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Characterization Techniques Common to DNA Nanotechnology

Characterization Techniques Common to DNA Nanotechnology. “Measure what is measurable, and make measurable what is not so.” --Galileo Galilei. Matthew Mancuso BEE 7600, Professor Dan Luo

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Characterization Techniques Common to DNA Nanotechnology

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  1. Characterization Techniques Common to DNA Nanotechnology “Measure what is measurable, and make measurable what is not so.” --Galileo Galilei Matthew Mancuso BEE 7600, Professor Dan Luo Department of Biomedical Engineering, Cornell University Presented Thursday February 10rd, 2011 Photo 51, x-ray scattering data produced by Rosalind Franklin which prompted the discovery of the double helix, taken in 1952

  2. Before We Get Started…Imaging is Characterization too! Bigger machines aren’t always better… But they certainly don’t hurt

  3. Characterization [is] the use of external techniques to probe into the internal structure and properties of a material. --Wikipedia, http://en.wikipedia.org/wiki/Characterization_(materials_science) What is Characterization? Traditional Biotech Fluorescence Techniques Light Scattering Techniques Dynamic Light Scattering Gel Electrophoresis Fluorescence Spectroscopy Forster Resonance Energy Transfer X-Ray Scattering Polymerase Chain Reaction Hot out of the Lab: Pacific Biosciences!

  4. “Big things move slow” Purpose / Uses • Determine size • Separate by size • Separate by charge Gel Electrophoresis Disadvantages / Limitations 200BP 100BP • Difficult to account for • shape • Requires reasonable • quantities of analyte 46BP http://www.molecularstation.com/molecular-biology-techniques/gel-electrophoresis/

  5. Measuring something my making more of it… Polymerase Chain Reaction (PCR) Disadvantages / Limitations Purpose / Uses • Relatively slow • Slightly different Conditions for different DNAs • Make more DNA • Run product through • a gel after • Detect small quantities http://en.wikipedia.org/wiki/File:PCR.svg http://philos.biol.mun.ca/~b4241/PCR/PCR%20procedure.html

  6. Measuring something while making more of it… Using DNA Reporter Probes Using Nonspecific DNA Dye Real Time PCR Purpose / Uses Disadvantages / Limitations • DNA binding dye has no specificity • Probes require targeted reagents • Great for Detection of DNA • Provides real time Quantification

  7. Fluorescence Techniques http://scienceblogs.com/purepedantry/2007/11/two_photon.php http://en.wikipedia.org/wiki/Stokes_shift http://color101.blogspot.com/

  8. Purpose / Uses • Quantify molecules • Determine Presence • Determine Ratios Fluorescence Spectroscopy Disadvantages / Limitations • Requires fluorophores • Provides limited information http://docinthemachine.com/2006/10/23/how-the-auofluorescence-system-woks-description/

  9. “NOT Fluorescence Resonance Energy Transfer” Forster Resonance Energy Transfer Disadvantages / Limitations Purpose / Uses • Requires specific fluorophores • Sensitive in terms of position and intensity • Quantify interaction • Quantify conformational • changes http://en.wikipedia.org/wiki/F%C3%B6rster_resonance_energy_transfer http://biomedcentral.inist.fr/index_affiche_revue.php?Affiche=affiche_html&ui=1746-4811-22&format=html&journal=10088&PHPSESSID=18d644553233e4240bee686306b25c9c

  10. Physical Methods:Rayleigh Scattering Rayleigh scattering (named after the British physicist Lord Rayleigh) is the elastic scattering of light or other electromagnetic radiation by particles much smaller than the wavelength of the light, which may be individual atoms or molecules. --Wikipedia, http://en.wikipedia.org/wiki/Rayleigh_scattering http://www.kollewin.com/blog/electromagnetic-spectrum/ http://en.wikipedia.org/wiki/Dynamic_light_scattering

  11. Purpose / Uses • Measures diffusion • Determine particle size • Determine size distribution Dynamic Light Scattering Disadvantages / Limitations • Assumes Spheres • Uses Autocorrelation Function (I hate the autocorrelation function) http://www.kollewin.com/blog/electromagnetic-spectrum/ http://en.wikipedia.org/wiki/Dynamic_light_scattering

  12. X-ray Scattering and Crystallography Disadvantages / Limitations Purpose / Uses • Complex sample prep • Complex technique • Requires x-ray source • (often synchrotron) • Determine size • Determine structure http://www.pbs.org/wgbh/nova/photo51/pict-01.html http://en.wikipedia.org/wiki/Bragg's_law http://people.ccmr.cornell.edu/~uli/pages/saxs.htm

  13. Purpose / Uses • Determine size • Separate by size • Separate by charge Pacific Biosciences Disadvantages / Limitations • Determine size • Separate by size • Separate by charge http://www.olympusmicro.com/primer/techniques/fluorescence/tirf/tirfintro.html http://reference.findtarget.com/search/Single%20molecule%20real%20time%20sequencing/

  14. Questions?

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