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A single-step competitive binding assay for mapping of single DNA molecules
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
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2012 (English)In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 417, no 1, 404-408 p.Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
2012. Vol. 417, no 1, 404-408 p.
Keyword [en]
DNA mapping, Nanofluidic. channels, Competitive assay, Single DNA molecules, Fluorescence microscopy
National Category
Natural Sciences
Identifiers
URN: urn:nbn:se:uu:diva-169364DOI: 10.1016/j.bbrc.2011.11.128ISI: 000299491600069OAI: oai:DiVA.org:uu-169364DiVA: diva2:506719
Available from: 2012-02-29 Created: 2012-02-28 Last updated: 2017-12-07Bibliographically approved

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Persson, Fredrik

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