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A short focused pulse of light was used to selectively cut lambda-phage DNA molecules at specific restriction sites. Lambda DNA (48.5 kbp) was stretched and placed in a solution containing a restriction enzyme (Sma 1), caged magnesium ions (using a DM-Nitrophen complex), and a chelating agent (EDTA). When a pulse of UV light was directed at a particular location on the stretched DNA molecule, magnesium ions were released into solution. A series of binding reactions then occur in which the enzyme and the chelating agent compete for free [Mg.sup.2+] ions. Since Sma 1 functions only in the presence of [Mg.sup.2+], as is true of most endonucleases, the site(s) in the vicinity of the pulse (typically ~6 [micro]m) were cut while other sites (three total for this DNA/enzyme pair) were not. The ratio of the concentration of the chelating agent to that of the magnesium ions was used to control the radius of this reaction zone with higher ratios leading to smaller, localized reaction areas. This optically based reaction mechanism could be useful to understand single molecule enzymatic kinetics, and when coupled with other DNA analysis techniques, this could be used to construct complex genotyping and sequencing devices that would analyze parts of single DNA molecules.

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 (~3.75 wt % linear polyacrylamide in Tris-HCl) is used to obtain fully stretched configurations (21 [micro]m) of fluorescently stained [lambda]-DNA molecules. We also present an optimized microelectrode design with pointed electrodes and an electrode spacing of 20 [micro]m for stretching DNA molecules with an ac field (1 MHz, 3 x [10.sup.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.