Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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METHODS AND APPARATUSES .F.OR STRETCHING
POLYMERS
This application claims the benefit of U.S. Provisional Application No.
60/149,020,
filed August 13, 1999, which is incorporated herein by reference in its
entirety.
1. FIELD OF THE INVENTION
The present invention relates to the general field of polymer
characterization. More
particularly, the invention relates to the use of structures to stretch a
polymer or to select a
polymer on the basis of length in a chip.
2. BACKGROUND OF THE INVENTION
Macromolecules are involved in diverse and essential functions in living
systems.
The ability to decipher the functions, dynamics, and interactions of
macromolecules is
dependent upon an understanding of their chemical and three-dimensional
structures. These
three aspects - chemical and three-dimensional structures and dynamics - are
interrelated.
For example, the chemical composition of a protein, and more particularly the
linear
arrangement of amino acids, explicitly determines the three-dimensional
structure into
which the polypeptide chain folds after biosynthesis (Kim & Baldwin (1990)
Arm. Rev.
Biochem. 59: 631-660), which in turn determines the interactions that the
protein will have
with other macromolecules, and the relative mobilities of domains that allow
the protein to
function properly.
Biological macromolecules are either polymers or complexes of polymers.
Different types of macromolecules are composed of different types of monomers,
i.e.,
twenty amino acids in the case of proteins and four major nucleobases in the
case of nucleic
acids. A wealth of information can be obtained from a determination of the
linear, or
primary, sequence of the monomers in a polymer chain. For example, by
determining the
primary sequence of a nucleic acid, it is possible to determine the primary
sequences of
proteins encoded by the nucleic acid, to generate expression maps for the
determination of
ANA expression patterns, to determine protein expression patterns, and to
understand how
mutations in genes correspond to a disease state. Furthermore, the
characteristic pattern of
distribution of specific nucleobase sequences along a particular DNA polymer
can be used
to unequivocally identify the DNA, as in forensic analysis. To this end, fast,
accurate and
inexpensive methods of characterizing polymers, and particularly nucleic
acids, are being
developed as a result of the endeavor of the Human Genome Project to sequence
the human
genome.
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A challenge to the characterization of the linear sequence of monomers in a
polymer
chain has come from the natural tendency of polymers in most media to adopt
unpredictable, coiled conformations. The average amount of such coiling is
dependent on
the interaction of the polymer with the surrounding solution, the rigidity of
the polymer, and
the energy of interaction of the polymer with itself. In most cases, the
coiling is quite
significant. For example, a ~.-phage DNA, theoretically 16 pm long when
stretched out so
that the DNA is in the B conformation, has a random coil diameter of
approximately 1 p,m
(Smith et al. (1989) Science 243:203-206).
DNA and many other biopolymers can be modeled as uniform elastic rods in a
worm-like chain in order to determine their random coil properties (Austin et
al. (1997)
Physics Today 50(2):32-38). One relevant parameter is the persistence length,
P, the length
over which directionality is maintained, which is given by:
P=x/kBT ( 1 )
where x is the elastic bending modulus (Houseal et al. (1989) Biophys. J.
56:507-516), kg is
the Boltzmann constant, and T is temperature (Austin et al. (1997) Physics
Today 50(2):32-
38). A longer persistence length means that the polymer is more rigid and more
extended.
Under physiological conditions, P=50 nm for DNA. While larger than the
molecular
diameter of 2.5 nm, the persistence length is many orders of magnitude smaller
than the
actual length of a typical DNA molecule such as a human chromosome, which is
about 50
mm long. From the persistence length, the overall coil size, R, can be
calculated (Austin et
al. (1997) Physics Today 50(2):32-38) as follows:
(RZ)=2PL (2)
where L is the contour length of the DNA molecule. In the case of chromosomal
DNA,
R~70pm. Clearly, it is much easier to analyze information on an extended piece
of DNA
that is 5 cm long than on a piece of DNA that has a coil size of 70pm.
The force necessary to stretch polymers such as DNA is not very large. The
worm-
like chain model allows the polymer to be considered to be like a spring, and
the force (FS)
needed to extend it close to its full natural length can be calculated (Austin
et al. (1997)
Physics Today 50(2):32-38) as follows:
FS~kBT/P (3)
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where all of the parameters are defined as above. Below FS, the relationship
between the
force applied and the amount of stretching is roughly linear; above FS,
applying more force
results in little change in the stretching (Smith et al. (1992) Science
258:1122-1126;
Bustamante (1994) Science 265:1599-1600). Hence, full stretching is
essentially attained
by applying FS. In the case of DNA, the force required to stretch it from its
coiled
conformation to its full length, which stretched conformation retains the B
conformation is
about 0.1 pN. Such a small force could, in principle, be obtained from
virtually any source,
including shear forces, electrical forces, and gravitational forces.
The danger in stretching DNA comes not in breaking the covalent bonds, which
requires at least 1 nN of force (Grandbois et al. (1999) Science 283:1727-
1730), but in over-
stretching. It has been observed that, when 70 pN of force is applied, DNA
adopts a super-
relaxed form, called "S-DNA", having nearly twice the length of normal B-form
DNA
having the same number of base pairs (Austin et al. (1997) Physics Today
SO(2):32-38).
Others have reported this transition at a force of 50 pN (Marko & Siggia
(1995)
Macromolecules 28:8759-8770). The length of S-DNA is less consistent than that
of B-
DNA stretched to its natural length and is more dependent on the exact force
applied
(Cluzel et al. (1996) Science 271:792-794), varying linearly with applied
force from 1.7 to
2.1 times the length of B-DNA. Since it may not be possible to know the exact
force
applied, it is desirable to avoid stretching DNA into its S-form. Therefore, a
force having a
range of about two orders of magnitude, from about 0.1 pN to 25 pN, is capable
of
consistent and predictable stretching of DNA to its fully extended B-form.
In addition, the force must be applied fast enough to keep the polymer from
recoiling. The natural relaxation time of a polymer, i, depends on the
solvent, as follows
(Marko (1998) Physical Review E 27:2134-2149):
~~LzP~/ksT (4)
where ~ is the viscosity of the solvent and the other parameters are as
defined above. In the
case of DNA at physiological conditions, the relaxation time is about 6
seconds, which can
be increased to 20 seconds in a solution with a viscosity of 220 cp (Smith et
al. (1999)
Science 283:1724-1727) or by running the DNA in a confined space to lengthen P
and
change the viscous drag (Bakajin et al. (1998) Phys. Rev. Let. 80:2737-2740).
Relaxation
time is also a function of the extent of stretching (Hatfield & Quake (1999)
Phys. Rev. Let.
82:3548-3551), so the values calculated above are a lower bound on the actual
relaxation
time.
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Regardless of the exact value of the relaxation time, the polymer must be
stretched
out on a shorter time scale. In the case of flow through a channel, in which
the stretching
comes from fluid strain on the polymer, the appropriate time scale for
stretching is the
reciprocal of the strain rate. The strain rate is defined as de/dt=dvx/dx,
where x is the flow
direction and vx is the x-component of the velocity. The multiple of the
strain rate and the
relaxation time is known as the Deborah number, Dew de/dt, and can be used to
determine
whether the stretching will be maintained (Smith & Chu (1998) Science 281:1335-
1340). If
De is much greater than one, then the strain force predominates and the
polymer will remain
stretched. If De is much smaller than one, then the natural relaxation process
dominates and
the polymer will not remain stretched. When other stretching forces are
involved,
dimensionless values can be derived from other appropriate time scales, such
as the
Weissenberg number in extensional flow (Smith et al. (1999) Science 283:1724-
1727).
Previous techniques used to stretch DNA involved immobilization of at least
one
end of the molecule on a surface, followed by manipulation of the other end,
stretching with
physical forces followed by immobilization, or running through a gel with
constricted
dimensions. Early attempts to stretch DNA for size measurement were conducted
by
Houseal et al. (1989, Biophys. J. 56:507-516). Contacting a DNA solution with
a gold
surface resulted in satisfactory binding, but use of the Kleinschmidt
procedure, which is
used extensively in electron microscopy to spread DNA molecules on a protein
monolayer,
resulted in a number of molecules remaining coiled instead of being stretched.
Another
attempt was made to stretch DNA by "gently" smearing it using a pipettor, a
technique that
is difficult to automate (PCT Publication No. WO 93/22463).
More sophisticated schemes have been devised for the immobilization of one end
of
DNA and other polymers on surfaces. In general, they involve the modification
of a surface
to expose reactive groups such as hydroxyl, amine, thiol, aldehyde, ketone, or
carboxyl
groups, or to attach such coupling structures as avidin, streptavidin, and
biotin. Examples
of these techniques are found in PCT Publication No. 97/06278; U.S. Pat. No.
5,846,724;
and Zimmermann & Cox (1994) Nucl. Acids Res. 22:492-497. Often, these
techniques
involve the use of a silane (Bensimon et al. (1994) Science 265:2096-2098).
Once the polymer is immobilized on one end, stretching is possible since the
forces
may be aligned perpendicular to the attachment surface. One common method is
to use a
receding meniscus to align the polymer, a process sometimes referred to as
"molecular
combing." In this technique, a second fluid is introduced that is
substantially immiscible
with the first, forming a meniscus at the interface. The original fluid is
then gradually
removed by mechanical, thermal, electrical, or chemical means or simply by
evaporation
and is replaced by the new fluid. As the interface moves, the polymer is
aligned
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perpendicular to the interface by surface tension and therefore, becomes
stretched. The
force of stretching by this method is expressed as a function of the diameter
D of the
polymer (D = 2.2 nm for double-stranded DNA) and the surface tension y
(Bensimon et al.
(1994) Science 265:2096-2098): F=y~D.
With an air/water interface, y is 0.07 N/m, giving a force of about 40 pN for
DNA, which is
clearly in the desired range. If the second fluid is properly chosen to
discourage polymer
movement, the polymer remains fixed in place indefinitely. Furthermore,
adjacent
polymers attached to the same surface all become aligned in the same
direction. The two
fluids involved, while often solvents of the polymer, can be only partial
solvents and one
can even be air. The degree of stretching is dependent on the modification of
the surface
(Bensimon, D. et al. (1995) Phys. Rev. Lett. 74(23):4754-4747), but is
consistent for any
given surface treatment. Variations of this technique have been employed (U.5.
Pat. No.
5,851,769; PCT Publication No. WO 97/06278; Bensimon et al. (1994) Science
265:2096-
2098; U.S. Pat. No. 5,840,862; Cox & Zimmermann (1994) Nucl. Acids Res. 22:492-
497).
Nevertheless, this technique cannot be easily adapted to a high-throughput
operation, since
the immobilization is a rate-limiting step and further modification of the
polymer is more
difficult after the immobilization.
An alternative way to manipulate DNA immobilized at one end involves the use
of
an optical trap. In this technique, a laser beam ("optical tweezers") imparts
momentum to a
DNA molecule through emitted photons. By shifting the position of the photons,
i.e.,
moving the beam, an extremely precise change can be induced in the direction
of travel of
the DNA (U.5. Pat. No. 5,079,169; Chu (1991) Science 253:861-866). Hence, a
DNA
molecule can be stretched using optical tweezers. The technique offers the
advantage of
being able to vary the force used for stretching and has been used to verify
reptation theory
(Perkins et al. (1994) Science 264:819-822). However, the laser can only hold
one
molecule in place at a time and has to be realigned for each subsequent
molecule, making it
unattractive for high-throughput analyses.
