Single-Molecule Target Sequence Detection Using Extensional Flow

We use planar extensional flow to trap and extend single molecules of double-stranded genomic DNA for detection of target sequences along the DNA backbone. Fluorescently tagged enzymatic probes are bound sequence-specifically to fluorescently labeled double-stranded λ-DNA. The complexes are introduced into a microfluidic cross slot, where the tagged DNA molecules are trapped and elongated at the stagnation point of the planar extensional flow. The degree of elongation can be controlled using the flow strength in the device. Beads bound along the stretched DNA may be directly observed, and their locations along the backbone determined, using fluorescence microscopy. Previously, we have demonstrated the feasibility of this method for detection of specific sequences, for single-molecule kinetic and binding studies, as well as for single-molecule sorting (R. Dylla-Spears, L.L. Sohn, and S.J. Muller, AIChE Annual Meeting, Salt Lake City, November 2007). We now compare the measured bead positions to the expected EcoRI target sequences on λ-DNA to determine the method's precision and accuracy at molecular extensions of 70% and 90% of the contour length.