Detection of Sequence Variations within Populations using Non-Symmetric PCR at the Near Digital Level
· Novelmethods for identifying mutational loads in target DNA sequences usingsingle-tube, asymmetric PCR reactions on mixed biological samples (specificallyenabled for mtDNA)
·Allowsfor amplification of targets and detection of multiple mutations in a singlePCR reaction· Overcomeslimitations of prior analysis methods which can obscure the presence ofmutations due to target DNA having methylated stretches of nucleic acids
The methods allow single-tube asymmetric PCR amplification and fluorescent detection of mutational loads within non-nuclear and genomic DNA contained in complex, mixed samples. Though the methods are broadly applicable to diagnostic and analytical assays, our invention has been enabled for the detectionof mitochondrial DNA (mtDNA) mutations within: cytochrome c oxidase subunit 2 (CO2), NADHdehydrogenase subunit 1 (ND1) and the hyper variable 2 (HV2) of humanmitochondria. The mtDNA of mice, rats, and the Nile rat (used to studydiabetes) have also been amplified using this technology for various studies.
The technologies for licensing are novel methodsfor asymmetric amplification and fluorescence detection of the mutational loadin nucleic acid target sequences. Thesemethods utilize single-tube, multiplex polymerase chain (PCR) reactions onmixed samples which each contain one or more target specific primer pairs for DNAamplification. These reactions also contain 1 or more probe pair sets thathybridize to sites within the target and each have covalently attached to it either afluorescent compound or a non-fluorescent complementary quenchermoiety. The SignalingProbe will not fluoresce unless bound to the amplified single-strand targetsequence and its signal is eliminatedwhenever both the fluorescent and quencher probes are bound to their adjacent sites on the target sequence.
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