U.S. patent application number 14/485119 was filed with the patent office on 2015-03-19 for system and method for presenting large dna molecules for analysis.
The applicant listed for this patent is University of Chicago, University of Leiden, Wisconsin Alumni Research Foundation. Invention is credited to Juan DePablo, Juan Hernandez-Ortiz, Kyubong Jo, Kristy Kounovsky-Shafer, Theo Odijk, David C. Schwartz.
Application Number | 20150075984 14/485119 |
Document ID | / |
Family ID | 51945992 |
Filed Date | 2015-03-19 |
United States Patent
Application |
20150075984 |
Kind Code |
A1 |
Schwartz; David C. ; et
al. |
March 19, 2015 |
SYSTEM AND METHOD FOR PRESENTING LARGE DNA MOLECULES FOR
ANALYSIS
Abstract
Systems and methods for presenting nucleic acid molecules for
analysis are provided. The nucleic acid molecules have a central
portion that is contained within a nanoslit. The nanoslit contains
an ionic buffer. The nucleic acid molecule has a contour length
that is greater than a nanoslit length of the nanoslit. An ionic
strength of the ionic buffer and electrostatic or hydrodynamic
properties of the nanoslit and the nucleic acid molecule combining
to provide a summed Debye length that is greater than or equal to
the smallest physical dimension of the nanoslit.
Inventors: |
Schwartz; David C.;
(Madison, WI) ; Kounovsky-Shafer; Kristy;
(Kearney, NE) ; Hernandez-Ortiz; Juan; (Medellin,
CO) ; Odijk; Theo; (Leiden, NL) ; DePablo;
Juan; (Chicago, IL) ; Jo; Kyubong; (Seoul,
KR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Wisconsin Alumni Research Foundation
University of Leiden
University of Chicago |
Madison
Leiden
Chicago |
WI
IL |
US
NL
US |
|
|
Family ID: |
51945992 |
Appl. No.: |
14/485119 |
Filed: |
September 12, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61877570 |
Sep 13, 2013 |
|
|
|
Current U.S.
Class: |
204/450 ;
204/600 |
Current CPC
Class: |
B01L 2300/0816 20130101;
B01L 3/502761 20130101; G01N 33/48721 20130101; B01L 2200/0663
20130101; G01N 27/44791 20130101; B01L 2300/0896 20130101 |
Class at
Publication: |
204/450 ;
204/600 |
International
Class: |
G01N 27/447 20060101
G01N027/447 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH
[0002] This invention was made with government support under
HG000225 awarded by the National Institutes of Health and 0832760
awarded by the National Science Foundation. The government has
certain rights in the invention.
Claims
1. A micro-fluidic device comprising: a first microchannel; a
second microchannel; a nanoslit extending between the first and
second micro channels, the nanoslit providing a fluid path between
the first and the second microchannels; a nucleic acid molecule
having a first end portion, a second end portion, and a central
portion positioned between the first end portion and the second end
portion; and an ionic buffer within the nanoslit and the first and
second microchannel; the first microchannel including a first
cluster region adjacent to a first end of the nanoslit and the
second microchannel including a second cluster region adjacent to a
second end of the nanoslit, the first cluster region containing the
first end portion, the second cluster region containing the second
end portion, and the nanoslit containing the central portion, the
nucleic acid molecule having a contour length that is greater than
a nanoslit length of the nanoslit, and an ionic strength of the
ionic buffer and electrostatic or hydrodynamic properties of the
nanoslit and the nucleic acid molecule combining to provide a
summed Debye length that is greater than or equal to a nanoslit
height or a nanoslit width, wherein the nanoslit height or nanoslit
width is the smallest physical dimension of the nanoslit.
2. The micro-fluidic device of claim 1, wherein the nanoslit has a
nanoslit width of less than or equal to 1 .mu.m or a nanoslit
height of less than or equal to 100 nm.
3. The micro-fluidic device of claim 1, wherein the nanoslit length
is less than or equal to half a contour length of the nucleic acid
molecule.
4. The micro-fluidic device of claim 1, wherein at least one of the
microchannels has one or more of the following: a microchannel
width of about 20 .mu.m, a microchannel length of about 10 mm, and
a microchannel height of about 1.66 .mu.m.
5. The micro-fluidic device of claim 1, the device further
comprising a temperature adjustment module.
6. The micro-fluidic device of claim 1, wherein the ionic buffer
has a temperature of less than or equal to 20.degree. C.
7. The micro-fluidic device of claim 1, wherein the ionic buffer
further comprises a viscosity modifier.
8. The micro-fluidic device of claim 1, wherein the nucleic acid
molecule has a relaxation time of at least about 30 seconds.
9. The micro-fluidic device of claim 1, wherein the nucleic acid
molecule is a DNA molecule.
10. The micro-fluidic device of claim 1, wherein the nucleic acid
molecule has a contour length that is at least two times greater
than the nanoslit length of the nanoslit.
11. A method of stretching a nucleic acid molecule in an ionic
buffer, the method comprising: positioning the nucleic acid
molecule such that a central portion of the nucleic acid molecule
occupies a nanoslit, a first end portion of the nucleic acid
molecule occupies a first cluster region adjacent to a first end of
the nanoslit, and a second end portion of the nucleic acid molecule
occupies a second cluster region adjacent to a second end of the
nanoslit, the nanoslit, the first cluster region, and the second
cluster region including the ionic buffer, the nucleic acid
molecule having a contour length that is greater than a length of
the nanoslit, and an ionic strength of the ionic buffer and
electrostatic or hydrodynamic properties of the nanoslit and the
nucleic acid molecule combining to provide a summed Debye length
that is greater than or equal to a nanoslit height or a nanoslit
width, wherein the nanoslit height or nanoslit width is the
smallest physical dimension of the nanoslit.
12. The method of claim 11, wherein positioning the nucleic acid
molecule includes threading the nucleic acid molecule through the
nanoslit.
13. The method of claim 11, wherein positioning the nucleic acid
molecule includes electrokinetically driving the central portion of
the nucleic acid molecule into the nanoslit.
14. The method of claim 11, wherein the nanoslit has a nanoslit
width of less than or equal to 1 .mu.m or a nanoslit height of less
than or equal to 100 nm.
15. The method of claim 11, wherein the nanoslit length is less
than or equal to half a contour length of the nucleic acid
molecule.
16. The method of claim 11, wherein at least one of the
microchannels has one or more of the following: a microchannel
width of about 20 .mu.m, a microchannel length of about 10 mm, and
a microchannel height of about 1.66 .mu.m.
17. The method of claim 11, wherein the ionic buffer has a
temperature of less than or equal to 20.degree. C.
18. The method of claim 11, wherein the ionic buffer further
comprises a viscosity modifier.
19. The method of claim 11, wherein the nucleic acid molecule has a
relaxation time of at least about 30 seconds.
20. The method of claim 11, wherein the nucleic acid molecule is a
DNA molecule.
21. The method of claim 11, the method further comprising imaging
at least a portion of the central portion.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Patent Application Ser. No. 61/877,570 filed on Sep. 13, 2013, the
disclosure of which is incorporated by reference herein as if set
forth in its entirety.
BACKGROUND OF THE INVENTION
[0003] The field of the invention is nucleic acid molecule
manipulation. More particularly, the invention relates to
stretching nucleic acid molecules in order to better present
portions of the nucleic acid molecules for inspection by various
techniques.
[0004] Much of the human genome is comprised of DNA sequences that
are present in multiple copies. Although such elements play an
important role in biological regulation and evolution, their
presence troubles current DNA sequencing approaches. Accordingly,
serious issues arise when trying to complete the sequencing of
human, or cancer genomes because short analyte molecules, currently
used by major sequencing platforms, often present redundant
sequence data. Like trying to assemble a jigsaw puzzle with pieces
bearing no uniquely discernible features, such sequence data make
it difficult to assemble the sequence of an entire genome.
Furthermore, our ability to assess genomic alterations within
populations as mutations, or polymorphisms is also limited. To meet
this challenge, genomewide analysis.sup.1-3 systems are now
featuring modalities that present large, genomic DNA
analytes.sup.3,4 for revealing genomic alterations through
bioinformatic pipelines. Achieving utility for genome analysis
using nanoconfinement approaches requires integration of system
components that are synergistically poised for dealing with large
data sets. Such components include sample preparation, molecular
labeling, presentation of confined DNA molecules, and detection,
complemented by algorithms incorporating statistical considerations
of experimental error processes for data analysis..sup.5-8
[0005] While a number of approaches to confine DNA molecules have
been examined and implemented in the past few years,.sup.5,10-15
few elongate DNA molecules close to their contour length. Kim et
al.,.sup.9 for example, elongated .lamda.-DNA within
poly(dimethylsiloxane) (PDMS) replicated nanochannels (250
nm.times.400 nm) and achieved a stretch of 0.88 using ultralow
ionic strength conditions (0.06 mM). To our knowledge, it was the
longest stretch reported for DNA molecules within nanochannels,
using low ionic strength buffers. In different work, Reisner et
al..sup.11 used 50 nm fused silica nanochannels with higher ionic
strength conditions (.about.5 mM) to elongate DNA molecules up to
0.83. Although the stretch with these two approaches was higher
than 0.8, both techniques exhibit limitations. The approach of
Reisner et al. is demanding in that it requires fabrication of
extreme nanoconfinement devices, smaller than the molecular
persistence length,.sup.16 to elongate DNA molecules close to the
molecular contour length, thereby increasing the complexity of the
molecular loading process.
[0006] Accordingly, a need exists for an approach to stretching a
nucleic acid molecule that overcomes the aforementioned
drawbacks.
SUMMARY OF THE INVENTION
[0007] The present invention overcomes the aforementioned drawbacks
by providing a microfluidic device and a method of stretching a
nucleic acid molecule.
[0008] In accordance with the present disclosure, the micro-fluidic
device can include a first microchannel, a second microchannel, a
nanoslit, a nucleic acid molecule, and an ionic buffer. The
nanoslit can extend between the first and second microchannels. The
nanoslit can provide a fluid path between the first and second
microchannels. The nucleic acid molecule can include a first end
portion, a second end portion, and a central portion positioned
between the first end portion and the second end portion. The ionic
buffer can be within the nanoslit and the first and second
microchannel. The first microchannel can include a first cluster
region adjacent to a first end of the nanoslit and the second
microchannel can include a second cluster region adjacent to the
second end of the nanoslit. The first cluster region can contain
the first end portion. The second cluster region can contain the
second end portion. The nanoslit can contain the central portion.
The nucleic acid molecule can have a contour length that is greater
than a nanoslit length of the nanoslit. An ionic strength of the
ionic buffer and electrostatic or hydrodynamic properties of the
nanoslit and the nucleic acid molecule can combine to provide a
summed Debye length that is greater than or equal to a nanoslit
height or a nanoslit width. The nanoslit height or nanoslit width
can be the smallest physical dimension of the nanoslit.
[0009] In accordance with the present disclosure, the method of
stretching a nucleic acid molecule in an ionic buffer can include
positioning the nucleic acid molecule such that a central portion
of the nucleic acid molecule occupies a nanoslit, a first end
portion of the nucleic acid molecule occupies a first cluster
region adjacent to a first end of the nanoslit, and a second end
portion of the nucleic acid molecule occupies a second cluster
region adjacent to a second end of the nanoslit. The nanoslit, the
first cluster region, and the second cluster region can include the
ionic buffer. The nucleic acid can have a contour length that is
greater than a length of the nanoslit. An ionic strength of the
ionic buffer and electrostatic or hydrodynamic properties of the
nanoslit and the nucleic acid molecule can combine to provide a
summed Debye length that is greater than or equal to a nanoslit
height or a nanoslit width. The nanoslit height or the nanoslit
width can be the smallest physical dimension of the nanoslit.
