FLUORESCENCE-BASED CLASSIFICATION OF MICROSATELLITES USING A SINGLE-WAVELENGTH SEMIAUTOMATIC SEQUENCER - GENOTYPE ASSIGNMENT AND IDENTITY TESTS BY ANALYSIS OF COMIGRATING PEAK PROFILES

Citation
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
Citations number
34
Categorie Soggetti
Biochemical Research Methods
Journal title
ISSN journal
01730835
Volume
16
Issue
10
Year of publication
1995
Pages
1875 - 1880
Database
ISI
SICI code
0173-0835(1995)16:10<1875:FCOMUA>2.0.ZU;2-I
Abstract
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.