Heteroduplex Analysis

Heteroduplex formation results when wild-type and mutant alleles are coamplified, denatured, and allowed to reanneal in a post-PCR annealing step, usually heating and cooling.41 Some of the strands pair with the complementary strand from the same allele and form homoduplexes. However, some strands pair with a strand from the other allele and form heteroduplexes. Because the heterodu-plexes have mismatched base pairs between strands, they form a partially open dsDNA sequence that migrates more slowly in electrophoresis than the fully annealed homoduplexes.

Two types of heteroduplex structures can be formed.42 When the mismatch consists of one or more single-base mutations,small open areas of dsDNA called "bubble-type" heteroduplexes are formed. When the mismatch is formed by insertions or deletions between the two alleles, a pronounced bending of the dsDNA is produced and referred to as a "bulge-type" heteroduplex. Bulge-type heterodu-plexes markedly affect the mobility of the dsDNA, whereas bubble-type heteroduplexes may be difficult to detect in polyacrylamide gels. Detection of single base-pair mismatches can be enhanced in two ways. Electrophoresis can be performed with mutation detection enhancement (MDE) gels, an altered form of polyacrylamide that enhances separation of heteroduplexes. The post-PCR introduction of a known sequence with a short deletion to form a bulge-type heteroduplex enhances the separation of sequences with base-pair mismatches in a process known as universal heteroduplex generation (UHG).

Examples of Applications of Heteroduplex Analysis

1. HIV subtyping

2. CFTR gene mutation analysis in cystic fibrosis

3. NF1 gene mutation analysis in neurofibromatosis type 1

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