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Heteroduplex Analysis Using the DCode System

). The mutations from exon 10 were a heterozygous ΔF508, three base deletion (-CTT), a heterozygous compound sample Q493 (C to T) and ΔF508 (-CTT), a homozygous ΔF508 (-CTT) and a heterozygous ΔI507, three base deletion (-ATC). Samples were provided by Dr. L. Silverman, University of North Carolina School of Medicine (Chapel Hill, NC). Genomic DNA from both wild-type and mutant samples were amplified by PCR to create end products of 289 bp for exon 7 and 369 bp for exon 10.

A 16 cm x 20 cm, 0.75 mm thick, 1x DEM gel made up in 0.6x TBE buffer (54 mM Tris, 54 mM boric acid, 1.2 mM EDTA) was used. 1.5 liters of 0.6x TBE buffer was added to the lower electrophoresis tank and 350 ml of 0.6x TBE buffer was added to the upper chamber in the DCode system. 5 l (200300 ng) of each amplified sample was mixed with 5 l 2x gel loading dye (70% glycerol, 0.05% bromophenol blue, 0.05% xylene cyanol, 2 mM EDTA) and electrophoresed on the DCode system at 100 V for 16 hours at room temperature. After electrophoresis, the gel was stained in 50 g/ml ethidium bromide in 0.6x TBE buffer for 5 minutes and destained in buffer for 10 minutes. The gel was imaged under UV transillumination.


Results and Discussion
Figure 1 shows the mutant and wild-type samples from the cystic fibrosis gene run on the DCode system. The mutant samples from exon 7 (Figure 1.A) in lanes 2 and 3 resolve into two heteroduplex bands (upper bands) and a homoduplex band (lower band). The two heteroduplex bands in the mutant samples from exon 10 (Figure 1.B) in lanes 2, 3 and 5 are resolved from the homoduplex bands. The ΔF508 homozygous mutant in lane 4 has no heteroduplex bands, and the homoduplex bands mig
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