[0010] The foregoing and other aspects and advantages of the
invention will appear from the following description. In the
description, reference is made to the accompanying drawings which
form a part hereof, and in which there is shown by way of
illustration a preferred embodiment of the invention. Such
embodiment does not necessarily represent the full scope of the
invention, however, and reference is made therefore to the claims
and herein for interpreting the scope of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0011] FIG. 1 shows microchannel-nanoslit device supporting the
formation of molecular dumbbells. (A) PDMS device adhered to
cleaned glass coverslip, immersed in buffer (not shown), for
electrokinetic loading of DNA molecules. (B) Dumbbells form when
loaded T4 DNA molecules (166 kb; 74.5 .mu.m, dye adjusted contour
length) exceed the nanoslit length (28 .mu.m); molecule ends
flanking nanoslits become relaxed coils within the microchannels
(lobes), thereby enhancing the stretch of intervening segments
within the nanoslits to (0.85.+-.0.16, I=0.51 mM); traces show
fluorescence intensity variations along molecular backbones. (C)
.lamda.-DNA molecules (48.5 kb, 21.8 .mu.m) are too short to form
dumbbells and are thus completely confined within the nanoslits; a
lower stretch (S/L=0.62.+-.0.08, I=0.48 mM) is further evidenced by
uneven fluorescence intensity profiles.
[0012] FIG. 2 shows simulated nanoslit geometry. An snapshot of a
T4-DNA molecule (166 kb, L=74.5 .mu.m) forming a dumbbell is shown.
Simulated slit lengths were 10, 20, and 30 .mu.m; the results were
equivalent due to the fact that the molecule stretch is independent
of molecular weight. The results presented here were calculated
using a 20 .mu.m long nanoslit.
[0013] FIG. 3 describes a stretch as a function of ionic strength
for T4 DNA (grey bullets ( ); error bars show SD on means, N=51-101
molecules) dumbbells showing good concordance between experiments,
simulation, and Odijk theory. White bullets ( ) show .lamda.
concatemer data from FIG. 5, and measurements using an internal
standard (see text). Successive dilutions of 1.times.TE buffer
varied ionic strength: 1.0, 0.74, 0.51, 0.45, 0.23, and 0.11 mM.
Triangles (.DELTA.) show results from BD simulations without
considering hydrodynamic interactions (FD). Boxes (.quadrature.)
show results from BD simulations with fluctuating hydrodynamics
interactions (HI). Dotted lines correspond to de Gennes and Odijk
scaling predictions. The shaded region encompasses the Odijk
scaling between an effective h.times.h channel and a 3h.times.h
slit.
[0014] FIG. 4 is a stretch along the nanoslit axial and width
directions as a function of nanoslit axial position for T4 DNA
dumbbells at I=0.51 mM. The predicted stretches are shown for HI
(continuous lines) and FD (dotted lines) chains. Snapshots of an HI
chain (top) and an FD chain (bottom) are included.
[0015] FIG. 5 is a stretch of .lamda.-DNA concatemer dumbbells as a
function of size: experiment compared to BD simulation. Arrows link
experimental and simulation results to graphical outputs and a
montage of micrographs; error bars show SD on the means (dots) for
N=9-93 molecules. Cartoon shows a vertical line delineating a
nanoslit; horizontal lines indicate nanoslit boundaries. Dumbbell
lobes enlarge with increasing molecular size for a given
slit/microchannel geometry and show a compelling similarity to
simulation.
[0016] FIG. 6 shows dumbbell relaxation times as a function of
molecule size for T4 (white bullets ( ), N=105 molecules),
.lamda.-DNA concatemers (black bullets ( ), N=4-21 molecules), and
M. florum DNA (grey bullets ( ), N=11-59 molecules) digested with
the restriction enzyme Apal. Each circle represents a mean
relaxation time (R.sub.t) for a given molecule size (146 kb-582 kb;
x-axis error bars show 95% confidence intervals). Linear regression
fit to the log-log plot shows an exponent of 1.23.+-.0.09
(R.sup.2=0.82). The exponent error is determined with a consistency
test that includes each point's mean and x-axis error. (Inset)
Dumbbell dynamics for a T4 DNA molecule imaged as a movie shown
here as compiled time slices (arrow a shows one slice; 0.440 s per
slice).
DETAILED DESCRIPTION OF THE INVENTION
[0017] All referenced patents, applications, and non-patent
literature cited in this disclosure are incorporated herein by
reference in their entirety. If a reference and this disclosure
disagree, then this disclosure is controlling.
[0018] This disclosure provides a micro-fluidic device. The
microfluidic device can include a first microchannel, a second
microchannel, a nanoslit, a nucleic acid molecule, and an ionic
buffer. The nanoslit can extend between the first and second
microchannels. The nanoslit can provide a fluid path between the
first and second microchannels. The nucleic acid molecule can have
a first end portion, a second end portion, and a central portion
positioned between the first end portion and the second end
portion. The ionic buffer can be within the nanoslit and the first
and second micro channel. The first micro channel can include a
first cluster region adjacent to a first end of the nanoslit and
the second microchannel can include a second cluster region
adjacent to the second end of the nanoslit. The first cluster
region can contain the first end portion. The second cluster region
can contain the second end portion. The nanoslit can contain the
central portion. The nucleic acid molecule can have a contour
length that is greater than a nanoslit length of the nanoslit. An
ionic strength of the ionic buffer and electrostatic or
hydrodynamic properties of the nanoslit and the nucleic acid
molecule can combine to provide a summed Debye length that is
greater than or equal to a nanoslit height or a nanoslit width. The
nanoslit height or the nanoslit width can be the smallest physical
dimension of the nanoslit.
[0019] This disclosure also provides a method of stretching a
nucleic acid molecule in an ionic buffer. The method can include
positioning the nucleic acid molecule such that a central portion
of the nucleic acid molecule occupies a nanoslit, a first end
portion of the nucleic acid molecule occupies a first cluster
region adjacent to a first end of the nanoslit, and a second end
portion of the nucleic acid molecule occupies a second cluster
region adjacent to a second end of the nanoslit. The nanoslit, the
first cluster region, and the second cluster region can include the
ionic buffer. The nucleic acid can have a contour length that is
greater than a length of the nanoslit. An ionic strength of the
ionic buffer and electrostatic or hydrodynamic properties of the
nanoslit and the nucleic acid molecule can combine to provide a
summed Debye length that is greater than or equal to a nanoslit
height or a nanoslit width. The nanoslit height or the nanoslit
width can be the smallest physical dimension of the nanoslit.
[0020] Referring to FIG. 1(A), a micro-fluidic device can include a
replica 10, such as a PDMS replica, including microchannels 12 and
nanoslits 14. The device can be mounted on a substrate 16. The
device can have electrodes 18.
[0021] Referring to FIG. 1(B), the microfluidic device can contain
stretched nucleic acid molecules 20 in the dumbbell configuration.
The fluorescence intensity 22 is shown next to the corresponding
nucleic acid molecule 20. The lack of variation in fluorescence
intensity 22 across the length of the nucleic acid molecule 20
indicates good stretching.
[0022] Referring to FIG. 1(C), the microfluidic device shown
contains nucleic acid molecules 24 that are shorter than the
nanoslit length, are not in the dumbbell configuration, and are not
fully stretched. The fluorescence intensity 26 is shown next to the
corresponding nucleic acid molecule. the larger variation in
fluorescence intensity 26 across the length of the nucleic acid
molecule 20 indicates poor stretching.
[0023] In certain embodiments, the nanoslit can be configured to
contain a central portion of the nucleic acid molecule when the
first cluster region holds a first clustered end portion of the
nucleic acid molecule and the second cluster region holds a second
clustered end portion of the nucleic acid molecule. The central
portion of the nucleic acid molecule can be located generally
between the first and second clustered ends of the nucleic acid
molecule. As used herein, "clustered ends" or a "clustered
configuration" refers to a configuration of the nucleic acid
molecule that is not stretched out and contains random coils in at
least a portion of the respective end of the nucleic acid molecule.
As used herein, a "cluster region" refers to a space within a
microchannel that is occupied by a clustered end. The cluster
region will inherently be the same size or smaller than its
respective microchannel.
[0024] In certain embodiments, the nanoslit can have physical
dimensions as follows. The nanoslit can have a smallest physical
dimension that is on the order of 1 nm to about 1 .mu.m. The
nanoslit can have a nanoslit width of between 200 nm and 10 .mu.m.
The nanoslit can have a nanoslit width of less than or equal to 1
.mu.m. The nanoslit can have a nanoslit length of less than or
equal to 30 .mu.m. The nanoslit can have a nanoslit height of
between 20 nm and 200 nm. The nanoslit can have a nanoslit height
of less than or equal to 100 nm. In certain embodiments, the
nanoslit can have a substantially uniform nanoslit width, a
substantially uniform nanoslit height, or both over the entire
nanoslit length.
[0025] In certain embodiments, the nanoslit can have a length of
less than or equal to a contour length of the nucleic acid molecule
or less than or equal to half a contour length of the nucleic acid
molecule.
[0026] In certain embodiments, the nanoslit can have a smallest
physical dimension of at least 50 nm. In certain embodiments, the
nucleic acid molecule is positioned in a nanoslit having a smallest
physical dimension of at least about 50 nm. In other words, the
device may not require that a cavity of less than 50 nm be
fabricated. Moreover, when a nucleic acid molecule is positioned
within, threaded through, or electrokinetically driven into a
nanoslit of this embodiment, the smallest passage that it will
encounter can be a passage of 50 nm.
[0027] In certain embodiments, the ionic buffer can have an ionic
strength that suitably combines with the nanoslit and nucleic acid
molecule to provide a summed Debye length that is greater than or
equal to a nanoslit height or a nanoslit width, wherein the
nanoslit height or nanoslit width is the smallest physical
dimension of the nanoslit. In certain embodiments, the ionic buffer
can have an ionic strength that provides a Debye length of the
nanoslit or the nucleic acid that is at least about 25% of a
nanoslit height or a nanoslit width, wherein the nanoslit height or
nanoslit width is the smallest physical dimension of the nanoslit.
In certain embodiments, the ionic buffer can have an ionic strength
of less than or equal to about 0.75 mM.
[0028] In certain embodiments, the ionic buffer can include
Tris-HCl, EDTA, 2-mercaptoethanol, POPE, or a combination
thereof.
[0029] In certain embodiments, the ionic buffer can include a
viscosity modifier. The viscosity modifier can be sucrose.
[0030] In certain embodiments, the first microchannel, the second
microchannel, or both can have physical dimensions as follows. The
microchannels can have a smallest physical dimension that is on the
order of 1 .mu.m to about 1 mm. The microchannels can have a
microchannel width of between 1 .mu.m and 1 mm. The microchannels
can have a microchannel width of about 20 .mu.m. The microchannels
can have a microchannel length of between 100 .mu.m and 20 cm. The
microchannels can have a microchannel length of about 10 mm. The
microchannels can have a microchannel height of between 20 nm and
100 .mu.m. The microchannels can have a microchannel height of
about 1.66 .mu.m.
[0031] In certain embodiments, the device can further comprise a
temperature adjustment module. The temperature adjustment module
can be used to adjust the temperature of the nucleic acid molecule,
the ionic buffer, or both. The temperature adjustment module can be
used to raise or lower the temperature in order to slow the motion
dynamics of the nucleic acid molecule. In certain embodiments, the
ionic buffer can have a temperature of less than or equal to
20.degree. C.