A third method of stretching DNA involves electrophoresis of either a DNA
immobilized at one end to move the unattached end of the molecule away from
the fixed
end and subsequently attaching the fixed end to a surface with avidin, or a
DNA unattached
at both ends and then attaching both ends to a surface with avidin (Kabata et
al. (1993)
Science 262:1561-1563; Zimmerman & Cox (1994) Nucl. Acids Res. 22:492-497). No
attempt was made to characterize the quality of the stretching using this
technique.
Furthermore, this technique shares the disadvantages of the previously-
mentioned
techniques (with respect to post-immobilization processing).
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DNA has also been stretched by electrophoresis without fixing one end of the
molecule. As part of a near-field detection scheme for sequencing
biomolecules, DNA has
been elongated by electrophoresis both in a gel and in solution, using
electrical forces to
move the DNA in position for reading (LT.S. Pat. No. 5,538,898). However, no
data were
given to determine the quality of the stretching of large polymers, and the
technique is
limited to analyzing approximately 3 megabases at a time.
An extension of this idea involves the use of dielectrophoresis, or a field of
alternating current, to stretch DNA. Washizu and Kurosawa ( (1990) IEEE
Transactions on
Industry Applications 26:1165-1172) have demonstrated that DNA will stretch to
its full
length in its B-DNA form in a field having strength 106 V/m and a frequency of
400 kHz or
more. At certain lower frequencies (around 10 kHz), the DNA will also stretch
fully, but in
a direction perpendicular to the field rather than parallel to it. This
technology has been
applied to sizing DNA by creating a gap with a tapered width between
electrodes such that
the DNA will align where the gap width equals the length of the DNA. It has
also been
found that this technique will not stretch single-stranded DNA due to
differences in solvent
interactions from double stranded DNA (Washizu et al. (1995) IEEE Transactions
on
Industry Applications 31:447-456). One disadvantage to this technique is that,
due to the
presence of induced dipoles along the length of the DNA, samples agglomerate
readily, and
in a heterogeneous sample, it is difficult to accurately identify the
components. In addition,
these experiments must be performed in deionized water in order to avoid the
unwanted
effects of Joule heating and electro-osmotic flow, presenting a sample
preparation difficulty
since most DNA exists in salt solutions or other solvents.
Gravitational forces have also been used to stretch DNA (U.5. Pat. No.
5,707,797;
Windle (1993) Nature Genetics 5:17-21). In this technique, drops of DNA from
the sodium
dodecyl sulfate lysing of cells were allowed to run down a slide held at an
angle. The effect
of gravity was enough to stretch out the DNA, even to its over-stretched S-DNA
form. The
DNA was then immobilized on the slide, making processing, e.g., fluorescent
labeling, prior
to stretching relatively difficult.
Church et al. have developed another method for polymer characterization that
involves measuring physical changes at an interface between two pools of media
as a
polymer traverses that interface (U.S. Pat. No. 5,795,782). This method is
relatively
inflexible. For example, the ion channel embodiment for nucleic acid
characterization
(Church et al. (1999) Science 284:1754-1756) works only for single stranded
DNA. An
interface usable for a wide variety of polymers has yet to be developed.
A method for measuring the length of DNA was developed by Kambara et al. (U.5.
Pat. No. 5,356,776). This method involves electrophoresis of DNA through a
gel; when the
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DNA reaches a portion of the gel no more than a few microns in diameter, it is
forced into a
straight line, where detection of fluorescent labels on each end of the DNA is
accomplished.
In another embodiment, the DNA is immobilized on one end in an aperture,
stretched by
electrophoresis, and a label on the other end of the molecule is detected. The
use of a gel in
S this method necessitates a higher voltage than in solution to move DNA, and
the end .
labeling precludes most other characterization of the DNA. In addition, long
DNA
molecules tend to become entangled in a gel. ~A modification of
electrophoresis procedures,
known as pulsed-field electrophoresis (Schwartz & Koval (1989) Nature 338:520-
522),
allows the full stretching of longer pieces of DNA by moving the electric
field. However,
this technique takes a substantially longer time to run because of the field
variation and
shares the other disadvantages of electrophoresis.
A hybrid of gel based and solution methods for stretching DNA was developed by
Schwartz et al. ((1993) Science 262:110-113). DNA was placed in a free molten
agarose
solution, stretched by gravity, and then fixed in place by the gelling
process. An enzyme
was also added during gelling to cut the DNA at specific sites. This method is
effective in
creating restriction maps, however, predictable stretching in an agarose
medium is difficult
and the adaptation of the technique to high-throughput methods of analyzing
uncut DNA is
problematic.
Other techniques for characterizing particles do not rely on stretching. For
example,
a method developed by Schwartz (U.5. Pat. No. 5,599,664; EP 0391674) allows
sizing and
massing by subjecting a particle to a force and measuring conformational and
positional
changes. In the case of polymers, the force is usually applied to a coiled
conformation.
Another method for sizing and sorting DNA molecules (Chou et al. (1999) Proc.
Natl.
Acad. Sci. USA 96:11-13) involves a device that operates on a micron scale.
The device
uses the integral fluorescence signal from coiled DNA passing a detector to
conduct the
analysis. Schmalzing et al. ((1998) Analytical Chemistry 70:2303-2310; (1997)
Proc. Natl.
Acad. Sci. USA 94:10273-10278) developed microfabricated devices for DNA
analysis,
including sequencing which employ small-scale versions of traditional
techniques, such as
electrophoresis, and do not rely on DNA stretching.
In order to accurately determine the linear sequence of information in
biopolymers,
it is necessary to stretch the biopolymer so that individual units are
distinguishable.
Although many techniques have been developed that stretch biopolymers, and
particularly
DNA, they all have drawbacks, such as uniformity and reproducibility of
stretching, ease of
handling the biopolymer, and applicability to all types and sizes of
biopolymers.
Furthermore, none of them are applicable to rapid analysis of information,
such as is
necessary to sequence large pieces of DNA on a reasonable time scale. Clearly,
there is a
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need for methods and apparatuses for reliably stretching polymers such that
the linear
sequence of information therein can be determined more rapidly and accurately
in order to
elucidate complex genetic function and diagnose diseases and genetic
dysfunctions.
Citation of a reference herein shall not be construed as indicating that such
reference
is prior art to the present invention.
3. SUMMARY OF THE INVENTION
In a first embodiment, the present invention relates to an integrated
apparatus for
stretching at least one polymer in a fluid sample comprising an elongation
structure,
wherein said elongation structure comprises a tapered channel, said tapered
channel
decreasing linearly in width from a first end to a second end, and wherein
said at least one
polymer, when present, moves along said tapered channel in a direction from
said first end
to said second end; whereby when said at least one polymer in said fluid
sample moves
along said tapered channel, a shear force is applied to said at least one
polymer.
This embodiment of the present invention is useful for stretching polymers,
particularly DNA, for further analysis.
In a second embodiment, the present invention relates to an integrated
apparatus
comprising: (a) at least one polymer in a fluid sample; and (b) an elongation
structure for
stretching said at least one polymer, wherein said elongation structure
comprises a tapered
channel, said tapered channel decreasing linearly in width from a first end to
a second end,
and wherein said at least one polymer, when present, moves along said tapered
channel in a
direction from said first end to said second end, whereby when said at least
one polymer in
said fluid sample moves along said tapered channel, a shear force is applied
to said at least
one polymer.
In a third embodiment, the present invention relates to an integrated
apparatus for
stretching at least one polymer in a fluid sample comprising an elongation
structure,
wherein said elongation structure comprises a tapered channel, said tapered
channel
decreasing in width at a greater than linear rate from a first end to a second
end, and
wherein said at least one polymer; when present, moves along said tapered
channel in a
direction from said first end to said second end; whereby when said at least
one polymer in
said fluid sample moves along said tapered channel, a shear force is applied
to said at least
one polymer.
This embodiment of the present invention is also useful for stretching
polymers,
particularly DNA, for further analysis.
In a fourth embodiment, the present invention relates to an integrated
apparatus
comprising: (a) at least one polymer in a fluid sample; and (b) an elongation
structure for
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stretching said at least one polymer, wherein said elongation structure
comprises a tapered
channel, said tapered channel decreasing in width at a greater than linear
rate from a first
end to a second end, and wherein said at least one polymer, when present,
moves along said
tapered channel in a direction from said first end to said second end; whereby
when said at
least one polymer in said fluid sample moves along said tapered channel, a
shear force is
applied to said at least one polymer.
In a fifth embodiment, the present invention relates to an integrated
apparatus for
stretching at least one polymer in a fluid sample comprising an elongation
structure,
wherein said elongation structure comprises a tapered channel, said tapered
channel
decreasing in width from a first end to a second end, and wherein said at
least one polymer,
when present, moves along said tapered channel in a direction from said first
end to said
second erid; whereby when said at least one polymer in said fluid sample moves
along said
tapered channel, a shear force is applied to said at least one polymer,
wherein said shear
force produces a shear rate that is constant.
This embodiment of the present invention is useful for stretching polymers,
particularly DNA, for further analysis.
In a sixth embodiment, the present invention relates to an integrated
apparatus
comprising: (a) at least one polymer in a fluid sample; and (b) an elongation
structure for
stretching said at least one polymer, wherein said elongation structure
comprises a tapered
channel, said tapered channel decreasing in width from a first end to a second
end, and
wherein said at least one polymer, when present, moves along said tapered
channel in a
direction from said first end to said second end; whereby when said at least
one polymer in
said fluid sample moves along said tapered channel, a shear force is applied
to said at least
one polymer, wherein said shear force produces a shear rate that is constant.
In a seventh embodiment, the present invention relates to an integrated
apparatus for
stretching at least one polymer in a fluid sample comprising an elongation
structure,
wherein said elongation structure comprises a central channel for holding
fluid and a
plurality of side channels for holding fluid connected to said central
channel; and wherein
said at least one polymer, when present, moves along said central channel in
an elongation
direction.
This embodiment of the present invention is useful for stretching polymers,
particularly DNA, for further analysis.
In an eighth embodiment, the present invention relates to an integrated
apparatus for
stretching at least one polymer in a fluid sample comprising: (a) an
elongation structure;
(b) a delivery channel leading into and out of said elongation structure for
delivering said at
least one polymer sample in said fluid to said elongation structure; and (c)
means for
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causing said at least one polymer in said fluid sample, when present, to move
within said
elongation structure, wherein said elongation structure comprises a central
channel for
holding fluid and a plurality of side channels for holding fluid connected to
said central
channel; and wherein, when said at least one polymer is present, said means
for causing
causes said at least one polymer to move along said central channel in an
elongation
direction.
This embodiment of the present invention is useful for stretching polymers,
particularly DNA, for further analysis.
In a ninth embodiment, the present invention relates to an integrated
apparatus for
stretching DNA in a fluid sample comprising: (a) an elongation structure; (b)
means for
delivering said DNA in said fluid sample to said elongation structure; and (c)
means for
causing said DNA in said fluid sample, when present, to move within said
elongation
structure, wherein said elongation structure comprises a central channel for
holding fluid
and a plurality of side channels for holding fluid connected to said central
channel; and
wherein, when said DNA is present, said means for causing causes said DNA to
move along
said central channel in an elongation direction.
