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Confocal Laser Scanning Microscopy of DNA Electrophoresis in Microchannels

Zheng Chen, University of Michigan, 3212 H.H Dow 2300 Hayward St., Ann Arbor, MI 48109, David T Burke, Department of Human Genetics, University of Michigan, 3212 H.H Dow 2300 Hayward St., Ann Arbor, MI 48109, and Mark A. Burns, Department of Chemical Engineering, Department of Biomedical Engineering, University of Michigan, 3212 H.H Dow 2300 Hayward St., Ann Arbor, MI 48109.

An accurate separation within a short distance is important for microfabricated genomic analysis systems. Electrophoretic separation of DNA has been performed in microfabricated systems including capillary electrophoresis with linear matrices to separations with cross-linked gel techniques. Recently, research has been conducted to understand DNA electrophoretic behavior in micro-scale separations, such as investigating single DNA molecule interaction with periodic obstacles (i.e. an array of pillars) within a fluid flow. Consequently, rapid advances in device design and processes can be achieved from these fundamental studies. However, essentially all proposed mechanisms for separation with a sieving matrix are based on detection of DNA bands in the plane parallel to the electric field during electrophoresis, regardless of size scale (i.e., macro or micro).

We have used confocal laser scanning microscopy (CLSM) to detect and analyze DNA electrophoretic migration in microchannel electrophoresis. DNA electrophoresis in both in-situ polymerized cross-linked polyacrylamide and linear polyacrylamide (LPA) solution was studied. Significant differences are observed between these two systems not only from conventional imaging (x-y plane) but also that of the cross section (x-z plane) in terms of planar distribution of DNA molecules. The observation suggests an inhomogeneous structure of in-situ polymerized cross-linked polyacrylamide in a microfabricated glass channel, consistent with the microscopic research with SEM. The observations also show the phenomenon of DNA band migration to the vertical center along z-axis. These observations may lead to more efficient design of the polymerization and detection system.