Single-molecule sequence detection via microfluidic planar extensional flow at a stagnation point

Abstract

We demonstrate the use of a microfluidic stagnation point flow to trap and extend single molecules of double-stranded (ds) genomic DNA for detection of target sequences along the DNA backbone. Mutant EcoRI-based fluorescent markers are bound sequence-specifically to fluorescently labeled ds lambda-DNA. The marker-DNA complexes are introduced into a microfluidic cross slot consisting of flow channels that intersect at ninety degrees. Buffered solution containing the marker-DNA complexes flows in one channel of the cross slot, pure buffer flows in the opposing channel at the same flow rate, and fluid exits the two channels at ninety degrees from the inlet channels. This creates a stagnation point at the center of a planar extensional flow, where marker-DNA complexes may be trapped and elongated along the outflow axis. The degree of elongation can be controlled using the flow strength (i.e., a non-dimensional flow rate) in the device. Both the DNA backbone and the markers bound along the stretched DNA are observed directly using fluorescence microscopy, and the location of the markers along the DNA backbone is measured. We find that our method permits detection of each of the five expected target site positions to within 1.5 kb with standard deviations of <1.5 kb. We compare the method's precision and accuracy at molecular extensions of 68% and 88% of the contour length to binding distributions from similar data obtained via molecular combing. We also provide evidence that increased mixing of the sample during binding of the marker to the DNA improves binding to internal target sequences of dsDNA, presumably by extending the DNA and making the internal binding sites more accessible.

Figure 1

(a) Cross-slot trapping scheme. Tagged λ-DNA complexes are introduced through one channel, opposed by buffer containing no fluorescent particles. Flow exits through the two outlet channels to one fixed-height and one variable height reservoir, adjustment of which permits trapping at the stagnation point. Device depth is 130 µm. Image area for DNA studies is an 80-µm square centered at the stagnation point. (b) Streak image showing stagnation point and particle path lines arising from planar extensional flow. (c) Strain rate map for flow in an 800-µm-wide cross slot, calculated using 2D fluid dynamics simulations with v_max = 892 µm/s. Contours represent lines of constant ε̇, spaced 0.25 s⁻¹ apart. Strain rate remains constant to within 95% of the peak strain rate over a 100-µm radius about the stagnation point.

Figure 2

Representative fluorescence images of marker-DNA complexes trapped at the stagnation point and stretched in planar extensional flow (De = 3.9), with corresponding fluorescence intensity profiles. Peaks in intensity indicate marker positions. Dashed vertical lines denote expected target locations for EcoRI on fully extended λ-DNA. Scale bar is 5 µm.

Figure 3

Binding distributions for mutant-EcoRI markers bound to stained λ-DNA stretched in planar extensional flow. The number of markers measured is (a) N = 132 at De = 1 and (b) N = 263 markers at De = 3.9. Solid line is a best fit to the data using a linear combination of Gaussians. Dashed vertical gray lines indicate expected target locations of EcoRI on fully extended λ-DNA.

Figure 4

Comparison of best Gaussian fits from binding distributions obtained from slide-stretched DNA (solid gray: 79% extension, broken gray: 92% extension) and flow-stretched DNA (solid black: De = 1, broken black: De = 3.9). Dashed vertical gray lines indicate expected target locations of EcoRI on fully extended λ-DNA.

Figure 5

Comparison of binding distributions obtained from (a) N = 130 markers bound to DNA with no mixing during incubation, and (b) N = 133 markers bound to DNA with continuous mixing during incubation. Solid lines depict Gaussian best fits to the data, while dashed vertical gray lines indicate expected target locations of EcoRI on fully extended λ-DNA. Statistics from best fits are provided in Table S-1. (c) Unmixed population (open circles) is distributed differently among the five target sites compared to mixed population (filled circles).