Restriction mapping in nanofluidic devices

Abstract

We have performed restriction mapping of DNA molecules using restriction endonucleases in nanochannels with diameters of 100-200 nm. The location of the restriction reaction within the device is controlled by electrophoresis and diffusion of Mg2+ and EDTA. We have successfully used the restriction enzymes SmaI, SacI, and PacI, and have been able to measure the positions of restriction sites with a precision of approximately 1.5 kbp in 1 min using single DNA molecules.

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 × 100 μm cross-section) with 10 nanochannels of ≈100 nm × 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 ΔVn (≈2 V), and the voltage across the microchannels is ΔVμ (≈2 V). During DNA imaging, no voltages were applied.

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.

Fig. 3.

Time-resolved restriction mapping of λ-DNA in nanochannels. (a) Restriction of three λ-DNA (48.5 kbp) molecules by using SmaI in channels of ≈120 nm × 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 λ-DNA molecules by using SacI in channels of 140 nm × 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,λ-DNA ladder; lane 2, long-range PFGE ladder; lanes 3 and 4, digestion product after Pacl.

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.