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Nano Research & Applications
ISSN: 2471-9838
April 26-27, 2018
Rome, Italy
NanoMat 2018
Page 40
17
th
Edition of International Conference on
Emerging Trends in
Materials Science and
Nanotechnology
S
ingle-molecule sequencing methods have been developed to
analyze DNA directly without the need for amplification. Here,
we present a new approach to sequencing single DNAmolecules
using atomic force microscopy (AFM). In our approach, four
surface-conjugated nucleotides were examined sequentially
with a DNA polymerase-immobilized AFM tip. By observing
the specific rupture events upon examination of a matching
nucleotide, we could determine the template base bound in the
polymerase’s active site. The subsequent incorporation of the
complementary base in solution enabled the next base to be
read. Additionally, we observed that the DNA polymerase could
incorporate the surface-conjugated dGTPwhen the applied force
was controlled by employing the force-clamp mode. MicroRNAs
(miRNAs) play key roles in controlling various cellular processes,
and the expression levels of individual miRNAs can be
considerably changed in pathological conditions such as cancer.
Accurate and precise quantification of miRNA at the single-cell
level will lead to a better understanding of miRNA function. Here,
we present a direct and sensitive detection method for a specific
miRNA using AFM. A hybrid binding domain (HBD)-tethering tip
enabledmaturemiRNAs tobe located individuallyonanadhesion
force map. By scanning several sections of a micron-sized DNA
spot, we were able to quantify the copy number of miR-134
from a single neuron and demonstrated that the expression was
increased upon the cell activation. Additionally, we visualized
individual miR-134s on fixed neurons after membrane removal
and observed 2-4 miR-134s in the area of 1.0x1.0 m2 of soma.
The number increased to 8-14 in stimulated neurons, and this
change matches with the ensemble-averaged increase in copy
number.
Figure 1:
Three steps for the single DNA sequencing with AFM
Recent Publications
1. Hyun-Seo Koo, Ikbum Park, Yoonhee Lee, Hyun
Jin Kim, Jung Hoon Jung, Joo Han Lee, Youngkyu
Kim, Joung-Hun Kim and Joon Won Park (2016)
Visualization and quantification of microRNA in a
single cell by AFM. Journal of the American Chemical
Society 138(36):11664.
2. YoonheeLee,YoungkyuKim,DonggyuLee,Dhruvajyoti
Roy and Joon Won Park (2016) Quantification of
fewer than ten copies of a DNA biomarker without
amplification or labeling. Journal of the American
Chemical Society 138(22):7075.
3. Woong Kim, Nara Kim, Joon Won Park and Zee
Hwan Kim (2016) Nanostar probes for tip-enhanced
spectroscopy. Nanoscale 8(2):987.
4. Dae Heon Kim, Jae-Eun Lee, Zheng-Yi Xu, Kyeong
Rok Geem, Yun Kwon, Joon Won Park, Inhwan
Hwang (2015) Cytosolic targeting factor AKR2A
Single DNA sequencing and visualization of
miR-134 in nerve cells with force-based AFM
Joon Won Park
Pohang University of Science and Technology, South Korea
Joon Won Park, Nano Res Appl, Volume:4
DOI: 10.21767/2471-9838-C1-007