432463 Sensitive, Multiplexed Electrophoretic miRNA Detection Using Peptide Nucleic Acids in Micelle-Elfse

Tuesday, November 10, 2015: 2:45 PM
Ballroom E (Salt Palace Convention Center)
James W. Schneider1, Johnathan M. Goldman1, Bruce Armitage2 and Danith Ly2, (1)Chemical Engineering, Carnegie Mellon University, Pittsburgh, PA, (2)Chemistry, Carnegie Mellon University, Pittsburgh, PA

miRNAs are short, noncoding nucleic acids that are biomarkers for a growing number of disease states, including breast cancer, Alzheimer's disease, and heart disease. Because they are only 20-22 bases in length, and are present in trace quantities, they are difficult to detect by conventional PCR and array methods. Here, we present an electrophoretic detection system that uses synthetic γ-substituted peptide nucleic acids, which bind miRNA with great specificity and stability. Low detection limits are achieved using an ultrabright probe in the form of a DNA oligomer saturated with intercalating dye. The micelle-ELFSE method has been extended to kilobase-length DNA to provide ultrabright fluorophores for sub-attomole detection of miRNA in less than 3 minutes. The use of surfactant running buffers, rather than gels, makes the method compatible with high amounts of serum (10-100 mg/ml) with minimal peak distortion. Finally, the use of gamma-substituted peptide nucleic acid probes gives great binding stability and specificity, allowing for miRNA detection in a sandwich format. The method can easily distinguish members of the closely related let-7 series. We will discuss efforts to concentrate miRNA by ITP-based sample stacking as well as theoretical limits of peak capacity for multiplexed detection in capillary and microchip electrophoresis. We will also discuss applications in early-stage cancer screening and other medical applications of this rapid, selective miRNA detection scheme.

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