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

We demonstrate the potential use of stagnation point flows for DNA target sequence detection on single, genomic-length DNA molecules. Such a method may be particularly useful for elucidating patterns of, for example, single nucleotide polymorphisms, which may be located thousands of base pairs apart. Many single-molecule sequence-detection assays require two steps First, DNA must be incubated with site-specific probes capable of binding to the target sites of interest. Second, the marker-laden DNA molecules must subsequently be stretched so that the positions of the bound probes along the length of the molecule can be determined.;We have created a fluorescent marker using a mutant EcoRI restriction endonuclease (K249C) that enables prolonged, direct visualization of specific sequences on genomic lengths of double-stranded (ds) DNA. The marker consists of a biotinylated enzyme, attached through the biotin-avidin interaction to a fluorescent nanosphere Control over biotin position with respect to the enzymes binding pocket is achieved by biotinylating the mutant EcoRI at the mutation site. Biotinylated enzyme is incubated with dsDNA and NeutrAvidin-coated, fluorescent nanospheres under conditions that allow enzyme binding but prevent cleavage. Marker-laden DNA is then fluorescently stained and stretched on polylysine-coated glass slides so that the positions of the bound markers along individual DNA molecules can be measured. We demonstrate the marker's ability to bind specifically to its target sequence using both bulk gel-shift assays and single-molecule methods.;We have also demonstrated the use of a microfluidic stagnation point flow to trap and extend single dsDNA molecules of genomic length for detection of target sequences along the DNA backbone. Mutant EcoRI-based fluorescent markers are bound sequence-specifically to fluorescently labeled ds A-DNA. The marker-DNA complexes are introduced into a microfluidic cross slot consisting of flow channels that intersect at ninety degrees. Buffer 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 interior target sequences of dsDNA, presumably by stretching the DNA and making the interior binding sites more accessible.