Restriction mapping


Restriction mapping is the process of generating a map of a DNA molecule, either linear or circular (a plasmid), indicating where the sites for certain restriction enzymes ('restriction sites') are using the data from restriction digests with those enzymes.

An example with a linear DNA molecule:

A linear DNA molecule is digested with the restriction enzyme HindIII to yield 6.2kb and 0.8kb fragments. This tells us that the overall size of the molecule is 7kb and the restriction site of HindIII (H) is 0.8kb of the way in:

----------|---------------------------
0.8kb...H...........6.2kb.............

A second single-digest is performed of the same molecule, this time by the enzyme Sal I, yielding 5.8kb and 1.2kb fragments. There are two possible restriction maps for this second digest [A and B]:

--------------|----------------------- .... [A]
.....1.2kb...S..........5.8kb.........


-----------------------|-------------- .... [B]
..........5.8kb.........S.....1.2kb...

If possibility A is correct, then a double-digest of HindIII and Sal I should yield this restriction map:

---------|----|-----------------------
0.8kb..H....S..........5.8kb........
...........0.4kb.....

If possibility B is correct, then a double-digest of HindIII and Sal I should yield this map instead:

--------|--------------|--------------
0.8kb.H......5kb....S.....1.2kb...

The only way to know is by performing a double digest with both restriction enzymes, and identifying the size of fragments garnered.


An example with a circular DNA plasmid:

A plasmid is 11kb in size. On their own, EcoR1, Taq1 and BamH1 each produce a single 11kb fragment - i.e. they cut the plasmid once to linearise it.

On double-digest with EcoR1 and Taq1, 6kb and 5kb fragments are produced. There are two possible restriction maps from this double-digest, A and B (the plasmid is linearised in these diagrams for ease of understanding; imagine that the end of the line links up to the beginning to make a circle):

|---------------------------------------------|-------------------------------------------------------------........[A]
E....................5kb............................T.................................6kb....................................

|---------------------------------------------|-------------------------------------------------------------........[B]
T.....................5kb............................E................................6kb....................................

On double-digest with EcoR1 and BamH1, 8kb and 3kb fragments are produced. Again, there are two possible restriction maps from this double-digest, C and D:


|----------------------|------------------------------------------------------------------------------------........[C]
E.........3kb...........B..............................................8kb....................................................


|----------------------|------------------------------------------------------------------------------------........[D]
B.........3kb...........E..............................................8kb....................................................

On double-digest with Taq1 and BamH1, 9kb and 2kb fragments are produced. Again, two possible restriction maps, E and F:


|--------------|--------------------------------------------------------------------------------------------........[E]
T.....2kb.....B..............................................9kb..............................................................

|--------------|--------------------------------------------------------------------------------------------........[F]
B.....2kb.....T..............................................9kb..............................................................

The question is, how do we map these sites relative to each other? The answer is below:

|----------------------|-----------------|------------------------------------------------------------------
E.........3kb...........B......2kb........T...................................6kb.........................................