Biology Reference
In-Depth Information
Restriction-site mapping
is a relatively simple technique that provides a physi-
cal map of the sites in the DNA at which different restriction enzymes cut. One
method for constructing restriction maps involves digesting the DNA with a
series of different endonucleases in separate reactions. The products of each
digestion are electrophoresed on agarose or polyacrylamide gels. DNA marker
fragments of known size are electrophoresed in lanes adjacent to the DNA
being examined to provide estimates of the lengths of the DNA fragments gen-
erated. DNA molecular markers of known size are available from commercial
sources. The DNA is stained with EtBr, and the bands that were produced are
examined under UV light and photographed.
After the single digestions are done, the DNA can be digested simultane-
ously with two restriction enzymes. Again, the size of the digestion products
is analyzed by gel electrophoresis, using size markers and the samples of the
first digest for comparison. If the products of the first and second digests are
electrophoresed in adjacent lanes on the gel, it is possible to detect small dif-
ferences in migration rate. Maps are built up from these data by a process of
trial and error and basic logic. Based on the sizes of the DNA fragments gener-
ated, it is possible to define the
order
of the restriction sites and the approxi-
mate
distances
between them. The resolution of map distances depends on the
accuracy with which the sizes of the DNA fragments can be estimated relative
to those of the size markers. However, restriction maps are rarely accurate to
<
100-200 bp.
Restriction maps of DNA provide the experimenter with useful information
for additional experiments. Furthermore, such restriction site maps can be used
as in systematics or population genetics studies (see Chapters 12 and 13). You
will use many of the techniques described in this chapter for other purposes,
including preparing a genomic library, as described in Chapter 6.
General References
Ausubel, F. M., R. Brent, R. E., Kingston, D. M., Moore, J. G. Seldman, and J. A., Smith, et al., Eds.,
Current Protocols in Molecular Biology
. Updated regularly since 1987. Humana Press, Totowa, NJ.
Online versions are also available so that up-to-date protocols are provided.
Barker, K., (2005).
At the Bench: A Laboratory Navigator
, 2nd ed. Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, NY.
Berger, S.L., and Kimmel, A.R., Eds., (2005).
Guide to Molecular Cloning Techniques. Methods in
Enzymology
, Vol. 152, Academic Press, San Diego, CA. An electronic version is available.
Brown, T.A., Ed., (1998).
Molecular Biology LabFax, Volume 1: Recombinant DNA
(2nd ed.).
Academic Press, San Diego, CA.
Brown, T.A., Ed., (2002).
Essential Molecular Biology. A Practical Approach
, Vols. I and II (2nd ed.).
IRL Press, Oxford, UK.
