1. Characterization Techniques Common to DNA Nanotechnology Matthew Mancuso BEE 7600, Professor Dan Luo Department of Biomedical Engineering, Cornell University Presented Thursday February 10 rd, 2011 “Measure what is measurable, and make measurable what is not so.” --Galileo Galilei 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. What is Characterization? Traditional Biotech Traditional Biotech 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)http://en.wikipedia.org/wiki/Characterization_(materials_science) Light Scattering Techniques Light Scattering Techniques Hot out of the Lab: Pacific Biosciences! Gel Electrophoresis Gel Electrophoresis Polymerase Chain Reaction Polymerase Chain Reaction Forster Resonance Energy Transfer Forster Resonance Energy Transfer Dynamic Light Scattering Dynamic Light Scattering X-Ray Scattering X-Ray Scattering Fluorescence Techniques Fluorescence Techniques Fluorescence Spectroscopy Fluorescence Spectroscopy

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

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

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

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

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

9. Forster Resonance Energy Transfer http://biomedcentral.inist.fr/index_affiche_revue.php?Affiche=affiche_html&ui=1746-4811- 22&format=html&journal=10088&PHPSESSID=18d644553233e4240bee686306b25c9c http://en.wikipedia.org/wiki/F%C3%B6rster_resonance_energ y_transfer Purpose / Uses Purpose / Uses Disadvantages / Limitations Disadvantages / Limitations Quantify interactionQuantify interaction Quantify conformationalQuantify conformationalchanges Requires specific fluorophores Requires specific fluorophores Sensitive in terms of position and intensitySensitive in terms of position and intensity “NOT Fluorescence Resonance Energy Transfer”

10. Physical Methods: Rayleigh Scattering http://en.wikipedia.org/wiki/Dynamic_light_scattering http://www.kollewin.com/blog/electromagnetic-spectrum/ 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.Lord Rayleighscatteringlightelectromagnetic --Wikipedia, http://en.wikipedia.org/wiki/Rayleigh_scatteringhttp://en.wikipedia.org/wiki/Rayleigh_scattering

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

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

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

14. Questions?