Single molecule DNA sequencing in submicrometer channels: state of the art and future prospects

Sauer, Markus; Angerer, Bernhard; Ankenbauer, W.; Földes-Papp, Zeno; Göbel, Florian; Han, K.-T.; Rigler, Rudolf; Schulz, Andreas; Wolfrum, J.; Zander, C.

doi:10.1016/s0168-1656(00)00413-2

Cited by 84 publications

(66 citation statements)

References 43 publications

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“…[1][2][3][4][5][6][7] For instance, in certain implementations of exonucleolytic sequence analysis it is desirable to link an individual long DNA strand to a surface ͑e.g., a bead͒ without manual intervention. 6,7 The ability to engineer such tasks will be greatly improved by predictive computational tools for polymer chains in microfluidic geometries.…”

Section: Introductionmentioning

confidence: 99%

Effect of confinement on DNA dynamics in microfluidic devices

Jendrejack

Schwartz

Graham

et al. 2003

The Journal of Chemical Physics

166

193

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The dynamics of dissolved long-chain macromolecules are different in highly confined environments than in bulk solution. A computational method is presented here for detailed prediction of these dynamics, and applied to the behavior of ϳ1-100 m DNA in micron-scale channels. The method is comprised of a self-consistent coarse-grained Langevin description of the polymer dynamics and a numerical solution of the flow generated by the motion of polymer segments. Diffusivity and longest relaxation time show a broad crossover from free-solution to confined behavior centered about the point HϷ10S b , where H is the channel width and S b is the free-solution chain radius of gyration. In large channels, the diffusivity is similar to that of a sphere diffusing along the centerline of a pore. For highly confined chains (H/S b Ӷ1), Rouse-type molecular weight scaling is observed for both translational diffusivity and longest relaxation time. In the highly confined region, the scaling of equilibrium length and relaxation time with H/S b are in good agreement with scaling theories. In agreement with the results of Harden and Doi ͓J. Phys. Chem. 96, 4046 ͑1992͔͒, we find that the diffusivity of highly confined chains does not follow the scaling relation predicted by Brochard and de Gennes ͓J. Chem. Phys. 67, 52 ͑1977͔͒; that relationship does not account for the interaction between chain and wall.

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Section: Introductionmentioning

confidence: 99%

Effect of confinement on DNA dynamics in microfluidic devices

Jendrejack

Schwartz

Graham

et al. 2003

The Journal of Chemical Physics

166

193

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“…Labeled nucleotide triphosphates and DNA polymerase enzyme were then washed in and out of the flow cell while the known locations of the DNA templates were monitored for the appearance of fluorescence. With this technique we show that DNA polymerase is active on surfaceimmobilized DNA templates and can incorporate nucleotides with high fidelity.A confounding factor in previous attempts to sequence single DNA molecules with fluorescence microscopy has been an inability to control background fluorescence and fluorescent impurities (20,23). In this work we used a combination of evanescent wave microscopy and single-pair fluorescence resonance energy transfer (spFRET; refs.…”

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confidence: 99%

“…A confounding factor in previous attempts to sequence single DNA molecules with fluorescence microscopy has been an inability to control background fluorescence and fluorescent impurities (20,23). In this work we used a combination of evanescent wave microscopy and single-pair fluorescence resonance energy transfer (spFRET; refs.…”

mentioning

confidence: 99%

“…Other proposals turn to nature for inspiration and seek to combine optical techniques with enzymes that have been fine-tuned by evolution to operate as machines that assemble and disassemble DNA with inherent single-base resolution (6,11,12). Although there have been single molecule studies of DNA polymerase (16,17), RNA polymerase (18,19), and exonuclease (20,21), measuring the activity of these enzymes with single-base resolution has been an elusive goal. We took advantage of the exquisite discrimination and fidelity of DNA polymerase to image sequence information in a single DNA template as its complementary strand is synthesized.…”

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confidence: 99%

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Sequence information can be obtained from single DNA molecules

