Technologies

The technologies for licensing are novel methods for asymmetric amplification and fluorescence detection of the mutational load in nucleic acid target sequences. These methods utilize single-tube, multiplex polymerase chain (PCR) reactions on mixed samples which each contain one or more target specific primer pairs for DNA amplification. These reactions also contain 1 or more probe pair sets that hybridize to sites within the target and each have covalently attached to it either a fluorescent compound or a non-fluorescent complementary quencher moiety. The Signaling Probe will not fluoresce unless bound to the amplified single-strand target sequence and its signal is eliminated whenever both the fluorescent and quencher probes are bound to their adjacent sites on the target sequence.

Mutations in mitochondrial DNA (mtDNA) can result as a side-effect of age, environmental hazards, genetic susceptibility, diet, drug exposure or a combination of causes. These mutations have been correlated with human diseases such as diabetes, Huntington’s, cancer, Parkinson’s, bipolar disorder, chronic fatigue syndrome, amyotrophic lateral sclerosis and Alzheimer’s. However, no specific point mutation has been linked to the onset of a disease. It is hypothesized that the buildup of random mutations over time in the multiple genomes of the mitochondria leads to dysfunction of the organelle and then onset of disease. In order to be able to observe the accumulation of mutations over time, current techniques require analysis of typically 1 to <10 mtDNA molecules which is expensive to carryout.

The technologies for licensing are novel methods for asymmetric amplification and fluorescence detection of the mutational load in nucleic acid target sequences including non-nuclear DNA (e.g. mtDNA, chloroplast DNA, episomal DNA) as well as RNA, cDNA, and genomic DNA. These methods utilize single-tube, multiplex polymerase chain (PCR) reactions on mixed samples which each contain one or more target specific primer pairs for DNA amplification. These reactions also contain 1 or more probe pair sets that hybridize to adjacent sites within the target, though probe pairs need not be next to each other, and each have covalently attached to it either a fluorescent compound ( “Signaling Probe”) or a non-fluorescent complementary quencher moiety (e.g. dabcyl or Black Hole Quencher; “Quencher Probe”). The Signaling Probe will not fluoresce unless bound to the amplified single-strand target sequence. The signal is eliminated by the fluorophore and quencher moiety whenever both probes are bound to their adjacent sites on the target sequence.

The mutational load for the target is determined by analyzing the differences in fluorescence for the hybridization curves. Signals can be acquired for analysis either as the reaction temperature is decreased (annealing) or increased (melting). The Signaling Probes and Quencher Probes are both mismatch tolerant and can hybridize to target sequences that contain 1 or more substitutions where probes having greater variation from the target’s complementarity lowers the melting temperature (T _m ) of the probe-target hybrid. The T _m differences can be used to differentiate the mutations accumulating in the target DNA sequence.