Fluorescence-based classification of microsatellites using a single-wavelength semiautomatic sequencer: genotype...

Microsatellite analysis, based on fluorescein labeling and reading through a semiautomatic single wavelength sequencer, is described. Pairs of labeled polymerase chain reaction (PCR) samples, mixed in equimolar proportion, were electrophoresed and the specific peaks read in a single gel lane. Identity was asserted when peaks overlapped in a unique fluorescent signal which, compared with individual sample profiles, had a twofold intensity. Classification was achieved by blending individual PCR products to 'locus specific allelic ladders' (composite samples containing a repertory of fragments allelic to a given locus) and by noticing the specific peak enhancement. The resulting protocol of analysis assigned no size and classified allelic forms by tandem repeat number. Applied to a large repertory of PCR products and compared with manual electrophoresis, this protocol proved to be reliable and reduced times and costs of genotype analysis. Analysis of comigrating peak profiles is highly objective and provides convincing evidence for diagnostics and identity tests.