Abstract
Production of accurate DNA products is essential for many biological applications including drug development and delivery, synthetic biology, molecular engineering and epigenetics. It is difficult to build long DNA chains accurately and repeatably with conventional methods such as phosphoramidite method and polymerase chain assembly. DNA aqueous enzymatic assembly is a promising alternative. Multiple DNA fragments can be reliably and consistently assembled into one product using engineering enzymes in an aqueous medium. Digital Microfluidics uses asymmetric electric fields to simultaneously manipulate multiple reagent droplets on an array of electrodes. This platform is biocompatible and easily controllable. It has been used for point-of-care testing and biological assay protocols while increasing throughput and minimizing reagent waste. It is a prime candidate for performing DNA assembly. This work successfully demonstrates assembly of single and double-stranded oligonucleotides on digital microfluidic devices in air and oil. Two DNA bricks were assembled in air and in oil to form a product 250 base pairs long. Each brick consisted of three annealed 80-nucleotide long oligonucleotides. Three, four and five single-stranded oligonucleotides (each between 28 and 50 nucleotides long) were assembled in air to obtain products of 125, 150, and 175 base pairs long respectively. On-chip incubations were performed at room temperature and 37°C by integrating an external heater into a commercially available DMF control system. In these experiments, droplets were surrounded by an oil medium to eliminate evaporation. DNA products were compared to benchtop controls to verify their success.
Publication Date
11-7-2024
Document Type
Dissertation
Student Type
Graduate
Degree Name
Engineering (Ph.D.)
College
Kate Gleason College of Engineering
Advisor
Michael Schertzer
Advisor/Committee Member
Patricia Iglesias Victoria
Advisor/Committee Member
Kathleen Lamkin-Kennard
Recommended Citation
Houchaimi, Sari, "Digital Microfluidic Oligonucleotide Synthesis: Automated assembly of DNA products in air and oil with integrated on-chip incubation" (2024). Thesis. Rochester Institute of Technology. Accessed from
https://repository.rit.edu/theses/11927
Campus
RIT – Main Campus