[0032] In certain embodiments, the micro-fluidic device can include
one or more electrodes. The electrode or electrodes can be arranged
substantially parallel to the nanoslit or nanoslits and can be used
to electrokinetically drive the nucleic acid molecule into the
nanoslit.
[0033] In certain embodiments, the nucleic acid molecule can have a
relaxation time of at least about 30 seconds. In certain
embodiments, the nucleic acid molecule can have a contour length
that is greater than a nanoslit length of the nanoslit or at least
two times greater than a nanoslit length of the nanoslit.
[0034] In certain embodiments, the nucleic acid molecule can be a
DNA molecule.
[0035] In certain embodiments, positioning the nucleic acid
molecule can include threading the nucleic acid molecule through
the nanoslit, electrokinetically driving the central portion of the
nucleic acid molecule into the nanoslit, or a combination
thereof.
[0036] In certain embodiments, the methods can further include
imaging at least a portion of the central portion of the nucleic
acid molecule. Imaging can include microscopy, such as fluorescence
microscopy, and the like.
[0037] As discussed throughout this disclosure, large DNA molecules
may be presented for analysis in a "dumbbell" configuration. In the
described approach, for example, a DNA molecule in an ionic buffer
is caused to pass through a nanoslit within a micro-fluidic device.
This may result in a configuration of the molecule in which a
central portion of the molecule (i.e., a portion of the molecule
within the nanoslit) is stretched toward a linear configuration and
the opposite ends of the molecule (i.e., portions of the molecule
outside the nanoslit) form a cluster configuration--i.e., a
"dumbbell" configuration. Such presentation of DNA, in accordance
with this disclosure, may take advantage of entropic, elastic and
hydrodynamic forces to stretch the DNA, and may be useful, for
example, in order to conduct various analyses on the stretched
central portion of the relevant molecule (i.e., the "bar" of the
dumbbell). To support such analyses, it may be useful to implement
apparatus and procedures that ensure that the portion of the DNA
molecule within a nanoslit reaches (or at least approaches) the
"Odijk" regime (i.e., a noted plateau in the extension of a
confined DNA molecule) and exhibits an appropriately long
relaxation time, in order to facilitate execution of the desired
protocol(s).
[0038] Through significant modeling and experimentation, it has
been determined that notable improvement in the degree of stretch
and the relaxation time of DNA molecules may be effected using
carefully selected combinations of micro-fluidic device
configurations and ionic solution characteristics. For example, use
of a low strength ionic environment in conjunction with
appropriately scaled nanoslits in a micro-fluidic device may
facilitate achievement of a more fully stretched configuration of a
subject DNA molecule than has been previously possible. Such
environment/scaling may beneficially manage the delicate balance
between electrostatic and hydrodynamic interactions responsible for
conformations of the observed molecules.
[0039] As one example, it has been determined that micro-fluidic
devices exhibiting nanoslit length of less than half the contour
length of the relevant molecule may deliver relaxation time on the
order of minutes, a marked improvement over molecules that do not
employ the dumbbell configuration described herein. This may
strongly facilitate experimental observation of the molecule.
Accordingly, for certain applications, it may be beneficial to
configure micro-fluidic devices with nanoslits having a length of
no more than half of the contour length of a relevant DNA molecule.
Notably, under certain configurations and conditions, once the
nanoslit length has been appropriately adjusted with respect to a
reference molecule, the same micro-fluidic device may be utilized
for relatively uniform presentation of molecules of any size (e.g.,
molecules with contour lengths (L) exceeding the contour length of
the reference molecule). For example, with respect to particular
molecules (e.g., .lamda. bacteriophage (New England Biolabs) 48.5
kb (L=16.5 .mu.m/21.8 .mu.m), T4 bacteriophage (Wako Chemicals) 166
kb (L=56.3 .mu.m/74.5 .mu.m), .lamda.-concatemers (New England
Biolabs, concatemer ladder, size range=137.4-582.0 kb), M. florum
(Apal digest: 252 kb, L=85.7 .mu.m/113.2 .mu.m; 541 kb L=184.2
.mu.m/243.2 .mu.m), each in solution containing 4% (v/v)
2-mercaptoethanol, 0.1% (w/v) POPE (Applied Biosystems) and TE
buffer (1.times.: 10 mM Tri-HCL and 1 mM EDTA pH 7.9) ranging from
0.01.times. to 0.1.times.) (L, above, indicates unstained/stained
contour length, respectively), a micro-fluidic device may be
beneficially fabricated to include 1 .mu.m wide.times.100 nm high
by 28 .mu.m long nanoslits. In this way, the length of the
nanoslits is generally less than half the (stained or unstained)
contour length of the tested molecules and enhanced relaxation
times may be achieved accordingly.
[0040] Micro-fluidic devices, such as those described above, may
also include microchannels at either end of the noted nanoslits. In
certain embodiments, the microchannels for the example device
described above may be fabricated with dimensions of 20 .mu.m
wide.times.1.66 .mu.m high.times.10 mm long. Fabrication of these
micro-fluidic devices (and others contemplated by the disclosure)
may utilize various known techniques, such as reactive ion etching
on silicon wafers, with PDMS replicas being created by soft
lithography and made hydrophilic by 02 plasma treatment.
[0041] In addition or as an alternative to the above-described
nanoslit configuration, the stretch of subject molecules may also
be beneficially enhanced by providing the nucleic acid in a reduced
ionic strength buffer. For example, in contrast to various current
theories, it has been discovered that Odijk regime stretching may
be obtained by providing effective confinement equal (or at least
comparable) to the persistence length of a relevant DNA molecule
(taking into account, in certain embodiments, electrostatic
considerations). To this end, decrease of ionic strength may
beneficially increase chain persistence lengths and enhanced
effective confinement, which is induced by the increased Debye
length of the micro-fluidic device's surface (itself also enhanced
by appropriately low ionic strength). For example, reduced ionic
strength and appropriately configured nanoslits may result in the
Debye lengths of the device and/or the persistence length of the
molecule being comparable (or equal) to nanoslit height (e.g.,
.about.100 nm, for the device described above). In this way,
because the effective confinement is comparable (or equal) to the
chain persistence length, the Odijk regime may be achieved.
Therefore, use of decreased ionic strength buffer with appropriate
micro-fluidic device configurations (e.g., nanoslit dimensions) may
result in longer relaxation times, thereby better facilitating
imaging-based genomic analysis and other investigation. As such, it
may be appropriate to design and fabricate micro-fluidic devices
(e.g., with respect to nanoslit dimensions) and to select buffer
ionic strength (e.g., with a view toward increasing Debye length)
based upon the persistence length of the relevant DNA molecule,
rather than (or in addition to) focusing on the effective DNA
diameter. For example, with respect to the example device discussed
above (i.e., with 1 .mu.m wide.times.100 nm high by 28 .mu.m
nanoslits), a TE buffer may be utilized having final concentrations
of 0.006% for 2-mercaptoethanol and 0.00015% for POPE.
[0042] As an additional measure, in certain embodiments, addition
of sucrose to low ionic strength solutions (as discussed above)
and/or an appropriately timed decrease of temperature may increase
solution viscosity and thereby further extend relaxation time.
[0043] In practice, therefore, subject DNA may be threaded through
nanoslits on a micro-fluidic device via timed electrical pulses,
resulting in the above-described "dumbbell" configuration. As noted
above, in certain embodiments, the nanoslits may be
configured/manufactured based upon relevant characteristics of the
DNA molecules (e.g., molecule contour length and/or persistence
length), and an appropriately low ionic strength buffer may be
utilized (e.g., 0.11 mM or similar strength, as necessary to
provide a device Debye length comparable to nanoslit height).
Notably, the lower ionic strength buffer (e.g., 0.11 mM), in
combination with the appropriately scaled nanoslits and the elastic
forces generated by the induced dumbbell configuration, may greatly
enhance DNA elongation, even to the point of a fully stretched
presentation. As also discussed throughout the disclosure, this
enhancement may result, for example, from hydrodynamic interactions
of the DNA dumbbells and entropic recoil of the dumbbell lobes as
well as the enhancement of electrostatic interactions via reduced
ionic strength conditions. In certain instances, sucrose may also
be added to the low ionic solution and/or temperature may be
decreased (e.g., shifted after loading) to increase solution
viscosity and thereby further extend the relaxation time.
[0044] Past efforts using 250 nm.times.400 nm PDMS replicated
nano-channels and 0.06 mM ionic strength buffer have delivered
stretch of 0.88 for .lamda. DNA. The Debye length under these
conditions is approximately 40 nm. Accordingly, the summed Debye
length (the Debye length of the device roof, the device floor, the
surface of the nucleic acid molecule facing the device roof, and
the surface of the nucleic acid molecule facing the device floor,
i.e., 4 times the Debye length) is about 160 nm, which is short of
the smallest physical dimension of 250 nm. Likewise, efforts with
50 nm fused silica nano-channels and .about.5 mM ionic strength
buffer have delivered stretch of up to 0.83 for DNA molecules. The
Debye length under these conditions is approximately 1.34 nm.
Accordingly, the summed Debye length is about 5.34 nm, which is
short of the smallest physical dimension of 50 nm. In contrast, use
of one embodiment of the disclosed combination of appropriately
scaled nanoslit dimensions (e.g., as tuned to relevant aspects of
the target molecule) and appropriately low ionic strength buffers
(e.g., as selected for enhanced scaling of the device Debye
lengths) may usefully deliver stretch of 1.06 or higher.
[0045] The analysis of very large DNA molecules intrinsically
supports long-range, phased sequence information, but requires new
approaches for their effective presentation as part of any genome
analysis platform. Using a multipronged approach that marshaled
molecular confinement, ionic environment, and DNA elastic
properties buttressed by molecular simulations we have developed an
efficient and scalable approach for presentation of large DNA
molecules within nanoscale slits. Our approach relies on the
formation of DNA dumbbells, where large segments of the molecules
remain outside the nanoslits used to confine them. The low ionic
environment, synergizing other features of our approach, enables
DNA molecules to adopt a fully stretched conformation, comparable
to the contour length, thereby facilitating analysis by optical
microscopy. Accordingly, a molecular model is proposed to describe
the conformation and dynamics of the DNA molecules within the
nanoslits; a Langevin description of the polymer dynamics is
adopted in which hydrodynamic effects are included through a
Green's function formalism. Our simulations reveal that a delicate
balance between electrostatic and hydrodynamic interactions is
responsible for the observed molecular conformations. We
demonstrate and further confirm that the "Odijk regime" does indeed
start when the confinement dimensions are of the same order of
magnitude as the persistence length of the molecule. We also
summarize current theories concerning dumbbell dynamics.
[0046] Here, electrokinetic loading of large DNAs into nanoslits
offers new routes to stretching of random coils and presentation as
analyte arrays. Nanoslits, or channels with aspect ratios>1,
realize genomically scalable nanoconfinement conditions that
facilitate acquisition of large data sets. Nanoslits also allow
inexpensive fabrication through large-scale replication of
disposable devices from electron-beam fabricated masters. Moreover,
low-ionic strength conditions increase a DNA molecule's persistence
length, thereby leading to nanoconfinement of DNA in devices that
are compatible with the inherent geometric limitations of silastic
materials..sup.5,9 In the first generation of "Nanocoding," the
mapping of confined DNA molecules was carried out with
sequence-specific labels..sup.5 The value of such mapping data for
genomic analysis was shown to depend on marker density.sup.6 and
molecular stretch S/L (where S is the apparent length of a molecule
and L is its contour length).
