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Affinity-Based Optical DNA Mapping in Nanochannels
- 1. FROM ANTIBIOTIC RESISTANCE PLASMIDS TO THE HUMAN GENOME AFFINITY-BASED OPTICAL DNA MAPPING IN NANOCHANNELS Fredrik Westerlund Chemical Biology Biology and Biological Engineering Chalmers University of Technology
- 2. Chalmers 2 • Nanofluidics • Optical DNA mapping • Background • “Our” DNA mapping assay • Antibiotic Resistance Plasmids • Human Genome Outline Müller, V. & Westerlund, F. Optical DNA mapping in nanofluidic devices: principles and applications. Lab on a Chip 17, 579–590 (2017).
- 3. • Fabricate nanochannels (down to ≈ 100*100 nm) • Enclose a DNA molecule (typically >50 kbp) and stretch by confinement (no handles) • Stained with fluorescent dyes (YOYO) • Visualize with fluorescence microscopy Low salt High salt Nanofluidics and DNA
- 4. deGennes regime: Odijk regime: F. Persson and J. O. Tegenfeldt, Chem. Soc. Rev., 2010, 39, 985–999. Theory – Polymer Physics
- 5. • Extension (r) scales linearly with contour length (L, number of bases). • A position X % into the image corresponds to a position X % into the contour length. > Sequence information X X Important!
- 6. Riehn et al, PNAS. 2005 Restriction Mapping
- 7. Visualize the sequence of intact genomic (Mbp) DNA • Scaffolding for base-by-base sequencing technique • Standard sequencing techniques have short read lengths (<1kb) • Detect structural gene variations • Copy number variations • Deletions • Insertions • Inversions • Translocations • Fast and efficient identification of bacteria and bacterial plasmids Optical DNA mapping
- 8. Chalmers 8 • Overuse in health care and agriculture • 25 years without new antibiotics The prognosis1 is one person dying every third second due to no longer treatable bacterial infections by 2050 Antibiotic Resistance 1 The Review on Antimicrobial Resistance. Tackling drug-resistant infections globally: final report and recommendations. 2016.
- 9. Chalmers 9 • Plasmids are responsible for the spread of antibiotic resistance • Transfer of plasmids via conjugation • Susceptible bacteria -> multiresistant DNA stored in the chromosome or as plasmids Bacteria and Their Plasmids Needed: Fast, efficient and amplification free techniques to identify plasmids coding for antibiotic resistance
- 10. Chalmers 7 Optical DNA Mapping – The Assay Nyberg L. K. et al. Scientific Reports, 2016, 6, 30410 Müller V. et al. ACS Infectious Disease, 2016, 2, 322-328
- 11. Chalmers 11 • Competitive binding between Netropsin and YOYO One step labeling Optical DNA Mapping – The Assay Bis-intercalating fluorophore AT-specific, non-fluorescent minor grove binder Nyberg L. et al. Biochemical and Biophysical Research Communications. 2012;417:404–408 Nilsson, A. N. et al. Nucleic Acids Research 42, e118 (2014).
- 12. Chalmers 12 Data Processing Optical DNA Mapping – The Assay Ambjörnsson group, Lund University
- 13. Chalmers 13 • Idea = compare experimental barcode with theoretical barcodes from a sequence database • Convert ATGCCGCCTGCTAATCG theoretical barcode • Unique match? Identifying Plasmids from a Sequence Database Experimental barcode R100 Theoretical barcode R100Experimental barcode pUUH Theoretical barcode pUUH Nyberg L. K. et al. Scientific Reports, 2016, 6, 30410
- 14. Chalmers 14 • Aim: Trace plasmids based on their size + barcode • P-value calculated for comparison of exp. vs exp. barcodes • In total 9 samples from 4 different neonates selected • Different strains, species, and time points • One plasmid responsible for entire outbreak? Tracing Plasmids from a Nosocomial Outbreak Müller V. et al. ACS Infectious Disease, 2016, 2, 322-328
- 15. Chalmers 15 Tracing Plasmids from a Nosocomial Outbreak • Main Findings • Shared plasmid of ~ 80 kbp found in each isolate • Assay allowed: • Monitor plasmid content • Detect spread between strains, species and patients Müller V. et al. ACS Infectious Disease, 2016, 2, 322-328
- 16. Chalmers 16 • Gene not possible to detect directly from barcode • Needs to be labeled • Problem = specificity • Solution = CRISPR/Cas9 Extending the Assay – Gene Detection • Enzyme involved in the adaptive immune system of some bacteria and archaea • 23 bp recognition site • Can be custom designed! Müller, V. et al. Scientific Reports 6, 37938 (2016).
- 17. Chalmers 17 Extending the Assay – Gene Detection Light/Mechanically induced breaks Site-specific Cas9 induced breaks • Few linearized plasmids • Breaks at random locations • Many linearized plasmids • Breaks at site specific locations Müller, V. et al. Scientific Reports 6, 37938 (2016).
- 18. Chalmers 18 Extending the Assay – Gene Detection Müller, V. et al. Scientific Reports 6, 37938 (2016).
- 19. Chalmers 19 • Assay evaluated on multiresistant bacteria • Detection limit established for stating the presence of a gene • Assay capable of detecting (resistance) genes on plasmids? Direct Gene Identification on Single Plasmid Molecules
- 20. 2017-10-13 Chalmers 20 Summary Plasmids • Finding the size and number of plasmids • Providing an ID for tracing or detection • Identifying a gene and assigning it to a specific plasmid
- 21. 2017-10-13 Chalmers 21 Group • Vilhelm Müller • Lena Nyberg • Karolin Frykholm • Sriram KK • Santosh K Bikkarolla • John Andersson Sahlgrenska Hospital • Christina Åhren • Nahid Karami Acknowledgments Lund University • Tobias Ambjörnsson • Saair Quaderi • Christoffer Pichler • Albertas Dvirnas Uppsala University • Linus Sandegren • Fredrika Rajer Karolinska Hospital • Christian Giske • Muhammad Kabir • Tsegaye Sewunet BioNanoGenomics • Alex Hastie • Denghong Zhang • Saki Chan