1 / 36

John Lund , Declan Ryan, Ranjana Mehta, Maryam Rahimi and Babak A. Parviz

Direct Electronic Identification of Oligonucleotides with Inelastic Electron Tunneling Spectroscopy. John Lund , Declan Ryan, Ranjana Mehta, Maryam Rahimi and Babak A. Parviz Center of Excellence in Genomic Sciences Microscale Life Sciences Center University of Washington USA.

makaio
Télécharger la présentation

John Lund , Declan Ryan, Ranjana Mehta, Maryam Rahimi and Babak A. Parviz

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. Direct Electronic Identification of Oligonucleotides with Inelastic Electron Tunneling Spectroscopy John Lund, Declan Ryan, Ranjana Mehta, Maryam Rahimi and Babak A. Parviz Center of Excellence in Genomic Sciences Microscale Life Sciences Center University of Washington USA

  2. Sequencing the Human Genome Uses the entire sequencing capacity of a large center (~4 in the USA) Enables personalized medicine

  3. What do we need to detect? DNA 4 possible bases Human genome: ~ 3 billion bases long

  4. All-Electronic Sequencing STM Tip Sequencing technique: Stretch ss-DNA on a conductive surface (e.g. graphite, atomically flat gold, etc) b. Perform a rough scan with a scanning tunneling microscope to locate the molecule on the surface c. Follow the molecule on the surface with computer controlled STM tip and decipher the bases Attributes of the technique: Single molecule (no PCR necessary) No labels; no chemical modification/manipulation of the DNA Can be performed in principle on very long strands (thousands to millions of bases) Can be parallelized by using a multiple probe system Can be very fast depending on the STM system and algorithms used. DNA Conductive Substrate 3D AFM image of  phage ss-DNA completely elongated on HOPG with molecular combing

  5. How Fast Can STMs Work? Carbon atoms on the surface of HOPG imaged at tip speed of 40000 nm/s This is equivalent to reading a whole bacterial genome in 10 seconds.

  6. Inelastic Tunneling Spectroscopy Electron Tunneling Current V Science 1974

  7. Inelastic Tunneling Spectroscopy I Electron Tunneling Current Low V V d2I/dV2 V

  8. Inelastic Tunneling Spectroscopy I Electron Tunneling Current High V V New Tunneling Pathway d2I/dV2 V

  9. Molecular Extension • Our stretching approach employs molecular combing to orient DNA molecules on atomically flat surfaces • The interaction of the DNA and surface is tuned using coordinating ions or self-assembled monolayers • DNA molecules are stretched by a receding meniscus between a substrate DNA Molecule Droplet Meniscus Substrate

  10. Experimental Details • We verified our technique with two phage genome systems • The Hind III digest of  phage DNA, which yields 8 fragments with effective size range of 125 bp to 23 kb • Virion X174 DNA is ss, covalently closed, circular, and 5,386 bases in length • The ds- phage DNA was disrupted to ss-DNA by heating for 5 minutes and immediately cooling on ice • MgCl2 is used to mediate adhesion between the DNA and freshly-cleaved graphite surface

  11. Procedure

  12. Procedure

  13. Procedure

  14. Procedure

  15. Results 3D AFM image of bare HOPG before combing DNA

  16. 3D AFM image of  phage ds-DNA completely elongated on HOPG with molecular combing. The DNA goes over multiple domains on the graphite surface.

  17. 3D AFM image of coiled  phage ss-DNA deposited on HOPG prior to molecular combing.

  18. 3D AFM image of  phage ss-DNA completely elongated on HOPG after the completion of the molecular combing procedure.

  19. STM results

  20. Tunneling spectroscopy on gold Pt/Ir STM tip A’s Tunneling current Gold substrate

  21. Spectroscopy on poly A’s

  22. Spectroscopy on poly C’s

  23. Spectroscopy on poly G’s

  24. Spectroscopy on poly T’s

  25. Deviation from blank gold

  26. Confirmation of IETS

  27. Measurement on stretched dsDNA 20 nm

  28. Tip steering approach

  29. Conclusions • All-electronic genome sequencing requires cost-effective and reproducible methods for extension of DNA on atomically flat surfaces • Molecular combing offers a simple and cost-effective method for stretching DNA on surfaces • IETS is a promising method for identifying DNA bases on conductive substrates using STM • We have measured IETS spectra on 5-mer DNA bases on gold and will apply our approach to sequencing strands of DNA in the future

  30. Acknowledgments • Our Research Group Members • Postdoctoral Research Fellows • Declan Ryan • Maryam Rahimi • Ranjana Mehta • Xiaorong Xiang (now at Intel) • Graduate Students • Jianchun Dong • Harvey Ho • Sam Kim (with D. Meldrum) • John Lund • Coretta Maremma • Chris Morris • Ehsan Saeedi • Angela Shum • Andrei Afanasiev • Jean Wang (with Lih Lin) • Undergraduate Students • Lisa Oh • James Etzkorn • Funding for our group: • National Institutes of Health (NIH) • Gordon and Betty Moore Foundation • National Science Foundation (NSF) • National Academies Keck Future Initiative (NAKFI) • Defense Advanced Research Project Agency (DARPA) • Office of Naval Research (ONR) • University Initiative Fund (UIF) at UW • UW Technology Gap Innovation Fund (TGIF)

  31. Undigested  phage ds-DNA on HOPG AFM images

  32. ds-DNA Hind III digest on HOPG with 10 mM MgCl2 AFM images

  33.  phage ss-DNA Hind III digest on HOPG with 10 mM MgCl2 AFM images

  34. STM imaging of ssDNA on HOPG

More Related