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10.1117/2.1200702.0622
Immobilization and stretching
of DNA molecules in a
microchannel
Venkat Dukkipati, Ji Hoon Kim, Stella Pang, and
Ronald Larson
A new technique can immobilize and stretch a large number of DNA
molecules for single-molecule DNA analysis applications.
DNA-protein interactions drive the cellular machinery for maintaining and transcribing DNA. To study the motion and kinetics of proteins along a DNA strand at the single-molecule
level, it is critical that the DNA molecules be stretched and
immobilized. However, existing stretching and immobilization
techniques, such as optical tweezers1 and molecular combing,2
either produce very few stretched molecules or molecules that
do not readily allow normal DNA-protein interactions. Hence
the interest in improving these techniques. Advances in the ability to stretch and immobilize large numbers of DNA molecules
in micro- and nanofluidic channels will provide a powerful tool
for single-molecule DNA sequencing and DNA-protein interaction analysis, gene mutation detection, and for the manufacture
of DNA-based electronic devices.
We recently developed a DNA immobilization technique—
protein-assisted DNA immobilization (PADI)—that can deposit
a large number of stretched and immobilized DNA molecules
in a microchannel, as shown in Figure 1. The PADI technique
works as follows. DNA-interacting proteins, such as restriction
enzymes and RNA polymerases (RNAPs), are first allowed to
bind to DNA in bulk solution at nonspecific segments. This
solution is then introduced into a microchannel whose hydrodynamic flow stretches the DNA-protein complex. When the
complex diffuses to the channel surface, its protein moiety adsorbs on the surface, resulting in immobilization of stretched
DNA molecules inside the microchannel. Figure 1 shows a large
number of λ-DNA molecules stretched and immobilized in a
100µm-wide and 1µm-deep microchannel. PADI thus allows immobilization of thousands of stretched DNA molecules from solutions of very low concentration (pM and fM). In principle,
Figure 1. Image of λ-DNA molecules stretched and immobilized in
the presence of T7 RNA polymerase (10nM) in a microfluidic device.
Reproduced by kind permission of the American Chemical Society from
Nanoletters 6, 2499–3504, 2006.3
the PADI technique can be used to immobilize double-stranded
DNA of any sequence and size. We expect that its application to
single-molecule detection in microchannels will prove inexpensive and more sensitive than DNA microarrays and nanowirebased detection methods.
PADI has several attractive features. It avoids overstretching
of DNA molecules. The degree of attachment of DNA to the
substrate can be controlled by changing the protein concentration without changing the substrate material. The number of
DNA molecules immobilized onto the substrate is time- and
concentration-dependent and can be controlled simply by varying the pumping time as well as the concentration. And stretching and immobilization are achieved at physiological pH.
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Using total internal reflection fluorescence microscopy
(TIRFM), single-protein molecules bound to DNA are imaged
in Figure 2. Combining TIRFM with PADI clearly could allow
large-scale DNA mapping with fluorescent tags such as peptide
nucleic acids for single-molecule DNA sequencing and gene mutation detection.
Controlling the degree of attachment of the DNA backbone to
the surface is critical for such applications. This can be achieved
by modifying protein concentrations. At high concentrations,
the DNA is firmly attached to the surface as shown in Figure
3(a). Lowering the concentration leads to fewer proteins bound
to DNA, resulting in loose DNA attachment to the surface as
shown in Figure 3(b).
Optical mapping is a single-molecule DNA sequencing
method that involves digesting the stretched DNA with restriction enzymes, followed measuring the length of the resulting
Figure 2. T7 DNA molecules (green) stretched and immobilized with
the assistance of T7 RNA polymerase (red). Arrows indicate positions
of bound T7 RNA polymerases. Scale bar = 2.5µm. Reproduced by
kind permission of the American Chemical Society from Nanoletters
6, 2499–3504, 2006.3
Figure 3. T7 DNA molecules are immobilized at (a) 5nM and (b)
0.5nM T7 RNA polymerase concentrations followed by DNA photocleavage upon exposure to illumination. Reproduced by kind permission of the American Chemical Society from Nanoletters 6, 2499–
3504, 2006.3
Figure 4. DNA fragment after digestion with the Sma I restriction enzyme. Scale bar = 2.5µm. Reproduced by kind permission of the American Chemical Society from Nanoletters 6, 2499–3504, 2006.3
Figure 5. RNA transcripts labeled with Alexa Fluor 546 uridine
triphosphate (red) formed along YOYO-stained T7 DNA (green). Scale
bar = 5µm. Reproduced by kind permission of the American Chemical
Society from Nanoletters 6, 2499–3504, 2006.3
DNA fragments. Figure 4 shows fragments of λ-DNA molecules
after digestion with the restriction enzyme Sma I.
We have also carried out single-molecule transcription. The
RNA transcripts were detected by incorporating fluorescently
labeled uridine triphosphate into the growing RNA chain. Figure 5 shows the transcripts as bright red dots along the stretched
DNA molecules stained with a YOYO nucleic acid dye.
In summary, we have developed a novel DNA immobilization method to stretch and immobilize DNA molecules onto a
substrate. Our PADI technique can also be applied to singlemolecule DNA sequencing and DNA-protein optical mapping.
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Author Information
Venkat Dukkipati, Ji Hoon Kim, Stella Pang, and
Ronald Larson
University of Michigan
Ann Arbor, MI
References
1. M. D. Wang, H. Yin, R. Landick, J. Gelles, and S. M. Block, “Stretching DNA with
optical tweezers,” Biophys. J. 72(3), pp. 1335–1346, 1997.
2. A. Bensimon, A. Simon, A. Chiffaudel, V. Croquette, F. Heslot, and D. Bensimon, “Alignment and sensitive detection of DNA by a moving interface,” Science 265(5180), pp. 2096–2098, 1994.
3. V. R. Dukkipati, J. H. Kim, S. W. Pang, and R. G. Larson, “Protein-assisted
stretching and immobilization of DNA molecules in a microchannel,” Nano
Lett. 6(11), pp. 2499–2504, 2006.
c 2007 SPIE—The International Society for Optical Engineering