Heteroduplex migration analysis

Figure 37-10 A schematic of heteroduplex migration analysis. When amplified DNA from a heterozygous specimen is denatured and cooled, the fragments reanneal in four combinations. Electrophoresis on a specialized polyacrylamide gel reveals the presence of heteroduplexes by extra band(s) appearing above the homoduplex band.

Alternatively, the denatured single strands can be made to reanneal to form double-stranded helices. Complementary strands will hybridize to each other. However, if there are sequence differences between two strands, one from each allele, they remain unpaired in the heteroduplex and, as a result, form open loops that reduce migration in the electrophoretic gel. This is the basis of heteroduplex analysis, in which distinct electrophoretic patterns are seen for different alleles, similar to that seen in SSCP (01). 

DNA and RNA with different sequences and mutations show different electrophoretic migration behavior. There are simple electrophoretic methods, which can be used to detect mutations and minimal differences within the sequences of the PCR product. Popular methods are single-strand conformational polymorphism and heteroduplex analysis. 

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