[0047] Through a concerted experimental and theoretical approach
outlined in previous work,.sup.5 we reasoned that engaging DNA
"dumbbell" conformations within our nanoslits would greatly enhance
DNA stretching through entropic, elastic, and hydrodynamic forces.
In this paper, we define a DNA dumbbell as comprising two relaxed
coils (lobes) within a microchannel flanking intervening polymer
segments residing within a nanoslit (FIG. 1). Our experiments
indeed show that molecular dumbbells increase DNA stretch within
nanoslits up to the full molecule contour length using the same
ionic strength and "spacious" confinement conditions (slit
dimensions: 100 nm.times.1000 nm) as in previous experiments..sup.5
More importantly, DNA dumbbells overcome limitations of current
approaches, including ionic strengths below 0.06 mM, or severe
confinement (below 50 nm). A combination of the lobes' entropic
recoil, hydrodynamic interactions, and electrostatic interactions,
mediated by low-ionic strength conditions, produces tension across
the DNA molecule backbone within the nanoslit, further elongating
the molecule. For the first time, a dumbbell conformation allows
the elongation of DNA molecules within nanoslits demonstrating
stretch up to 1.06.+-.0.19. Our results indicate that the "Odijk
regime" is achieved once the persistence length is equal to the
effective confinement (including electrostatic considerations), in
apparent contradiction to other theories that suggested that the
effective DNA diameter is the relevant parameter for the de
Gennes-Odijk transition..sup.11 In addition, we find that once the
contour length of the molecule is longer than twice the nanoslit
length, the dumbbell's relaxation time is on the order of minutes,
and increases with lobe size. The stretch remains independent of
the molecular weight. Such molecular presentation greatly enhances
the entrapment of stretched molecules (i.e., out-of-equilibrium
metastable states), thereby making this approach a practical
component for genome analysis systems.
[0048] Recently, Yeh et al..sup.17 also performed experiments on
confined DNA molecules in combined micro- and nanoscale devices
similar to those employed by Kim et al..sup.9 They observed that
under some circumstances, long DNA was able to form dumbbells. They
explained their observations in terms of quasistatic arguments,
highlighting an entropy-driven single molecule tug-of-war (TOW)
scheme that enables study of the statics and the dynamics of
entropic recoil under strong confinement. In this work we show that
this quasi-static regime, corresponding to symmetric lobes within
the microscale confinement, has a vanishing probability of
appearance. The confined molecules are under nonequilibrium
conditions, and the uneven size of the lobes controls molecular
recoil. By taking account of nonequilibrium conditions, we show
that several mechanisms can control molecule dynamics and dumbbell
lifetimes.
[0049] Materials and Experimental Methodology
[0050] Device Fabrication and Setup. Microchannel-nanoslit device
masters were fabricated by electron beam lithography using the JEOL
JBX-5DII system (CNTech, UW-Madison). Nanoslits (1 .mu.m
wide.times.100 nm high.times.28 .mu.m long) were etched into a
silicon wafer by CF4 reactive ion etching and modified SU8
microchannels (20 .mu.m wide.times.1.66 .mu.m high.times.10 mm
long) were overlaid (see FIG. 1). PDMS replicas were created by
soft lithography, made hydrophilic by O.sub.2 plasma treatment, and
stored in distilled water for 24 h then the devices were utilized
for a couple of months. Nanoslit devices were mounted on
acid-cleaned negatively charged glass surfaces..sup.1 Platinum
electrodes (wire, 0.013'' diameter) were placed in a diagonal
orientation, nearly parallel to the nanoslits, in the buffer
chamber, a glass surface affixed to the bottom of a Plexiglas
holder, and attached to Kepco (model BOP 100-1M) bipolar
operational power supply. DNA solutions were loaded into the
microchannels using capillary action, and devices were immersed in
buffer [TE with final concentrations of 2-mercaptoethanol (0.006%)
and POPE (0.00015%; Applied Biosystems)] for 20 min, allowing
buffer equilibration before measurements. After the device is
immersed, DNA molecules were electrokinetically driven into the
nanoslits, timed before they completely exited, so that they were
trapped as dumbbells.
[0051] DNA Samples and Stretching.
[0052] DNA samples, stained with YOYO-1
(1,1-[1,3-propanediylbis[(dimethyliminio)-3,1-propanediyl]]bis[4-[(3-meth-
yl-2 (3H)benzoxazolylidene)methyl]-quinolinium iodide).sup.5
(Molecular Probes), included [(unstained/stained contour length),
L; assuming an intercalation rate of 1 dye/4 bp]
.lamda.-bacteriophage (New England Biolabs) 48.5 kb (L=16.5
.mu.m/21.8 .mu.m), T4 bacteriophage (Wako Chemicals) 166 kb (L=56.3
.mu.m/74.5 .mu.m), .lamda.-concatemers (New England Biolabs, A
concatemer ladder, size range=137.4-582.0 kb), Mesoplasma florum
(Apal digest: 252 kb, L=85.7 .mu.m/113.2 .mu.m; 541 kb, L=184.2
.mu.m/243.2 .mu.m. DNA solutions also contained 4% (v/v)
2-mercaptoethanol, 0.1% (w/v) POPE (Applied Biosystems) and TE
buffer (1.times.: 10 mM Tris-HCl and 1 mM EDTA pH 7.9) ranging from
0.01.times. to 0.1.times.; ionic strength was determined by
conductivity using a NaCl standard..sup.9
[0053] Image Capture and Analysis.
[0054] YOYO-1-stained molecules were imaged (Manual Collect
softwares) using a Hamamatsu CCD camera (Orca-ER), coupled to a
Zeiss 135 M epifluorescence microscope (63.times. Zeiss
Plan-Neofluar oil immersion objective), illuminated by an argon ion
laser (488 nm; 8 .mu.W to 200 .mu.W measured at nosepiece) for
stretch and relaxation time experiments. A more sensitive camera
(Andor iXon-888 EMCCD) was used to image the relaxation kinetics of
T4 dumbbell molecules. Images were analyzed using ImageJ.sup.18 to
subtract background using the "rolling ball" algorithm.sup.19
segment by thresholding the molecule from the background and
measuring molecular fluorescence intensities and length.
[0055] Mesoplasma florum Preparation.
[0056] M. florum.sup.20 was grown in ATCC 1161 at 30.degree. C.
then pelleted. Cells were washed with a solution of 10 mM Tris-HCl,
pH 7.6, and 1 M NaCl then pelleted and resuspended. Warmed cells,
37.degree. C., were mixed with 1:1 (v/v) with 1% low melting
temperature agarose and dispensed in an insert tray.
Inserts.sup.21,22 were pooled in a 50 mL conical tube and incubated
in 6 mM Tris-HCl pH 7.6, 1 M NaCl, 100 mM EDTA, 1%
N-lauroylsarcosine, and 20 .mu.g/mL RNase, overnight at 37.degree.
C. Inserts were then transferred to 0.50 M EDTA pH 8.0, 1%
N-lauroylsarcosine, with 1 mg/mL Proteinase K and incubated
overnight at 50.degree. C. followed by 0.1 mM phenylmethylsulfonyl
fluoride then dialyzed 10 times with 0.50 M EDTA, pH 9.5. Inserts
were twice dialyzed against 1.times.TE, then dialyzed in
0.1.times.TE for electroelution.
[0057] Determination of Surface Charge Density.
[0058] Surface charge density was estimated using electroosmotic
flow measurement in the nanoslit device with two ports.
Electroosmotic flow was measured in a setup similar to that
described by Huang et al..sup.23 Ports were cut into an oxygen
plasma treated nanoslit device with a standard razor blade.
Platinum electrodes, spaced 20 mm apart, were placed in the ports
and connected to an EC-105 power supply (EC Apparatus Corporation)
with a 195 SZ resistor, between second reservoir and the ground. A
multimeter was connected directly across the resistor to measure
the potential drop as an external electrical potential was applied.
Twenty millimolar phosphate buffer, pH 7.0, was added to load and
flush the system then 10 mM phosphate buffer pH 7.0 is added,
followed by application of .about.100 V (3 min); the voltage
polarity was then reversed for an additional 3 min. A linear fit
identified the intercept (time, t) between the forward and reverse
bias for each set of experiments. The electroosmotic mobility
(.mu..sub.EOF) was calculated by
.mu. EOF = L C Et ( 1 ) ##EQU00001##
where L.sub.C is the microchannel length, E is the electric field,
and t is time.
[0059] From the electroosmotic mobility, the charge density on a
surface (.sigma..sub.e) is
.sigma..sub.c=.zeta..di-elect cons..di-elect cons..sub.0.kappa.
exp(r.sub.w.kappa.) (2)
where .zeta. is zeta potential, .kappa..sup.-1 is Debye length,
r.sub.w is the normal distance from the surface, .di-elect cons.
the relative permittivity, and co is the permittivity of a vacuum.
Accordingly, the surface density of the device interior was found
to be 1.1 to 1.3 e/nm.sup.2.
[0060] Bead Diffusion Under Nanoconfinement.
[0061] YG carboxyl terminated beads (24 nm; Molecular Probes) in
0.20 mM and 10 mM NaCl, .kappa..sup.-1=22 and 3 nm, respectively)
within nanoslits were imaged using Total Internal Reflection
Fluorescence Microscopy (TIRF) microscopy using a Zeiss TIRF 100X
1.46 NA objective and 135TV inverted microscope. The optical train
comprised: 488 nm illumination (argon-ion laser, Coherent); quarter
wave plate; Galilean telescope (40 mm and 200 mm focal length
lenses (Edmund Industrial Optics)); broadband filter 485/20
(Semrock); and 525/50 excitation filter (Chroma); beam was then
mapped by a 125 mm field length convex lens onto the objective.
TIRF excitation produced a penetration depth of .about.70 nm (less
than nanoslit depth; 100 nm); images passed through a 525/50
emission filter (Chroma) onto an Andor iXon-888 camera, running
Andor SOLIS software, which were then background subtracted with a
"rolling ball" algorithm for shading correction;.sup.19 a Kalman
stack algorithm was implemented to decrease image noise. The
periodicity of bead fluorescence intensity fluctuations was
analyzed using a Fast Fourier Transform (FFT) for discerning maxima
peaks.
[0062] DNA Model and Simulation Approach
[0063] Brownian dynamics (BD) simulations were performed to
simulate the DNA dumbbell conformation within the nanoslits.
Long-range hydrodynamic interactions were included through a
Green's function formalism and calculated with the O(N) General
Geometry Ewald-like Method (GGEM)..sup.24-34 There is a combination
of confinement effects for the dumbbell conformation. A molecule in
a slit experiences a de Gennes' regime (confinement
size.about.R.sub.g).sup.32 in the microchannel, and within the
nanoslit width, an Odijk regime (confinement
size.about.l.sub.p).sup.16,33,34 in the nanoslit height. This
combination of regimes places a number of restrictions on the model
to be used to describe the slits considered in this work (see FIG.
2).
[0064] Available descriptions of DNA range from detailed atomistic
models,.sup.35 to mesoscale models that use multiple sites to
define a nucleotide,.sup.36-39 to coarse grained models that
describe multiple nucleotides in terms of individual beads (and
springs)..sup.40-42 Notable examples include the Kratky-Porod model
with a continuous worm-like chain (WLC) model, bead-spring models
that use Marko and Siggia interpolation,.sup.43-47 and nonlinear
elastic spring (FENE)-based models..sup.26,27,48 The appropriate
model must resolve the length scales of the nanoconfinement without
a finite discretization of the persistence length, because
characteristic times for segmental diffusion are several orders of
magnitude smaller than characteristic chain-diffusion times.
