DNA purification from crude samples for human identification using gradient elution isotachophoresis

Abstract

Gradient elution isotachophoresis (GEITP) was demonstrated for DNA purification, concentration, and quantification from crude samples, represented here by soiled buccal swabs, with minimal sample preparation prior to human identification using STR analysis. During GEITP, an electric field applied across leading and trailing electrolyte solutions resulted in isotachophoretic focusing of DNA at the interface between these solutions, while a pressure-driven counterflow controlled the movement of the interface from the sample reservoir into a microfluidic capillary. This counterflow also prevented particulates from fouling or clogging the capillary and reduced or eliminated contamination of the delivered DNA by PCR inhibitors. On-line DNA quantification using laser-induced fluorescence compared favorably with quantitative PCR measurements and potentially eliminates the need for quantitative PCR prior to STR analysis. GEITP promises to address the need for a rapid and robust method to deliver DNA from crude samples to aid the forensic community in human identification.

Recent progress in nucleic acids isotachophoresis

“…(F) The voltage was removed, the pressure was increased, and the purified and concentrated DNA was delivered past the LIF detector for quantification and into a clean PCR tube for qPCR and short tandem repeat analysis. Reprinted with permission from …”

“…They designed and established the origami paper analytic device (oPAD) based on the principle of traditional Japanese paper (F) The voltage was removed, the pressure was increased, and the purified and concentrated DNA was delivered past the LIF detector for quantification and into a clean PCR tube for qPCR and short tandem repeat analysis. Reprinted with permission from [77] folding [85]. Consequently, they combined oPAD with ITP to perform focusing of DNA sample.…”

Recent advances in enhancing the sensitivity of electrophoresis and electrochromatography in capillaries and microchips (2012–2014)

“…Strychalski et al used gradient elution ITP (GEITP) for DNA purification from crude samples for human identification using STR analysis . In GEITP, a controlled variable pressure driven counterflow (CF) was used to control the focusing interface position while excluding other particulate and contaminations from the capillary, which is useful as it can be used to prevent PCR inhibitors from focusing with the DNA.…”

Exploring Gradients in Electrophoretic Separation and Preconcentration on Miniaturized Devices

“…Ross and coworkers have contributed to gradient elution ITP (GEITP) [28]. Compared to conventional ITP, it includes applied pressure-driven flow as a counterforce to the electrophoretic movement, generating precise position control and avoiding introduction of contaminates.…”

“…Electrophoretic-based separation and preconcentration schemes were initially drawn from capillary electrophoretic methods. Since then, electrophoresis has exerted a powerful influence and has led to a variety of derivations and branches, including field-amplified sample stacking [15][16][17][18][19][20][21], isotachophoresis [22][23][24][25][26][27][28][29][30], free flow electrophoresis [31][32][33][34][35] and gradient focusing [36][37][38][39][40][41][42]. Among them, gradient techniques are of special interest.…”