The diagnostic and prognostic importance of genetic mutations in cancer has provided impetus for the development of simplified, sensitive and robust methods for their detection. Intergen is developing novel pyrene-containing oligonucleotides as probes for use in mutation detection. The key advantage in the use of pyrene as a reporter is that the formation of pyrene dimers results in strong excimer fluorescence with an emission maximum different from that of pyrene monomer. Oligonucleotide probes can be designed such that two pyrenes, positioned on the adjacent ends of two oligonucleotides, come into dose contact only after both oligonucleotides hybridize to the correct sequence. There is virtually no background, in the portion of the spectrum where pyrene excimers emit light, even in the presence of high concentrations of unhybridized starting probes. These characteristics are ideal for developing a homogeneous """"""""closed-tube"""""""" assay whereby all reactants are added to a single tube (i.e., probes, PCR primers, enzyme) that is allowed to react and read for positive or negative excimer fluorescence. Preliminary studies have demonstrated that this technology can accurately detect point mutations in a synthetic strand of DNA. We will assess the superiority of pyrene-based mutation detection over current methods and (in Phase II) develop diagnostic assays for the detection of early stage cancer.
The widespread use of fluorescent detection systems has created a market for novel and improved fluorochromes. The spectral characteristics of pyrene make it highly competitive in this market. Further, we believe that the improved characteristics of pyrene-based mutation detection systems will give them a significant advantage over current mutation detection systems contemplated for use as screens for early stage cancer in high-risk individuals (e.g., smokers).
