Integrated view of genome structure and sequence of a single DNA molecule in a nanofluidic device

Marie, Rodolphe; Pedersen, Jonas Nyvold; Bauer, David L.V.; Rasmussen, Kristian Hagsted; Yusuf, Mohammed; Volpi, Emanuela V.; Flyvbjerg, Henrik; Kristensen, Anders; Mir, Kalim U.

doi:10.1073/pnas.1214570110

Cited by 93 publications

(114 citation statements)

References 39 publications

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“…This extension exceeds all previously reported results obtained in nanochannels with similar dimensions and salt concentrations [39,40] and, hence, may be of practical interest: Research on genomic-length DNA confined inside nanofluidic structures has increased our understanding of biological interactions of DNA and enables coarse-grained optical mapping of the sequence of intact individual DNA molecules [41][42][43][44][45][46]. Stretching DNA to nearly its contour length remains a challenge, however.…”

Section: Prl 113 268301 (2014) P H Y S I C a L R E V I E W L E T T Econtrasting

confidence: 45%

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Thermophoretic Forces on DNA Measured with a Single-Molecule Spring Balance

et al. 2014

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Section: Prl 113 268301 (2014) P H Y S I C a L R E V I E W L E T T Econtrasting

confidence: 45%

“…Stretching DNA to nearly its contour length remains a challenge, however. It requires either very narrow nanochannels [47,48], extreme salt concentrations [49], or crossed flows [46]. Very narrow channels are difficult both to fabricate and to load with DNA.…”

Section: Prl 113 268301 (2014) P H Y S I C a L R E V I E W L E T T Ementioning

confidence: 99%

Thermophoretic Forces on DNA Measured with a Single-Molecule Spring Balance

et al. 2014

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“…The latter scaling is based on a Flory exponent ν = 3/5; the more accurate estimate X ∼ D (ν − 1)/ν ∼ D −0.7015 is in excellent agreement with simulation data for long chains in the extended de Gennes and de Gennes regimes. [12][13][14][15]17 It is important to recognize that, while the extension of the confined chain is the simplest observable metric and quite relevant for genomic applications of confined DNA, [18][19][20][21][22][23][24] this single thermodynamic variable is likely insufficient to resolve many of the open questions surrounding different regimes. Rather, we require measurements of both the average chain extension and its variance, the latter being related to the effective spring constant of the chain.…”

Section: Introductionmentioning

confidence: 99%

Mixed confinement regimes during equilibrium confinement spectroscopy of DNA

Gupta

Sheats²,

Muralidhar

et al. 2014

The Journal of Chemical Physics

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We have used a combination of fluorescence microscopy experiments and Pruned Enriched Rosenbluth Method simulations of a discrete wormlike chain model to measure the mean extension and the variance in the mean extension of λ-DNA in 100 nm deep nanochannels with widths ranging from 100 nm to 1000 nm in discrete 100 nm steps. The mean extension is only weakly affected by the channel aspect ratio. In contrast, the fluctuations of the chain extension qualitatively differ between rectangular channels and square channels with the same cross-sectional area, owing to the "mixing" of different confinement regimes in the rectangular channels. The agreement between experiment and simulation is very good, using the extension due to intercalation as the only adjustable parameter.

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“…These are the same forces as those making rubber bands and O-rings Composite photograph of individual double-stranded DNA molecules fluorescently dyed in a sequence-specific manner (barcoding) (7) and flow-stretched for visual inspection (8). Green vertical lines show three such molecules, each about 2 Mbp long, stretched in nanoslits by opposing flows.…”

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confidence: 99%

“…The top orange line shows 16 repeats: an impossible counting task for established sequencing methods, but trivial here, done with a single molecule. Insertions, deletions, and inversions were also found (8). Blue chromosomes: a single molecule that looked interesting in this visual inspection was retrieved from the nanofluidic device and whole genome amplified for further analysis.…”

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confidence: 99%

How to get into that “room at the bottom”

Flyvbjerg¹

2014

Proc. Natl. Acad. Sci. U.S.A.

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"There's plenty of room at the bottom," as Richard Feynman famously formulated it in 1959 (1). He considered the problem of manipulating and controlling things "at the bottom" of the length scale, from Ångströms to micrometers. The fame came retroactively with the rise of nanotechnology (2). By then, the advantage of studying small objects in small laboratories was clear: in a microscopic laboratory space, one can do things that are impossible in larger laboratories, such as inspecting a long DNA molecule visually (Fig. 1). In a larger volume of fluid, Brownian motion makes such a long, flexible molecule tangle up in a bundle, unless one pulls at its ends, which requires handles of a kind. It is done, but unpractical for inspection of many molecules, as they all would need handles. It seems simpler to flow the DNA molecule into a nanochannel so narrow that the molecule is fully stretched. Unfortunately, this is not simple, because there's plenty of friction in that room at the bottom and an entropic barrier at its doorstep. This is a bottleneck for a promising complement to state-of-the-art DNA sequencing technologies. They could use the coarse-grained map of genomes in Fig. 1 to guide the assembly of sequenced DNA fragments (ref. 3, section 6.1). This bottleneck is the reason why the denaturation maps shown in Fig. 1 were produced in a nanoslit requiring human dexterity and not in a nanochannel. The effectively 2D slit is a compromise between the 3D volume, in which DNA bundles up, and the effectively 1D nanochannel that automatically stretches the DNA molecule. The slit gives us what we want, but not in the way we want it: it proves the concept optimally, but the required dexterity is not readily automated and scaled by parallelization. In PNAS, Berard et al. (4) avoid this bottleneck altogether with their convex lens-induced nanoscale templating (CLINT), which is a clever extension of a single-molecule imaging technique called convex lens-induced confinement (CLIC) (5). Instead of threading DNA molecules into nanochannels, Berard et al. form the nanochannels around their intended contents. This is no small trick, considering the intended contents are stretched DNA molecules. The molecules must be first stretched out and only then enclosed in the nanochannels. A chicken-and-egg problem?The authors' trick is to harness the very entropic forces that oppose attempts to thread a DNA molecule into a nanochannel from its end. These are the same forces as those making rubber bands and O-rings Fig. 1. Composite photograph of individual double-stranded DNA molecules fluorescently dyed in a sequence-specific manner (barcoding) (7) and flow-stretched for visual inspection (8). Green vertical lines show three such molecules, each about 2 Mbp long, stretched in nanoslits by opposing flows. The downward flows end in a microchannel (greenish, at bottom). Orange dashed lines show enlarged details of the stretched molecules: two barcode patterns of fluorescence and dark patches. The top orange line shows 16 repeats: an impos...

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Integrated view of genome structure and sequence of a single DNA molecule in a nanofluidic device

Cited by 93 publications

References 39 publications

Thermophoretic Forces on DNA Measured with a Single-Molecule Spring Balance

Thermophoretic Forces on DNA Measured with a Single-Molecule Spring Balance

Mixed confinement regimes during equilibrium confinement spectroscopy of DNA

How to get into that “room at the bottom”

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