REAL-TIME GENE QUANTIFICATION WITH INTERNAL STANDARDS
DESCRIPTION (provided by applicant): 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.
PROPOSED COMMERCIAL APPLICATION:
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.
Small Business Information at Submission:
IDAHO TECHNOLOGY 390 WAKARA WAY SALT LAKE CITY, UT 84108
Number of Employees: