Single-Molecule DNA Flow-Stretching Force Spectroscopy with Higher Resolution Using Dark-Field Microscopy

Lee, Ryanggeun; Jeon, Young Hoon; Lee, Jong–Bong

doi:10.3938/jkps.75.350

Cited by 2 publications

(1 citation statement)

References 14 publications

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“…Subjection of a magnetic bead to this force also results in the stretching of the attached DNA molecule. A 15-50 kb long double-stranded DNA (dsDNA) is typically used in such experiments and the imaging of the bead is usually performed by dark field microscopy, allowing one to achieve high signal-to-noise ratios [80]. Due to the bead's spherical symmetry, its centroid position can be easily tracked with a precision of 1-10 nm using a sub-pixel fitting [81].…”

Section: Magnetic Bead-based Approachmentioning

confidence: 99%

DNA Flow-Stretch Assays for Studies of Protein-DNA Interactions at the Single-Molecule Level

2022

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Protein-DNA interactions are the core of the cell’s molecular machinery. For a long time, conventional biochemical methods served as a powerful investigatory basis of protein-DNA interactions and target search mechanisms. Currently single-molecule (SM) techniques have emerged as a complementary tool for studying these interactions and have revealed plenty of previously obscured mechanistic details. In comparison to the traditional ones, SM methods allow direct monitoring of individual biomolecules. Therefore, SM methods reveal reactions that are otherwise hidden by the ensemble averaging observed in conventional bulk-type methods. SM biophysical techniques employing various nanobiotechnology methods for immobilization of studied molecules grant the possibility to monitor individual reaction trajectories of biomolecules. Next-generation in vitro SM biophysics approaches enabling high-throughput studies are characterized by much greater complexity than the ones developed previously. Currently, several high-throughput DNA flow-stretch assays have been published and have shown many benefits for mechanistic target search studies of various DNA-binding proteins, such as CRISPR-Cas, Argonaute, various ATP-fueled helicases and translocases, and others. This review focuses on SM techniques employing surface-immobilized and relatively long DNA molecules for studying protein-DNA interaction mechanisms.

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Section: Magnetic Bead-based Approachmentioning

confidence: 99%

DNA Flow-Stretch Assays for Studies of Protein-DNA Interactions at the Single-Molecule Level

2022

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Application of dark-field microscopy for detection of DNA and protein

Tang¹

2022

HSET

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Dark-field microscopy (DFM) can be used to observe living cells and microorganisms. In contrast to a bright-field microscopy, the entire field of view in DFM is dark by default and the dark background provides a high contrast. And when the researchers placed the target samples in the path of the light cone, it appeared bright because it was on an almost black background, so it shows outstanding detail. DFM is a technique to improve the contrast of specimens with poor imaging quality under conventional illumination. After the direct light is blocked by the opaque aperture in the condenser, the light passing through the sample at an inclined angle is reflected into the microscopy objective lens through diffraction, refraction and reflection, producing a bright sample image. The DFM is preferred for opaque, low transparency and low contrast specimens, especially when staining is not a viable option. DFM has the unique advantages of being easy to modify and effective, so this technology shows a diverse of different applications. Herein, this research outlines the characteristics of DFM and analyzes its application in DNA and protein detection.

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Single-Molecule DNA Flow-Stretching Force Spectroscopy with Higher Resolution Using Dark-Field Microscopy

Cited by 2 publications

References 14 publications

DNA Flow-Stretch Assays for Studies of Protein-DNA Interactions at the Single-Molecule Level

DNA Flow-Stretch Assays for Studies of Protein-DNA Interactions at the Single-Molecule Level

Application of dark-field microscopy for detection of DNA and protein

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