Using Atomic Force

Microscopy to Study DNA

Structure
Michele Pugh
Senior Pratt Engineering Undergraduate Fellow
Mechanical Engineering
Associate Professor Piotr Marszalek
March 30, 2005
 Goals
• Can techniques of atomic force microscopy can be
used to examine DNA structure?
• Observe DNA structure and detect damage to DNA
molecules using two techniques using an atomic
force microscope
– Imaging
– Force spectroscopy
• Become proficient in both imaging and force
spectroscopy (“stretching”)
• Develop consistent procedures for both techniques
 AFM imaging of DNA
As the cantilever tip
scans the sample
surface, the
microscope produces
a topographical image
of the sample by
measuring the
deflection of the tip
Sample preparation for imaging
• Mica surface is treated
with APTES and the DNA
and proteins are diluted
with TE buffer and MgCl2
solution
• The DNA-protein solution
heated to normal body
temperature to induce the
proteins to bind to the
DNA
• DNA solution deposited
on the treated mica and
incubated at room
temperature
 Imaging DNA with attached proteins
• DNA in yellow at a
higher “elevation” than
the mica surface
• Proteins in white at a
higher “elevation” than
the DNA
• Sample preparation is
very important in order
to minimize the
appearance of
aggregates and foreign
particles in the images
 Using force spectroscopy to
measure the elasticity of DNA
• By recording data about
the displacement of the
sample and the deflection
of the cantilever tip, the
length and tension of the
DNA molecule can be
determined
• Elasticity curve of control
DNA is different than that
of DNA damaged by
radiation
Sample preparation for “stretching”
• A much higher
concentration of DNA is
needed in order to
increase the chances that
the cantilever will pick up
a molecule
• DNA diluted in TE buffer
with NaCl solution to
stabilize the molecule
• DNA solution deposited
onto pure gold substrate
Measuring elasticity of control
DNA
4300

3800

3300

2800
Force (pN)

2300

1800

1300

800

300

-200
-100 100 300 500 700 900 1100 1300
Extension (nm)
 What control DNA should look
like…
2400

1900

1400
Force (pN)

900

400

-100
-100 400 900 1400 1900 2400 2900 3400 3900 4400 4900
Extension (nm)

Obtained by Gwangrog Lee

 Measuring elasticity of DNA
exposed to gamma radiation
350

300

250

200
Force (pN)

150

100

50

0
-100 100 300 500 700 900 1100 1300 1500
-50

-100
Extension (nm)
 Conclusions
• Small mechanical manipulations of DNA
molecules can be used to study the
structure of the molecule
• Techniques of atomic force microscopy
can be used to detect changes in the
structure of DNA molecules associated
with radiation damage
 Possible applications of this
research
• Detection of DNA damage induced by
environmental agents, such as UV or
gamma radiation DNA, specifically
those that cause skin cancer
• Detection of spontaneous mutations
in DNA molecules
• Better visualization of the DNA and
repair protein complex
 Acknowledgements

Dr. Piotr Marszalek

Dr. Qinming Zhang
Gwangrog Lee
Dean Martha Absher and the Pratt
Fellows Program