In recent years, rising importance is being placed on high quality sample preparation, particularly when expensive downstream genomic analysis techniques such as sequencing and genome mapping are involved. However, limited tools exist to verify the quality of DNA/RNA beyond UV absorbance measurements to determine purity, fluorescence assays to determine yield, and electrophoretic methods to determine size. New alternatives to electrophoresis could significantly facilitate sample preparation QC, particularly in applications where improvements in speed, sensitivity, and performance are necessary or where new metrics could be useful. In Phase I, we developed an instrument called PicoSep that integrated inline isotachophoresis (ITP) preconcentration and single molecule free solution hydrodynamic separation to create one of the most sensitive DNA sizing platforms seen to date. Separation occurs through a unique hydrodynamic mechanism that enables the separation of both large (100 kb+) and small DNA (20 bp) using only pressure and a buffer- filled microchannel or capillary (0.5 - 20 m ID), sizing performance unmatched by any single electrophoresis platform. During this work, we also discovered a novel, electrode-free concentration method called counter flow concentration (CFC). Using CFC or ITP, we can enhance sensitivity up to PCR levels. In this Phase II proposal, we will develop a high-throughput PicoSep instrument and leverage PicoSep's unique properties to facilitate sample preparation in two critical applications. First, we will develop a kit for high molecular weight DNA sizing to replace slow and insensitive pulsed field gel electrophoresis analysis in long-read sequencing and genome mapping. Second, we will develop a kit for RNA integrity analysis. RNA integrity is the dominant metric used to determine RNA quality and degradation. PicoSep's single molecule sensitivity will enable detection of minute amounts of degradation with near zero sample consumption. These sizing-based kits will serve as the initial products while future kits will leverage PicoSep's other single molecule capabilities to determine advanced DNA/RNA metrics such as damage from abasic sites and oxidation.
Rising importance is being placed on high quality sample preparation, particularly when expensive genomic analysis techniques such as sequencing are involved or when valuable clinical samples are being tested. However, limited analytical tools exist to verify the quality of DNA and RNA. We will create a powerful new single molecule analysis platform for sample preparation quality control called PicoSep that is more sensitive, more accurate, more informative, and uses less sample than any existing method.
| Friedrich, Sarah M; Burke, Jeffrey M; Liu, Kelvin J et al. (2017) Molecular rheotaxis directs DNA migration and concentration against a pressure-driven flow. Nat Commun 8:1213 |
