A single-step competitive binding assay for mapping of single DNA molecules
Journal article, 2012

Optical mapping of genomic DNA is of relevance for a plethora of applications such as scaffolding for sequencing and detection of structural variations as well as identification cif pathogens like bacteria and viruses. For future clinical applications it is desirable to have a fast and robust mapping method based on as few steps as possible. We here demonstrate a single-step method to obtain a DNA barcode that is directly visualized using nanofluidic devices and fluorescence microscopy. Using a mixture of YOYO-1, a bright DNA dye, and netropsin, a natural antibiotic with very high AT specificity, we obtain a DNA map with a fluorescence intensity profile along the DNA that reflects the underlying sequence. The netropsin binds to AT-tetrads and blocks these binding sites from YOYO-1 binding which results in lower fluorescence intensity from AT-rich regions of the DNA. We thus obtain a DNA barcode that is dark in AT-rich regions and bright in GC-rich regions with kilobasepair resolution. We demonstrate the versatility of the method by obtaining a barcode on DNA from the phage T4 that captures its circular permutation and agrees well with its known sequence.

Single DNA

restriction maps

resolution

molecules

Nanofluidic. channels

distamycin

channels

DNA mapping

complexes

Competitive assay

dyes

Fluorescence microscopy

netropsin

double-stranded dna

Author

Lena Nyberg

Chalmers, Chemical and Biological Engineering, Physical Chemistry

Fredrik Persson

University of Gothenburg

Chalmers, Physics

J. Berg

Chalmers, Chemical and Biological Engineering

Johanna Bergström

Chalmers, Chemical and Biological Engineering

Emelie Fransson

Chalmers, Chemical and Biological Engineering

L. Olsson

Chalmers, Chemical and Biological Engineering

M. Persson

Chalmers, Chemical and Biological Engineering

Antti Stålnacke

Chalmers, Chemical and Biological Engineering

Jens Wigenius

Chalmers, Chemical and Biological Engineering, Physical Chemistry

J. O. Tegenfeldt

University of Gothenburg

Fredrik Westerlund

Chalmers, Chemical and Biological Engineering, Physical Chemistry

Biochemical and Biophysical Research Communications

0006-291X (ISSN) 1090-2104 (eISSN)

Vol. 417 1 404-408

Areas of Advance

Nanoscience and Nanotechnology (2010-2017)

Life Science Engineering (2010-2018)

Subject Categories

Biochemistry and Molecular Biology

Chemical Sciences

DOI

10.1016/j.bbrc.2011.11.128

PubMed

22166208

More information

Latest update

4/18/2019