Duplication and deletion analysis by fluorescent real-time PCR-based genotyping

• Published in: Clinica chimica acta 2006, Vol.363 (1), p.138-146
• Main Authors: Ruiz-Ponte, C., Carracedo, A., Barros, F.
• Format: Article
• Language: English
• Subjects: Deletions; Duplications; Fluorescence; Gene Deletion; Gene Dosage; Gene Duplication; Genotype; Humans; Melting curves; Molecular Diagnostic Techniques - methods; Polymerase Chain Reaction - methods; Polymorphism, Single Nucleotide; Real-time PCR; Reproducibility of Results; Sensitivity and Specificity; SNPs; Temperature
• ISSN: 0009-8981; 1873-3492
• DOI: 10.1016/j.cccn.2005.05.044

Gene dosage determination is an increasingly important field for the study of genome variation and organization. In parallel, the advances in our understanding of the genetic basis of disease have produced an exponential increase in the demand for molecular diagnostic analyses. Although efforts have been spent on increasing both the accuracy and the throughput of the gene dosage analysis, the success has been limited. A large number of suitable methods has been proposed; most are based on quantitative real-time PCR or amplification of multiple targets. A new approach exploits the differences between fluorescent signals of SNP alleles in heterozygous samples to assess duplications. The SNP typing-dependent fluorescent signal allelic asymmetry is an intrinsic characteristic of a SNP typing assay and can lead to a simple and cost-effective gene dosage method. This strategy provides sufficient throughput and sensitivity for duplication analysis. There are advantages and disadvantages of real-time methodology when applying the approach to the molecular diagnostic field.