scholarly journals Stretching DNA to twice the normal length with single-molecule hydrodynamic trapping

Lab on a Chip ◽  
2020 ◽  
Vol 20 (10) ◽  
pp. 1780-1791
Author(s):  
Yan Jiang ◽  
Theodore Feldman ◽  
Julia A. M. Bakx ◽  
Darren Yang ◽  
Wesley P. Wong
Keyword(s):  
Fluorescence Imaging ◽  
Single Molecule ◽  
High Speed ◽  
Force Spectroscopy ◽  
Dna Molecules ◽  
Normal Length ◽  
Single Dna Molecules ◽  
Hydrodynamic Trapping

High-speed hydrodynamic trapping enables combined surface-free force spectroscopy and fluorescence imaging of single DNA molecules at extreme forces.

scholarly journals Stretching DNA to twice the normal length with single-molecule hydrodynamic trapping

2019 ◽  
Author(s):  
Yan Jiang ◽  
Theodore Feldman ◽  
Julia A.M. Bakx ◽  
Darren Yang ◽  
Wesley P. Wong
Keyword(s):  
Single Molecule ◽  
High Speed ◽  
Force Spectroscopy ◽  
Soft Materials ◽  
Covalent Attachment ◽  
Extended State ◽  
Single Molecule Fluorescence ◽  
Uv Curable ◽  
Force Application ◽  
Hydrodynamic Trapping

AbstractSingle-molecule force spectroscopy has brought many new insights into nanoscale biology, from the functioning of molecular motors, to the mechanical response of soft materials within the cell. To expand the single-molecule toolbox, we have developed a surface-free force spectroscopy assay based on a high-speed hydrodynamic trap capable of applying extremely high tensions for long periods of time. High-speed single-molecule trapping is enabled by a rigid and gas-impermeable microfluidic chip, rapidly and inexpensively fabricated out of glass, double-sided tape and UV-curable adhesive. Our approach does not require difficult covalent attachment chemistries, and enables simultaneous force application and single-molecule fluorescence. Using this approach, we have induced a highly extended state with twice the contour length of B-DNA in regions of partially intercalated double-stranded (dsDNA) by applying forces up to 250 pN. This highly extended state resembles the hyperstretched state of dsDNA, which was initially discovered as a structure fully intercalated by dyes under high tension. It has been hypothesized that hyperstretched DNA could also be induced without the aid of intercalators if high-enough forces were applied, which matches our observation. Combining force application with single-molecule fluorescence imaging is critical for distinguishing hyperstretched DNA from single-stranded DNA that can result from peeling. High-speed hydrodynamic trapping is a powerful yet accessible force spectroscopy method that enables the mechanics of biomolecules to be probed in previously difficult to access regimes.

scholarly journals Somatic mutation landscapes at single-molecule resolution

2021 ◽  
Author(s):  
Stefanie V. Lensing ◽  
Peter Ellis ◽  
Federico Abascal ◽  
Iñigo Martincorena ◽  
Robert J. Osborne
Keyword(s):  
Single Molecule ◽  
Single Cells ◽  
Error Rates ◽  
Dna Molecules ◽  
Base Pairs ◽  
Single Dna Molecules ◽  
Sequencing Library ◽  
Somatic Mutagenesis ◽  
Qpcr Quantification ◽  
Duplex Sequencing

Abstract Somatic mutations drive cancer development and may contribute to ageing and other diseases. Yet, the difficulty of detecting mutations present only in single cells or small clones has limited our knowledge of somatic mutagenesis to a minority of tissues. To overcome these limitations, we introduce nanorate sequencing (NanoSeq), a new duplex sequencing protocol with error rates <5 errors per billion base pairs in single DNA molecules from cell populations. The version of the protocol described here uses clean genome fragmentation with a restriction enzyme to prevent end-repair-associated errors and ddBTPs/dATPs during A-tailing to prevent nick extension. Both changes reduce the error rate of standard duplex sequencing protocols by preventing the fixation of DNA damage into both strands of DNA molecules during library preparation. We also use qPCR quantification of the library prior to amplification to optimise the complexity of the sequencing library given the desired sequencing coverage, maximising duplex coverage. The sample preparation protocol takes between 1 and 2 days, depending on the number of samples processed. The bioinformatic protocol is described in:https://github.com/cancerit/NanoSeqhttps://github.com/fa8sanger/NanoSeq_Paper_Code

Single-molecule DNA visualization using AT-specific red and non-specific green DNA-binding fluorescent proteins

The Analyst ◽  
2019 ◽  
Vol 144 (3) ◽  
pp. 921-927 ◽  
Author(s):  
Jihyun Park ◽  
Seonghyun Lee ◽  
Nabin Won ◽  
Eunji Shin ◽  
Soo-Hyun Kim ◽  
...  
Keyword(s):  
Dna Binding ◽  
Single Molecule ◽  
Fluorescent Proteins ◽  
Physical Map ◽  
Dna Molecules ◽  
Single Dna Molecules

