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In this paper, we demonstrate immobilization and stretching of single lambda-phage DNA molecules within microfluidic systems using ac fields. We present a novel "thiol-on-gold"-based immobilization technique for fixing one specific end (3' end) of a DNA molecule onto a gold electrode. A polymer-enhanced medium (approximately 3.75 wt % linear polyacrylamide in Tris-HCl) is used to obtain fully stretched configurations (21 microm) of fluorescently stained lambda-DNA molecules. We also present an optimized microelectrode design with pointed electrodes and an electrode spacing of 20 microm for stretching DNA molecules with an ac field (1 MHz, 3 x 10(5) V/m). Finally, using these techniques, we immobilize a single DNA molecule at one electrode edge, stretch the molecule, and fix the other end at an adjacent electrode edge, forming a bridge between two electrodes within a microfabricated device.