The objective of this Fast-Track STTR is to develop a simple, inexpensive, closed-tube PCR method for genotyping and sequence variant scanning. Instead of covalently-labeled probes, one of two primers includes a tail with a probe element. After asymmetric PCR, this tail anneals internally to the same strand, """"""""snapping back"""""""", to form a stem/loop structure. Both this stem and the full length PCR product form DNA duplex regions that can be melted. When a dye is present that fluoresces with duplex DNA, melting analysis of the stem allows localized genotyping, and PCR product melting screens for any sequence differences between the two PCR primers. Phase I specific aims are: 1. Demonstrate robust Snapback genotyping of all six SNP types. 2. Demonstrate robust heterozygote scanning with Snapback primers. Progression to Phase II depends on complete genotyping of all SNP types in plasmids and genomic DNA, as well as successful demonstration of genotyping and scanning from the same melting curve. High-resolution melting instruments (HR-1, LightScanner, HR-AMP) software, and custom DNA dyes are available from other projects for use with Snapback primers. The following Phase II specific aims will extend the robustness and utility of the method with a focus toward commercialization: 1. Synthesize an optimal DNA dye for Snapback genotyping and scanning. 2. Predict the melting temperatures of Snapback hairpins under natural PCR conditions. 3. Develop Snapback assays for clinical targets (warfarin dosing and cystic fibrosis). 4. Develop Snapback multiplexing. Advantages of Snapback primers for genotyping and scanning include a homogeneous assay (no need for sample transfer, reagent additions, or automation), closed-tube analysis (no contamination risk), nondestructive analysis, simultaneous scanning and genotyping, and speed (rapid intra-molecular hybridization, PCR in 15 min, melting in 1-2 min). In most research and clinical applications, the need for sequencing is drastically reduced. For many diseases, it is difficult and expensive to screen for all possible sequence variants that may contribute to the disease and/or modify therapy. We propose a simple solution (DNA melting) targeted toward rapid laboratory diagnosis and personalized medicine, applicable to genetic disease, oncology, and infectious agents. Both known (genotyping) and unknown (scanning) sequence variants can be identified in <30 min.
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