Characterization Techniques Common to DNA Nanotechnology

Matthew Mancuso BEE 7600, Professor Dan LuoDepartment of Biomedical Engineering, Cornell University Presented Thursday February 10rd, 2011

“Measure what is measurable, and

make measurable

what is not so.”

--Galileo GalileiPhoto 51, x-ray scattering data produced by

Rosalind Franklin which prompted the discovery of the double helix, taken in 1952

Before We Get Started…Imaging is Characterization too!

Bigger machines aren’t always better…But they certainly don’t hurt

What is Characterization?

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)

Light ScatteringTechniques

Hot out of the Lab: Pacific Biosciences!

Gel Electrophoresis

Polymerase ChainReaction

Forster Resonance Energy Transfer

Dynamic LightScattering

X-RayScattering

Fluorescence Techniques

FluorescenceSpectroscopy

Gel Electrophoresis“Big things move slow”

Purpose /Uses

http://www.molecularstation.com/molecular-biology-techniques/gel-electrophoresis/

46BP

100BP

200BPDisadvantages /Limitations

• Determine size• Separate by size•Separate by charge

•Difficult to account for

shape• Requires reasonable

quantities of analyte

Polymerase Chain Reaction (PCR)Measuring something my making more of it…

http://en.wikipedia.org/wiki/File:PCR.svg

Purpose /Uses

Disadvantages /Limitations

• Make more DNA• Run product through

a gel after•Detect small quantities

• Relatively slow• Slightly different

Conditions for

different DNAshttp://philos.biol.mun.ca/~b4241/PCR/PCR%20procedure.html

Real Time PCRMeasuring something while making more of it…

Using DNA Reporter ProbesUsing Nonspecific DNA Dye

Purpose /Uses

Disadvantages /Limitations

• Great for Detection of DNA•Provides real time Quantification

• DNA binding dye has no specificity• Probes require targeted reagents

Fluorescence Techniques

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

Fluorescence SpectroscopyPurpose /

Uses

Disadvantages /Limitations

• Quantify molecules• Determine Presence• Determine Ratios

• Requires

fluorophores• Provides limited

information

http://docinthemachine.com/2006/10/23/how-the-auofluorescence-system-woks-description/

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_energy_transfer

Purpose /Uses

Disadvantages /Limitations

• Quantify interaction•Quantify conformational

changes

• Requires specific

fluorophores• Sensitive in terms of

position and intensity

“NOT Fluorescence Resonance Energy Transfer”

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.

--Wikipedia, http://en.wikipedia.org/wiki/Rayleigh_scattering

• Measures diffusion• Determine particle size

• Determine size distribution

Dynamic Light Scattering

http://en.wikipedia.org/wiki/Dynamic_light_scattering http://www.kollewin.com/blog/electromagnetic-spectrum/

Purpose /Uses

Disadvantages /Limitations

• Assumes Spheres• Uses Autocorrelation

Function (I hate the

autocorrelation function)

X-ray Scattering and Crystallography

http://people.ccmr.cornell.edu/~uli/pages/saxs.htm

Purpose /Uses

Disadvantages /Limitations

• Determine size•Determine structure

•Complex sample prep• Complex technique•Requires x-ray source

(often synchrotron)http://en.wikipedia.org/wiki/Bragg's_lawhttp://www.pbs.org/wgbh/nova/photo51/pict-01.html

Pacific BiosciencesPurpose /

Uses

Disadvantages /Limitations

• Determine size• Separate by size•Separate by charge

• Determine size• Separate by size•Separate by charge

http://reference.findtarget.com/search/Single%20molecule%20real%20time%20sequencing/http://www.olympusmicro.com/primer/techniques/fluorescence/tirf/tirfintro.html

Questions?