Real-time PCR has greatly improved the ease, accuracy and precision of quantitative PCR by allowing reaction kinetics to be observed, measured and recorded as PCR is occurring. We have successfully commercialized the LightCycler, a real-time PCR instrument that combines a rapid thermal cycler with a fluorimeter. We have developed sequence specific probe systems that rely on fluorescent signal to detect PCR product. These hybridization probes can also be used to detect polymorphisms after PCR by probe melting. In phase I, we combined a method of competitive quantitative end-point PCR with the ability of hybridization probes to differentiate single nucleotide polymorphisms by probe melting temperature. We also developed a mathematical method of melting curve analysis that can be used to quantify allele frequencies in heterogeneous samples. This analytical method can accurately quantify allele frequencies as low as 1 percent in pooled samples. This method has great potential in the area of high throughput Single Nucleotide Polymorphism (SNP) analysis. In particular, quantification of allele frequencies in pooled samples for disease association studies, quantification of allelic patterns of gene transcription, and quantification of mutation load. In phase II, we propose to develop a commercial instrument and analysis software for quantitative melting curve analysis using a standard 384-well microplate format. The proposed instrument is not a thermal cycler. Instead it will analyze PCR products generated on any 384-well PCR thermocycler by hybridization probe melting. This strategy will result in a low-cost, quantitative instrument that is compatible with standard high-throughput PCR instruments and automated workstations commonly used in many labs. Instrumentation and analysis software will be validated by high throughput quantification of mutant-to-wildtype mitochondrial allele frequencies in individuals harboring variable loads of mitochondrial disease muations.
Idaho Technology is looking to expand into the growing field of Single Nucleotide Polymorphism (SNP) analysis. We recognize the need for high throughput capabilities that are compatible with other PCR thermocyclers and automated DNA workstations. We propose to bring to market an instrument that offers these features, and incorporates quantitative features not currently available with other methods of SNP analysis.