Kratky-Porod or higher resolution models are computationally
demanding (there is a time scale separation of 8 orders of
magnitude between the bead and chain diffusion times). At the other
end of the spectrum, a continuous WLC model describing 10-20
persistence lengths in terms of a single spring does not have the
resolution required to describe nanoslit confinement.
[0065] Yeh et al..sup.17 performed simulations of bead-spring
chains connected by springs to describe their experiments on DNA
dumbbells. Starting from a WLC representation of the springs,
however, they modified the law until agreement was observed between
the model and experiments. It is unclear, however, whether such an
approach would be able to describe large DNA molecules over a wide
range of conditions and whether it would be truly predictive. A
good compromise, and one that we adopt in this work, is provided by
the Underhill-Doyle (UD) model..sup.49-51 The UD model was
originally developed for a .theta.-solvent; in this work we include
excluded volume forces and hydrodynamic interactions to describe
good-solvent conditions and to generate Zimm scaling. The polymer
molecule, dissolved in a viscous solvent, is represented by a
bead-spring chain consisting of N.sub.b beads connected through
N.sub.s=N.sub.b-1 springs. The conditions of our confined systems
are such that the Reynolds number is zero, and inertia is
neglected. The force balance on each bead requires
f.sub.l.sup.h+f.sub.l.sup.s+f.sub.l.sup.v+f.sub.l.sup.w+f.sub.l.sup.b=0,
for l=1, . . . ,N.sub.b (3)
for bead l, f.sub.l.sup.h is the hydrodynamic force, f.sub.l.sup.v
is the bead-to-bead excluded volume force, f.sub.l.sup.w is the
bead-wall excluded volume force, f.sub.l.sup.b is the Brownian
force, and f.sub.l.sup.s is the UD spring force.
[0066] This model, developed using a constant stretch mechanical
ensemble, is used for the connectivity between adjacent molecule
beads. This model is defined as follows:.sup.49-51
f.sup.s=[a.sub.1(1-{circumflex over
(r)}.sup.2).sup.-2+a.sub.2(1-{circumflex over
(r)}.sup.2).sup.-1+a.sub.3+a.sub.4(1-{circumflex over (r)}.sup.2)]x
(4)
where {circumflex over (r)}=r/q.sub.0, q.sub.0 is the maximum
spring extension, and r=|x|, x=(x, y, z).
[0067] The coefficients of this polynomial expansion are defined
by
a 1 = 1.0 ( 5 ) a 2 = - 7 .chi. ( 6 ) a 3 = 3 32 - 3 4 .chi. - 6
.chi. 2 ( 7 ) a 4 = ( 13 / 32 ) + ( 0.8172 .chi. ) - ( 14.79 .chi.
2 ) 1 - ( 4.225 .chi. ) + ( 4.87 .chi. 2 ) ( 8 ) ##EQU00002##
where .chi.=N.sub.p,s.sup.-1 and N.sub.p,s is the number of
persistence lengths per spring.
[0068] In the development of the model, Underhill and Doyle.sup.49
did an error estimation of the spring law as a function of
N.sub.p,s, and found that it reproduces DNA behavior with a maximum
error of 1% for N.sub.p,s.gtoreq.4. We selected the maximum length
resolution of the UD model given by N.sub.p,s=4.
[0069] For the nonbonded bead-bead interactions, we use a Gaussian
excluded volume potential. Neutron scattering data for dilute
solutions of linear polymers in good solvent conditions indicate
ideal chain behavior at small distances along the chain and good
solvent behavior at long distances..sup.52-57 We consider the
increase in energy due to the overlap of two submolecules (or
molecular blobs). Each submolecule is considered to have a Gaussian
probability distribution with second moment
S.sub.s.sup.2=N.sub.p,sl.sub.p.sup.2/6, where N.sub.p,s is the
number of persistence lengths per spring. Considering the energy
penalty due to overlap of two Gaussian coils, one arrives at the
following expression for the excluded volume potential between two
beads of the chain:.sup.53,55
.PHI. v = 1 2 k B T .omega. v N p , s 2 ( 3 4 .pi. S s 2 ) 3 / 2
exp [ - 3 r 2 4 S s 2 ] ( 9 ) ##EQU00003##
where f.sup.v=-.gradient..PHI..sup.v, .omega..sub.v is the excluded
volume parameter related to the DNA effective diameter,.sup.54,55
k.sub.B is the Boltzmann constant, and T is the temperature.
[0070] A repulsive Lennard-Jones potential.sup.58,59 is used to
describe bead-wall excluded volume interactions, where the
Euclidean distance is replaced by the wall normal direction.
[0071] The dynamics of the bead-spring DNA molecules are described
by evolving the configurational distribution function. The
diffusion equation for that function has the form of a
Fokker-Planck equation; the force balance described above
corresponds to the following system of stochastic differential
equations of motion for the bead's positions:.sup.55,60,61
dR = [ U 0 + 1 k B T D F + .differential. .differential. R D ] dt +
2 B dW ( 10 ) ##EQU00004##
where R is a vector containing the 3N.sub.b coordinates of the
beads that constitute the polymer chain, with x.sub.i denoting the
Cartesian coordinates of bead 1.
[0072] The vector U.sub.o of length 3N.sub.b represents the
unperturbed velocity field, i.e., the velocity field in the absence
of any polymer molecule. The vector F has length 3N.sub.b, with
f.sub.l denoting the total non-Brownian, nonhydrodynamic force
acting on bead 1. Finally, the 3N.sub.b independent components of
dW are obtained from a real-valued Gaussian distribution with zero
mean and variance dt. The motion of a bead of the chain perturbs
the entire flow field, which in turns influences the motion of
other beads. These hydrodynamic interactions (HI) enter the polymer
chain dynamics through the 3.times.3 block components (D.sub.l.mu.)
of the 3N.sub.b.times.3N.sub.b diffusion tensor, D=k.sub.BTM (M is
the mobility tensor), which may be separated into the bead Stokes
drag and the hydrodynamic interaction tensor,
.OMEGA..sub.l.mu.;
D l .mu. = [ .delta. .xi. .delta. l .mu. + ( 1 - .delta. l .mu. )
.OMEGA. l .mu. ] ( 11 ) ##EQU00005##
[0073] Here .delta. is a 3.times.3 identity matrix,
.delta..sub.l.mu. is the Kronecker delta, and is the bead friction
coefficient. The Brownian perturbation is coupled to the
hydrodynamic interactions through the fluctuation-dissipation
theorem: D=BB.sup.T. The characteristic length, time, and force
scales describing the system are set by the bead hydrodynamic
radius a, the bead diffusion time .xi.a.sup.2/k.sub.BT, and
k.sub.BT/a, respectively. The bead friction coefficient .xi. is
related to the solvent viscosity .eta. and a through Stokes' law,
i.e., .xi.=6.pi..eta.a.
[0074] In conventional Green's function-based methods, M is
computed explicitly; the resulting matrix-vector operation to
determine the fluid velocity requires O(N.sup.2) operations.
Additionally, for nonperiodic domains, appropriate boundary
conditions must be included in order to correctly calculate the
velocity; for example, u(x)=0 for no-slip boundaries. Jendrejack et
al..sup.2,57,62-64 enforced the boundary conditions with solutions
using finite element methods (FEMs), where the quadratic scaling
limits analysis to small systems. Hernandez-Ortiz et al..sup.65
generalized a method developed by Mucha et al..sup.66 that scales
as O(N.sup.1.66 log N), but is restricted to slit geometries. There
are other approaches that allow the calculation of MF by O(N log N)
calculations in periodic domains. For instance, there are Ewald sum
and particle-mesh Ewald (PME) methods that are based on the
Hasimoto.sup.67 solution for Stokes flow driven by a periodic array
of point forces. In this work, the fluid velocity (MF) is
calculated using the O(N) GGEM introduced by Hernandez-Ortiz et
al..sup.25-31,68 GGEM yields MF without explicit construction of M
and, when combined with Fixman's.sup.69,70 midpoint integration
algorithm and Fixman's.sup.71 Chebyshev polynomial approximation
for BdW, it allows us to evolve the chains in time through an
efficient O(N) matrix free formulation..sup.26-29 Details of this
method and its implementation are described below.
[0075] The ionic strength influences DNA conformations through
electrostatic interactions between the charges on the DNA phosphate
backbone and interactions with nanoslit walls. These interactions
are screened over the Debye length (.kappa..sup.-1), defined by
.kappa..sup.2=2N.sub.Ae.sup.2I/.di-elect cons..sub.0.di-elect
cons.k.sub.BT (where N.sub.A is Avogadro's number, e is the
electronic charge, I is the ionic strength, .di-elect cons..sub.0
is the permittivity of free space, and s is the dielectric constant
of water). As the Debye length increases (from 10 to 30 nm) due to
the decrease in ionic strength (1.0 to 0.11 mM), the persistence
length of the molecule increases due to backbone like-charge
repulsions, and due to the decrease in the effective height of the
channel (which is in turn due to surface-DNA charge repulsions).
Odijk.sup.34 and Skolnick and Fixman.sup.72 (OSF) have estimated
theoretically how the persistence length (l.sub.p) of a worm-like
polyelectrolyte coil is affected by a short-ranged electrostatic
potential. Baumann et al. confirmed their theoretical predictions
through experiments on large DNA molecules,.sup.73 achieving a
quantitative prediction with an expression of the form
l p = l p , 0 + ( 0.0324 M I ) nm ( 12 ) ##EQU00006##
where l.sub.p,0 is the intrinsic persistence length corresponding
to fully screened electrostatic contributions (l.sub.p,0=50
nm).
[0076] In our experiments, the persistence length of the dumbbell
molecules ranges from 82.4 to 358 nm. Although predictions of the
OSF theory have raised concerns,.sup.74 OSF is known to give the
correct scaling for the persistence length with respect to the
ionic strength..sup.73,75 As alluded to earlier, the ionic
environment also plays a major role in the confinement because the
surface of the device has a charge density of 1.1 to 1.3
e/nm.sup.2, with its own Debye length. Our BD simulations do not
include electrostatic interactions with the walls directly;
instead, the model was parameterized to account for the change in
persistence length, and the wall-excluded volume was modified
according to the Debye length. Note that we are currently
implementing a full HI-electrostatic DNA model to account for these
effects more accurately, and results will be presented in the
future. The model parameterization was performed using experimental
data for .lamda.-DNA in the bulk; we use L=21 .mu.m, R.sub.g=0.7
.mu.m, S=1.5 .mu.m, l.sub.p=53 nm at I=10.798 mM, and a Zimm
diffusion coefficient (HI chains) of D.sub.Z=0.0115 .mu.m.sup.2/s
in a 43.3 cP solvent at 23.degree. C. (Note that the actual
viscosity (.eta..sub.s) is much lower).
[0077] Scaling arguments were then used to find the model
parameters at different ionic strengths:
R.sub.g.about.L.sup.3/5l.sub.p.sup.1/5.omega..sup.1/5 and
D.sub.Z.about..eta..sub.S.sup.-1R.sub.g.sup.-1 (13)
where .omega..about..kappa..sup.-1+.kappa..sup.-1
log(V.sub.eff.kappa..sup.-1) is the effective diameter of
DNA,.sup.76,77 and V.sub.eff is an effective DNA line
charge.sup.78,79. The UD model was subsequently parameterized to
produce the necessary scaling dictated by the ionic strength and
persistence length; thus, the range of the bead-bead excluded
volume was modified to .omega.l.sub.p.sup.2 to follow the scaling
given in eq 13,.sup.54 while the bead-wall excluded volume range
was increased in order to account for the wall Debye length.
