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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.

SUBMITTER: Dylla-Spears R

PROVIDER: S-EPMC3322643 | biostudies-literature | 2010 Jun

REPOSITORIES: biostudies-literature

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

Dylla-Spears Rebecca R Townsend Jacqueline E JE Jen-Jacobson Linda L Sohn Lydia L LL Muller Susan J SJ

Lab on a chip 20100331 12

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 ...[more]

PMID: 20358051

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Project description:In this study, we show the importance of extensional rheology, in addition to the shear rheology, in the choice of blood analog solutions intended to be used in vitro for mimicking the microcirculatory system. For this purpose, we compare the flow of a Newtonian fluid and two well-established viscoelastic blood analog polymer solutions through microfluidic channels containing both hyperbolic and abrupt contractions∕expansions. The hyperbolic shape was selected in order to impose a nearly constant strain rate at the centerline of the microchannels and achieve a quasihomogeneous and strong extensional flow often found in features of the human microcirculatory system such as stenoses. The two blood analog fluids used are aqueous solutions of a polyacrylamide (125 ppm w∕w) and of a xanthan gum (500 ppm w∕w), which were characterized rheologically in steady-shear flow using a rotational rheometer and in extension using a capillary breakup extensional rheometer (CaBER). Both blood analogs exhibit a shear-thinning behavior similar to that of whole human blood, but their relaxation times, obtained from CaBER experiments, are substantially different (by one order of magnitude). Visualizations of the flow patterns using streak photography, measurements of the velocity field using microparticle image velocimetry, and pressure-drop measurements were carried out experimentally for a wide range of flow rates. The experimental results were also compared with the numerical simulations of the flow of a Newtonian fluid and a generalized Newtonian fluid with shear-thinning behavior. Our results show that the flow patterns of the two blood analog solutions are considerably different, despite their similar shear rheology. Furthermore, we demonstrate that the elastic properties of the fluid have a major impact on the flow characteristics, with the polyacrylamide solution exhibiting a much stronger elastic character. As such, these properties must be taken into account in the choice or development of analog fluids that are adequate to replicate blood behavior at the microscale.

Dataset Information

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

Publications

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

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