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Nucleic Acids Res. 2014 Sep;42(15):e118. doi: 10.1093/nar/gku556. Epub 2014 Jul 10.

Competitive binding-based optical DNA mapping for fast identification of bacteria--multi-ligand transfer matrix theory and experimental applications on Escherichia coli.

Author information

1
Department of Astronomy and Theoretical Physics, Lund University, Sölvegatan 14A, 223 62 Lund, Sweden.
2
Division of Chemistry and Biochemistry, Department of Chemical and Biological Engineering, Chalmers University of Technology, Kemivägen 10, 412 96 Göteborg, Sweden.
3
Department of Astronomy and Theoretical Physics, Lund University, Sölvegatan 14A, 223 62 Lund, Sweden Department of Applied Physics, Chalmers University of Technology, Kemivägen 10, 412 96 Göteborg, Sweden.
4
Division of Solid State Physics, Department of Physics, Lund University, PO 118, 221 00 Lund, Sweden.
5
Department of Infectious Diseases, Sahlgrenska Academy, University of Gothenburg, Guldhedsgatan 10A, 413 46 Göteborg, Sweden.
6
Department of Applied Physics, Chalmers University of Technology, Kemivägen 10, 412 96 Göteborg, Sweden.
7
Division of Chemistry and Biochemistry, Department of Chemical and Biological Engineering, Chalmers University of Technology, Kemivägen 10, 412 96 Göteborg, Sweden fredrik.westerlund@chalmers.se.

Abstract

We demonstrate a single DNA molecule optical mapping assay able to resolve a specific Escherichia coli strain from other strains. The assay is based on competitive binding of the fluorescent dye YOYO-1 and the AT-specific antibiotic netropsin. The optical map is visualized by stretching the DNA molecules in nanofluidic channels. We optimize the experimental conditions to obtain reproducible barcodes containing as much information as possible. We implement a multi-ligand transfer matrix method for calculating theoretical barcodes from known DNA sequences. Our method extends previous theoretical approaches for competitive binding of two types of ligands to many types of ligands and introduces a recursive approach that allows long barcodes to be calculated with standard computer floating point formats. The identification of a specific E. coli strain (CCUG 10979) is based on mapping of 50-160 kilobasepair experimental DNA fragments onto the theoretical genome using the developed theory. Our identification protocol introduces two theoretical constructs: a P-value for a best experiment-theory match and an information score threshold. The developed methods provide a novel optical mapping toolbox for identification of bacterial species and strains. The protocol does not require cultivation of bacteria or DNA amplification, which allows for ultra-fast identification of bacterial pathogens.

PMID:
25013180
PMCID:
PMC4150756
DOI:
10.1093/nar/gku556
[Indexed for MEDLINE]
Free PMC Article

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