( e 5' clamped amplicon contained three m...)A Multiple Mutation Model System as a Test Development and Training Tool for Denaturing Gradient Gel Electrophoresis

e 5' clamped amplicon contained three melting domains with a ten degree difference at it's 3' end and a 2 degree difference at the 5' end. DGGE detected 7 of the 16 mutations with the majority of the detected mutations located in the lowest melting domain.(Figure 2b) The presence of a HinFI restriction enzyme site 5' to the lowest melting domain allowed the design of two new primers which excluded this lowest melting domain and yielded a more uniform melting profile. The first primer re-design used the original 5' GC-clamped primer and a new non-clamped primer overlapping the HinFI site (termed the 5' HinFI amplicon) (Figure 3a). The second re-design used a non-clamped version of the 5' primer and a 3' GC-clamped primer proximal to the HinFI site (termed the 3' HinFI amplicon) (Figure 3b). A third primer pair was synthesized to include the region of exon 4 excluded by the other primer designs (termed the 3' overlap amplicon) (Figure 3c). DGGE of the 5' HinFI amplicon detected 8 of the 12 mutants known to be present in the amplified product. DGGE of the 3' HinFI amplicon detected 9 of the 12 mutants known to be present. DGGE of the 3' overlap amplicon detected all 5 of the mutations known to be present in the amplicon. Figure 4 depicts a typical gel result following electrophoresis of exon 4 fragments through a DGGE gel. The numbers above the lanes correspond to the mutations detected.

Discussion
DGGE compares favorably to other gene scanning techniques with respect to detection rate, robustness and size of analyzed fragment. An important advantage of DGGE is the possibility to design primers and gel conditions to maximize the detection of alterations in advance.
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