Theoretical Considerations on Nanoconfined DNA Dumbbells
[0078] An analytical theory is used to provide interpretation for
the dynamical behavior of the nanoconfined DNA molecules.
[0079] Free Energy of a Lobe.
[0080] If we momentarily neglect the opening of the nanoslit, we
may view the DNA chain within one lobe of the dumbbell as a long
flexible coil of contour length s restricted by a hard smooth wall.
The partition function of a coil with two ends fixed is known to be
given by a Gaussian function in free space minus its mirrored
version induced by an image charge.sup.80-82 (if the chain is
ideal). This is because its value must reduce to zero at the wall.
Integrating over the configuration of one end point, one derives
the partition function G(z;s), where z is the distance of the other
end of the lobe to the wall and the lobe consists of s/A Kuhn
segments of length A=2l.sub.p. G is actually a function of
z/s.sup.1/2A.sup.1/2 only which for zs.sup.1/2A.sup.1/2 reduces
to.sup.81
G ( z ; s ) = ( 2 z 2 .pi. s A ) 1 / 2 ( 14 ) ##EQU00007##
[0081] The area of the opening of the nanoslit is D.times.h (hD).
Equation 14 is strictly valid if z>h. Here, h=O(l.sub.p), so the
DNA within the nanoslit (with zero or few back folds) is joined to
the lobe with z>.about.h by a short intervening section of DNA
whose description is challenging. The free energy of the latter
maybe neglected, however, so the free energy of the lobe is
expressed as
F 1 ( z ; s ) = - k B T ln G ( h ; s ) = constant - k B T ln h + 1
2 k B T ln s ( 15 ) ##EQU00008##
[0082] If the DNA of total contour length L translocates through a
nanopore instead of a nanoslit, eq 15 then leads to a total free
energy
F L = constant + 1 2 k B T ln ( L - s ) s ( 16 ) ##EQU00009##
as argued by Sung and Park..sup.83
[0083] If the lobes are asymmetric, there is a force
f 1 = - .differential. F L .differential. s = - ( L - 2 s ) k B T 2
( L - s ) s ( 17 ) ##EQU00010##
on the DNA driving it out of the nanopore. A similar force should
play a dominant role when the DNA translates through a nanoslit in
the deflection regime, provided there are two lobes. The effect of
excluded volume is rather weak; it merely changes the numerical
coefficient in eq 15..sup.81
[0084] Symmetrical Dumbbell.
[0085] It is of interest to study the equilibrium of the
symmetrical dumbbell. If we suppose the nanoslit is long and we
neglect electrostatics, we may write the total free energy of the
DNA
F Total = k B T ln s + l s 2 k B T 4 g ( L - s ) ( 18 )
##EQU00011##
from eq 15.
[0086] We have added an ideal chain term for the stretched DNA
spanning the nanoslit of length l.sub.s (l.sub.s.sup.2gL). A long
chain slithers back and forth along the channel and has a global
persistence length gl.sub.s. Therefore, the force on the DNA
f s = - .differential. F Total .differential. s = - k B T s - l s 2
k B T 4 g ( L - s ) 2 ( 19 ) ##EQU00012##
is never equal to zero; an exactly symmetrical dumbbell
conformation cannot exist in equilibrium.
[0087] The two lobes must retract into the nanoslit. The
counterintuitive nature of the free energy of a single lobe has
been emphasized before by Farkas et al..sup.84 An isolated chain
experiences a deflection force away from a wall (s is constant but
z becomes larger in eq 14). However, for a lobe attached to a
section of DNA within the nanoslit, z=h is held fixed and s is
variable. We note that the entropic force arising from the lobes in
eq 19 is generally quite weak.
[0088] Excluded-Volume Effect and Nondraining Limit.
[0089] How well do the physical properties of the DNA samples used
in FIG. 6 conform to asymptotic regimes? The excluded volume
parameter z.sub.el is a measure of the excluded-volume effect
between two Kuhn segments.sup.85
z.sub.el=0.183.omega.L.sup.1/2l.sub.p.sup.-3/2 (20)
where the DNA effective diameter is .omega.=74.9 nm at I=0.51
mM.
[0090] The total persistence length equals 113.5 nm from eq 12.
Hence, z.sub.el ranges from 2.5 to 5.0 for the DNA samples in FIG.
6 (molecule sizes ranging from 146 to 582 kb). The excluded volume
effect may regarded as close to asymptotic (z.sub.el1).
[0091] If a DNA molecule is regarded as a wormlike chain with a
hydrodynamic diameter d.apprxeq.2 nm, the draining properties
depend on the parameters L/2l.sub.p and d/2l.sub.p.apprxeq.0.01.
Yamakawa and Fujii have developed a theory for the translational
friction coefficient in their classic work..sup.86 Here, the DNA
coils turn out to be long enough so that their hydrodynamics is,
effectively, in the nondraining limit.
[0092] Nanoconfinement-Mediated Ejection.
[0093] In the case where there is only a single lobe, the DNA is
ejected from the nanoslit because there is a substantial free
energy difference between the nanoconfined DNA and its equivalent
in the remaining lobe..sup.87-89 Burkhardt computed the coefficient
C.sub.1 in the expression for the free energy of a DNA chain in a
nanoslit numerically..sup.90
F cL = C 1 k B Tx l p 1 / 3 ( D - 2 / 3 + h - 2 / 3 ) ( 21 )
##EQU00013##
where C.sub.1=1.1036 and x=L-s. This clearly often overwhelms the
contribution from the lobe (eq 15) and the force
f.sub.s=-.differential.F.sub.cL/.differential.x on the chain is
constant.
[0094] The DNA is forced out of the nanoslit; the force f must
overcome the hydrodynamic friction on the DNA, which may be viewed
effectively as a straight rod under the ionic conditions imposed
here. The coefficient of friction in the longitudinal dimension may
be written as.sup.53
.zeta. .apprxeq. 2 .pi..eta. s x ln ( h / d ) ( 22 )
##EQU00014##
[0095] This is independent of D because the upper cutoff in the
hydrodynamics is the smaller scale h, which itself is much larger
than d. Therefore to a first approximation, the equation of motion
of the sliding DNA may be expressed as
.zeta. ( t ) x ( t ) t = - f s ( 23 ) ##EQU00015##
[0096] The lobe increases in size as the DNA is ejected, but the
frictional force on it may be neglected in eq 23. From the previous
section, we know its size R(s) scales as s.sup.3/5 so that we have
dR/dt=-3/5(R(t)/s(t)) dx(t)/dt. Moreover, in the nondraining limit,
the coefficient of friction on the expanding lobe is .eta.R(t) so
the lobe friction is a higher order term. Another issue is how well
bulk hydrodynamics applies within the slit. There is evidence for a
possible breakdown of this assumption for very tight silica
nanoslits (h equal to about 20 nm)..sup.91,92 In our case, the PDMS
nanoslits, which are less tight, are also expected to be smoother
although we feel a thorough investigation of the magnitude of the
friction is warranted in the future.
[0097] Equation 23 is readily solved and leads to a parabolic
equation as has been presented before.sup.87-89
x 2 = l s 2 ( 1 - t .tau. s ) ( 24 ) .tau. s = .pi..eta. s l s 2 f
s ln ( h / d ) ( 25 ) ##EQU00016##
[0098] It has been assumed that the DNA fills the entire nanoslit
at t=0. If we set h=0.1 .mu.m, D=1 .mu.m, =0.1135 .mu.m, L=28
.mu.m, =1 cP, and d=2 nm, we compute a force Ifs'=13k.sub.BT/.mu.m
and an ejection time .tau..sub.s=12 s. The latter agrees well with
the time the T4 DNA molecule is ejected from the nanoslit in
experiments. A tentative conclusion is that bulk hydrodynamics
indeed applies within the nanoslit. By contrast, the experimental
friction on the DNA in the square silica nanochannels of Mannion et
al..sup.89 was found to be five times higher than predicted by an
expression analogous to eq 22. This discrepancy is unexplained.
[0099] Lobe Translocation.
[0100] Numerous computational and analytical studies have been
devoted to the translocation of a flexible polymer chain through a
nanopore, as has been reviewed recently..sup.93 In our experiments,
the DNA stretch is very high within the nanoslit, so we think it is
plausible that the translocation dynamics of the DNA lobe should be
quite similar to that in a nanopore device. A full analysis of all
chain fluctuations will be needed to bear this out in the
future.
[0101] Often the time .tau..sub.1 a chain needs to translocate
through a nanopore scales as a power law in terms of the number of
segments N, i.e.,
.tau..sub.1.about.N.sup..beta. (26)
[0102] A main objective has been to compute .beta. precisely, but
this has engendered considerable controversy..sup.93 This is beyond
the scope of this work, although we have summarized several
representative predictions for .beta. in Table 1.
TABLE-US-00001 TABLE 1 Exponent .beta. of the Lobe Translocation
Time .tau..sub.1~ N.sup..beta. as a Function of the Number of
Segments N.sup.a free-draining nondraining unbiased without memory
effects 1 + 2.nu. = 2.2.sup.94 3.nu. = 1.8.sup.94 with memory
effects 2 + .nu. = 2.6.sup.95 1 + 2.nu. = 2.2.sup.95 forced without
memory effects 2.nu. = 1.2.sup.97 3.nu. - 1 = 0.8.sup.97 with
memory effects (1 + 2.nu.)/(1 + .nu.) = 1.38.sup.97 3.nu./(1 +
.nu.) = 1.13.sup.97 .sup.aIn forced translocation, the time is
inversely proportional to the force. The excluded-volume exponent
.nu. is chosen here to be equal to 3/5.
[0103] In the case of unbiased translocation, Chuang et al..sup.94
argued that the polymer chain cannot be viewed as a single particle
diffusing across an entropic barrier given by eq 15. The diffusion
through the nanopore is collective and Rouse-like across a distance
R.about.s.sup.v, the size of the lobe. The translocation time
.tau..sub.1 should then scale as NR.sup.2(N).about.N.sup.1+2v (see
entry in Table 1). With hydrodynamic interactions, the frictional
factor proportional to N reduces to N.sup.v.about.R.
[0104] Recently, it has been proposed that this simple scenario
should be amended..sup.95 The presence of the nanopore (or
nanoslit) implies the dynamics of translocation is strongly
inhomogeneous. The diffusion of segments across the pore causes an
imbalance in tension between the two lobes. The translocation time
affected by these memory effects becomes effectively longer (see
Table 1).
[0105] When the extending force f on a lobe is large enough (f
R(s)>k.sub.BT), the translocation becomes forced, and
.tau..sub.1 is inversely proportional to f. We have corroborated
the entry in Table 1 for the case without memory effects because it
disagrees with an earlier estimate..sup.96 Our argument is based on
the rate of dissipation dF/dt. On the one hand, this equals the
velocity of the chain V.sub.i at the opening of the nanopore times
the force f reeling the lobe in. In view of the fact that the
radius of the lobe R.about.s.sup.v, we know that
V.sub.i=-ds/dt=-(s/vR) dR/dt. On the other hand, the rate of
dissipation in the Rouse limit within the contracting lobe is given
by Nf.sub.0(dR/dt), where f.sub.0=.zeta..sub.0(dR/dt) is the
typical force on a segment with a friction coefficient of
.zeta..sub.0. The typical velocity of a segment is dR/dt. The two
rates must be identical, thus leading to the entry in Table 1.
