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

Artikel i vetenskaplig tidskrift
Författare A. N. Nilsson
Gustav Emilsson
Lena Nyberg
C. Noble
Liselott Svensson-Stadler
Joachim Fritzsche
Edward R.B. Moore
J. O. Tegenfeldt
T. Ambjornsson
Fredrik Westerlund
Publicerad i Nucleic Acids Research
Volym 42
Nummer/häfte 15
Sidor e118
ISSN 0305-1048
Publiceringsår 2014
Publicerad vid Institutionen för biomedicin, avdelningen för infektionssjukdomar
Sidor e118
Språk en
Länkar dx.doi.org/10.1093/nar/gku556
https://gup.ub.gu.se/file/177404
Ämneskategorier Molekylärbiologi

Sammanfattning

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: aP-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.

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