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Restriction mapping in nanofluidic devices

Robert Riehn ^*, Manchun Lu , Yan-Mei Wang ^*, Shuang Fang Lim ^*, Edward C. Cox , and Robert H. Austin ^*, 

Departments of^*Physics andMolecular Biology, Princeton University, Princeton, NJ 08544

View larger version (28K): [in a new window]   Fig. 1. Schematic of the device used in the experiments. Fluidic components were first fabricated on a fused silica substrate and later sealed with a fused silica coverslip. We linked two microfluidic channels (a and b, 1 µm x 100 µm cross-section) with 10 nanochannels of 100 nm x 100 nm (c). The solution in the "loading" microchannel (b) contained DNA and EDTA, and the "exit" microchannel (a) contained Mg2+. Both channels contained restriction enzyme. Both DNA and Mg2+ were moved through the device by electrophoresis using four electrodes. The voltage applied across the length of the nanochannels is marked Vn (2 V), and the voltage across the microchannels is Vµ (2 V). During DNA imaging, no voltages were applied.

View larger version (35K): [in a new window]   Fig. 2. Images of 100-µm-long nanochannels containing a Mg2+-sensitive dye. (Scale bar: 5 µm.) The image in a was recorded without voltages, and b was acquired with 1 V over the nanochannel. Magnesium entered the nanochannels from the left, and the images were averaged for 40 s. c is the ratio of images a/b taken along the nanochannels.

View larger version (66K): [in a new window]   Fig. 3. Time-resolved restriction mapping of -DNA in nanochannels. (a) Restriction of three -DNA (48.5 kbp) molecules by using SmaI in channels of 120 nm x 120 nm cross-section. The DNA is stretched to 40% of its contour length. (Left) Individual 10-ms frames. (Right) Time traces, in which each line corresponds to intensity along the nanochannel in a single frame. From the known DNA sequence, we expect fragments of 19.4, 12.2, 8.3, and 8.6 kbp, in that order. (b) Restriction of three -DNA molecules by using SacI in channels of 140 nm x 180 nm cross-section. (Left) Individual 10-ms frames. (Right) Time traces. The DNA is expected to stretch to 25-30% of its contour length in channels of these dimensions. We expect fragments of 22.6, 0.9, and 24.8 kbp. The smallest 0.9-kbp segment is in general not visible. (c) Cutting 61-kbp DNA with PacI. The top panels are time traces of cutting in roughly 120 nm wide nano-channels, where we expect a stretch of 40%. (Right) Pulsed-field gel electrophoresis separation of the digestion product of an unpurified 61-kbp DNA and cloning vector. Lane 1,-DNA ladder; lane 2, long-range PFGE ladder; lanes 3 and 4, digestion product after Pacl.

View larger version (22K): [in a new window]   Fig. 4. The absolute cut positions from 29 molecules with two and three cuts. The line is a fit to the histogram by using the sum of three Gaussian distributions.

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