A microfabricated device for sizing and sorting DNA molecules

Abstract

We have demonstrated a microfabricated single-molecule DNA sizing device. This device does not depend on mobility to measure molecule size, is 100 times faster than pulsed-field gel electrophoresis, and has a resolution that improves with increasing DNA length. It also requires a million times less sample than pulsed-field gel electrophoresis and has comparable resolution for large molecules. Here we describe the fabrication and use of the single-molecule DNA sizing device for sizing and sorting DNA restriction digests and ladders spanning 2-200 kbp.

Figure 1

Optical micrograph of T-channel device. The large channels have lateral dimensions of 100 μm, which narrow down to 5 μm at the T junction. The depth of the channels is 3 μm. In early prototypes, we found that because of the large aspect ratio (100 μm in width by 3 μm in depth), some of the elastomer channels would bow and pinch off by sealing directly to the glass. This problem was remedied in later versions by adding support pillars to the mask that would prop up the large channels and prevent bowing. (Inset) Magnified view of T junction. The channels are 5 μm wide at this point. Note the high fidelity of the elastomer replica.

Figure 2

Histogram of HindIII digest of λ DNA. The peaks represent (from right to left) fragments of length 23 kbp, 9 kbp, 6 kbp, 4 kbp, and an unresolved combination of 2 kbp and 2.3 kbp.

Figure 3

Precision and resolution of HindIII digest. The histogram of Fig. 2 was fit with 5 Gaussians to estimate the precision and resolution of the measurement. (Left) The known sizes of the restriction fragments are compared with the fitted peak locations. The measurements are linear with a precision of a few percent. (Right) The widths of the peaks determine the resolution of the measurement. The coefficient of variation is the peak’s SD divided by its height and is an indication of the fractional resolution. Resolution improves with longer molecules. In both graphs, the error bars are smaller than the size of the data point symbols.

Figure 4

Analysis of λ ladder. To test the upper length limit of the device, a λ ladder was analyzed. Peaks corresponding to 50, 100, 150, and 200 kbp can clearly be resolved. (Inset) The peak height measurement is linear even out to 200 kbp.