Chemical and Biomolecular Engineering, Department of


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A DISSERTATION Presented to the Faculty of The Graduate College at the University of Nebraska In Partial Fulfillment of Requirements For the Degree of Doctor of Philosophy, Major: Interdepartmental Area of Engineering (Chemical & Biomolecular Engineering), Under the Supervision of Professors Anuradha Subramanian and David S. Hage. Lincoln, Nebraska: April, 2011

Copyright 2011 Joel R. TerMaat


The de novo synthesis of genes is emerging as a powerful tool in biotechnology. The ability to synthesize genes of any desired sequence opens the door to seemingly unlimited research possibilities. Major advances have been made recently in de novo gene synthesis, with Polymerase Chain Assembly (PCA) routinely used to construct functional sequences from short single-stranded oligonucleotides. However, current PCA techniques are lacking in speed and fidelity. Additionally, substantial undesired reactants/products are present in the final reaction. A novel process and accompanying workstation has been developed that incorporates rapid PCA synthesis coupled with subsequent affinity purification of the synthesis mixture. The system enables fast and accurate PCA synthesis and isolation of the full length DNA of interest.

In the synthesis step, the desired sequence is assembled and PCR amplified in a fast thermocycler to generate a high yield of product with minimal runtime and errors. A traditional 2-step PCA-PCR approach is utilized to assemble and amplify the full-length gene. Alternatively, integration of PCA and PCR into a single rapid reaction is also employed, working reliably up to about 1 kb. For the synthesis of genes longer than 1.5 kb, a convergent rapid synthesis strategy is proposed in which the full-length sequence is assembled by a series of synthesis steps from smaller fragments. In this work, a variety of genes ranging from 600 bp up to 3.8 kb in length are synthesized by rapid PCA techniques.

The second section of the workstation employs two affinity columns to isolate the desired full-length product from shorter unwanted reactants/products inherent in the PCA reaction. During PCR amplification, labels are incorporated into the desired product on both ends via PCR primers. Undesired products contain only one of these labels, or no label at all. The first column interacts with one of the labels to partially purify the mixture. The intermediate product is then subsequently purified via the second column to isolate the full-length sequence. An initial prototype workstation was developed, while the 2nd generation instrument consisted of process refinements using two antibody columns for immunoaffinity purification.