In a tenth embodiment, the present invention relates to an integrated
apparatus
comprising: (a) at least one polymer in a fluid sample; (b) an elongation
structure for
stretching said at least one polymer, wherein said elongation structure
comprises a central
channel for holding fluid and a plurality of side channels for holding fluid
connected to said
central channel.
In an eleventh embodiment, the present invention relates to an integrated
apparatus
for stretching at least one polymer in a fluid sample comprising an elongation
structure,
wherein said elongation structure comprises a channel with at least one bend,
and wherein
said at least one polymer, when present, moves along said channel.
This embodiment of the present invention is useful for stretching polymers,
particularly DNA, for further analysis.
In a twelfth embodiment, the present invention relates to an integrated
apparatus for
stretching DNA in a fluid sample comprising: (a) an elongation structure; and
(b) means
for delivering said DNA in said fluid sample to said elongation structure,
wherein said
elongation structure comprises a channel with at least one bend, and wherein
said DNA,
when present, moves along said channel.
In a thirteenth embodiment, the present invention relates to an integrated
apparatus
comprising: (a) at least one polymer in a fluid sample; and (b) an elongation
structure for
stretching said at least one polymer, wherein said elongation structure
comprises a channel
with at least one bend.
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In a fourteenth embodiment, the present invention relates to an integrated
apparatus
for stretching at least one polymer in a fluid sample comprising an elongation
structure,
wherein said elongation structure comprises a tapered channel along which said
at least one
polymer, when present, moves in a flow direction, and wherein said channel
comprises a
plurality of obstacles to motion of said at least one polymer.
This embodiment of the present invention is useful for stretching polymers,
particularly DNA, for further analysis.
In a fifteenth embodiment, the present invention relates to an integrated
apparatus
for stretching at least one polymer in a fluid sample comprising an elongation
structure,
wherein said elongation structure comprises a central channel along which said
at least one
polymer, when present, moves in a flow direction and a plurality of side
channels connected
to said central channel, and wherein said central channel further comprises a
plurality of
obstacles to motion of said at least one polymer.
In a sixteenth embodiment, the present invention relates to an integrated
apparatus
for stretching at least one polymer in a fluid sample comprising an elongation
structure,
wherein said elongation structure comprises a channel with at least one bend
along which
said at least one polymer, when present, moves in a flow direction, and
wherein said
channel comprises a plurality of obstacles to motion of said at least one
polymer.
In a seventeenth embodiment, the present invention relates to an integrated
app~'atus for stretching at least one polymer in a fluid sample comprising an
elongation
structure, wherein said elongation structure comprises a channel along which
said at least
one polymer, when present, moves in a flow direction, and wherein said channel
comprises
a plurality of posts, at least one of said posts having a non-quadrilateral
polygonal cross
sectional shape.
The fifteenth, sixteenth and seventeenth embodiments of the present invention
are
useful for stretching polymers, particularly DNA, for further analysis.
In an eighteenth embodiment, the present invention relates to an integrated
apparatus for stretching at least one polymer in a fluid sample comprising an
elongation
structure, wherein said elongation structure comprises a channel along which
said at least
one polymer, when present, moves in a flow direction, and wherein said channel
comprises
a plurality of obstacles to motion of said at least one polymer, said
plurality of obstacles
being positioned as a series of rows, each said row positioned perpendicular
to said flow
direction, and each successive row offset from a previous row, whereby at
least a portion
not equal to a multiple of 1/2 of one of said obstacles overlaps an extension
of a gap formed
by two adjacent obstacles in said previous row along said flow direction.
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This embodiment of the present invention is useful for stretching polymers,
particularly DNA, for further analysis.
In a nineteenth embodiment, the present invention relates to an integrated
apparatus
comprising: (a) at least one polymer in a fluid sample, every said polymer
having a
diameter greater than or equal to a minimum diameter; and (b) an elongation
structure for
stretching said at least one polymer, wherein said elongation structure
comprises a channel
along which said at least one polymer, when present, moves in a flow
direction, and
wherein said channel comprises a plurality of obstacles to motion of said at
least one
polymer, said plurality of obstacles positioned as a series of rows, each said
row positioned
perpendicular to said flow direction, and each adjacent pair of obstacles in
each of said
series of rows is separated by a distance greater than 50 times said minimum
diameter.
This embodiment of the present invention is useful for stretching polymers,
particularly DNA, for further analysis.
In a twentieth embodiment, the present invention relates to an integrated
apparatus
for stretching at least one polymer in a fluid sample comprising an elongation
structure,
wherein said elongation structure comprises a channel along which said at
least one
polymer, when present, moves in a flow direction, and wherein said channel
comprises a
plurality of obstacles to motion of said at least one polymer, said plurality
of obstacles
decreasing in size along said flow direction.
This embodiment of the present invention is also useful for stretching
polymers,
particularly DNA, for further analysis.
In a twenty-first embodiment, the present invention relates to an integrated
apparatus for stretching DNA comprising an elongation structure, wherein said
elongation
structure comprises a tapered central channel, said tapered central channel
comprising a first
end and a second end, and wherein said DNA, when present, moves along said
tapered
central channel in a direction from said first end to said second end, wherein
said elongation
further comprises a plurality of side channels connected to said tapered
central channel,
wherein said tapered central channel comprises at least one bend; and wherein
said tapered
central channel comprises a plurality of obstacles to motion of said DNA.
In a twenty-second embodiment, the present invention relates to an integrated
apparatus for stretching DNA comprising an elongation structure, said
elongation structure
comprising: (a) a first tapered channel, said first tapered channel comprising
a first end, a
second end, and a plurality of posts between said first end and said second
end in a
staggered arrangement comprising a number of rows between 12 and 15, said
first tapered
channel decreasing in width at an angle of 26.6°, said angle being
defined at said first end
with respect to a constant-width channel, said first end having a width
between 0.5 and 5
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Vim, said posts having a cross sectional area equal to 1.5 ~mz and separated
by a gap equal
to 0.5 Vim; and (b) a second tapered channel, said second tapered channel
connected to said
first tapered channel at said second end and decreasing in width such that a
shear force
producing a constant shear rate is applied to said DNA, when present, to a
width between
0.5 and S pm, said second tapered channel having a length between 1 and 3 mm.
In a twenty-third embodiment, the present invention relates to a method for
stretching at least one polymer comprising the steps of: (a) delivering said
at least one
polymer to an elongation structure, said elongation structure comprising a
tapered channel
with a first end and a second end; and (b) moving said at least one polymer
along said
tapered channel from said first end to said second end, whereby said tapered
channel causes
a shear force that produces a constant shear rate to be applied to said at
least one polymer as
said at least one polymer moves along said tapered channel.
The method encompassed by this embodiment of the present invention is useful
for
stretching polymers, particularly DNA, for further analysis.
In a twenty-fourth embodiment, the present invention relates to a method for
stretching at least one polymer comprising the steps of: (a) delivering said
at least one
polymer to an elongation structure, said elongation structure comprising a
linearly tapered
channel with a first end and a second end; and (b) moving said at least one
polymer along
said tapered channel from said first end to said second end.
In a twenty-fifth embodiment, the present invention relates to a method for
stretching at least one polymer comprising the steps of (a) delivering said at
least one
polymer to an elongation structure, said elongation structure comprising a
tapered channel
with a first end and a second end, said tapered channel decreasing at a
greater than linear
rate from said first end to said second end; and (b) moving said at least one
polymer along
said tapered channel from said first end to said second end.
In a twenty-sixth embodiment, the present invention relates to a method for
stretching at least one polymer comprising the steps of: (a) delivering said
at least one
polymer to an elongation structure, said elongation structure comprising a
central channel
holding fluid and a plurality of side channels holding fluid connected to said
central
channel, said central channel comprising a first end and a second end; and (b)
moving said
at least one polymer along said central channel from said first end to said
second end.
The methods of the twenty-fourth, twenty-fifth, and twenty-sixth embodiments
of
the present invention are useful for stretching polymers, particularly DNA,
for further
analysis.
In a twenty-seventh embodiment, the present invention relates to a method for
stretching at least one polymer comprising the steps of: (a) delivering said
at least one
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polymer to an elongation structure, said elongation structure comprising a
channel with at
least one bend, said channel comprising a first end and a second end; and (b)
moving said at
least one polymer along said channel from said first end to said second end.
The method encompassed by this embodiment of the present invention is useful
for
stretching polymers, particularly DNA, for further analysis.
In a twenty-eighth embodiment, the present invention relates to a method for
stretching at least one polymer comprising the steps of: (a) delivering said
at least one
polymer to an elongation structure, said elongation structure comprising a
channel and a
plurality of obstacles to motion of said at least one polymer within said
channel, said central
channel comprising a first end and a second end; and (b) moving said at least
one polymer
along said channel from said first end to said second end, wherein said
plurality of obstacles
to motion decrease in size along a direction from said first end to said
second end.
The method encompassed by this embodiment of the present invention is useful
for
stretching polymers, particularly DNA, for further analysis.
In a twenty-ninth embodiment, the present invention relates to a method for
stretching at least one polymer comprising the steps of (a) delivering said at
least one
polymer to an elongation structure, said elongation structure comprising a
channel and a
plurality of obstacles to motion of said at least one polymer within said
channel, said central
channel comprising a first end and a second end; and (b) moving said at least
one polymer
along said channel from said first end to said second end, wherein at least
one of said
obstacles has a non-quadrilateral polygonal cross-sectional shape.
The method encompassed by this embodiment of the present invention is useful
for
stretching polymers, particularly DNA, for further analysis.
In a thirtieth embodiment, the present invention relates to a method for
stretching at
least one polymer comprising the steps of: (a) delivering said at least one
polymer to an
elongation structure, said elongation structure comprising: (i) a tapered
central channel
with at least one bend, said tapered central channel comprising a first end
and a second end;
(ii) a plurality of side channels connected to said tapered central channel;
and (iii) a
plurality of obstacles to motion of said at least one polymer within said
tapered central
channel; and (b) moving said at least one polymer along said central channel
from said first
end to said second end.
The method encompassed by this embodiment of the present invention is useful
for
stretching polymers, particularly DNA, for further analysis.
In a thirty-first embodiment, the present invention relates to an integrated
apparatus
for stretching at least one polymer in a fluid sample comprising an elongation
structure,
wherein said elongation structure comprises a channel along which said at
least one
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polymer, when present, moves in a flow direction, and wherein said channel
comprises at
least one step that decreases the depth, z, of the channel from a first end to
a second end.
In a thirty-second embodiment, the present invention relates to an integrated
apparatus comprising an elongation structure comprising a channel, said
channel
comprising at least one step that decreases the depth, z, of said channel from
a first end to a
second end, said channel comprising at least one polymer in a fluid sample,
said channel
being configured such that a shear force is applied to said at least one
polymer as it moves
in a direction from said first end to said second end.
In a thirty-third embodiment, the present invention relates to an integrated
apparatus
for stretching at least one polymer in a fluid sample comprising an elongation
structure, said
elongation structure comprising: (a) a first channel, said first channel
comprising a first end
and a second end; and (b) a second channel, said second channel comprising a
third end and
a fourth end, said third end being connected to said first channel at said
second end, along
which said at least one polymer, when present, moves in a flow direction, and
wherein said
1 S first channel decreases in width from said first end to said second end at
a rate different
from the rate at which said second channel decreases in width from said third
end to said
fourth end.