Two-color DNA physical map for efficient identification of single DNA molecules.

scholarly journals Single molecule force spectroscopy studies of DNA denaturation by T4 gene 32 protein

2004 ◽  
Vol 18 (2) ◽  
pp. 203-211 ◽  
Author(s):  
Mark C. Williams ◽  
Kiran Pant ◽  
Ioulia Rouzina ◽  
Richard L. Karpel
Keyword(s):  
Dna Binding ◽  
Optical Tweezers ◽  
Single Molecule ◽  
Protein Interactions ◽  
Force Spectroscopy ◽  
Melting Transition ◽  
Dna Molecules ◽  
Force Extension ◽  
Single Molecule Force Spectroscopy ◽  
Gene 32

Single molecule force spectroscopy is an emerging technique that can be used to measure the biophysical properties of single macromolecules such as nucleic acids and proteins. In particular, single DNA molecule stretching experiments are used to measure the elastic properties of these molecules and to induce structural transitions. We have demonstrated that double‒stranded DNA molecules undergo a force‒induced melting transition at high forces. Force–extension measurements of single DNA molecules using optical tweezers allow us to measure the stability of DNA under a variety of solution conditions and in the presence of DNA binding proteins. Here we review the evidence of DNA melting in these experiments and discuss the example of DNA force‒induced melting in the presence of the single‒stranded DNA binding protein T4 gene 32. We show that this force spectroscopy technique is a useful probe of DNA–protein interactions, which allows us to obtain binding rates and binding free energies for these interactions.

Membrane-based actuation for high-speed single molecule force spectroscopy studies using AFM

2010 ◽  
Vol 39 (8) ◽  
pp. 1219-1227 ◽  
Author(s):  
Krishna Sarangapani ◽  
Hamdi Torun ◽  
Ofer Finkler ◽  
Cheng Zhu ◽  
Levent Degertekin
Keyword(s):  
Single Molecule ◽  
High Speed ◽  
Force Spectroscopy ◽  
Single Molecule Force Spectroscopy ◽  
Spectroscopy Studies

Photobleaching of Asymmetric Cyanines Used for Fluorescence Imaging of Single DNA Molecules

2001 ◽  
Vol 123 (33) ◽  
pp. 7985-7995 ◽  
Author(s):  
Claire Kanony ◽  
Björn Åkerman ◽  
Eimer Tuite
Keyword(s):  
Fluorescence Imaging ◽  
Dna Molecules ◽  
Single Dna Molecules

Counting Single DNA Molecules in a Capillary with Radial Focusing

2003 ◽  
Vol 56 (3) ◽  
pp. 149 ◽  
Author(s):  
Jinjian Zheng ◽  
Edward S. Yeung
Keyword(s):  
Single Molecule ◽  
Detection Efficiency ◽  
Signal To Noise Ratio ◽  
Sampling Rate ◽  
Ccd Camera ◽  
Counting Efficiency ◽  
Spherical Particles ◽  
Dna Molecules ◽  
Single Dna Molecules ◽  
Relative Standard

For single-molecule detection, usually a small detection volume of 10 pL or less is used to improve the signal-to-noise ratio. Detection of every molecule in a sample requires that the sample be driven through a well-defined volume to facilitate laser excitation. We report a novel approach to count single DNA molecules with nearly 100% efficiency. By applying an electric field across a 40 cm long, 75 × 75 µm2 square capillary together with hydrodynamic flow from cathode to anode, we were able to concentrate more than 95% of DNA molecules into a 10 µm region at the centre of the capillary. The YOYO-1 labelled λ-DNA molecules were imaged with an intensified CCD camera. We found that the single DNA molecule detection efficiency in a 10–17 M solution was 114 ± 21%. The mobility of the DNA molecules after radial focusing was relatively constant, with relative standard deviations ranging from 0.8% to 1.4%. This allowed us to match the sampling rate to the length of the detection window to maximize counting efficiency. Analysis of a 40.2 nL injected plug of 2 × 10–14 M λ-DNA gave a result of 492 ± 73 molecules, which agreed well with the estimated value of 484. This method should be generally useful for counting deformable molecules or non-spherical particles at extremely low concentrations.

scholarly journals Dynamic force spectroscopy of single DNA molecules

1999 ◽  
Vol 96 (20) ◽  
pp. 11277-11282 ◽  
Author(s):  
T. Strunz ◽  
K. Oroszlan ◽  
R. Schafer ◽  
H.-J. Guntherodt
Keyword(s):  
Force Spectroscopy ◽  
Dynamic Force ◽  
Dna Molecules ◽  
Dynamic Force Spectroscopy ◽  
Single Dna Molecules
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