Bio-Rad DCode™ Universal Mutation Detection System - Manual (Page 13)

Fig. 4.1. A 299 base-pair betaglobin sequence (exon 1).
Figure 4.2 shows the melting profile for the betaglobin sequence graphed using the 50%
melted probability. The 50% melting probability is typically used for analyzing melt profiles
for denaturing gel electrophoresis. From the melting profile, it can be seen that there are two
low-temperature melting regions and one high-temperature melting region in the middle of the
sequence. A 40 base-pair GC clamp is needed to create a region of high melting temperature
above 80 °C. The GC clamp places the sequence of interest into the lowest melting region and
the mutations in this region can be detected by denaturing gradient gels.
2,3
Fig. 4.2. The melting profile for the example betaglobin sequence.
The GC clamp can be added to the 5' or 3' end of the sequence. In this example, the
40 base-pair GC clamp is added to the 3' end of the sequence using lower case letters
(Figure 4.3).
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