A TWO-DIMENSIONAL MICROFLUIDIC SEPARATION PLATFORM FOR HIGH-THROUGHPUT GENETIC MUTATIONAL ANALYSIS

Abstract

A principal challenge to upgrading genetic-based disease prevention and management is the establishment of new mutation detection strategies that surpass the performance and capabilities of traditional gel-based approaches. The need to screen individuals for the presence of mutations across a large number of genes requires the development of technology that can more rapidly and more cost-effectively identify DNA sequence heterogeneity.

In this work, we demonstrate the feasibility of microfluidic separation platforms to transform traditional genetic screening methods by implementing these techniques in miniaturized systems to achieve far superior throughput than what is realized in contemporary clinical laboratories. Furthermore, to maximize the cost-effectiveness of these microfluidic systems, we have explored the use of inexpensive polymer materials as the substrates for these systems. In the initial part of this work, fabrication methods for polymeric microfluidic separation platforms were developed. The initial system consisting of a hybrid polydimthylsiloxane-silicon structure was used to demonstrate the capability of microfluidic platforms to perform field-effect electroosmotic flow control at enhanced efficiencies.

Polycarbonate microfluidic devices were developed to demonstrate temperature gradient gel electrophoresis (TGGE) for the first time in a miniaturized platform. TGGE analyses of model mutant DNA fragments, each containing a single base substitution, were achieved using both single and 10-channel parallel measurements in a microfluidic platform. When compared to conventional capillary systems, the polymer microfluidic TGGE system described here demonstrated similar separation efficiency at one tenth of the analysis time, thus displaying the potential of this technology to dramatically increase the throughput of TGGE analyses.

Finally the conception and realization of a parallel two-dimensional microfluidic platform for genetic mutation screening is described here. The system provides for online coupling of an initial size-based gel electrophoresis separation with a secondary parallel sequence-based TGGE separation. Such a separation strategy allows for the highthrouput analysis of complex genomic samples such as a mixture of multiplexed polymerase chain reaction (PCR) products. The comprehensive two-dimensional mutation analysis performed using this microfluidic system demonstrated the capability to detect mutations in DNA fragments at speeds approximately one to two orders of magnitude higher than is possible with conventional gel-based gene scanning methods.