David Broemeling

Nonlinear electrophoretic response yields a unique parameter for separation of biomolecules

We demonstrate a unique parameter for biomolecule separation that results from the nonlinear response of long, charged polymers to electrophoretic fields and apply it to extraction and concentration of nucleic acids from samples that perform poorly under conventional methods. Our method is based on superposition of synchronous, time-varying electrophoretic fields, which can generate net drift of charged molecules even when the time-averaged molecule displacement generated by each field individually is zero. Such drift can only occur for molecules, such as DNA, whose motive response to electrophoretic fields is nonlinear. Consequently, we are able to concentrate DNA while rejecting high concentrations of contaminants. We demonstrate one application of this method by extracting DNA from challenging samples originating in the Athabasca oil sands.

An instrument for automated purification of nucleic acids from contaminated forensic samples

Forensic crime scene sample analysis, by its nature, often deals with samples in which there are low amounts of nucleic acids, on substrates that often lead to inhibition of subsequent enzymatic reactions such as PCR amplification for Short Tandem Repeat (STR) profiling. Common substrates include denim from blue jeans, which yields indigo dye as a PCR inhibitor, and soil, which yields humic substances as inhibitors. These inhibitors frequently co-extract with nucleic acids in standard column or bead-based preps, leading to frequent failure of STR profiling. We present a novel instrument for DNA purification of forensic samples that is capable of highly effective concentration of nucleic acids from soil particulates, fabric, and other complex samples including solid components. The novel concentration process, known as Synchronous Coefficient of Drag Alteration, is inherently selective for long-charged polymers such as DNA, and therefore is able to effectively reject known contaminants. We present an automated sample preparation instrument based on this process, and preliminary results based on mock forensic samples.

A Novel Applicator Design for Contact-Free Delivery of Aqueous Reagent to Individual Biopsies in a Tissue Microarray

The use of molecular techniques to inform diagnosis, prognosis, and treatment design will play an important role in the future of medicine. Each new test, however, represents a new cost to the health care system, and significant effort is required to move new techniques to the clinical setting in the most cost-effective and efficient manner. Consequently, there is a compelling need for technological improvements that will facilitate clinical application of novel assays, by reducing cost and complexity in implementation. This is particularly important in cancer pathology. Here, we present a novel applicator technology that enables staining of individual biopsies in a tissue microarray (TMA) to provide low-cost, multiplexed biomarker testing at the level of intact tissue. The applicator is designed to deliver tens of nanoliters of aqueous reagent to arrayed tissue biopsies kept under a layer of oil-based Liquid Coverslip without contacting the biopsies. A pin consisting of concentric stainless steel electrodes separated by a hydrophobic insulator provides a balance between hydrophobicity and hydrophilicity to hold a reagent droplet on the tip of the pin, whereas a small electrical current passed through the droplet spanning the electrodes is used for drop sensing. This design is more amenable to repeatable manufacturing than a previous prototype, which in initial testing demonstrated successful immunohistochemical and in situ hybridization staining of individual biopsies in a TMA, but was difficult to produce. This new design was tested to investigate the factors affecting its operation, in terms of the volume of reagent picked up and its ability to successfully deliver reagent to the biopsies.