DNA and RNA sample preparation and quantitation with on-chip isotachophoresis (ITP)
PI: Juan G. Santiago
We have developed a novel method to purify nucleic acids from complex samples which exploits the electrophoretic mobility of nucleic acids (and not their affinity to a liquid or solid phase, as with all other methods). Rogacs et al., (2014) reviews and summarizes this innovation including over 20 journal papers from our group. Although these papers are fairly recent (in the last ~6 years), they have been collectively cited more than 1300 times. Our ITP-based sample preparation technique provides a dynamic range of extraction from 0.1 picogram to 1 microgram of nucleic acid; a high degree of automation for nucleic acid sample preparation; simultaneous on-line quantitation; and seamless integration with downstream assays. We have successfully demonstrated our ITP-based sample preparation on a wide variety of complex samples including whole blood; Malaria parasites in blood; bacteria in blood; cell-free nucleic acids in serum and plasma; bacteria in clinical urine samples of patients presenting with urinary tract infections; >12 mammalian cell cultures; and a wide range of tissue samples including liver, kidney, brain, and fixed-formalin paraffin embedded (FFPE) samples. The technology has been spun off into a startu- company called Purigen Biosystems (purigenbio.com)
Figure 1: In its simplest implementation, ITP purification consists of dispensing untreated raw lysate directly into a reservoir, initiation ITP, and extracting purified nucleic acid from a second downstream reservoir.
Figure 2: ITP extraction of DNA from blood lysate. (a) schematic showing how nucleic acids is focused into an ITP interface between a leading electrolyte ion (LE) and a trailing electrolyte ion (TE), while excluding impurities such as proteins.
Figure 3: Example experiment showing extraction, focusing, and quantification of nucleic acids from a human whole blood sample. The images are taken from Marshall et al. (2014).
Garcia-Schwarz, G., A. Rogacs, S.S Bahga, and J.G. Santiago, "On-chip isotachophoresis for separation of ions and purification of nucleic acids," 61, 3890, Journal of Visualized Experiments, 2012.(click here for pdf)
Marshall, L.A., Rogacs, A., Meinhart, C.D., and Santiago, J.G. "An Injection Molded Microchip for Nucleic Acid Purification from 25 Microliter Samples using Isotachophoresis," Journal of Chromatography A, 2014.(click here for pdf)
Rogacs, A., Marshall, L.A., and Santiago, J.G. "Purification of Nucleic Acids using Isotachophoresis," 1335, pp. 105-120, Journal of Chromatography A, 2014.