Heteroduplex Analysis HET or HA

In a mixture of wild-type and mutant DNA molecules, heteroduplexes are formed by heat denaturation and reannealing. Heteroduplexes formed between different alleles migrate with different mobilities when subjected to electrophoresis side by side in a nondenaturing polyacrylamide gel.65 Sequence differences are detected by visually comparing the migration patterns obtained. A level of detection of approximately 80-90%, similar to that obtained in SSCP analysis, is attained with DNA fragments of 200-600 base pairs in length.

In one modification of HET, capillary-based conformation-sensitive gel elec-trophoresis (capillary CSGE), mutation detection is transferred from acrylamide gel to capillary electrophoresis.66 This modification is capable of detecting every possible single base heteroduplex, all short insertions and deletions (1-4 base pairs), as well as 16 of 22 substitutions of up to approximately 350 base pairs. Multiple PCR products can be resolved by using different fluorescent labels. CSGE can be automated and offers the potential for high-throughput analysis.

Other modifications of HET that enhance heteroduplex resolution have been developed.67 One modification involves PCR of the region of interest followed by hybridization of the PCR product to a synthetic molecule called a ''universal heteroduplex generator'' (UHG). This method was used in a study of apolipo-protein E mutations to induce heteroduplex formation. Heteroduplex formation was visualized on a nondenaturing minigel with ethidium bromide. The method was found to be simpler, faster, and more reliable than the current method of choice (RFLP with Hha restriction), and is suitable for high-throughput screening.67

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