Accessing DNA by low voltage alternating current Joule effect heating

Abstract

A DNA release sample preparation method based on the use of low voltage alternating currents (LVACs) to generate Joule effect heating (JEH) is reported. This is a simple cell disruption strategy that offers internal, homogenous, rapid and low power consumption heating for the access of analytical grade DNA in seconds. A 100 μL JEH microreactor with a parallel and symmetric two electrode arrangement for uniform field generation was fabricated by machining and used to characterise JEH and DNA release from human epithelia, yeast ( Saccharomyces cerevisiae) and Gram-positive bacteria ( Enterococcus faecium) cell types. A 1 kHz sinusoidal low voltage (e.g . 10 Vrms) alternating current was used to reduce electrode:sample interactions. Following 96 °C JEH treatment, effective DNA release was identified by PicoGreen ® quantification for all three cell types. The JEH treated sample material was further successfully used, without purification, as a PCR template. Exposure to JEH-mediated 96 °C temperatures for a 1 s duration was used to provide PCR-grade DNA template material from S. cerevisiae and E. faecium cells, and a 10 s duration was used for human epithelia cells. However, prolonged (>1 min) exposure to 96 °C JEH-mediated temperatures resulted in diminished DNA returns and the production of components that interfered with the PCR reaction. Further miniaturisation of the LVAC JEH cell by microfabrication was considered, and a JEH microreactor designs were evaluated by FLOTHERM v3.2 thermal modelling. Thermal isolation, using a free-standing cavity structure was identified as an excellent means to enable rapid heating (220 °C s −1) with low power consumption (0.2 W).