Microscale and fluorescence-based techniques/ systems to monitor biological changes in real-time

The long-term objective of this work is to develop an integrated continuous-flow microfluidic device for the analysis of genetic changes in DNA. Initially, this required the development of new techniques to simulate and then quantify DNA damage using existing laboratory instrumentation. Many analytical protocols exist for the quantification of varied types of DNA damage, which span a range of complexity and sensitivity. As an efficient alternative to the existing procedures, this work demonstrates the application of quantitative polymerase chain reaction (qPCR) and high-resolution DNA melting analysis (HRMA) to the detection and quantification of intramolecular DNA damage and/or strand breaks. These proven molecular biology methods are essentially single-step processes. When implemented with a third-generation saturating DNA dye, we have demonstrated that high sensitivity can be obtained with both qPCR and HRMA. This work reinforces the applicability of these techniques in the real-time analysis of biological changes using existing laboratory instrumentation. In order to begin the next step of miniaturizing these protocols, we have refined a microfluidic fabrication protocol. Using these optimized processing conditions to manufacture prototype microfluidic devices, we have successfully achieved on-chip DNA amplification, which is the most critical component of the qPCR methods that we have developed for DNA damage analysis. By further integrating a fluorescence-imaging functionality into this system (which is beyond the scope of this current project), this microfluidic system will provide a rapidity and sensitivity of DNA damage quantification that does not currently exist