Bio-Rad CHEF Mapper® XA System - Manual (Page 45)

The importance of using the appropriate switch interval when analyzing pulsed field data
is apparent from Figure 7.3. An Mlu I digest of human DNA was run using three different
pulsed field conditions prior to blotting and hybridization. Each of the gels resolved a differ-
ent size range of DNAs, as indicated by the marker sizes in the figure. Under conditions where
only fragments from 200 to 600 kb were resolved, four bands were visible on the autoradio-
gram. When longer switch intervals were employed, so that fragments up to 1,600 kb are
separated, three bands are visible. In this experiment the central band appears darker than the
other two. Often, however, they are of uniform intensity and provide no clue to the doublet
lurking there. Using conditions which resolve bands above 2 mb, only a smear is seen on the
autoradiogram, in a position corresponding to the massed S. cerevisiae chromosomes. Thus, to
obtain accuracy in both sizing and in establishing the number of bands in Southern blots, gel
conditions that maximize resolution in the area containing the bands of interest must be used.
Numerous other factors can influence the ability to detect and accurately size bands of
interest in pulsed field gels. The most common reasons for failure include:
• The DNA samples are degraded by nuclease either during the sample preparation or dur-
ing the restriction digestion. Degradation during preparation produces plugs which yield
small DNA when run directly after preparation. Test each new batch of samples for con-
taminating nuclease by performing a mock digestion and running the products on a pulsed
field gel. The mock digestion consists of incubation at the same temperature and time in
a restriction digestion reaction minus the restriction enzyme (i.e., DNA+buffer+BSA).
Samples which are free of nuclease will have almost all the DNA remaining in the plug
or moving with the limiting mobility region of large unresolved DNA.
• Incorrect gel conditions are used. Size markers should indicate that the conditions used
(especially voltage and switch time) were appropriate for resolving the size region of
interest. Each new digest must be analyzed under a variety of gel conditions. Generally,
it is best to start with the conditions which give as broad a range of sizes as possible for
a rough estimate of the number and sizes of bands (this is often facilitated by using a
switch time ramp). Then gels which focus on a narrower size range can be used to more
precisely determine the sizes of the fragments.
• Incomplete restriction digestion can result from protein remaining in the samples or from
incomplete removal of the agents used to prepare the DNA (EDTA, protease, detergent).
In addition, because many of the enzymes used to generate large fragments are sensitive
to methylation, it can be extremely difficult to cut particular methylated sites. Using DNA
prepared from different sources (e.g., fibroblasts instead of blood) can often give differ-
ent length fragments due to tissue-specific methylation patterns.
• Sample overloading. The apparent size of fragments in pulsed field gels is strongly depen-
dent on the amount of DNA loaded in the gel. With increasing amounts of DNA, bands
are retarded in their migration, and indicated larger than true sizes. Figure 7.4 shows
hybridization to two different amounts of the same restriction digest run side by side.
Two distinct bands are visible in lane 1. As expected, an increased amount of DNA leads
to broadening of both bands. Furthermore, the midpoint of each of the broad bands is
shifted higher in the gel. This is particularly clear for the band near the bottom of the
autoradiogram. A lighter exposure of the pattern in lane 2 indicates a larger size for each
of the two bands than is seen in lane 1.
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