In a thirty-fourth embodiment, the present invention relates to an integrated
apparatus for stretching at least one polymer in a fluid sample comprising an
elongation
structure, said elongation structure comprising: (a) a first channel having a
width equal to
10 pm and a depth equal to 1 pm, said first channel comprising a first end, a
second end,
and a plurality of posts between said first end and said second end in a
staggered
arrangement comprising between at least 12 to 15 rows, said plurality of posts
terminating
at said second end and each post in said plurality of posts having a cross-
sectional area of
between 1 ~mz and 25 pmz; and (b) a second channel, said second channel
comprising a
third end and a fourth end, said third end being connected to said first
channel at said
second end, said second channel decreasing in width at a rate of 1/x2 from
said third end to
said fourth end, said total width decreasing from 10 ~m to 1 pm, wherein x is
the distance
along the length of said second channel, the length of said second channel
being equal to 5
wm~ said second channel comprising one step at said third end that reduces the
depth of said
second channel to 0.25 pmz, wherein said at least one polymer, when present,
moves along
said first channel and said second channel in a flow direction.
In a thirty-fifty embodiment, the present invention relates to an integrated
apparatus
for selectively stretching at least one polymer in a fluid sample on the basis
of length,
comprising an elongation structure, wherein said elongation structure
comprises: (a) a first
channel, said first channel comprising a first end, a second end, and a
plurality of posts in a
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staggered arrangement between said first end and said second end, each post in
said
plurality of posts being situated at a distance no less than L from said
second end; and (b) a
second channel, said second channel comprising a third end and a fourth end,
said third end
being connected to said first channel at said second end, said second channel
decreasing in
width from said third end to said fourth end, along which said at least one
polymer, when
present, moves in a flow direction.
In a thirty-sixth embodiment, the present invention relates to an integrated
apparatus
for stretching a plurality of polymers having varying lengths in a fluid
sample, comprising
an elongation structure, wherein said elongation structure comprises: (a) a
first channel,
said first channel comprising a first end and a second end; (b) a second
channel, said second
channel comprising a third end and a fourth end, said third end being
connected to said first
channel at said second end, said second channel decreasing in width from said
third end to
said fourth end; and (c) a plurality of posts in a staggered arrangement in
said first channel
and said second channel, along which said plurality of polymers, when present,
move in a
flow direction.
In a thirty-seventh embodiment, the present invention relates to a method for
stretching at least one polymer, comprising moving said at least one polymer
along an
elongation structure, said elongation structure comprising a first channel,
said first channel
comprising a first end and a second end, and a second channel, said second
channel
comprising a third end and a fourth end, said third end connected to said
first channel at
said second end, wherein said first channel decreases in width from said first
end to said
second end at a rate different from the rate at which said second channel
decreases in width
from said third end to said fourth end.
In a thirty-eighth embodiment, the present invention relates to a method for
stretching at least one polymer having a length greater than or equal to L in
a fluid sample
comprising moving said at least one polymer along an elongation structure,
said elongation
structure comprising a first channel, said first channel comprising a first
end, a second end,
and a plurality of posts in a staggered arrangement between said first end and
said second
end, each post in said plurality of posts being situated at a distance L from
said second end,
and a second channel, said second channel comprising a third end and a fourth
end, said
third end being connected to said first channel at said second end, said
second channel
decreasing in width from said third end to said fourth end, wherein a polymer
having a
length greater than or equal to L is stretched and a polymer having a length
less than L is
not stretched.
In a thirty-ninth embodiment, the present invention relates to a method for
stretching
a plurality of polymers having varying lengths in a fluid sample comprising
moving said
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plurality of polymers along an elongation structure, said elongation structure
comprising:
(a) a first channel, said first channel comprising a first end and a second
end; (b) a second
channel, said second channel comprising a third end and a fourth end, said
third end being
connected to said first channel at said second end, said second channel
decreasing in width
from said third end to said fourth end; and (c) a plurality of posts in a
staggered
arrangement in said first channel and said second channel.
In a fortieth embodiment, the present invention relates to a method for
stretching at
least one polymer, comprising moving said at least one polymer along an
elongation
structure, said elongation structure comprising: (a) a first channel having a
width equal to
10 ~m and a depth equal to 1 pin, said first channel comprising a first end, a
second end,
and a plurality of posts between said first end and said second end in a
staggered
arrangement comprising between at least 12 to 15 rows, said plurality of posts
terminating
at said second end and each post in said plurality of posts having a cross-
sectional area of
between 1 ~mz and 25 ~mz; and (b) a second channel, said second channel
comprising a
third end and a fourth end, said third end being connected to said first
channel at said
second end, said second channel decreasing in width at a rate of 1/x2 from
said third end to
said fourth end, said total width decreasing from 10 ~m to 1 Vim, wherein x is
the distance
along the length of said second channel, the length of said second channel
being equal to 5
Vim, said second channel comprising one step at said third end that reduces
the depth of said
second channel to 0.25 pmz.
4. BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows examples of various structures that fall within the scope of the
invention.
FIG. 2(a-m) shows (a) several embodiments of stretching structures involving
funnels,
posts, branches, and serial structures; (b) an enlarged example of two-funnel
structures with
posts in serial; (c) several embodiments of complex post arrangements and
branched
structures; (d) an embodiment of a structure containing serial and parallel
structures; (e) an
asymmetric branched structure; (f) an structure having a combination of small
obstacles
which define small gaps; (g) a structure having a combination of polygons,
bars, and posts;
(h) an asymmetric bent structure; (i) an enlarged view of a branched structure
having posts;
(j) a large funnel structure with support posts; (k) a funnel structure with
posts; (1) funnel
structures with a linear increase in flow rate with and without posts; and (m)
a summary of
some of the funnel structures encompassed by the present invention.
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FIG. 3 shows an embodiment of the shear-stretching regime using a constantly-
tapered
channel.
FIG. 4 shows an embodiment of the shear-stretching regime in which the shear
rate
$ drastically increases as flow proceeds down the length of the channel.
FIG. 5 shows an embodiment of the shear-stretching regime using a tapered
channel
designed to impart a constant shear force.
FIG. 6 shows an embodiment of the shear-stretching regime in which the shear
comes from
the addition of fluid from side channels.
FIG. 7 (a) shows how shear force is imparted in a narrowing channel, with
local
components of rotational and extensional force nearly equal; (b) shows how
shear force is
imparted when addition of fluid creates the force, with extensional force
exceeding the
rotational force.
FIG. 8 shows an embodiment of the shear-stretching regime in which shear comes
from
both a narrowing channel and the presence of side channels.
FIG. 9 (a) shows the "racetrack effect" of fluid on the outside of a bend
taking longer to
pass around the corner than fluid on the inside; (b) shows how the "racetrack
effect" can
lead to the uncoiling of a polymer in a bend.
FIG. 10 displays an embodiment of the tortuosity regime, in which the channels
follow a
sine wave shape.
FIG. 11 displays an embodiment of the tortuosity regime in which the channels
follow a
zig-zag shape.
FIG. 12 displays an embodiment of the tortuosity regime in which the channels
follow right
angles in a "snake" shape.
FIG. 13 shows how a tortuous channel can be used for multiple detection of the
same
polymer as it travels down a channel.
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FIG. 14 shows how a polymer can stretch in an embodiment of the obstacle field
regime
with gradated sizing of obstacles.
FIG 15 shows the coordinate frame for an elongation structure.
FIG. 16 (a) shows an embodiment of the obstacle field regime with square-grid
alignment
of circular obstacles; (b) shows an embodiment of the obstacle field regime
with an offset-
grid alignment of circular obstacles.
FIG. 17 shows an embodiment of the obstacle field regime with close spacing of
rectangular
obstacles of an exaggerated aspect ratio.
FIG. 18 shows an embodiment of the obstacle field regime with close spacing of
circular
obstacles.
FIG. 19 shows an embodiment of the obstacle field regime with three gradated
sizes of
circular obstacles.
FIG. 20 shows a configuration for consistent unraveling, delivery, and
stretching of DNA of
varying sizes.
FIG. 21 shows a configuration of a preferred embodiment of a structure for
stretching DNA
that combines a post field, a funnel that tapers as 1/x2 , wherein x is the
distance along the
length of the funnel, and a step that reduces the channel depth.
FIG. 22 shows a schematic of a molecular size sorting device, wherein signals
of molecules
of length L or greater can be easily distinguished from signals of molecules
of length less
than L.
FIG. 23 shows a schematic of a device that stretches molecules of all lengths,
such that
signals from all of them are uniformly detected.
FIG. 24 shows a sensitive optical apparatus that utilizes confocal
fluorescence illumination
and detection.
FIG. 25 demonstrates one embodiment of the overall polymer analysis system.
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FIG. 26 shows DNA in various stretching states in the entrance to a constant-
shear channel.
FIG. 27 (a-g) show a SO kb DNA being stretched out in a tapered channel.
FIG. 28 shows a DNA measured at 537 kb stretched out in a channel.
FIG. 29 shows a histogram displaying experimentally determined DNA lengths.
FIG. 30 shows histograms of experimentally determined lengths of phage lambda
DNA
from the structure of FIG. 20 (a) without posts, and (b) with posts.
5. DETAILED DESCRIPTION OF THE INVENTION
5.1 INTRODUCTION
The present invention provides structures that allow polymers of any length,
including nucleic acids containing entire genomes, to be stretched into a
long, linear
conformation for further analysis. Polymers are loaded into a device and run
through the
structures, propelled by, inter alia, physical, electrical or chemical forces.
Stretching is
achieved by, e.g., applying shear forces as the polymer passes through the
structures,
placing obstacles in the path of the polymer, or a combination thereof.
Because the forces
are applied continuously, it is possible to stretch out polymers to a length
that is equal to or
greater than the active area of the apparatus, i.e., where information about
the polymer is
collected as the polymer is analyzed. For example, if a video camera or laser
illuminated
volume is focused on the region of the chip where spreading occurs, we can
monitor
unlimited lengths of DNA molecules, i.e., much larger than the video image or
the laser
illumination volume. Since multiple molecules may be stretched in succession,
extremely
high throughput screening, e.g., screening of more than one molecule per
second, is
achieved.
Extended polymers or ensembles of polymers are characterized. Extended,
labeled
polymers are moved past at least one station, at which labeled units of the
polymers interact
with the station to produce an object-dependent impulse. As used in this
application,
"moves past" refers to embodiments in which the station is stationary and the
extended
polymer is in motion, the station is in motion and the extended polymer is
stationary, and
the station and extended polymer are both in motion.
Although the invention may be used for characterizing any polymer, it is
preferable
that the polymers have a predominantly, though not necessarily exclusively,
linear or
single-chain arrangement. Examples of such polymers include biological
polymers such as
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deoxyribonucleic acids, ribonucleic acids, polypeptides, and oligosaccharides.
The
polymers may be heterogeneous in backbone composition, thereby containing any
possible
combination of individual monomer units linked together, e.g., peptide-nucleic
acids
(PNA), which have amino acids linked to nucleic acids. In a preferred
embodiment, the
polymers are homogeneous in backbone composition and are, e.g., nucleic acids,
polypeptides or oligosaccharides. The term "backbone" is given its usual
meaning in the
field of polymer chemistry. A nucleic acid as used herein is a biopolymer
comprised of
nucleotides, such as deoxyribose nucleic acid (DNA) or ribose nucleic acid
(RNA). A
protein or polypeptide as used herein is a biopolymer comprised of amino
acids. In the
most preferred embodiment, the extended object is a double-stranded DNA
molecule.
