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Our group focuses on developing molecular assays for clinical applications in inherited diseases and pharmacogenetics. The clinical genetic tests we have validated extend from single mutation detection to complete analysis of large multi-exon genes, such as the genes responsible for cystic fibrosis (CFTR; 27 exons) and hereditary hemorrhagic telangiectasia (HHT; 2 genes, 24 exons). Mutation detection by melting curve analysis, methylation-specific PCR for imprinting disorders, multiplex analysis using bead arrays (Luminex) technology, full gene scanning methods (temperature gradient capillary electrophoresis and high-resolution melting), and deletion analysis by multiplex ligation-dependent probe amplification (MLPA) are some of the methods used in our laboratory. We are also interested in developing direct molecular haplotyping techniques that determine the chromosomal association of multiple genetic variants in individual samples. We have applied our diverse technologies to haplotype mutations of the CFTR gene [R117H association with the poly(T) and poly(TG) repeat tracts in IVS-8] and the β-2 adrenergic receptor (ADRB2). Our research and development group is committed to developing more powerful diagnostic tools for clinical testing to provide better patient care. We also focus on result interpretation, such as identifying and evaluating rare sequence variants with uncertain clinical significance.
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The Technique
Melting analysis with fluorescent-labeled hybridization probes is a simple method to detect variants and is used routinely in our research and development and clinical labs. The probes we describe in our paper allow the analysis of several variants in a single melting analysis. The loci-scanning probe (LSProbe) anneals to several noncontiguous sequences of a template, and a bulge-loop is formed in the template, allowing continuous binding of the LSProbes. As with other probes, LSProbe stability is affected by the presence of mismatches between probe and template, allowing variants to be detected by melting analysis. Our paper extends the application of noncontiguous probes that we previously described for molecular haplotyping to use in multiplex genotyping. Additionally, we describe this approach with both end-labeled hybridization probes and unlabeled probes detected by a double-stranded dye.
Multiplex genotyping by melting analysis of loci-spanning probes: β-globin as an example, p.193.
