NON-CHROMATOGRAPHIC PURIFICATION OF SYNTHETIC BIO-OLIGOMERS

Synthetic oligonucleotides and peptides have found wide applications in industry and academic research labs. There are ~60 peptide drugs on the market and over 500 under development. The global annual sale of peptide drugs in 2010 was estimated to be $13 billion. There are three oligonucleotide-based drugs on market; among them, the FDA newly approved Kynamro was predicted to have a $100 million annual sale. The annual sale of oligonucleotides to academic labs was estimated to be $700 million. Both bio-oligomers are mostly synthesized on automated synthesizers using solid phase synthesis technology, in which nucleoside or amino acid monomers are added sequentially until the desired full-length sequence is reached. The additions cannot be complete, which generates truncated undesired failure sequences. For almost all applications, these impurities must be removed. The most widely used method is HPLC. However, the method is slow, expensive, labor-intensive, not amendable for automation, difficult to scale up, and unsuitable for high throughput purification. It needs large capital investment, and consumes large volumes of harmful solvents. The purification costs are estimated to be more than 50% of total production costs. Other methods for bio-oligomer purification also have drawbacks, and are less favored than HPLC for most applications. To overcome the problems of known biopolymer purification technologies, we have developed two non-chromatographic purification methods. They are (1) catching failure sequences by polymerization, and (2) catching full-length sequences by polymerization. In the first method, a polymerizable group is attached to the failure sequences of the bio-oligomers during automated synthesis; purification is achieved by simply polymerizing the failure sequences into an insoluble gel and extracting full-length sequences. In the second method, a polymerizable group is attached to the full-length sequences, which are then incorporated into a polymer; impurities are removed by washing, and pure product is cleaved from polymer. These methods do not need chromatography, and all drawbacks of HPLC no longer exist. Using them, purification is achieved by simple manipulations such as shaking and extraction. Therefore, they are suitable for large scale purification of oligonucleotide and peptide drugs, and also ideal for high throughput purification, which currently has a high demand for research projects involving total gene synthesis. The dissertation will present the details about the development of the techniques. Chapter 1 will make an introduction to oligodeoxynucleotides (ODNs), their synthesis and purification. Chapter 2 will describe the detailed studies of using the catching failure sequences by polymerization method to purify ODNs. Chapter 3 will describe the further optimization of the catching failure sequences by polymerization ODN purification technology to the level of practical use. Chapter 4 will present using the catching full-length sequence by polymerization method for ODN purification using acid-cleavable linker. Chapter 5 will make an introduction to peptides, their synthesis and purification. Chapter 6 will describe the studies using the catching full-length sequence by polymerization method for peptide purification.

An Experience-Driven Pedagogy for the Instruction of Software Testing in Computer Science

In the realm of computer programming, the experience of writing a program is used to reinforce concepts and evaluate ability. This research uses three case studies to evaluate the introduction of testing through Kolb's Experiential Learning Model (ELM). We then analyze the impact of those testing experiences to determine methods for improving future courses. The first testing experience that students encounter are unit test reports in their early courses. This course demonstrates that automating and improving feedback can provide more ELM iterations. The JUnit Generation (JUG) tool also provided a positive experience for the instructor by reducing the overall workload. Later, undergraduate and graduate students have the opportunity to work together in a multi-role Human-Computer Interaction (HCI) course. The interactions use usability analysis techniques with graduate students as usability experts and undergraduate students as design engineers. Students get experience testing the user experience of their product prototypes using methods varying from heuristic analysis to user testing. From this course, we learned the importance of the instructors role in the ELM. As more roles were added to the HCI course, a desire arose to provide more complete, quality assured software. This inspired the addition of unit testing experiences to the course. However, we learned that significant preparations must be made to apply the ELM when students are resistant. The research presented through these courses was driven by the recognition of a need for testing in a Computer Science curriculum. Our understanding of the ELM suggests the need for student experience when being introduced to testing concepts. We learned that experiential learning, when appropriately implemented, can provide benefits to the Computer Science classroom. When examined together, these course-based research projects provided insight into building strong testing practices into a curriculum.