Accurate DNA microsatellite and microRNA detection with a biological nanopore
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[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT REQUEST OF AUTHOR.] Nanopore-based detection has been used in many genetic, epigenetic, proteomic, and bio-mechanism studies. In this report, we introduced two designed strategies for detecting DNA and micro-RNA with nanopore platform. First, we focused on a specific type of DNA denoted as DNA microsatellite. Microsatellite is a tandem DNA repeat unit containing one to six bases. The expansion of microsatellite always associates severe genetic disorders, such as Huntington disease (HD), Fragile X syndrome and varies kinds of cancer. In this study, we focused on a hereditary genetic disorder-Myotonic Dystrophy type I (DM1), which is caused by the expansion of CTG repeats in 3'UTR region within dystrophia myotonica protein kinase (DMPK) gene. Patients suffered from this disease always exhibited muscle atrophy as well as cognitive disorders. For now, the commonly used diagnostic approaches for DM1 are PCR and Southern blot. However, the efficiency and accuracy of PCR are restricted by the sequence length, and Southern blot is limited by the sample loads. Although DNA sequencing develops rapidly in recent years, the precision of detecting long tandem repeats still remains a problem. Also, sequencing is not necessary for diagnostic purpose. In this report, we presented a novel approach using nanopore technology. The double strand DNAs bound to Hg2+ to form different numbers of nanolocks. By adding an overhang sequence to the target strand, long double strand DNAs were able to unzip outside the nanopore and generate distinguishable duration histogram. In this case, we successfully linked the lengths of CTG repeats with the numbers of nanolocks
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