Development of an integrated microdevice for multiplex genetic polymorphism analysis

Abstract

Polymerase chain reaction (PCR) has been considered as a gold standard method of nucleic acids analysis in the fields of clinical diagnostics, microbiological detection, forensics, environmental screening, and food safety testing. Recently, many efforts have been dedicated to transforming the conventional PCR platform into lab-on-a-chip based miniaturized system due to the advantages of high-speed analysis, high-throughput capability, low reagent consumption, and portability. In this thesis, I have focused on the devel-opment of an integrated genetic analysis microdevice for on-site multiplex single nucleotide polymorphism (SNP) typing. For application to on-site genetic analysis using PCR, the process for separation and detection of resultant PCR products should be integrated with the PCR function on a single chip format. In addition, the fluorescence-based optical instrumentation and a peripheral device for detection and operation of the microsystem need to be miniaturized to realize the portable genetic analysis system. In Chapter 3, an allele-specific polymerase chain reaction (AS PCR) and capillary electrophoresis (CE) were integrated into a 4-in single chip platform, which was then applied for on-site identification of HANWOO (Korean indigenous beef cattle). The glass-based microchip consists of four layers: a microchan-nel plate for microfluidic control, a Pt-electrode plate for a resistance temperature detector (RTD), a poly(dimethylsiloxane) (PDMS) membrane and a manifold glass for microvalve function. The AS PCR for multiplex SNP typing was conducted on a 2-μL PCR reactor. After AS PCR, the amplicons were moved to the CE injection channel by electrophoretic force, and then separated along the CE separation channel through the sieving matrix. At the end of the separation channel, the miniaturized fluorescence detector could gain the fluorescence signal from the amplicons, showing the electropherogram of the AS products. The operations of sample loadin...