Paul Zoller and Theresa Redila-Flores
Bio-Rad Laboratories, Hercules, California
Heteroduplex Analysis (HA) is a commonly used mutation screening method due to its simplicity. The technique is based on conformational differences in double-stranded DNA caused by the formation of heteroduplex molecules.1 Heteroduplex molecules have a mismatch in the double-strand, causing a distortion in its usual conformation. This distortion or altered conformation can be detected on polyacrylamide gels due to slower migration than the corresponding homoduplex molecules. Heteroduplexes are generated during PCR* of a heterozygous individual or by adding mutant and wild-type DNA in the same PCR reaction or by denaturing and renaturing a mixture of mutant and wild-type DNA. Both mutant and wild-type samples are run on the same gel to analyze differences in mobility. Heteroduplex molecules with as little as one mismatch can show a different mobility in a gel from homoduplex molecules. Polyacrylamide analogs have been developed, such as DEMTM (Detection Enhancing Matrix) from Bio-Rad, which enhance the ability to detect mutations in heteroduplex samples when compared to conventional polyacrylamide gels.2
In this experiment, we show that Heteroduplex Analysis on the DCode universal mutation detection system can be used to analyze mutations in the cystic fibrosis gene.
The test samples consist of wild-type and mutant DNA samples from the cystic fibrosis gene exons 7 and 10. The heterozygous mutations from exon 7 were 1154insTC, two base insertion (+TC) and ΔF311, three base deletion (-TTC). 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 migrate the same as the wild-type. Under these conditions, it was not possible to resolve the two homoduplex bands of the ΔF508 mutant. This mutation may be detected if heteroduplexes are formed by mixing the mutant and wild-type samples together, then denaturing and renaturing the DNA.
The ability to resolve the heteroduplex bands in the mutant samples makes it possible to distinguish between the mutant and wild-type samples. Heteroduplex Analysis can be used as a method for screening heterozygous mutations in the cystic fibrosis gene using the DCode system.
1. Nagamine, C.M., Chan, K. and Lau, Y-F.C., Hum. Genet., 45, 337-339 (1989).
2. White, M., Carvalho, M., Derse, D., OBrien, S. and Dean, M., Genomics, 12, 301-306 (1992).
*The Polymerase Chain Reaction (PCR) process is covered by patents owned by Hoffmann-LaRoche. Use of the PCR process requires a license.
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