Master of Science (MS)
Biomedical, Chemical & Materials Engineering
Customized production of synthetic DNA from oligonucleotides is in high demand. However, current technologies are costly and labor-intensive. A microfluidic technology can significantly decrease cost and labor. The purpose of this study was to develop a gene assembly protocol that was utilized on the Mondrian™ SP digital microfluidic device. The fragment of the human influenza virus hemagglutinin (HA) gene (339 bp) was assembled from 12 oligonucleotides by the Gibson assembly method and error corrected with CorrectASE™ enzyme twice. The samples were analyzed by Sanger sequencing to verify the final accuracy of the assembly. A complete automation of droplet generation and movement on digital microfluidic droplet technology was achieved in the study. The reactions were scaled down to 0.6-1.2 µL. Gibson assembly, PCR, and enzymatic error correction reactions were optimized and combined in a single protocol. The microfluidic assembly demonstrated approximately 3 errors/kb error frequency. Polymerase chain reaction supplemented with additional MgCl2, Phusion, and PEG 8000 provided amplification of the assembly and error correction products. The lowest error frequency of 0.3 errors/kb was achieved after one CorrectASE™ treatment. However, microfluidic error correction was not reliable due to CorrectASE™ interactions with the microfluidic surface, which need to be the subject of future work.
Khilko, Yuliya, "Development of DNA assembly and error correction protocols for a digital microfluidic device" (2017). Master's Theses. 4805.