As used herein with respect to individual units of a polymer, "linked" or
"linkage"
means two units are joined to each other by any physicochemical means. Any
linkage
known to those of ordinary skill in the art, covalent or non-covalent, is
embraced. Natural
linkages, e.g., amide, ester, and thioester linkages, which are those
ordinarily found in
nature to connect the individual units of a particular polymer, are most
common. However,
the individual units of a polymer stretched by the structures of the invention
may be joined
by synthetic or modified linkages.
A polymer is made up of a plurality of individual units, which are building
blocks or
monomers that are linked either directly or indirectly to other building
blocks or monomers
to form the polymer. The polymer preferably comprises at least two chemically
distinct
linked monomers. The at least two chemically distinct linked monomers may
produce or be
labeled to produce different signals. Different types of polymers are composed
of different
monomers. For example, DNA is a biopolymer comprising a deoxyribose phosphate
backbone to which are attached purines and pyrimidines such as adenine,
cytosine, guanine,
thymine, 5-methylcytosine, 2-aminopurine, hypoxantine, and other naturally and
non-
naturally occurnng nucleobases, substituted and unsubstituted aromatic
moieties. RNA is a
biopolymer comprising a ribose phosphate backbone to which are attached
purines and
pyrimidines such as those described for DNA but wherein uracil is substituted
for
thymidine. Deoxyribonucleotides may be joined to one another via an ester
linkage through
the 5' or 3' hydroxyl groups to form the DNA polymer. Ribonucleotides may be
joined to
one another via an ester linkage through the 5', 3' or 2' hydroxyl groups.
Alternatively,
DNA or RNA units having a 5', 3' or 2' amino group may be joined via an amide
linkage to
other units of the polymer.
The polymers may be naturally-occurring or non-naturally occurnng polymers.
Polymers can be isolated, e.g., from natural sources using biochemical
purification
techniques. Alternatively, polymers may be synthesized, e.g., enzymatically by
in vitro
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amplification using the polymerise chain reaction (PCR), by chemical
synthesis, or by
recombinant techniques.
The structures of the invention are used in conjunction with methods for
analyzing
the extended polymers by detecting signals referred to as object-dependent
impulses. An
"object-dependent impulse," as used herein, is a detectable physical quantity
which
transmits or conveys information about the structural characteristics of at
least one unit-
specific marker of an extended polymer. A unit-specific marker, as used
herein, can either
be a measurable intrinsic property of a particular type of individual unit of
the extended
polymer, e.g., the distinct absorption maxima of the naturally occurnng
nucleobases of
DNA (the polymer is intrinsically labeled), or a compound having a measurable
property
that is specifically associated with one or more individual units of a polymer
(the polymer is
extrinsically labeled). A unit-specific marker of an extrinsically labeled
polymer may be a
particular fluorescent dye with which all nucleobases of a particular type,
e.g., all thymine
nucleobases, in a DNA strand are labeled. Alternatively, a unit-specific
marker of an
extrinsically labeled polymer may be a fluorescently labeled oligonucleotide
of defined
length and sequence that hybridizes to and therefore "marks" the complementary
sequence
present in a target DNA. Unit-specific markers may further include, but are
not limited to,
sequence specific major or minor groove binders and intercalators, sequence-
specific DNA
or peptide binding proteins, sequence specific PNAs, etc. The detectable
physical quantity
may be in any form that is capable of being measured. For instance, the
detectable physical
quantity may be electromagnetic radiation, chemical conductance,
radioactivity, etc. The
object-dependent impulse may arise from energy transfer, directed excitation,
quenching,
changes in conductance (resistance), or any other physical changes. In one
embodiment, the
object-dependent impulse arises from fluorescence resonance energy transfer
("FRET")
between the unit-specific marker and the station, or the enviromnent
surrounding the
station. In preferred embodiments, the object-dependent impulse results from
direct
excitation in a confined or localized region, or epiillumination of a confocal
volume or slit-
based excitation is used. Possible analyses of polymers include, but are not
limited to:
determination of polymer length, determination of polymer sequence,
determination of
polymer velocity, determination of the degree of identity of two polymers,
determination of
characteristic patterns of unit-specific markers of a polymer to produce a
"fingerprint", and
characterization of a heterogeneous population of polymers using a statistical
distribution of
unit-specific markers within a sample population.
There are numerous methods and products available for analyzing polymers as
described in PCT Publication No. WO 98/35012, which is incorporated herein by
reference
in its entirety.
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Various methods for analyzing polymers differ in their potential sensitivity
and
resolution, i.e., the minimum distance between two unit-specific markers
wherein the unit-
specific markers are distinguishable. A low resolution technique is capable of
distinguishing unit-specific markers having a larger distance between them; a
high
resolution technique is capable of distinguishing unit-specific markers having
a smaller
distance between them. The resolution of a particular technique is determined
by the
characteristic distance through which the station may sense the particular
unit-specific
marker of the extended polymer. A shorter characteristic distance makes for
better
resolution. The lowest resolution techniques include monitoring of light
transmission and
directed excitation, which have a resolution of 50-100 nm or more (Tan &
Kopelman
(1996) Chem. Anal. Ser. 137: 407-475.). In contrast, the resolution ofFRET is
on the order
of the Forster radius, the distance between donors and acceptors at which the
most efficient
energy transfer occurs, which is typically on the order of 2-7 nm. The
distance between
0
adjacent base pairs in a fully-extended DNA molecule having the B-conformation
is 3.4 A,
or 0.34 nm. In its natural state in solution, DNA does not exist in its fully-
extended B-
conformation, but as a coil with a diameter on the order of 10 Vim. Therefore,
it is much
more difficult to resolve a plurality of unit-specific markers on a coiled DNA
molecule and
the molecule should be extended before analysis.
5.2 SHEAR FORCE AS A MEANS OF STRETCHING POLYMERS
When a polymer molecule reaches a physical obstruction, it will either pass by
without interaction or "hook" around the obstruction such that portions of the
chain remain
on each side of the obstacle. This does not mean the polymer is bonded to the
obstruction
or otherwise physically attached. The lopsidedness of the draping around the
obstacle
determines the rapidity with which the molecule proceeds down the favored
side. (See
Austin & Volkmuth, Analysis 1993 (21) 235-238.) In addition, localized
velocity gradients
are created at the obstacles, since the cross-sectional area available for
fluid flow is reduced.
As a result, the fluid flowing in between the obstacles moves faster than the
fluid before and
after. This creates a shear force acting on approaching molecules that serves
as a stretching
force on the polymer. When this effect is multiplied by having an entire field
of properly-
sized obstacles, the polymer stretches out to make it past all the obstacles
in the field. In a
preferred embodiment, the polymer is stretched out in a linear fashion.
Once the polymer has passed the array of obstacles and enters a channel in its
fully
extended form, where in a preferred embodiment it is analyzed, it will
naturally tend to
return to a lower-energy, more coiled conformation. To prevent this from
happening,
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channels are designed to provide a constant shear force on the polymer in a
narrowing
channel, causing it to remain in a stretched conformation.
A constant shear rate, or change in average velocity with distance in the
channel, is
defined as S:
s
aurax = s (s)
where x is the distance down a substantially rectangular channel, and a is the
average fluid
velocity in the x direction, which is computed from the overall fluid flow (Q)
and the cross-
sectional area, A, of the channel as follows:
u=Q/A (6).
In one embodiment where the channel cross-section is rectangular, the channel
may
1 s be defined by a constant height, H and width, W such that the cross-
sectional area A=HW,
and the average fluid velocity is given by:
a=Q/HW (7)
Applying the boundary condition that the fluid flow must be continuous (i.e.,
incompressible), Q is constant. Hence, a is inversely proportional to W. This
relationship
can be substituted into the original expression for S to determine a
relationship between the
shear rate and the width:
2s S = aurax=Q/H a/ax (1/W) _ (-Q/HWZ) (dW/dx) (8)
dW/dx = (-SH/Q)(Wz) (9)
Integrating this expression, it is found that:
W=(SHx/Q + C)-' (10)
where C is a constant of integration determined by the original width of the
channel
(boundary condition). This equation for the width of the channel is used to
define a channel
3s beyond a post structure. Similar calculations may readily be completed by
those of skill in
the art for non-rectangular channel shapes. When no net momentum transfer
occurs in the
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height axis, i.e., when the velocity profile in the z-axis has been
established, the shear rate
from the width profile results in a stretching force. Illustrating in the case
of a Newtonian
fluid, the stress tensor, iYZ, required to compute the force is easily
expressed in terms of the
shear rate:
F = f f -iyZdzdx = f f -~(du/dx)dzdx = f f ~Sdzdx, (11)
where ~ is the solution viscosity. In these equations, x is the direction of
motion, y is the
width, and z is the height. The surface over which the shear rate needs to be
integrated is
that of the channel wall, which results in:
F = ~HLS (12)
where L is the length of the channel wall, approximately the length of the
channel in which
the constant shear is maintained.
Therefore, an aqueous channel with 1 ~m depth, 1 mm length, and shear rate of
0.25/s gives a force of approximately 0.25 pN, adequate to stretch DNA, which
the
inventors have verified experimentally. Notably, this result confirms that the
constant-shear
channel not only maintains the extension of previously-stretched DNA, but also
contributes
to further stretching of DNA, or stretches DNA on its own.
In a preferred embodiment, the two general methods for achieving stretching
have
been combined. Gradated arrays of obstacles that are posts have been placed in
structures
which also impart shear forces on passing molecules, ensuring not only the
initial stretching
of the polymer by the obstacles, but also the maintenance of stretching after
the polymer has
traversed the obstacles.
5.3 STRUCTURES FOR STRETCHING POLYMERS
The structures for stretching DNA of the present invention ("elongation
structures")
comprise two components: a delivery region and a region of polymer elongation.
The
delivery region is a wider channel that leads into and out of the region of
polymer
elongation. The region of elongation comprises at least one of four main
components: (1)
funnels; (2) structures having branched channels; (3) channels with bends or
curves; and (4)
obstacles defining small gaps, wherein the obstacles can be, inter alia, posts
or steps. The
invention encompasses combinations of the four main components and variations
of the
main components themselves. A combination of two or more of the main component
features can give rise to additional designs that work well to extend and
stretch polymers,
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particularly DNA, in a controllable fashion. In addition, several of the same
design may be
repeated in parallel or in series.
Examples of structures (FIG. 1) that fall within the scope of the invention
include,
but are not limited to:
i) funnels with a non-linear increase in fluid velocity;
ii) funnels with a linear increase in fluid velocity;
iii) funnels with obstacles defining small gaps as the region of DNA
elongation;
iv) funnels with a non-linear increase in fluid velocity and obstacles
defining small
gaps;
v) funnels with a linear increase in fluid velocity and obstacles defining
small gaps;
vi) funnels with mixed obstacle sizes and gaps, including a gradient of
obstacles sizes
and gaps;
vii) branched structures having regions of increased fluid velocity from
converging
channels;
viii) branched structures having multiple regions of increased fluid velocity
from
multiple converging channels;
ix) branched structures having obstacles defining small gaps;
x) branched structures which have at least one funnel as one of the branches;
xi) branched structures with mixed obstacle sizes and gaps, including a
gradient of
obstacle sizes and gaps;
xii) structures which have obstacles which define small gaps and also bends or
curves;
xiii) structures which have obstacles defining small gaps which have a
periodicity (sine
patterns, boxcar repeats, zig-zags);
xiv) structures which have obstacles defining small gaps which are non-
quadrilateral
polygons; -
xv) structures having a mixture of obstacles which define small gaps, e.g., a
set of bars
defining small gaps juxtaposed to a field of sine patterns, or a field of
triangles,
circles, or stars;
xvi) structures having obstacles defining small gaps integrated with funnels,
branched
structures, or bends or curves;
xvii) structures having bends or curves in a funnel shape;
xviii) structures having bends or curves with obstacles defining small gaps;
xix) structures having regions of DNA elongation in series;
xx) structures having regions of DNA elongation in parallel;
xxi) structures having multiple delivery channels with respective regions of
elongation;
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xxii) structures having three-dimensional geometries involving embodiments of
the other
categories; and
xxiii) structures which are closed loops containing regions of DNA stretching.
