Bio-Rad DCode™ Universal Mutation Detection System - Manual (Page 15)

Fig. 4.5. The melting profiles for a mutant and wild-type sequence from a portion of the beta-
globin gene (exon 1).
Sometimes the addition of a GC-clamp to the end of a sequence does not work and
optimizing the location of the GC-clamp is required. Figure 4.6 shows the melting profile for
a 300 base-pair sequence with a 40 base-pair GC clamp attached on the 3' end using the 50%
probability. From the melting profile, it can be seen that there are two low melt regions (bases
1–100, 155–250) and a single high melt region in the middle of the sequence (bases 100–155).
This melt profile may cause mutations in the 170–260 base-pair region to be undetected on a
denaturing gradient gel. It is recommended to use a melt profile that is flat or is decreasing in
temperature from the GC clamp so that the region of interest is in the lowest melting domain.
In this example the sequence may have to be split up into two sequences. The GC clamp can
be added to the 3' end at base 155 for screening mutations in the 1–155 base region and added
to the 5' end at base 100 for screening mutations in the 155–250 base region.
Fig. 4.6. The melting profile of a 300 base-pair sequence with a 3' GC clamp attached.
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