Purification of nucleic acids using isotachophoresis

March 28, 2014
Authors: A. Rogacs, L. A. Marshall, J. G. Santiago

Abstract: Reviewed are methods of nucleic acid (NA) extraction and sample preparation using an electrophoretic purification and focusing method called isotachophoresis (ITP). ITP requires no special surface chemistries or geometric structures, and can be achieved in a compact system with no moving parts. ITP is also compatible with a wide range of samples and lysing methods. Described are general principles of ITP, considerations around the application of ITP to biological samples (e.g., blood, urine and saliva), ITP electrolyte design considerations for fast and selective NA purification, and examples of ITP compatible lysing methods. Several of the challenges associated with purification of NAs are presented as well as methods to address these. Lastly, specific examples of lysing methods and ITP chemistries are described for purification of NA including host and pathogenic DNA, pathogenic rRNA, and host micro-RNA from complex sample matrices.

Bacterial RNA - Rogacs_small

Bacterial RNA Extraction and Purification from Whole Human Blood Using Isotachophoresis

June 29, 2012
Authors: Rogacs, A., Qu, Y., and Santiago, J. G.,

Abstract: We demonstrate a novel assay for physicochemical extraction and isotachophoresis-based purification of 16S rRNA from whole human blood infected with Pseudomonas putida. This on-chip assay is unique in that the extraction can be automated using isotachophoresis in a simple device with no moving parts, it protects RNA from degradation when isolating from ribonuclease-rich matrices (such as blood), and produces a purified total nucleic acid sample that is compatible with enzymatic amplification assays. We show that the purified RNA is compatible with reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and demonstrate a clinically relevant sensitivity of 0.03 bacteria per nanoliter using RT-qPCR.

DNA from malaria - Marshall_small

Extraction of DNA from Malaria-Infected Erythrocytes Using Isotachophoresis

November 28, 2011
Authors: Marshall, L. A., Han, C. M., and Santiago, J. G.

Abstract: We demonstrate a technique for purification of nucleic acids from malaria parasites infecting human erythrocytes using isotachophoresis (ITP). We release nucleic acids from malaria-infected erythrocytes by lysing with heat and proteinase K for 10 min and immediately, thereafter, load sample onto a capillary device. We study the effect of temperature on lysis efficiency. We also implement pressure-driven counterflow during ITP extraction to extend focusing time and increase nucleic acid yield. We show that the purified genomic DNA samples are compatible with polymerase chain reaction (PCR) and demonstrate a clinically relevant limit of detection of 0.5 parasites per nanoliter using quantitative PCR.

Purification from Whole Blood_small

Purification of Nucleic Acids from Whole Blood Using Isotachophoresis

October 15, 2009
Authors: A. Persat, L.A. Marshall, and J.G. Santiago

Abstract: We present and demonstrate a novel technique for the purification of nucleic acids from biological samples using isotachophoresis (ITP). We demonstrate a simple and rapid method to achieve ITP-based extraction, preconcentration, and purification of DNA from nanoliter volumes of whole blood. We show that ITP purification yields genomic DNA samples which can be quantitated with fluorescence measurements and are immediately compatible with polymerase chain reaction (PCR) (e.g., a PCR-friendly solution free of significant inhibitors). We hypothesize ITP purification is applicable to processing of a wide range of complex biological samples.