Work Description

Title: Single-molecule microscopy image data and analysis files for "Ultra-specific and Amplification-free Quantification of Mutant DNA by Single-molecule Kinetic Fingerprinting" Open Access Deposited
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  • Single-Molecule Recognition through Equilibrium Poisson Sampling (SiMREPS) experiments were performed on an Olympus IX-81 objective-type TIRF microscope equipped with a 60X oil-immersion objective (APON 60XOTIRF, 1.49 NA) with both Cell^TIRF and z-drift control modules, and an EMCCD camera (IXon 897, Andor), using MetaMorph acquisition software (Molecular Devices). Transient binding of a fluorescent probe oligonucleotide to DNA molecules immobilized to the surface of a custom-built sample cell was monitored for 10 min under TIRF illumination by 640 nM laser light with a 500 ms exposure time (1200 total frames), and recorded as a stack of TIF images (movie). Movie files were analyzed using custom scripts written in MATLAB, and the QuB software suite (State University of New York at Buffalo) to: 1) identify the locations of immobilized candidate DNA molecules and extract a fluorescence intensity versus time trace for each, 2) determine the number of binding and dissociation events (Nb+d) and the median fluorescent probe bound (τbound,median) and unbound (τunbound,median) time for each candidate molecule, which together comprise the kinetic fingerprint of the candidate molecule, and 3) evaluate the kinetic fingerprint and data quality of each candidate to arrive at the final number of immobilized DNA molecules of a specific sequence.
  • This work contains the experimental data and associated analysis that are described in the research publication entitled "Ultra-specific and Amplification-free Quantification of Mutant DNA by Single-molecule Kinetic Fingerprinting". This work contains multiple zip files, each of which represents one of the principal experiment groups presented in the publication. Each experiment group contains movie and analysis files corresponding to various experimental conditions related to that experiment group.
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  • Other Funding Agency
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User Curation Notes
  • The MATLAB scripts are available upon request from Alex Johnson-Buck, see "Deposit_documentation.pdf".
Last modified
  • 03/26/2019
  • doi:10.7302/Z2CZ35DF
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Files (Count: 20; Size: 104 GB)


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