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Project Goals
Relevance to LLNL Mission
FY2006 Accomplishments and Results
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References
Bustamante, C., Z. Bryant, and S. B. Smith, 2003,
"Ten Years of Tension: Single-Molecule DNA
Mechanics," Nature 421, pp. 423-427.
Brewer, L. R., M. Corzett, and R. Balhorn, 1999,
"Protamine-Induced Condensation and Decondensation
of the Same DNA Molecule,"
Science 286, pp. 120-123.
Bianco, P. R., et al., 2001, "Processive Translocation
and DNA Unwinding by Individual
Recbcd Enzyme Molecules," Nature 409,
pp. 374-378.
Brewer, L., M. Corzett, E. Y. Lau, and
R. Balhorn, 2003, "Dynamics of Protamine 1
Binding to Single DNA Molecules," J. Biol.
Chem., 278, pp. 42403-42408.
Contact Us
George Dougherty
(925) 423-3088 |
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Single-Molecule Assay of DNA Integrity
DNA molecules are susceptible to
damage resulting from flow through
microchannels in microfluidic devices
and systems. This damage, which can
take the form of either single- or double-strand
breaks, is dependent on the size
of the channels, fluid flow speeds, and
the size of the DNA molecules that flow
through the device. This damage can
make it difficult to analyze the DNA
using processes such as DNA sequencing,
polymerase chain reaction (PCR),
and labeling of specific sequences with
fluorescent probes to search for specific
DNA molecules.
An assay to characterize the degree
of damage is important because it allows
one to optimize microfluidic device
parameters such as flow speed, aperture
size, and geometry. A single molecule
assay to characterize damage is particularly
useful for microfluidic devices
where the concentration of DNA is low,
such as low copy number DNA analysis.
In these cases, bulk assays, such as
electrophoresis, are not sensitive enough
to characterize the damage to the DNA.
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Figure 1. Two optical traps
(orange laser beams) are
used to hold, stretch, and
measure the elasticity of a
single lambda-phage DNA
molecule via 1-μm beads
attached to the ends. |
In this project we measured the
elasticity of single lambda-phage
DNA molecules (contour length
= 16.4 μm) before and after flow
through microfluidic devices. Optical
traps were used to stretch single DNA
molecules attached to 1-μm beads as
shown in Fig. 1. The displacement of
the bead in each optical trap varied
linearly with applied force, much like
a bead on a spring, and the image of
the bead on a quadrant photodiode detector
was used to accurately measure
the elastic tension the DNA molecule
experienced as the distance between
the traps increased. Figure 2 shows
the difference in elasticity between double-stranded DNA and single-stranded
DNA that forms the basis of
this assay. It takes much greater force
to stretch double-stranded DNA than
it does single-stranded DNA, and
this fact can be used to detect DNA
molecules that contain single-stranded
portions, due to single-strand nicks, or
where strands have been pulled apart
due to shearing forces.
 |
| Figure 2. Differences in the elasticity of double-stranded
DNA (dsDNA) and single-stranded DNA
(ssDNA). |
Project Goals
The project goals were to 1) identify
a DNA biosensor for testing; 2) perform
control measurements on DNA at very
low flow speeds; 3) test DNA integrity
using both gel electrophoretic and DNA
single-molecule elasticity measurements
to determine the degree of both
double- and single-strand breaks; and
4) optimize the flow speed and geometry
of the microfluidic device to
minimize DNA damage.
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Relevance to LLNL Mission
The expertise gained from this project
will enable LLNL to develop microfluidic devices for DNA biosensors with
a higher efficiency of detection. The
increased efficiency of DNA detection
will be the direct result of assessing the
damage to DNA samples after they pass
through microfluidic biosensors, and
then optimizing flow speed and biosensor
geometry to minimize this damage.
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FY2006 Accomplishments and Results
First we performed control measurements
on lambda-phage DNA during
flow through microfluidic devices using
both bulk- and single-molecule techniques.
We flowed lambda-phage DNA
through a "packed bed reactor" at flow
speeds between 0 and 10 μL/s. Gel
electrophoresis of the resultant DNA revealed
no double-strand breaks. This is
shown in Fig. 3. A 1-kb sizing standard
run in the left-most lane gives an idea what fragmentation of the original DNA
would have looked like.
 |
| Figure 3. Gel electrophoresis of lambda-phage
DNA molecules that have been run through
a "packed bed reactor" (courtesy E. Wheeler)
at flow rates of A) 0 μL/s, B) 1 μL/s, C) 5 μL/s,
and D) 10 μL/s. No double-strand breaks were
observed. The leftmost lane contains a 1-kb
sizing ladder. |
Next, we stretched individual lambda-phage DNA molecules that flowed
through a multichannel cell at very low
speeds (0.3 μL/s). Figure 4 shows the
characteristic "overstretching transition"
and is very similar to the graph shown
in Fig. 2. However, at higher flow rates
damage to the DNA was evidenced by
a dip in the overstretching transition
shown in Fig. 5. The dip is indicative of
damage to the molecule and results from
a portion of the molecule becoming
single-stranded. This data is supported
by Fig. 2, which indicates that there is
a large difference in the elasticity of
single- and double-stranded DNA at
forces close to 65 pN.
 |
 |
| Figure 4. Elasticity of a single lambda-phage
DNA molecule taken after an initial flow rate of
0.3 μL/s through a microchannel flow cell. |
Figure 5. Elasticity of a single lambda-phage DNA
molecule taken after an initial flow rate greater
than 1 μL/s through a microchannel flow cell,
showing a strong dip in the middle of the overstretching
transition. This dip shows that a portion
of the molecule is single-stranded, possibly due to
fluid shear forces. |
This technique represents a sensitive
way to measure damage to single
DNA molecules that occurs when
DNA flows through small channels at
high flow rates. Further work will be
required to obtain more quantitative
information about the extent of the
damage to the DNA.
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