2014 Theses Doctoral

Novel Engineering Approaches for DNA Sequencing and Analysis

Palla, Mirko

DNA sequencing is a fundamental tool in biological and medical research. DNA molecules contain the heritable genetic information in all living organisms and encode all the proteins in our body. Therefore, determination of DNA sequence is useful in basic biological research, evolutionary biology, as well as the applied biological fields, such as diagnostic or forensic research. High-throughput DNA sequencing is essential for personalized medicine. To achieve this dream, the price of genome sequencing should be dramatically decreased to a level that most people can afford. Despite the refinements of Sanger sequencing, the current genome sequencing cost remains formidable. Therefore, revolutionary advances in DNA sequencing technology are demanded. To overcome the limitations of the current sequencing technologies, a variety of new DNA sequencing methods have been investigated with the aim of eventually realizing the goal of the $1,000 genome, including sequencing by synthesis (SBS). In this thesis, we build upon current state-of-the-art sequencing technologies such as SBS to develop novel proof-of-principle technologies for high-throughput DNA sequencing; demonstrate a general platform for high sensitivity biomolecular detection; and briefly study DNA processing protein (e.g., helicase) functions at the atomistic level. The following is a summary of the resulting work presented herein: first, a new DNA sequencing technology development is presented that utilizes surface-enhanced Raman spectroscopy (SERS); second, a novel approach of SERS-based biosensing for quantitative detection of biomolecules is demonstrated; third, a rigorous mathematical development of an analytical model is described to predict experimental SERS signal intensity distributions for biomolecular quantification; fourth, a versatile SERS-based quantitative method is developed to monitor the catalyst-free click reaction efficiency for small molecule conjugation; fifth, theoretical work using molecular dynamics simulations for analyzing the mechanical behavior of a molecular motor involved in DNA processing are described; and finally, the thesis presents a proposed nanodevice to combine the SERS-SBS technology with our bioquantification method into one functional unit. Consequently, these research efforts provide a foundation for the novel use and integration of SERS-SBS into microfluidic systems for a wide range of applications, such as high-throughput DNA sequencing and genetic diagnostics, as well as the theoretical framework to investigate DNA polymerase function at the atomic level.