Detection of Sequence Variations within Populations using Non-Symmetric PCR at the Near Digital Level
- 總結
- · Novelmethods for identifying mutational loads in target DNA sequences usingsingle-tube, asymmetric PCR reactions on mixed biological samples (specificallyenabled for mtDNA)
- 技術優勢
- ·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
- 技術應用
- 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.
- 詳細技術說明
- 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.
- *Abstract
-
Mutations in mitochondrial DNA (mtDNA) canresult as a side-effect of age, environmental hazards, genetic susceptibility,diet, drug exposure or a combination of causes. These mutations have been correlatedwith human diseases such as diabetes, Huntington’s, cancer, Parkinson’s,bipolar disorder, chronic fatigue syndrome, amyotrophic lateral sclerosis andAlzheimer’s. However, no specific point mutation has been linked to the onsetof a disease. It is hypothesized that the buildup of random mutations over timein the multiple genomes of the mitochondria leads to dysfunction of theorganelle and then onset of disease. In order to be able to observe theaccumulation of mutations over time, current techniques require analysis oftypically 1 to <10 mtDNA molecules which is expensive to carryout.
The technologies for licensing are novel methodsfor asymmetric amplification and fluorescence detection of the mutational loadin nucleic acid target sequences including non-nuclear DNA (e.g. mtDNA,chloroplast DNA, episomal DNA) as well as RNA, cDNA, and genomic DNA. 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 adjacent sites within the target, though probe pairs need not benext to each other, and each have covalently attached to it either afluorescent compound ( “Signaling Probe”) or a non-fluorescent complementary quenchermoiety (e.g. dabcyl or Black Hole Quencher; “Quencher Probe”). The SignalingProbe will not fluoresce unless bound to the amplified single-strand targetsequence. The signal is eliminated by the fluorophore and quencher moietywhenever both probes are bound to their adjacent sites on the target sequence.
The mutational load for the target isdetermined by analyzing the differences in fluorescence for the hybridizationcurves. Signals can be acquired foranalysis either as the reaction temperature is decreased (annealing) orincreased (melting). The SignalingProbes and Quencher Probes are both mismatch tolerant and can hybridize totarget sequences that contain 1 or more substitutions where probes having greatervariation from the target’s complementarity lowers the melting temperature (Tm)of the probe-target hybrid. The Tmdifferences can be used to differentiate the mutations accumulating in thetarget DNA sequence.
- *IP Issue Date
- None
- *IP Type
- Utility
- 國家
- United States
- 申請號碼
- 9,637,790
- 國家/地區
- 美國
