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

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.

Distribution of distances between DNA barcode labels in nanochannels close to the persistence length

“…(14) to obtain the free energy corresponding to the probability densities from our experiments in the 43 nm channel. Following the harmonic approximation, we assumed that X min is the mode of the distribution (and thus the extension corresponding to the minimum value of the free energy, F min ) and then computed a FIG.…”

“…[1][2][3] Here, the DNA is stretched by confinement in a nanochannel 4,5 and the genomic information is then read by fluorescence microscopy, either using sequencespecific probes, [6][7][8][9][10][11] the absence of fluorescence due to localized melting of AT-rich regions, 12,13 or competitive binding. 14 Genome mapping in nanochannels uses intact genomic DNA molecules that are hundreds of kilobases (and even megabases) in contour length. These massive stretches of labeled DNA greatly facilitate the assembly of genomic maps for anchoring sequencing data, 2 and they are especially important for the analysis of structural variations.…”

Topological events in single molecules of E. coli DNA confined in nanochannels

“…4,7,8 The Irys system works by inserting labels by a nick protocol, but it is also possible to obtain coarse-grained genomic data by modifying the binding affinity of YOYO. 9,10 …”

Applications of Optical DNA Mapping in Microbiology

“…This labeling method was applied to identify a given strain of E. coli in a library of nine strains. The approach was based on fitting experimental barcodes of large pieces (50160 kb) of DNA to the theoretical barcodes of all of the full E. coli genomes available in the library (Figure 2E) (58). …”