Further examples of structures that fall within the scope of the invention are
shown
in FIG. 2(a-1). These include, several embodiments of stretching structures
involving
funnels, obstacles, branches, and serial structures; two funnel structures
with posts in serial;
embodiments of several complex post arrangements and branched structures; an
asymmetric
branched structure; a structure with a combination of small obstacles that
define small gaps;
a structure with a combination of polygonal, bar, and post obstacles; an
asymmetric bent
structure; a branched structure having posts; a large funnel structure with
support posts; a
funnel structure with posts; funnel structures with a linear increase in flow
rate both with
and without posts. FIG. 2(m) is a summary of some of the possible funnel
structures.
Typically, the elongation structures of the invention can have lengths of from
1 pm to 2 cm,
preferably from 1 pm to 1 mm, widths of from 2 pm to 1 mm, and depths of from
0.1 pm
to 10 pm.
Each of the four main components of a functional polymer elongation and
stretching
structure are described below.
Funnel structures. Funnel structures are tapered channels that apply shear
forces in a
regular and continuous manner as the polymer flows down the channel. The
particular
shear forces are defined by the type of channel structure and shape. In one
embodiment of
the invention, the channel is a tapered channel (FIG. 3) that begins at a
given width and
continuously decreases to a second width, creating an increasing shear force
in the funnel
portion of the channel defined by:
du/dx = (-Q/H)(dW/dx)(1/WZ) (13)
In one embodiment of the invention, the width decreases linearly so that dW/dx
is constant;
in this embodiment, the shear, du/dx, thus increases as W decreases. In this
embodiment,
the angle of the funnel as measured from the continuation of a straight wall
is preferably
between 1 ° and 75 °, with a most preferred value of
26.6° for DNA in a low viscosity
solution such as TE (10 mM TRIS, 1 mM EDTA) buffer, pH 8Ø Starting widths
for the
linear funnel embodiment preferably range from 1 micron to 1 cm, with ending
widths
preferably in the range of 1 nm to 1 mm depending on the polymer in question,
with most-
preferred values of 50 microns and 5 microns, respectively, for DNA.
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The channel could also be configured such that the width decreases at an
increasing
rate as fluid passes down the channel (FIG. 4), resulting in an increase in
shear as the
channel is traversed. Such tapers offer especially good protection against
natural relaxation
of the polymer, since as time passes and the molecules move down the channel,
they face
increasing counter-forces to their tendency to recoil. Furthermore, the
increasing force.
taper allows some design flexibility; any polymer that will encounter shear
forces large
enough to cause the polymer to stretch in the taper and will not encounter
shear forces large
enough to cause the polymer to break in the taper can be successfully run
through the taper
and stretched. There is no need to find the ideal or threshold force for the
polymer, only an
effective range. In embodiments involving pressure-driven fluid flow (see
Driving forces,
below), increasing shear also offers the greatest increase in velocity for a
given pressure
drop, since the final velocity is a function of the cross-sectional area and
the pressure drop
is a function of the cross-sectional area and length of channel. The same
small cross-
sectional area (and hence large velocity) can be reached in a shorter distance
(and hence
smaller pressure drop). In a preferred embodiment, the width of the funnel, W,
decreases as
'/(ax°+b), where n is any real number greater than 1, a is a nonzero
real number, b is a real
number, and x is distance along the length of the funnel (and the direction of
polymer flow).
Potential equations for the taper of increasing shear force funnels include
W=1/x2, W=1/x3,
etc.
In yet other embodiments, channels are designed such that the shear rate is
constant,
leading to a tapered channel such as that shown in FIG. 5. The value of the
constant shear
rate required to achieve an adequate force to completely stretch the polymer
over the course
of the channel will vary based on the length of that channel (refer to Eq.
(12)). Therefore,
0.01/s might be a reasonable shear rate in order to completely stretch a
polymer in a very
long, e.g. > 1 cm, channel, but might result in almost no polymer stretching
in a very short,
e.g. < 10 pm, channel. Lengths of channels may vary significantly, with
preferred values
from 10 ~m to 1 cm and the most preferred values in the range of 1 - 2 mm. In
one
embodiment, the channel is 1 mm long and the shear rate is 0.075/s.
The shear rate of the funnel can be determined by measuring the distance
between
~o down points on a strand of DNA. For example, concatamers of ~, DNA are used
as
standards for shear force measurements. A unique sequence on each concatamer
is
fluorescently tagged with a hybridization probe. The interprobe distance on
the concatamer
is thus the length of a single ~, DNA molecule (48 kilobases). The physical
distance
between the probes is determined using video microscopy or time-of flight
measurements.
The physical distance for ~, DNA in native solution is 14.1 pm. This value is
compared
with the actual measured physical distance. For instance, if the measured
distance is 15.0
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Vim, then the shear rate can be calculated from the amount of stretching that
the DNA has
experienced in the stretching structures. The predicted shear on the DNA, as
measured by
the velocity of the DNA and the dimensions of the channel (see Equation 10),
is matched
with the elongation of the DNA and its intrinsic non-linear stiffness.
Branched channels. A second aspect of the invention used to stretch and
elongate
polymers is to create branched structures, which cause either changes in fluid
flow rates or
changes in polymer directionality (see below in Structures with bends or
curves). Side
channels feed more fluid into a main channel, resulting in a change in fluid
velocity and
hence causing polymer stretching. A typical arrangement of branched channels
is shown in
FIG. 6. Side channels preferably have a combined cross-sectional area ranging
from about
1 % to 500% of the cross-sectional area of the main channel. Most preferably,
side channels
have a combined cross-sectional area of about 50% of the cross-sectional area
of the main
channel. In one embodiment, the side channels are present in a pattern that is
repeated,
which results in a dilution of the shear force at each individual entrance to
the main channel
and, hence, a closer approximation of a constant-shear situation. This
arrangement
highlights the advantages and disadvantages of the side channels. One
disadvantage of this
component of polymer elongation is that all of the force on the main channel
fluid is
dissipated in a relatively small region near the junction of the main channel
and the side
channels. Therefore, this configuration does not lead to a constant-force
situation.
However, an advantage of this component of polymer elongation is that, because
the
additional fluid in the side channels is moving in the same direction as the
fluid in the main
channel, the force is not purely shear force, but has a substantial
extensional flow
component. Pure shear, which is the force exerted by a tapered funnel on a
polymer, is a
superposition of extensional forces and rotational forces as shown in FIG.
7(a). The
extensional force on a polymer accelerates it in the direction of the fluid
flow, such that the
portion of the polymer located in the region of extensional flow moves faster
than the
portion still located in a more stagnant region, stretching out the polymer.
The rotational
force causes the polymer to spin or "tumble" in conformation, which can cause
stretched
potions of the polymer to fold up on themselves and recoil. In the embodiments
that have
stronger extensional forces, such as the side channel junction configuration
shown in FIG.
7(b), the polymer tends to accelerate away from the junction, which results in
lower
rotational forces, thus allowing for better stretching.
As will be appreciated by those of skill in the art, the channel dimensions
may be
modified and the flow rate increased in the same region of the chip. In fact,
a significant
increase in the flow rate followed by a constant-shear section is one way not
only to stretch
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out a polymer, but also to direct it away from the walls of the channel. One
arrangement
embracing this embodiment of the invention is shown in FIG. 8. In yet another
embodiment, additional flow is brought in only from one side of the main
channel, thereby
positioning a polymer traveling down the main channel toward one side. This
positioning
design could be used to ensure that a polymer is aligned to pass under a
narrow detector in a
broader channel.
Structures with bends or curves. The third aspect of the invention uses
tortuosity to
achieve stretching. As fluid flow encounters changes in its path, alignments
ranging from a
small bend to a right angle, the fluid on the outside of the curve or corner
will take longer to
go around the turn than the fluid on the inside of the curve or corner (FIG.
9(a)). This so-
called "racetrack effect" can help stretch out polymers. Such a bend does not
include a "T"
junction. In a rectangular section of a channel, a polymer may flow such that
it straddles
more than one fluid flow line, and since the fluid in each line travels at the
same velocity, it
retains its configuration. In contrast, when the distance traveled by each
fluid flow line
diverges at a bend or corner, the polymer is stretched locally by the velocity
differential.
Furthermore, the polymer tends to move toward the higher-velocity flow line,
so that even
if the channel curves back to regain its original direction, the polymer does
not fully recoil
because locally it is within the same flow line. A possible sequence of this
kind of
stretching is shown in FIG. 9(b). While this effect is insufficient to stretch
an entire long
molecule in a single set of turns, it can gradually uncoil specific regions,
and enough
repetition of a tortuous channel can stretch an entire molecule.
One of the gentler incarnations of the tortuosity regime is an embodiment
where the
configuration of the channel follows a sine wave pattern (FIG. 10). In another
embodiment,
the channel takes the form of a zig-zag shape (FIG. 11), or, in yet a further
embodiment,
even a "snake"-shape with only right-angle corners (FIG. 12), though this
severe of a corner
tends to cause stagnant flows and other undesirable fluid dynamics. For those
embodiments
where the channel has a zig-zag shape, each bend preferably has an angle
between 5 ° and
75 °; for DNA a preferred value of every such angle is 26.6
(effectively a 53.4° angle where
the zig-zag reverses). Such zig-zag shapes may be periodic, in which the angle
of the bends
is always the same, or may comprise a pattern of differential bends. The
period of
repetition for the zig-zags may vary from as little as 2 pm to 1 cm, with
preferred values of
20-50 ~m for DNA (1000 times the persistence length). For those embodiments
where the
channel has a sinusoidal shape, the amplitude to period ratios are preferably
between 0.01
and 5. The number of periods for any of these patterns may vary from 1 period
to 500, with
a preferred value of 10.
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In a further embodiment, tortuous channels are used to create multiple
detection
possibilities. When a detector, such as a position-dependent photomultiplier
tube arranged
in a 1 x 256 array, is situated along the direction of flow in the channel,
the tortuous channel
can be aligned so that it repeatedly crosses the detection zone at defined
locations. The
polymer being stretched is then observed at several locations, creating
redundancy and error
checking in the system. Such an arrangement is shown in Fig. 13, with fluid
traveling down
channel 111 passing through detection zone 110 at six locations, 112-117.
Obstacles definin sg mall yaps. The fourth aspect of structures which tend to
cause
stretching is the field of obstacles. As described more generally above,
obstacles induce
stretching both by reducing the available cross-sectional area of the channel
(causing local
strain on the molecules) and by acting as physical barriers which cannot be
passed by large
coils of polymer. One example is a configuration of posts that work to
actually stretch a
polymer and is shown in FIG. 14.
