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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.

Nilsson AN, Emilsson G, Nyberg LK, Noble C, Stadler LS, Fritzsche J, Moore ER, Tegenfeldt JO, Ambjörnsson T, Westerlund F - Nucleic Acids Res. (2014)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Astronomy and Theoretical Physics, Lund University, Sölvegatan 14A, 223 62 Lund, Sweden.

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Related in: MedlinePlus

Schematic illustration of the principle of the CB assay. YOYO-1 (yellow stars) and netropsin (gray circles) are simultaneously added to a DNA with AT- rich (red) and GC-rich (blue) regions. Netropsin binds preferentially to AT-rich regions preventing YOYO-1 to bind to these regions. When stretched in nanofluidic channels the DNA molecules show an emission intensity along the contour that reflects the underlying sequence with bright GC-rich and dark AT-rich regions.
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Figure 1: Schematic illustration of the principle of the CB assay. YOYO-1 (yellow stars) and netropsin (gray circles) are simultaneously added to a DNA with AT- rich (red) and GC-rich (blue) regions. Netropsin binds preferentially to AT-rich regions preventing YOYO-1 to bind to these regions. When stretched in nanofluidic channels the DNA molecules show an emission intensity along the contour that reflects the underlying sequence with bright GC-rich and dark AT-rich regions.

Mentions: As an alternative to DNA melting, we have recently demonstrated how competitive binding (CB) between YOYO and the natural antibiotic netropsin can be used to create optical maps of single, nanoconfined DNA molecules (16). Netropsin binds in the minor groove of DNA and has a very strong preference for binding to AT-rich sequences (17,18). When DNA is added to a mixture of YOYO and netropsin, the two molecules will compete for the AT-rich binding sites and the result is an emission intensity along the DNA contour that reflects the underlying sequence, where GC-rich regions appear bright and AT-rich regions dark (Figure 1). Proof-of-principle CB experiments were performed on commercially available DNA from lambda and T4 phages, demonstrating that the emission patterns observed reflect the underlying sequences in a predictable way (16).


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

Nilsson AN, Emilsson G, Nyberg LK, Noble C, Stadler LS, Fritzsche J, Moore ER, Tegenfeldt JO, Ambjörnsson T, Westerlund F - Nucleic Acids Res. (2014)

Schematic illustration of the principle of the CB assay. YOYO-1 (yellow stars) and netropsin (gray circles) are simultaneously added to a DNA with AT- rich (red) and GC-rich (blue) regions. Netropsin binds preferentially to AT-rich regions preventing YOYO-1 to bind to these regions. When stretched in nanofluidic channels the DNA molecules show an emission intensity along the contour that reflects the underlying sequence with bright GC-rich and dark AT-rich regions.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4150756&req=5

Figure 1: Schematic illustration of the principle of the CB assay. YOYO-1 (yellow stars) and netropsin (gray circles) are simultaneously added to a DNA with AT- rich (red) and GC-rich (blue) regions. Netropsin binds preferentially to AT-rich regions preventing YOYO-1 to bind to these regions. When stretched in nanofluidic channels the DNA molecules show an emission intensity along the contour that reflects the underlying sequence with bright GC-rich and dark AT-rich regions.
Mentions: As an alternative to DNA melting, we have recently demonstrated how competitive binding (CB) between YOYO and the natural antibiotic netropsin can be used to create optical maps of single, nanoconfined DNA molecules (16). Netropsin binds in the minor groove of DNA and has a very strong preference for binding to AT-rich sequences (17,18). When DNA is added to a mixture of YOYO and netropsin, the two molecules will compete for the AT-rich binding sites and the result is an emission intensity along the DNA contour that reflects the underlying sequence, where GC-rich regions appear bright and AT-rich regions dark (Figure 1). Proof-of-principle CB experiments were performed on commercially available DNA from lambda and T4 phages, demonstrating that the emission patterns observed reflect the underlying sequences in a predictable way (16).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Astronomy and Theoretical Physics, Lund University, Sölvegatan 14A, 223 62 Lund, Sweden.

Show MeSH
Related in: MedlinePlus