant DNA molecules (the staggered ends of each
piece allows for modified reannealing).
-There are three types of restriction enzymes (I, II, III) but only type II is used for
mapping. Type I does not create DNA fragments reproducibly; type III can cut
reproducibly but has a cognate methylase associated with type I and forms a
complex when in the same solution.
-Type II is the only useful one in recombinant technology because it cuts
reproducibly (restriction enzyme activity requires Mg and cleaves the phosphate
bond) and because it does not have a methylase associated with the other types of
restrictions enzymes; it is also the only one that creates staggered ends during the
5’ Protruding Ends/Tails
5’ – G(3’OH) 4 (5’PO )AATC – 3’
3’ – CTTAA(5’PO ) (3’OH)G – 5’
-the staggered ends are palindromic and allow for the reannealing of the ends, the
conditions for reannealing are different from those of digestion.
Palindrome Sequences & Mapping DNA Sequences
-Differ in length and sequence of recognition sites; the shorter recognition sites
(i.e. 4bp vs. 6bp)