11.2.2. Gel retardation assays (SSCP and DGGE)
Single Strand Conformation Polymorphism (SSCP) and Denaturing Gradient Gel Electrophoresis (DGGE) are two techniques that use electrophoresis to differentiate directly between variants in a population of sequences (Hauser et al., 2000; Stach et al., 2001). In SSCP the nucleic acids are made single-stranded, to fold into a preferred secondary structure. In DGGE, the nucleic acids are separated in an electrophoretic gel containing a salt gradient that will progressively denature the nucleic acids. In both methods, minor nucleotide differences between polymorphs in the population affect the migration of the DNA. Another technique with a similar philosophy is the heteroduplex mobility shift assay, where single nucleotide mismatches between a probe and target affect the mobility of the hybridised complex during electrophoresis, (Arens, 1999).
- Entire populations
can be screened for genetic complexity within the target sequence in a single
- Assay is limited to about 300 bases, requiring many assays to cover a genome.
- Protocols are complex, sensitive to procedural accuracy and subject to errors.
- The nature of the polymorphs requires further analysis.