Memory effects give rise to nontrivial exponents.sup.97 also
presented in Table 1.
Results and Discussion
[0106] Dumbbell Formation Completely Stretches DNA Molecules and
Requires Hydrodynamic Considerations.
[0107] Using experimental and simulation approaches, we explored
the idea that elastic and hydrodynamic contributions to DNA
stretch, originating from the coil itself (a dumbbell lobe), in
addition to contributions from just nanoconfinement, would greatly
enhance DNA elongation. We created DNA dumbbells within our
nanoslit device, shown in FIG. 1, by strategically threading DNA
molecules through nanoslits, using carefully timed electrical
pulses. Conditions were adjusted allowing DNA ends to occupy the
two microchannels bounding nanoslit entrances creating dumbbell
lobes comprising random coils. DNA stretch within nanoslit portions
of the device is estimated by fluorescence intensity measurements
comparing nanoslit versus microchannel portions of the same
molecule: S/L=l.sub.s f.sub.m/S.sub.mf.sub.s; where f.sub.m is the
integrated fluorescence intensity of the entire molecule, f.sub.s
and l.sub.s are the fluorescence intensity and length of the
molecular portion within a slit, and S.sub.m is the known length of
the molecule (.mu.m; dye corrected).
[0108] We expect ionic strength affecting DNA stretch by the
electrostatic contributions to persistence length, or polymer
stiffness, and the electrostatic environment presented by the
device..sup.5 Accordingly, we evaluated these collective effects on
DNA stretch by varying the buffer ionic strength enveloping both
sample and device. FIG. 3 shows DNA stretch, using T4 and
.lamda.-bacteriophage DNA, as a function of ionic strength,
I.di-elect cons.[0.11, 1.0] mM from experiments and from BD
simulations (I.di-elect cons.[0.5, 10] mM; see Materials and
Experimental Methodology). As the ionic strength decreases, DNA
stretch within a nanoslit increases, as previously reported by Jo
et al..sup.5 Here, however, the additional coupling of dumbbell
elastic forces greatly enhance DNA stretch by a substantial 37%
(S/L=0.85.+-.0.16; I=0.51 mM) over molecular nanoconfinement
without dumbbells (S/L=0.62.+-.0.08; I=0.47 mM). Further reduction
of ionic strength enables presentation of fully stretched
(S/L=1.06.+-.0.19; I=0.11 mM) DNA molecules. We further validate
these stretch estimations using .lamda.-DNA as an internal
fluorescence standard of known size, within slits, for normalizing
integrated fluorescence intensities of .lamda.-concatamer DNA
dumbbells (confined portions): (0.87.+-.0.14, N=231; I=0.48 mM),
which is similar to the previous value found for T4 DNA
(0.85.+-.0.16; I=0.51 mM). The stretch values found for T4 and
.lamda. experiments agreed (FIG. 3), indicating consistency and
reproducibility of the stretch measurement approaches.
[0109] FIG. 3 also shows the results of our theoretical predictions
by BD simulations, as compared to experiments. For completeness,
results are shown for calculations that include fluctuating
hydrodynamic interactions (HI), and calculations when such
interactions are neglected (free-draining model, FD). Note that
part of the chain is in the nanoslit, and here, HI are expected to
be screened and play a minor role. However, as the results in FIG.
3 indicate, HI significantly contributes to the dumbbell dynamics
and greatly influences molecular stretch. This can be explained by
the fact that Zimm dynamics of the lobes in the microchannel
(outside the slit) influence the dynamics of chain segments within
the intervening nanoslit. Two trends are discernible in the
simulation results: for I>1.0 mM the stretch is nearly constant,
and for I.ltoreq.0.74 mM a sudden increase is observed. Within this
latter range, the persistence length of the chain reaches values
comparable to the nanoslit height (.about.100 nm), thereby placing
the level of confinement in the Odijk regime. Note that the
confinement size, at these ionic strength conditions, is smaller
than 100 nm because the walls have their own ion cloud. At this
point, the underlying physics becomes complicated due to
interactions between the ion clouds associated with the chain and
walls. However, one major effect is the reduction of the effective
confinement size, i.e., the chain persistence length increases and
the "free" available space between the walls decreases. Our
simulations of stretch follow the experimentally observed trends,
but slightly under-predict (5%) the experimental data. We attribute
the discrepancy to the fact that full electrostatic interactions
are not included in our model. Also note that it is not possible to
use the current model for the two lowest ionic strength conditions
considered in experiments because the persistence length is higher
than the confinement (l.sub.p>100 nm). These points aside, the
simulations reveal the underlying physical phenomena behind
dumbbell-mediated stretch, and most importantly, the critical
interplay between HI acting at the lobes and the electrostatic
interactions helping to confine and elongate DNA molecules.
[0110] FIG. 4 provides a comparison of molecular stretch in
different directions, both in the presence and absence of HI.
Outside the nanoslits, the stretch in all directions, S.sub.1
(axial), S.sub.2 (perpendicular) and S. (confinement) is in the
range 30-32% (where S.sub.i=|max(x.sub.i)-min(x.sub.i)|.sub.i, for
the ith direction of the chain i). Thus, S.sub.i is the distance
between the two segments of the chain having the longest separation
in each direction. In contrast, the segment inside the nanoslits
exhibits distinct differences in the three directions when HI are
included. First, the stretch in the axial direction, S.sub.1, is
always higher with HI than without (FD chains). The HI S.sub.1
stretch is always around 5-7% below the total stretch, indicating
that it is the major contributor to the total stretch. The FD
S.sub.1 stretch, on the other hand, remains constant with ionic
strength in the range 55-60%. The S.sub.2 stretch in the nanoslits,
in the perpendicular direction, is in the range 20-25% without HI
(FD chains); similar to that observed outside the nanoslit. The HI
S.sub.2 stretch inside the nanoslits is 10-15%. This change in the
perpendicular stretch indicates a clear difference between the HI
and FD molecular conformations within the nanoslits. The FD chains
do not "feel" the dumbbell lobes, thereby allowing the chain to
perform a pseudorandom walk in the nanoslit width direction (bottom
chain in FIG. 4); in contrast, HI dumbbells exhibit a "collective"
behavior that increases the stretch in the axial direction and
impedes the chain from moving freely in the nanoslit width
direction; the net result is the creation of a "rigid" dumbbell
(top chain in FIG. 4). To summarize, the dumbbell conformation
leads to elongation of DNA molecules within a pseudonanochannel.
Electrostatic interactions, enhanced by our low ionic strength
conditions, accentuate the confinement of DNA molecules. Note that
Debye lengths range from 3 nm at 11 mM to 30 nm at 0.11 mM.
Importantly, at low ionic strength, the Debye length is comparable
to the nanoslit height, an effect that cannot be overlooked..sup.98
This electrostatic effect, combined synergistically with collective
HI and nanoconfinement, greatly enhances DNA stretch.
[0111] Debye Length Considerations.
[0112] Given these simulation results, which highlight
electrostatic contributions by the device walls to DNA stretch, we
experimentally investigated how the Debye length affects
nanoconfinement.sup.99 by studying the diffusion kinetics of
negatively charged latex beads (24 nm) within nanoslits using TIRF
microscopy (see Materials and Experimental Methodology). The idea
is that bead diffusivity would be measurably perturbed, as a
function of ionic strength, due to the accrued Debye lengths of the
device (22 nm, I=0.20 mM; 3 nm, I=10 mM) and the beads (24 nm). The
average periodicity was measurably different for 0.1997 mM and
9.987 mM NaCl, namely, 8.+-.3 s and 12.+-.4 s, respectively (N=16
beads), thereby implying that the Debye length effectively limits
the height of the nanoslit (i.e., bead diffusion is more confined
at lower ionic strengths). These observations confirm the sudden
decrease of chain motilities in the confined direction, once the
ionic strength is decreased. Simulated DNA motility (diffusion) in
the confined direction was .about.90 nm at the higher ionic
strength conditions, which shifted to a very small 1-5 nm at lower
ionic strength conditions.
[0113] How DNA Size Affects Dumbbell Stretching and Relaxation
Time.
[0114] FIG. 5 shows DNA stretch as a function of molecular size (97
kb-582 kb; I=0.51 mM), using a series of .lamda. concatemers. Note
that the same device can be used for uniform presentation of
molecules of any size, once the molecule contour length exceeds
twice the nanoslit length for ensuring confident dumbbell
formation. In the figure, experimental and simulated results are
included. For a dumbbell conformation, we calculate the mean
squared variation of the axial position of chain segments within
the nanoslit. Importantly, this mobility indicates how reliable an
optical measurement of labeled DNA features is inside the nanoslit;
the simulation results show a mobility of 150.+-.20 bp for I=11 mM,
and 100.+-.20 bp for I=0.51 mM. We note that ultimately the two
lobes of the dumbbell do not stabilize the conformation, even when
the dumbbell is symmetric (see the Theoretical Considerations
section).
[0115] The effective relaxation time of dumbbell molecules was
analyzed by loading the molecules in the same manner as in the
stretching experiments. In the dynamical experiments, however, some
molecules had to be imaged over a 7 h time course using attenuated
illumination to prevent photocleavage, which would destroy
dumbbells. Bright dumbbell lobes are thresholded in the image data
for their analysis, leaving invisible the connecting DNA backbones
within the nanoslits. The last time point at which a molecule was
observed determined the relaxation time of a dumbbell within a
slit; we then averaged all relaxation times for a given molecular
size, irrespective of relative lobe size. Molecules remaining after
completion of measurements were checked for spurious
surface-attachment by applying an electrical field; adhered
molecules are not included in our data sets. Also, molecules 100 kb
were excluded because they formed small lobes that rapidly relaxed.
The relaxation time is the translocation time of a single DNA lobe
plus the ejection time of the DNA chain out of the nanoslit. The
latter time turns out to be quite short, typically about 10 s. This
agrees well with our theoretical estimate of 12 s based on entropic
ejection; the viscosity of the aqueous solvent inside the nanoslit
would appear to be close to that of the bulk. The ejection time is
a simple, minor correction, which we have subtracted from the
relaxation time. The resulting translocation times are plotted in
FIG. 6 for the .lamda. concatemers (black), T4 (white), and M.
florum (grey) DNA molecules. The dependent variable is not the
actual molecular mass of the DNA molecules, but the molecular mass
of the two lobes of the dumbbell (MI) because we have subtracted
the DNA mass within the nanoslit from this. This correction is
significant for the lower masses. In FIG. 6, we have fitted the
lobe translocation time with a power law .tau..about.MI.sup.1.23
(If we had plotted the original relaxation times, the exponent
would have been 1.71). The dumbbell lobe fluorescence intensities
fluctuate over time until one lobe slips into the nanoslit (arrow
b), then the molecule transits the nanoslit into the bottom
microchannel and exits into the microchannel (arrow c). The details
of the inset of FIG. 6 for purposes of this application are less
critical than understanding that the inset is a time lapse of the
above-described motion.
[0116] Our exponent 1.23 rules out unbiased translocation (see
Table 1, where we also show that the DNA chains are effectively
nondraining and the excluded-volume effect is quite fully exerted).
It is comparable with the exponent 1.13 predicted for forced
translocation with hydrodynamic interactions in the nanopore
case..sup.97 At present, it is, however, difficult to rule out a
theory of translocation without memory effects. In the bulk, the
frictional properties of a long DNA chain may be nondraining.
