Restriction
mapping
Restriction mapping is a useful
way to characterise a particular DNA molecule. It enables us to locate and
isolate DNA fragments for further study and manipulation. The relative location
of different restriction enzyme sites to each other are determined by enzymatic
digest of the DNA with different restriction enzymes, alone and in various
combinations (ee restriction
enzymes web notes). The digested DNA is separated by gel electrophoresis
and the fragment sizes that have been generated are used to build the 'map'
of sites of the fragment. The map lets us know 'where we are' in the linear
DNA macromolecule.
Remember, not all DNA is linear.
For circular DNA molecules (like plasmids) restriction enzymes that cut
at a single site will generate a linear molecule, which will run as a single
band in gel electrophoresis.
Single and double digests
of DNA
Fig 7-24, Lodish et al. (4th ed)
After digestion with each enzyme,
and with both enzymes, the fragments are analysed by agarose gel electrophoresis.
The separated fragments are typically visualised with ethidium bromide/UV light and the sizes of the
fragments determined by comparison with standard DNA molecular weight markers.
Agarose gel electrophoresis
of uncut and digested DNA,
gel stained with ethidium
bromide and visualised with UV light
M: DNA molecular weight
markers
U: uncut DNA
E: EcoRI
digested DNA
H/S: HindIII
+ SalI digested DNA
Can you determine what type
of DNA is being analysed on this gel?
Another method used for restriction
mapping:
Analysing partial digest of
an end-labelled molecule
Fig 7-25, Lodish et al. (4th ed)
The digested DNA is separated
by gel electrohoresis and the DNA fragments visualised by autoradiography
(the gel is exposed to film). The positions of the multiple recognition
sites for enzyme II are inferred from
the lengths of the labeled pieces.
Using restriction enzyme maps for analysing
Recombinant DNA
- Checking the size of the
insert
- Checking the orientation
of the insert
- Determining pattern of
restriction sites within insert DNA
Detailed restriction
maps are available for each cloning vector
Checking insert and orientation
by restriction mapping
- the orientation of the
fragment can be determined by cutting with an enzyme that recognises an
internal restriction site within the insert and with one that recognises
a site in the polylinker of the vector. See the agarose gel example above that shows the
digest of a plasmid vector and recombinant DNA (different clones of the plasmid
vector with insert in the EcoRI site but in opposing orientations).
For a tutorial on how to draw
restriction maps, please click on this link How
to draw Restriction Maps