High-performance integrated DNA analysis microsystems

High-throughput genetic analysis is critical for understanding the processes by which living organisms function and for detecting and treating disease. Typical experimental genetic methods are labor-intensive, sensitive to contamination, inefficient and costly. The objective of my work is to develop and demonstrate microsystems that integrate the steps of genetic analysis into simple, robust and rapid processes.; The heart of my work was the integration of two key genetic analysis steps, PCR amplification and capillary electrophoretic (CE) analysis of the products on a monolithic microdevice. PCR chambers (280 nL) and integrated CE microchannels were chemically etched into a glass wafer. Microfluidic valves and hydrophobic vents were developed to load the chamber and to contain the sample within the reactor. This PCR-CE microdevice was successfully used to perform amplification from initial DNA template concentrations as low as 4--5 molecules in the 280-nL reactor volume. A similar microdevice was used to conduct stochastic PCR amplification and demonstrated sensitivity to the ultimate single molecule limit.; I next developed fully integrated microfabricated heaters and temperature sensors for the PCR-CE microdevice. Thin film temperature detectors were fabricated within the PCR chambers and heaters were microfabricated on the backside of the device. The resulting microsystem was capable of temperature transition rates up to 20°C/second, twice that of previous devices and orders of magnitude faster than conventional PCR thermal cyclers. The utility of this device was demonstrated by performing a sex determination assay directly from human genomic DNA.; Finally, to explore the capabilities of these integrated microsystems in point-of-care and field-capable applications, a portable DNA analysis instrument was developed. The instrument incorporates all the components necessary for running the PCR reaction and for performing electrophoresis and optical detection on a PCR-CE microdevice the size of a microscope slide. Detection of Escherichia coli pathovars and antibiotic resistance in Staphylococcus aureus cells was accomplished in a 30-minute assay with 2--3 cell sensitivity. My demonstration of fully portable systems for genetic analysis establishes their feasibility for rapid detection in the areas of public health, bioterrorism and biowarfare, clinical medicine, and forensics.