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Stanford University

Stanford Microfluidics Laboratory

Extraction of DNA from Malaria-Infected Blood

Lewis A. Marshall, Crystal M. Han, Juan G. Santiago

 

We developed a technique for purification of nucleic acids from malaria parasites infecting human erythrocytes using isotachophoresis (ITP). We used thermal and enzymatic lysis to release nucleic acids without introducing high concentrations of salts, and optimized ITP chemistry for selective extraction of DNA. We demonstrated our technique both using conventional glass capillary devices and novel printed circuit-board microfluidic devices developed by the Bachmann Group at UC Irvine.


Figure 1.DNA extraction process. A culture sample containing P. falciparum parasites infecting (within) red blood cells was diluted, mixed with proteinase K, and lysed at 95° C. During lysis, parasite cells released their DNA into the cell lysate. The cell lysate was pipetted directly into the microfluidic well containing the TE. An electric field was applied, and the DNA was pulled into a capillary, where it focused at the ITP interface. In this process, PCR inhibitors (including proteins) remain unfocused in or near the TE well. The focused ITP zone containing purified nucleic acids eventually reached the LE well, where it was extracted for off-chip PCR.

Integrated Processing on Printed Circuit Board Device

We demonstrated this extraction technique using both a conventional glass capillary and a novel printed circuit board chip developed by the Bachman laboratory. The printed circuit board device allowed us to perform the lysis procedure on-chip, rather than in a benchtop thermocycler. This PCB chips with integrated microfluidics and electronics provides the potential to integrate and automate sample preparation.

 

Figure 2. Printed circuit board device with microfluidic channel, and sample reservoirs with integrated heaters.

Lysis

We developed a protocol for lysing malaria parasites in blood with heat and enzymatic action by proteinase K. This allowed us to access the nucleic acids of the cells without introducing high concentrations of salt that would interfere with downstream ITP separation. To quantify our lysis method, we directly stained and counted the malaria parasites with fluorescent dye.

 

Figure 3. Lysis efficiency over maximum lysis temperatures between 56° and 95° C using a benchtop thermocycler, and at applied currents between 50 and 80 mA applied current using the on-PCB heater. We compared pre-lysis and post-lysis parasite density by manual cell counting on disposable Cell-Vu hemocytometers. Parasite cells were visualized using SYBR Gold. Error bars indicate propagated 90% confidence interval based on Student t-distribution (N = 14-18 at each temperature).

Extraction

We extracted nucleic acids from our cell lysate using isotachophoresis. We used an aqueous leading electrolyte containing chloride and a trailing electrolyte containing HEPES. Nucleic acids are strongly negatively charged at biological pH, and focus between the leading and trailing electrolytes. We applied up to +600 V to the LE well, depending on channel configuration, and grounded the TE well. We monitored the ITP zone using epifluorescent microscopy, as shown in Figure 4.

 

Figure 4. Image of nucleic acids eluting into the extraction well. Nucleic acids are labeled with SYBR Gold and imaged under an epifluorescence microscope.


Reference

Marshall, L. A., Han, C. M., and Santiago, J. G. (2011) Extraction of DNA from Malaria-Infected Erythrocytes Using Isotachophoresis, Anal Chem. (click here for pdf)

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