However, the polymer conformations are strongly inhomogeneous for a
lobe attached to a nanoslit or nanopore. It may be argued that a
portion of the chain conforms to Rouse dynamics, so the predicted
exponent would be somewhere between 0.8 and 1.2 (Table 1). Our
exponent 1.23 also appears to agree with the value 1.27 measured by
Storm et al..sup.100-102 for DNA translocating through a
silicon-oxide nanopore. However, their ionic strength was high (I=1
M), so the excluded volume was weak and their chains were close to
ideal (v=1/2 in Table 1). Our own (unpublished) analysis shows that
the top lobe in FIG. 6 is indeed being translocated into the
nanoslit under an external force, which appears to be constant. The
origin of this force is obscure at present; it cannot be of
entropic origin as discussed in the Theoretical Considerations, for
this force is much too weak. These mild forces lead to lengthy
translocation and relaxation times.
[0117] There is some debate or confusion in the
literature.sup.11,103-105 regarding the transition between de
Gennes and Odijk confinement regimes. The set of experiments
presented here help clarify one issue in that debate, because they
have been performed at very low salt concentrations. The decrease
of ionic strength has two major consequences: an increase of the
chain persistence length, and an enhanced, effective confinement
induced by the Debye length of the device's surface. Our
experimental observations show that once the effective confinement
is equal to the chain persistence length the Odijk regime is
achieved. This feature apparently contradicts other
conclusions,.sup.11 which suggested that the effective DNA
diameter, .omega., has a major effect on the de Gennes-Odijk
transition. However, the contradiction is apparent because the
ionic strength in ref..sup.11 is much higher than used here. Wang
et al..sup.106 have attempted to show how the results of
ref..sup.11 fit in with the intermediate regimes. FIG. 3 includes
the stretch predictions of de Gennes theory
(S/L.about.(.omega.l.sub.p).sup.1/3(Dh).sup.-1/3) and Odijk theory
(S/L.about.1-[(D/l.sub.p).sup.2/3+(h/l.sub.p).sup.2/3]), for D=1
.mu.m.times.h=100 nm nanoslit. Initially, one may infer from the
figure that the experiments do not follow any scaling regime;
however, we must recall that the dumbbell conformation emulates
nanochannel confinement. In other words, the DNA dumbbells "feel"
an effective, lower channel width. Once this effect is included,
the experiments follow the Odijk predictions that the shadow region
encompasses (FIG. 3); namely, an Odijk prediction for a 3h.times.h
nanoslit and for a h.times.h channel. As pointed out by T.
Odijk,.sup.16,33,34 his theory does not include severe
electrostatic interactions; accordingly, his method will slightly
under-predict stretch at the lower ionic strength conditions
considered here. We are currently developing an improved molecular
model to account for full electrostatic interactions..sup.25 Once
the dumbbells are formed and the molecule is presented in a fully
stretched manner, a natural question is to examine the mobility of
the chains within the nanoslit and the dumbbell's relaxation time.
However, the dumbbell dynamics reported here show relaxation times
that will support genomic analysis schemes using imaging, which
require consistently stretched DNA molecules. The addition of
sucrose to the low ionic strength solutions and the decrease of
temperature (i.e., shifted after loading) would increase solution
viscosity and extend the relaxation time of dumbbell molecules.
[0118] Modern genome analysis demands long-range sequence
information that is uniquely presented by large DNA molecules. As
such, the findings presented here, using tightly coupled
experimental and simulation approaches, have provided an
experimental and theoretical infrastructure for the design and
implementation of the newer genome analysis systems. These advances
may provide the means for fully leveraging the informational
advantages intrinsically offered by very long DNA molecules in ways
that will greatly enhance our understanding of genome
structures.
APPENDIX
[0119] General Geometry Ewald-like Method and O(N)
Algorithm.sup.25-31
[0120] The fluid velocity MF is calculated using the O(N) GGEM
introduced by Hernandez-Ortiz et al..sup.28 A brief description of
the GGEM starts with considering the Stokes system of equations for
a flow driven by a distribution of Nb point forces,
-.gradient.p(x)=.eta..gradient..sup.2u(x)=-.rho.(x)
.gradient.u(x)=0 (27)
where .eta. is the fluid viscosity and the force density is
.rho. ( x ) = l = 1 N b f l .delta. ( x - x l ) ( 28 )
##EQU00017##
where f.sub.l is the force exerted on the fluid at point
x.sub.l.
[0121] The solution of 27 can be written in terms of a
Stokeslet.sup.28,107 and combined into the MF product. If computed
explicitly, this product is a matrix-vector operation requiring
O(N.sup.2) calculations. GGEM determines the product implicitly for
any geometry (with appropriate boundary conditions) without
performing the matrix-vector manipulations. It starts with the
restatement of the force-density expression in eq 27,
.rho.(x)=.rho..sub.l(x)+.rho..sub.g(x) using a smoothing function
g(x), similar to conventional particle-mesh Ewald
methods..sup.108-110 This screening function satisfies
.intg..sub.all spaceg(x)dx=1 (29)
[0122] By linearity of the Stokes equation, the fluid velocity is
written as a sum of two parts, with separate solutions for each
force-density. The "local density"
.rho. 1 ( x ) = l = 1 N b f l [ .delta. ( x - x l ) - g ( x - x l )
] ( 30 ) ##EQU00018##
drives a local velocity, u.sub.l(x), which is calculated assuming
an unbounded domain:
u 1 ( x ) = l N b G 1 ( x - x l ) f l ( 31 ) ##EQU00019##
where G.sub.1(x) is composed of a free-space Greenis's function, or
Stokeslet, minus a smoothed Stokeslet obtained from the solution of
Stokes equations with the forcing term modified by the smoothing
function g(x).
[0123] For the Stokes equations, we found that a modified Gaussian
smoothing function defined by
g ( r ) = .alpha. 3 .pi. 3 / 2 ( - .alpha. 2 r 2 ) ( 5 2 - .alpha.
2 r 2 ) ( 32 ) ##EQU00020##
yields a simple expression for G.sub.l(x):
G 1 ( x ) = 1 8 .pi..eta. ( .delta. + xx r 2 ) erfc ( .alpha. r ) r
- 1 8 .pi..eta. ( .delta. - xx r 2 ) 2 .alpha. .pi. 1 / 2 ( -
.alpha. 2 r 2 ) ( 33 ) ##EQU00021##
[0124] Because G.sub.l(x) decays exponentially on the length scale
.alpha.-1, in practice the local velocity can be computed, as in
conventional Ewald methods, by only considering near-neighbors to
each particle l..sup.58,111
[0125] For the present work, the point-particle approximation is
not desired; in particular, as the chain size increases, the
probability that particles will overlap, having un-physical
velocities, increases. To avoid this problem, the bead hydrodynamic
radius, a, can be used to define a new smoothed-force density that
gives a non-singular velocity. This is achieved by replacing the
Stokeslet by a regularized Stokeslet, using the same modified
Gaussian with a replaced by .xi., with .xi..about.a.sup.-1,
yielding
G 1 R ( x ) = 1 8 .pi..eta. ( .delta. + xx r 2 ) [ erfc ( .xi. r )
r - erf ( .alpha. r ) r ] + 1 8 .pi..eta. ( .delta. - xx r 2 ) ( 2
.xi. .pi. 1 / 2 ( - .xi. 2 r 2 ) - 2 .alpha. .pi. 1 / 2 ( - .alpha.
2 r 2 ) ) ( 34 ) ##EQU00022##
where the superscript R stands for regularized force density. For
.xi..sup.-1=3a/(.pi.).sup.1/2, the maximum fluid velocity is equal
to that of a particle with radius a and the pair mobility remains
positive-definite..sup.28,112
[0126] The global velocity, u.sub.g(x), is due to the force
distribution .rho..sub.g(x), which is given by
.rho. g ( x ) = l = 1 N b f l g ( x - x l ) ( 35 ) ##EQU00023##
[0127] For a general domain, we find the solution to Stokes'
equation numerically, requiring that u.sub.l(x)+u.sub.g(x) satisfy
appropriate boundary conditions. At a no-slip boundary, we would
require u.sub.g(x)=-u.sub.l(x). For problems with periodic boundary
conditions, Fourier techniques can be used to guarantee the
periodicity of the global velocity u.sub.g(x). The periodicity on
the local velocity, u.sub.l(x), is obtained using the minimum image
convention.
[0128] In the present case, the global contribution is solved with
a FEM formulation, where 8-noded brick elements.sup.27,113 are used
for the velocity and constant elements are used for the
corresponding global pressure. The solution of the linear system is
done through a fast LU decomposition solver for sparse matrices,
SUPER-LU..sup.114,115 The LU decomposition of the matrix is only
done at the beginning of the simulation; during the time
advancement, the only necessary computation is the
back-substitution, making the GGEM algorithm highly efficient
(.about.O(N), given the sparse characteristic of the matrix). Given
the fact that the GGEM solution is independent of .alpha., the
appropriate selection of this parameter is based on the
optimization of the computational time. In the global calculation,
to reach an accurate solution, the mesh size must be smaller than
the scale of the smoothing function, which is .alpha..sup.-1.
Therefore, the mesh resolution scales as M.about..alpha..sup.3; the
cost of each back-substitution scales as M.sup.2, leading to a
total global cost that scales as .alpha..sup.6. In the local
calculation, the contribution of all pairs that lie within a
neighbor list determined by the decay of the local Green's function
must be calculated. The local Green's function decays over a
distance .alpha..sup.-1, so the number of neighbors for each
particle scales as N.alpha..sup.-3. The calculation must be
performed over all pairs, which is the number of particles times
the number of neighbors per particle, resulting in a local
calculation cost that scales as N.sup.2.alpha..sup.-3. Minimizing
the total (local and global) computational cost with respect to a
gives an optimal a that scales as .alpha..sub.opt.about.N.sup.2/9
and a total cost that scales as O(N.sup.4/3). If we had chosen a
different, linear, method for the solution (GMRES, Bi-conjugate
methods.sup.116), the global cost would have scaled as
.alpha..sup.3, leading to an optimal value of
.alpha..sub.opt.about.N.sup.1/3 and a total computational cost that
would scale as O(N).
[0129] Because GGEM yields MF without explicit construction of M,
it is desirable to time-integrate eq 10 without requiring this
product, i.e., a "matrix-free" formulation. Fixman.sup.69,70
proposed a method to time-integrate this system without needing to
evaluate .differential./.differential.RD:
R * = R ( t ) + 1 2 [ U 0 ( R ) + M ( R ) F ( R ) ] .DELTA. t + 1 2
2 D ( R ) B - 1 ( R ) .DELTA. W ( t ) R ( t + .DELTA. t ) = R ( t )
+ [ U 0 ( R * ) + M ( R * ) F ( R * ) ] .DELTA. t + 2 D ( R * ) B -
1 ( R ) .DELTA. W ( t ) ( 36 ) ##EQU00024##
[0130] The only remaining step is to evaluate B.sup.-1dW in a
matrix-free way. As also noted by Fixman,.sup.71 this can be done
by a Chebyshev polynomial approximation method that requires only
matrix-vector products, not the matrix itself. This approach has
already been implemented in unbounded or periodic
domains;.sup.26-28,30,62,64,117,118 with GGEM it can be directly
generalized to arbitrary domains.
[0131] The present invention has been described in terms of one or
more preferred embodiments, and it should be appreciated that many
equivalents, alternatives, variations, and modifications, aside
from those expressly stated, are possible and within the scope of
the invention.
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