A study of the origin of ‘shadow bands’ seen when typing dinucleotide repeat polymorphisms by the PCR

Dinucleotide repeat polymorphisms ('microsatellites') are usually typed by resolving the products of PCR amplification on denaturing acrylamide gels. With this methodology, an allele consists not of a single fragment, but rather of a ladder of fragments, typically separated by intervals of 2nt. Mechanisms that have been invoked to explain the generation of these 'shadow bands' include slipped strand mispairing occurring during the PCR and artefactual 'recombination' caused by out-of-register annealing of truncated PCR products. The D11S527 locus contains the microsatellite sequence (GT)n(CTGT)m. By performing direct sequencing of PCR products derived from individuals homozygous at D11S527, we show that these products vary in length due solely to variations in the length of the dinucleotide repeat tract. These results rule out PCR recombination and support slipped strand mispairing as the major mechanism for generation of shadow bands.