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The DNAbased quantitative analysis of geneticchimerism is becoming increasingly more important for molecular biology ingeneral and molecular medicine in particular. Useful genomic targets for theseanalyses are polymorphic sequences, but here the problem of a reliablequantification with high dynamic range is not yet satisfactorily solved. Tothis end we have combined the allelespecific amplification with a realtimePCRbased quantification for rapid allelotyping and chimerism analysis. Thesequence variations are discriminated by the 3end of the allelespecific primer.Amplification is monitored by SYBRGreen I fluorescence. We demonstrate theefficiency of this method for two clinically relevant targets: (i) the 10 bpinsertion/deletion polymorphism in the promoter of the factor VIIc (FVIIc) geneand (ii) the 4G/5G single nucleotide polymorphism in the promoter of theplasminogen activator inhibitor-1 (PAI-1) gene. Both polymorphisms areassociated with clinical risk factors. Allelotyping results were in completeagreement with those obtained by reference methods. Mixed chimeric DNA samplescould be quantified reliably with a dynamic range of 1:3000 for an easy target(FVIIc) and of 1:64 for a difficult target (PAI-1). Our protocol isparticularly useful for rapid, reliable and inexpensive genotyping andquantitative chimerism analysis without requiring expensive fluorophor dyelabelled probes.

biological chemistry

Detection and Quantification of Insertion/Deletion     Variations by Allele-Specific Real-Time PCR: Application for Genotyping and     Chimerism Analysis