Real-time PCR genotyping using displacing probes
TL;DR: A new real-time PCR genotyping approach that uses displacement hybridization-based probes: displacing probes that should make this method suitable for routine clinical testing and large-scale genetic screening.
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Abstract: Simple and reliable genotyping technology is a key to success for high-throughput genetic screening in the post-genome era. Here we have developed a new real-time PCR genotyping approach that uses displacement hybridization-based probes: displacing probes. The specificity of displacing probes could be simply assessed through denaturation analysis before genotyping was implemented, and the probes designed with maximal specificity also showed the greatest detection sensitivity. The ease in design, the simple single-dye labeling chemistry and the capability to adopt degenerated negative strands for point mutation genotyping make the displacing probes both cost effective and easy to use. The feasibility of this method was first tested by detecting the C282Y mutation in the human hemochromatosis gene. The robustness of this approach was then validated by simultaneous genotyping of five different types of mutation in the human beta-globin gene. Sixty-two human genomic DNA samples with nine known genotypes were accurately detected, 32 random clinical samples were successfully screened and 114 double-blind DNA samples were all correctly genotyped. The combined merits of reliability, flexibility and simplicity should make this method suitable for routine clinical testing and large-scale genetic screening.
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Citations
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•Journal Article
A novel MHC class-I-like gene is mutated in patients with hereditary haemochromatosis
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TL;DR: Wavelength-shifting molecular beacons are substantially brighter than conventional molecularBeacons that contain a fluorophore that cannot efficiently absorb energy from the available monochromatic light source, and their use improves and simplifies multiplex genetic analyses.
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A new class of homogeneous nucleic acid probes based on specific displacement hybridization
TL;DR: A new class of probes for homogeneous nucleic acid detection based on the proposed displacement hybridization that demonstrates that multiple targets can be distinguished in the same solution, even if they differ from one another by as little as a single nucleotide.