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Heteroduplex Analysis Using the DCode System

Paul Zoller and Theresa Redila-Flores
Bio-Rad Laboratories, Hercules, California


Introduction
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.


Method
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
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