1 S The obstacles can vary in cross-sectional shape and in cross-sectional
area. The
terms "cross-sectional shape" and "cross-sectional area," as used herein with
reference to
obstacles, and unless otherwise indicated, refer to the shape of the X-Y
projection and the
area of the X-Y plane of the obstacle, respectively, as shown in FIG. 1 S. In
particular
embodiments, the obstacles comprise square posts, round posts, elliptical
posts or posts
with a rectangular cross-section of any aspect ratio (including extremely long
"bars"); in
other embodiments, the obstacles comprise posts with a cross-section shaped as
a regular or
irregular non-quadrilateral polygon. In one preferred embodiment, the cross-
sectional shape
is triangular. In other preferred embodiments, these shapes are modified to
have a concave
edge on the edge that faces the direction from which the fluid is coming (such
as a shallow
U-shape). In still other embodiments, posts having a cross-sectional shape
wherein one
dimension is longer than the other preferably have an aspect ratio of 2 to 20,
more
preferably of 2 to 5.
Each of these obstacles may be placed at any angle to the direction of flow.
In
preferred embodiments, the obstacles are aligned with either a flat surface
perpendicular to
the direction of the flow, or at a 45 ° angle to the flow, though if
preferential positioning of
the polymer molecules is desired, other angles which physically direct
polymers toward a
destination would be used. Preferably, obstacles wherein one dimension is
longer than the
other are placed with their longer dimension perpendicular to the flow
direction. Another
factor in the layout of the obstacles is the grid on which they are placed. If
placed on a
repeating square matrix (FIG. 16(a)), certain fluid flow lines are almost
unaffected by the
obstacles, and unstretched or poorly stretched polymers may be able to track
along these
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flow lines and make it through the obstacle field without being stretched. To
prevent this,
each successive column is preferably offset to place the next obstacle where
the gap in the
previous column had been (FIG. 16(b)), forcing all flow lines to have
curvature and
inducing stretching on all passing molecules. The offset may also be less than
the full 50%
of the repetition unit so that every other column is not in the same alignment
as shown in
the figures; every fourth or sixth column may have an identical alignment, for
example, or
there may never be a repetition of alignment, as long as the flow lines at
some point are
forced to curve around an obstacle.
Besides alignment in the flow, there are two other parameters relevant for
obstacles:
the size of the passages between them, and the total Y-Z cross-sectional area
of the posts
relative to the Y-Z cross-sectional area of the channel (FIG. 1 S ), both of
which affect the
preferred obstacle size. The width of the passages between obstacles should
not be smaller
than the diameter of the stretched polymer, and is preferably not less than
approximately 50
times the diameter of the stretched polymer in order to increase the
probability that the
1 S polymer will be able to pass through the channel without becoming stuck in
the obstacle
field. An example of inadequate passage width leading to polymers not getting
through the
obstacles is shown in FIG. 17. On the other hand, the passages are preferably
not as wide as
the diameter of the coiled polymer, in which case the coil could pass through
the obstacle
field without having to stretch at all. Hence, the preferred spacing of the
obstacles is highly
dependent on the polymer being analyzed. In the case of long DNA with a chain
diameter
of 2 nm and a coiled diameter varying upward from about 1 pm, the passage
width is
preferably between 100 nm and 800 nm, with a most preferred value equal to 500
nm. ' For
polymers with a very small diameter, gels may be used in place of obstacle
fields, giving
pore sizes (equivalent to passage width in the fields) of 1 nm to 1000 nm.
The total Y-Z cross-sectional area occupied by the obstacles most directly
impacts
the velocity gradients that occur in between the obstacles, and which
encourage stretching.
Hence, it is preferable to have a larger ratio of obstacle Y-Z cross-sectional
area to total
channel Y-Z cross-sectional area (also known as the fill ratio, which when
expressed as a
percentage is given by 100 multiplied by the ratio of the total area of the
posts to the total
area of the channel) to maximize the velocity gradients. On the other hand,
forcing too
much material through a relatively small gap can lead to clogging if more than
one polymer
tries to enter a channel at the same time. Hence, to balance these competing
considerations,
the fill ratio is preferably between 33% and 95%. This is the ratio of
occluded area to total
area in a particular channel expressed as a percentage. For example, a post
having a 1 pmt
Y-Z cross-sectional area in a channel having a 3 ~mz Y-Z cross-sectional area
has a fill ratio
of 33%, while a 20 pmz post in a 21 pmt channel has a fill ratio of 95%. The
most
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preferred value for the fill ratio is between 50% and 80% for DNA. An example
of
obstacles too large, leading to clogging, is shown in FIG. 18.
In order to alleviate problems with polymers clogging small passages in the
post
field, differential passage widths are used in some embodiments of the
invention. In some
embodiments, this is accomplished by varying the size of the obstacles. In
other
embodiments, this is accomplished by varying the fill ratio. In still other
embodiments,
both obstacle size and fill ratio are varied. In such embodiments, polymers
first encounter
wide passages between obstacles and subsequently encounter passages of
decreasing widths
(FIG. 19), forcing them to gradually become more elongated in order to proceed
down the
smaller channels. In a preferred embodiment, passage widths are gradated from
about 5 pm
per passage to about 1 pm per passage in the flow direction. In another
embodiment, post
sizes are gradated from a cross-sectional area of about 10 pmz to about 1 pmz
in the flow
direction. In other embodiments, the obstacle cross-sectional area and passage
width may
be varied individually to achieve similar effects, i.e., the obstacle size may
change and the
passage size may remain constant, or the passage size may change and the
obstacle size may
remain constant. In a preferred embodiment, all obstacles have the same cross-
sectional
area, but the fill ratio increases in the flow direction. The cross-sectional
area of the posts
can vary from 0.1 ~m2 to 1 mm2, preferably from 0.1 pmt to 10 pmz, more
preferably from
1 pmz to 100 ~m2, even more preferably from 1 pmt to 25 ~m2, depending on the
size of the
polymer being stretched and the size of the channel used. Such pre-alignment
of polymers
serves to decrease the possibility of entanglement and hence provides more
predictable
stretching.
Obstacles can also be fabricated into the depth or z-dimension of the
structures, i.e.,
by introducing "steps" into the top and/or bottom of the channel to decrease
the depth.
Instead of having obstacles placed across a channel, as discussed above, the
entire channel
can change in depth, providing the same kind of barrier and shear forces
around the ba 'rner
as obstacles placed along the width of the channel. Furthermore, changes in
depth can be
relatively inexpensive to implement, as controlling the depth of etching on
the sub-micron
scale is generally easier than trying to create feature sizes on the sub-
micron scale using
photolithography. Without being bound by any theory, a significant change in
depth at a
specific location in essence creates the same effect as a single row of posts,
or as a funnel of
infinitely short length, x. To approximate a funnel in a fashion that is easy
to manufacture
using standard microfabrication techniques, the height change can be designed
to occur in
several steps along the length of the channel, instead of in one step at a
single location. In a
preferred embodiment, a single-step configuration reduces the height of the
channel by a
factor of five. In other embodiments, a configuration having at least one step
reduces the
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height of the channel from by about a factor of 2 to by about a factor of 100.
In still other
embodiments, the steps vary in height from about 0.1 pm to about 0.9 Vim.
Combinations of components. In further embodiments of the invention, the three
general aspects of structures, shear-inducing (i.e., tapered and branched
channels), tortuous,
and obstacle-filled, are used in combination. The constant-shear tapered
channel, for
example, is good not only at stretching in itself, but in maintaining
stretching in polymers
that have already been stretched by obstacle fields. A channel with a tortuous
contour can
also shrink in width following a constant-shear pattern to capitalize on both
effects. In
preferred embodiments, a gradated obstacle field or alignment structure is
used to pre-
stretch the polymer, followed by a section of fine obstacles, tortuous
patterns, or high shear
area to complete the stretching, and a constant-shear or increasing shear
section to maintain
the stretching until the detection point is reached.
Applicants have found that an especially effective structure is a combination
of an
obstacle field upstream of a tapered channel. The obstacle field serves to
uncoil the DNA
from its random coil configuration, presenting one end of the molecule
preferentially to the
downstream structure(s). It is advantageous for the obstacle field to be in a
wide region of
the channel where the flow velocity is relatively low such that the drag force
applied to a
molecule that becomes folded around or otherwise retained by one of the
obstacles is not
sufficient to break the molecule. As the molecule winds through the obstacle
field, one end
will tend to lead the rest of the molecule and enter the tapered channel
first. The molecule
will then be further stretched by the shear force of the flow through the
tapered channel.
Without being bound by any theory, applicants have found that the partial
uncoiling and end
presentation effected by the obstacle field combined with the stretching in
the tapered
channel is especially effective in accomplishing DNA stretching. Comparison of
experimental data from a tapered channel with an upstream post field to data
from a tapered
channel alone, shows that better stretching is achieved by the combination of
the post field
and tapered channel under similar conditions of flow and temperature (see
Example 2 and
FIGS. 29 (a) and (b)). The experimental data shows that, while a tapered
channel does
stretch DNA, a structure that combines a tapered channel with a post field
provides
significantly greater stretching on average and stretches a greater proportion
of the DNA.
In preferred embodiments, an obstacle field, step or alignment structure is
used to
pre-stretch and align the polymer, followed by a section of constant or
increasing shear or
elongation to complete and maintain the stretching until the detection region
is reached.
Preferably, the obstacle field is matched with a tapered channel in a way that
avoids
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contractile flow (i.e., decreasing velocity). Hence it is preferred that posts
or steps are
located in or terminate at a tapered portion of the channel.
In more preferred embodiments, the channel is a two-funnel structure, that is,
it has
two tandem regions with different degrees of tapering. An example of a two-
funnel
structure is shown in FIG. 20. In one embodiment, the two-funnel structure
further
comprises a post field in the first tapered region. In the two-funnel
configuration, stretching
of the polymer is completed in the second tapered region (right-most channel
region in FIG.
20). Pressure driven flow is the preferred driving force because of its
simplicity and ease
of application.
In a most preferred embodiment, the structure has a first channel region with
a
constant width of about 10 pm and a height of about 1 ~m in which is placed an
obstacle
field along the flow direction and leading into a second channel region that
is a funnel
whose width tapers as 1/x2, from a width of about 10 ~m to about 1 Vim, and
whose height is
reduced in a single step at the entrance to the funnel from about 1 pm to
about 0.25 pm
(FIG. 21). The ratio of the initial channel width to the final channel width
is preferably
greater than 10, and the length of the funnel portion is preferably less than
one-half the
initial width. The obstacle field preferably comprises at least between 12 and
15 rows of
posts having a cross-sectional area substantially equal to 1 ~,m, wherein the
rows have an
increasing fill ratio in the flow direction. In one embodiment, six rows have
an increasing
fill ratio from 0% to 50% in the flow direction, and the subsequent 12-15 rows
have a
constant fill ratio of 50%, wherein the centers adjacent rows of the
subsequent 12-15 rows
are at a distance of about 2 ~m (FIG. 21). In another embodiment, the rows
have a
continuously increasing fill ratio from 0% to 80% in the flow direction.
