An automatic ultramicro volume DNA ligation process using coplanar electrode type of electrowetting-on-dielectric (EWOD) microfluidic system was designed for economy of reagent. The droplets, containing DNA, a ligation enzyme and a multi-salt reaction buffer, served to complete the ligation using a developed EWOD system. Droplets of ultramicro volume (0.3 μL) were successfully generated from reservoirs between one plate with coplanar electrodes and another plate with a hydrophobic surface free of electrodes. In one successful cloning, the usage of total reagents in an ultramicro volume EWOD chip was 2.1 μL; no volume was wasted, for comparison with 85 % waste with the standard protocol and 80 % waste with a free-cover coplanar EWOD chip. The results show also that the entire process was accomplished without damage to the chip surface and without biomaterial annulment.
Because synthetic oligonucleotides have advantages of cost, purity and custom-made sequences relative to natural products, oligonucleotides are advantageously replacing genomic and cloned DNA as DNA probes. Modification of functional groups at the two ends of a probe for immobilization on a solid support remains, however, tedious and expensive. We have devised a rapid, efficient and flexible, and inexpensive method for synthesis in-situ of oligonucleotide probes on a magnetic bead. This procedure requires about 15 min at 25 °C with a DNA polymerase I Large Klenow Fragment. We used MB, with the synthesized probe on the surface, to detect the single nucleotide polymorphism (SNP) site in an oligonucleotide ligation assay to confirm the correct function of the synthesized probe.
In the DNA detection, this work demonstrated the feasibility of detecting 250 zM Escherichia coli O157:H7 eaeA target DNA by using a magnetic bead-based DNA detection assay with designed labeling strategy within 40-60 min. The magnetic beads (MB) were used as the solid support for the binding probe and isolated the target DNA from the sample. The detection signals could be amplified from the multi-layers biotin-streptavidin conjugated quantum dots (QDots) based on binding with specific designed biotinylated linker. This assay method would provide a simple, rapid, and ultra-sensitive detection method for DNA or other biomolecular analysis.
In the future, with a design consisting of electrode pathways in a flexible pattern and multiplex reservoirs, an EWOD digital microfluidic system with coplanar electrodes would be improved as an efficient parallel DNA-cloning system in construction of an artificial library or expression library. On the other hand, the EWOD system also be suggest operating the SNP detection via oligonucleotide ligation assay and the MB which carried in-situ oligonucleotide probe on the surface. Moreover, was also being expected that the detecting process developed for very low concentration DNA could be successfully achieved by using the EWOD system.

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