Screening of a highly polymorphic microsatellite for microheterogeneity in human identification.
Single-strand conformation polymorphism (SSCP) analysis combined with automated laser fluorescence detection is proposed as a comprehensive, rapid and sensitive method for screening sequence variation of the human beta-actin-related pseudogene (HUMACTBP2). Eleven sequenced alleles representing each type of known sequence variant of HUMACTBP2 locus were studied. Allelic variants of the same size but different sequence structures are easily resolved on the basis of their secondary conformation. Fifty ACTBP2 amplification products previously typed on a denaturing gel were repeatedly examined to determine the utility of SSCP analysis in terms of ease of interpretation and reproduction capabilities of the conformational patterns. Eleven sequenced ACTBP2 allelic variants were used as external conformation standards in polymerase chain reaction (PCR)-SSCP subtyping. This enabled identification of polymorphism in a particular length variant and therefore consistent discrimination between heterozygous samples appeared identical on denaturing gels. Of five "homozygous" samples, one was shown to be heterozygous for two distinct alleles of the same size but different sequences. Thus, the method provides a unique possibility for detecting false homozygotes. The technique complements both denaturing gel electrophoresis and DNA sequencing in studies on the overall variability of the ACTBP2 locus.
Miścicka-Sliwka
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The Ludwik Rydygier's University School of Medical Sciences, Forensic Medicine Institute, Bydgoszcz, Poland. grzybu@polbox.com
Grzybowski
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Czarny
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Woźniak
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D000199:Actins / Q000235:genetics*; D000483:Alleles; D004276:DNA, Satellite; D005069:Evaluation Studies as Topic; D018740:Genetic Heterogeneity; D005820:Genetic Testing; D014644:Genetic Variation; D005838:Genotype; D006801:Humans; D018895:Microsatellite Repeats; D016133:Polymerase Chain Reaction / Q000379:methods; D018807:Polymorphism, Single-Stranded Conformational; D011544:Pseudogenes
D000199:Actins; D004276:DNA, Satellite