( the DCode universal mutation detecti...)DGGE Analysis Using the DCode System, Rev B

Methods
The test samples consisted of a wild-type and three mutant DNA samples from the -globin gene. The three single-base heterozygous mutations were IVS1-1 (G >A), IVS1-6 (T >C) and IVS1-110 (G >A). Genomic DNA from both wild-type and mutant samples was amplified by PCR, creating an end product of 281 base pairs.

A 16 x 16 cm, 1 mm thick, 8% acrylamide/bis (37.5:1) gel with a parallel denaturing gradient range of 3555% in 1x TAE buffer (40 mM Tris, 20 mM acetic acid, 1 mM EDTA) was used. The gradient gel was cast using Bio-Rads Model 475 gradient delivery system. Mutant sample (200300 ng) was mixed with 5 l of 2x gel loading dye (70% glycerol, 0.05% Bromophenol Blue, 0.05% Xylene Cyanole, 2 mM EDTA) and electrophoresed on the DCode system at 130 V for 3 hr at a buffer temperature of 56C. After electrophoresis, the gel was stained in 50 g/ml ethidium bromide in 1x TAE buffer for 5 min and destained in buffer for 10 min. The gel was imaged under UV transillumination.

Results and Discussion
Figure 1 shows -thalassemia samples run on a parallel DGGE gel. The three heterozygous samples in lanes 13 were resolved into two heteroduplex and two homoduplex bands. The heteroduplex bands (upper bands) migrated slower than the corresponding homoduplex bands (lower bands). The wild-type sample in lane 4 was resolved as one band, and it migrated to the same distance as the wild-type band in the mutant samples. The ability to resolve the heteroduplex fragments and the mutant homoduplex fragment in the mutant samples makes it possible t
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