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(322e) Diffusion-Based Microfluidic PCR for “One-Pot” Analysis of Cells

Ma, S., Virginia Tech
Loufakis, D. N., Virginia Tech
Cao, Z., Virginia Tech
Chang, Y., Virginia Tech
Achenie, L., Virginia Tech
Lu, C., Virginia Tech

Genetic analysis starting with cell samples often requires multi-step processing including cell lysis, DNA isolation/purification, and polymerase chain reaction (PCR) based assays. When conducted on a microfluidic platform, the compatibility among various steps often demands complicated procedure and complex device structure. Here we present a microfluidic device that permits “one-pot” strategy for multi-step PCR analysis starting from cells (i.e. using one reactor for successive reactions without separation and purification). Taking advantage of the diffusivity difference, we replace the smaller molecules in the reaction chamber by diffusion while retaining DNA molecules inside. This simple scheme effectively removes reagents from the previous step to avoid interference and thus permits multi-step processing in the same reaction chamber. Our approach shows high efficiency for PCR and potential for a wide range of genetic analysis including assays based on single cells.

So far there have been three strategies to alleviate the impact of cell pretreatment on PCR: 1. Institute some type of isolation step to remove lysis reagent and undesired intracellular molecules while preserving nucleic acids. The involved isolation step increase the complexity of the procedure and chip design. 2. Use alternative lysis methods, such as freeze-thaw or heating, that interfere with PCR to a less degree than surfactants. These methods are less efficient than surfactant-based lysis. 3. Use Direct PCR kit based on Phusion polymerase that is tolerant to surfactant-based lysis reagents, but this commonly-used fluorescence-based quantification is impossible with the Phusion polymerase system. To summarize, microfluidic strategies that permit simple operation and device design and are compatible with Taq polymerase are still in high demand.

In this work, we demonstrate a simple scheme for conducting microfluidic PCR starting from cells, taking advantage of the difference in the diffusivity between genomic DNA and various reagent/intracellular molecules. Our microfluidic device had a simple structure that included a reaction chamber connected with two loading chambers on both sides. The connections between the reaction chamber and the two loading chambers could be cut off by closing two-layer valves 7. The lysis buffer and the PCR mix were introduced into the chamber by concentration-gradient-driven diffusion. During such diffusion, the new solution replaces the solution and molecules from the previous step without removing the slow-diffusing genomic DNA. The single chamber (“one-pot”) design drastically minimizes the complexity of the microfluidic device. We envision that this may be a general approach for on-chip multi-step assays on genomic DNAs.