Braslavsky

Hébert

Kartalov

et al. 2003

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

427

245

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The completion of the human genome draft has taken several years and is only the beginning of a period in which large amounts of DNA and RNA sequence information will be required from many individuals and species. Conventional sequencing technology has limitations in cost, speed, and sensitivity, with the result that the demand for sequence information far outstrips current capacity. There have been several proposals to address these issues by developing the ability to sequence single DNA molecules, but none have been experimentally demonstrated. Here we report the use of DNA polymerase to obtain sequence information from single DNA molecules by using fluorescence microscopy. We monitored repeated incorporation of fluorescently labeled nucleotides into individual DNA strands with single base resolution, allowing the determination of sequence fingerprints up to 5 bp in length. These experiments show that one can study the activity of DNA polymerase at the single molecule level with single base resolution and a high degree of parallelization, thus providing the foundation for a practical single molecule sequencing technology.T he Sanger method of DNA sequencing (1) and subsequent developments in automation (2) and computation (3) revolutionized the world of biological sciences and eventually led to the sequencing of the consensus human genome (4, 5). The successes of this and other genome projects have only whetted the appetite of the scientific community, and many applications of DNA sequencing have been proposed that will require cheaper, faster, or more sensitive sequencing technology than conventional methods currently provide. After the determination of the consensus human genome, there is a desire to sequence many individual human genomes to provide highresolution genotypes that can be used to determine the complex relationships among disease, pharmaceutical efficacy, and genetic variability (6-8). Similarly, aggressive technological innovation is required for the field of comparative genomics to reach its full potential (4). Finally, mRNA sequencing is valuable to determine exon splicing patterns (9) and as a tool to discover gene function from context-specific expression data (10).There have been many proposals to develop new sequencing technologies based on single molecule measurements, generally either by observing the interaction of particular proteins with DNA (6, 11-13) or by using ultra high-resolution scanned probe microscopy (14). Although none of these methods has been demonstrated experimentally, they are interesting because they promise high sensitivity, low cost, and in some cases a high degree of parallelization (15). Unlike conventional technology, their speed and read length would not be inherently limited by the resolving power of electrophoretic separation. Single molecule sensitivity might permit direct sequencing of mRNA from rare cell populations or perhaps even individual cells.A major obstacle in the development of single molecule sequencing schemes is that DNA has an extraordinarily ...

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“…Single molecules have been detected by means of fluorescence in relatively large capillaries, 4-6 micrometer 7-10 and even submicrometer sized channels. [11][12][13][14][15] Previous work in our laboratories reported on the detection of flowing fluorescent particles like bacteria and microspheres in a microcapillary mounted on a confocal fluorescence microscopy setup. 16 These results led to the design of a microfluidic biochip aimed at detection and sorting of relatively small particles in real time using the same confocal fluorescence microscope.…”

Section: Introductionmentioning

confidence: 99%

Design of a confocal microfluidic particle sorter using fluorescent photon burst detection

Kunst

Schots

Visser

2004

Review of Scientific Instruments

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An instrumental system is described for detecting and sorting single fluorescent particles such as microspheres, bacteria, viruses, or even smaller macromolecules in a flowing liquid. The system consists of microfluidic chips (biochips), computer controlled high voltage power supplies, and a fluorescence microscope with confocal optics. The confocal observation volume and detection electro-optics allow measurements of single flowing fluorescent particles. The output of the avalanche photodiode (single photon detector) is coupled to a real-time photon-burst detection device, which output can address the control of high voltage power supplies for sorting purposes. Liquid propulsion systems like electro-osmotic flow and plain electric fields to direct the particles through the observation volume have been tested and evaluated. The detection and real-time sorting of fluorescent microspheres are demonstrated. Applications of these biochips for screening of bacteriophages-type biolibraries are briefly discussed.

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Single molecule DNA sequencing in submicrometer channels: state of the art and future prospects

Cited by 84 publications

References 43 publications

Effect of confinement on DNA dynamics in microfluidic devices

Effect of confinement on DNA dynamics in microfluidic devices

Sequence information can be obtained from single DNA molecules

Design of a confocal microfluidic particle sorter using fluorescent photon burst detection

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