Real-time PCR has greatly improved the ease, accuracy and precision of quantitative PCR by allowing observation of the PCR product concentration at every cycle. To date, all real-time PCR quantification applications have been limited to quantification relative to an external standard curve. We have successfully commercialized the LightCycler(TM), a real-time PCR instrument that combines a rapid thermal cycler with a fluorimeter. We developed a sequence specific probe system for the LightCycler that brings two fluorophores closer together by hybridization during PCR so that resonance energy transfer occurs. We propose to develop two methods for real-time quantification with internal standards using hybridization probes. The first method uses a competitor with a unique hybridization probe binding site. The competitor will be distinguished from the target by using differently colored hybridization probes for the target and the competitor. The second method uses a competitor which differs from the target by only a single base. The target and the competitor will be distinguished by the differential melting of fluorescently labeled hybridization probes. These methods will provide investigators with the advantages of a homogenous, real-time PCR system while giving the added control that internal standards provide.
Idaho Technology is looking to expand the range of applications for its LightCycler technology. Competitive quantitative PCR is the gold standard for PCR quantification. Developing competitive quantitative PCR for the LightCycler would greatly increase the LightCycler's utility.
