FLUORESCENCE-BASED CLASSIFICATION OF MICROSATELLITES USING A SINGLE-WAVELENGTH SEMIAUTOMATIC SEQUENCER - GENOTYPE ASSIGNMENT AND IDENTITY TESTS BY ANALYSIS OF COMIGRATING PEAK PROFILES
A. Moscetti et al., FLUORESCENCE-BASED CLASSIFICATION OF MICROSATELLITES USING A SINGLE-WAVELENGTH SEMIAUTOMATIC SEQUENCER - GENOTYPE ASSIGNMENT AND IDENTITY TESTS BY ANALYSIS OF COMIGRATING PEAK PROFILES, Electrophoresis, 16(10), 1995, pp. 1875-1880
Microsatellite analysis, based on fluorescein labeling and reading thr
ough 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 sin
gle gel lane. Identity was asserted when peaks overlapped in a unique
fluorescent signal which, compared with individual sample profiles, ha
d a twofold intensity. Classification was achieved by blending individ
ual PCR products to 'locus specific allelic ladders' (composite sample
s containing a repertory of fragments allelic to a given locus) and by
noticing the specific peak enhancement. The resulting protocol of ana
lysis assigned no size and classified allelic forms by tandem repeat n
umber. Applied to a large repertory of PCR products and compared with
manual electrophoresis, this protocol proved to be reliable and reduce
d times and costs of genotype analysis. Analysis of comigrating peak p
rofiles is highly objective and provides convincing evidence for diagn
ostics and identity tests.