5.4 STRUCTURES FOR POLYMER SELECTION BY LENGTH
As described in the previous section, post fields can be used to produce non-
random
alignment of polymers and to effectively separate one end of the polymer chain
from the
random coil that is the equilibrium structure of the polymer in solution. If a
post field is
placed at a distance L from the mouth of a tapered channel, which can be of
any shape
desired to maintain or produce stretching, e.g., straight, constant shear, or
higher order
polynomial, the resulting structure can also be used to select molecules by
length. This
process is illustrated in FIG. 22.
FIG. 22 shows a schematic view of a post field constructed according to the
methods
described below (see Methods of fabricating structures), positioned before a
funnel region
of shear or elongational flow. Because the posts fill a portion of the
channel, fluid moving
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through the channel will experience a decrease in velocity as it moves from
the post region
into the straight section of the channel. This decrease in velocity produces a
contracting
flow, i.e., the polymer will re-coil in the region of decreased fluid
velocity. DNA molecules
that travel along the channel and become hooked around a post will be
stretched by the
flow. If the molecule has a length equal to or longer than the distance L from
the posts to
the start of the tapered region, it will be released from the post field into
the region of
elongational flow, in effect spanning the region of decreased fluid velocity
without
recoiling, and will remain stretched, as shown schematically by DNA molecule 1
in FIG.
22. If the molecule is shorter than L, e.g., DNA molecule 2 in FIG. 22, then
it will leave the
posts while still in the contracting flow region of the channel, where it will
contract rapidly
into an equilibrium coil. Therefore, a molecule having a length greater than
or equal to L
will be stretched and a molecule having a length less than L will not be
stretched. If a
detector is positioned at the exit from the funnel, as shown in FIG. 22, the
signals from
coiled molecules (length less than L) and stretched molecules (length greater
than or equal
to L) will be distinguishable. For example if the detector were monitoring
intercalator-
stained DNA, contracted molecules would produce a short, intense pulse,
whereas fully-
stretched molecules would produce a long, less intense signal. Thus it is
possible to
produce structures that separate mixed populations of polymers into two
groups, i.e., those
having lengths shorter than L and those having lengths equal to or longer than
L, by simply
setting L, the distance from the trailing end of the post field to the mouth
of the tapered
region, to a length that is substantially the same as the length of the
molecules from which
signal is to be detected.
In another embodiment, it may be desirable to stretch and uniformly detect
signal
from molecules of all lengths in a given population. This can be done by
eliminating the
region of contracting flow by, e.g., extending the post field of FIG. 22 into
the channel, as
shown in FIG. 23. Since the detector is located at the entrance to the channel
(as in FIG.
22), where the post field ends, all molecules will be stretched as they pass
the detector, and
therefore, signals from all molecules, regardless of their lengths, will be
detected. In these
embodiments, the flow remains constant because the area between the posts is
matched to
the channel area to which the post field extends.
5.5 DESIGN CONSIDERATIONS
Stretching considerations and types of structures to be used. Different
structures
give rise to different types of DNA stretching and elongation. There is
tethered stretching
and uniform stretching. Tethered stretching entails creating an unequal force
distribution on
one end of the molecule to create full extension in a flow profile. Tethered
stretching is
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straightforward to create using obstacles defining small gaps. Uniform
stretching, on the
other hand, is more complex and involves extensive modeling of polymer
dynamics.
Uniform stretching is defined as creating a uniform tension over each unit of
the DNA
molecule. Structures which are designed to create uniform stretching include
those with
constant shear forces in the x-direction of the design such as funnels with
non-linear
increases in flow rates.
Polymer size considerations. The structural designs are such that they are
scalable
and some are universal. Structures can be increased in size, and the relative
dimensions
changed, in order to accommodate polymer molecules of different lengths. Sizes
of interest
range from several kilobases to at least megabases of DNA, although there is
no upper limit
on the length of polymer molecules that can be accommodated. One megabase of
DNA has
a length greater than 300 microns. Channel dimensions can be made up to
several
millimeters. In this manner, whole chromosomes (ranging in size from 50 - 250
megabases) can be handled and stretched.
Configurations of channels on overall chip. The delivery channels leading to
the
regions of DNA elongation can include delivery channels which are parallel,
radial,
branched, interconnected, and closed loops. Delivery channels in the preferred
embodiment
are wide channels, i.e., 1 - 1000 microns, which lead to regions of DNA
stretching and
elongation.
Methods of fabricating structures. The preferred method to fabricate the
designed
structures is by lithography, such as e-beam lithography, deep-uv lithography,
photolithography, LIGA (acronym of the German words "Lithographie,"
"Galvanoformung," and "Abformung," meaning lithography, electroplating, and
molding),
and elastomeric molding. Two and three dimensional structures are fabricated
by these
techniques. Further methods to create three dimensional defined channels
include track-
etching and molding techniques.
Other methods to create nano-sized obstacles include methods that involve
chemical
means such as photodeposition of colloids, self assembly of localized
polymers, and cross-
linked networks of polymers. For example, a non-linear funnel with localized
deposition of
agarose gel in the funnel can create an environment of controlled stretching.
Delivery mechanisms. Structures intended to stretch out the polymer are not
the
only ones which may be useful to place in a channel. Structures designed to
position the
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polymer favorably in one part of a channel over another are useful in ensuring
that the
polymer is fed to a particular stretching structure or to a particular
detection zone. Besides
the adding of fluid to a single channel as mentioned above (see Branched
channels) the
positioning can also be accomplished by forcing flow lines closer together.
Polymers driven
by fluid flow (induced by any of the later-cited methods such as pressure
differential and
gravity) will principally follow the fluid flow lines (in electrophoresis for
charged
biopolymers, the polymer follows the field lines, which can be similarly
modified).
Random motion can cause portions of the chain to move to an adjacent flow
line. If the
flow lines encounter a constriction or obstacle, the flow lines become closer
together around
the obstacle, leading to a greater chance that the same lateral random motion
will cause a
change in flow lines. As the flow lines return to their original spacing on
the other side of
the structure (if the channel returns to its original width), velocity
gradients between the
flow lines tend to draw the polymer toward the faster flow lines. In this way,
the formerly
random distribution of polymer can be made to shift to something more regular.
In one
1 S embodiment, for example, a large triangle in the middle of a channel with
a side
perpendicular to the channel facing downstream tends to orient polymers toward
the center;
this is because polymers formerly near the walls tend to be pulled toward the
center by the
fluid moving laterally on the downstream side of the triangle. In other
embodiments, other
shapes are used to help in positioning, such as cross-shaped obstacles,
wedges, and obstacle
fields with offsets that tend to direct larger channels at a particular side
of the channel.
While it might seem intuitive that a channel with a simple bend in it should
have a
positioning effect, the velocity gradients involved are actually quite small
and the effect by
itself is quite modest.
Methods to improve stretching in structures. In further embodiments of the
invention, the effectiveness of the shear-inducing regimes is enhanced by
increasing the
viscosity of the solution. The actual force imparted by the constriction of a
channel is
proportional to the viscosity of the solution. In some embodiments, the
viscosity of the
solution is increased by the addition of one or more viscosity-modifying
components.
Glycerol (with a viscosity of nearly 900 cP at room temperature) can be added
to an
aqueous solution in concentrations as high as 70% (w/v) if it does not react
chemically with
the polymer. Sugars, such as sucrose, xylose, and sorbitol may also be added.
Water- .
soluble polymers, such as polyethylene glycol, may also be added. In the case
of DNA,
high molecular weight polyacrylamide, polyethylene oxide or long-chain length
polysaccharides (even at concentrations as low as 0.01% by weight) can
increase the
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viscosity of aqueous solutions without modifying the structure of the DNA
being
characterized.
The viscosity may also be modified by adding an amount of the polymer being
characterized, but which will not be detected by the detection zones of the
structures. For
example, if FRET is being performed on an extrinsically labeled DNA molecule,
then
additional DNA molecules that are not extrinsically labeled may be added to
the labeled
polymer solution in order to increase the viscosity. In this way, only labeled
molecules are
detected and the unlabeled DNA serves only to modify the viscosity of the
solution, but
does not interfere with signal generation from the labeled molecules.
In another embodiment, viscosity is increased by decreasing the temperature;
pure
water, for example, nearly doubles in viscosity as it approaches the freezing
point. In
addition to increasing the viscosity, a decrease in temperature is used to
minimize Brownian
motion and extend relaxation times. There is a substantial improvement in
stretching when
an aqueous buffer solution, such as 1X TE solution (10 mM TRIS, 1 mM EDTA), is
1 S changed from ambient temperature to 4°C.
Driving forces. The driving force for moving the polymer through the
structures can
come from any means, including physical, electrical, thermal, or chemical
forces. The
simplest driving force is allowing flow to be driven by capillary action as
the first contact is
made between the sample solution and the device. While the surface energies
involved can
provide a high velocity in the channel, control of the flow in this regime is
limited.
The use of chemical potential allows for indirect, and hence limited, control.
One
advantage of setting up a concentration gradient is to provide an extremely
slow, steady
flow rate. This is accomplished by creating a large excess of a species at one
side of the
structures and consuming the diffusing species after it induces fluid flow
through the
structures to the other side, with control based on the excess concentration.
The polymer
flows through the structures along with the fluid whose flow is induced by the
migrating
species.
A preferred embodiment directly controls the flow of the fluid. In such an
embodiment; a pressure head is established on the entrance side of the
structures,
encouraging the fluid to flow to the far side, opened to atmospheric pressure
or maintained
at reduced pressure. The pressure head may come from any device imposing a
physical
force, such as a syringe pump. Currently, syringe pumps dispense up to the 100
pL/s range,
and desired flow rates in a device may be under 1 pL/s, meaning that it may be
necessary to
create a "bypass channel" with a large cross-sectional area, thus increasing
the desired flow
rate of the device and allowing control with off the-shelf equipment, with the
loss only of
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some volume of sample. In another embodiment of the pressure control system,
in devices
with a pressure drop of less than atmospheric pressure, one end of the system
is pulled with
a vacuum, literally sucking material to be stretched through the structures.
The pressure
drop required to induce flow at a desired velocity is a function of the
channel geometry
(especially the minimum cross-sectional dimension) and that velocity, but is
typically
within an order of magnitude of 10 psi for 100 micron per second flow in a
millimeter-long,
micron-deep channel which is otherwise quite wide through most of the device.
In another
embodiment, a combination of a pressure head at a first end of the channel and
a vacuum at
a second end of the channel are used to propel a polymer from the first end to
the second
end.
In yet a further embodiment, the polymer is controlled through the fluid flow
by
setting up a temperature gradient on each side of the stretching zone. Natural
convection
then creates a fluid flow through the stretching zone. Since it is much harder
to create and
control temperature gradients on the micron scale on which these devices
operate, this
1 S method, like the chemical potential method, is preferably used for very
low fluid flow.
In still another embodiment, the flow of the polymer is controlled, for
charged
polymers such as DNA, by setting up an electric field which acts on the
charges on the
polymer and not necessarily on the surrounding fluid at all (if it is
uncharged). The electric
field is preferably established by the presence of two oppositely-charged
electrodes in
solution, but entire arrays of electrodes can be used to create more
complicated or uniform
field patterns. The polymers then follow electric field lines instead of flow
lines (in some
instances an inconsequential change, depending on the physical layout of the
chip and the
charge density of the solution). This can be damaging to stretching if the
surrounding
solution contains oppositely-charged objects which flow in the opposite
direction (electro-
kinetic flow), or surface charges on the wall of the channels causing flow of
ions along the
walls (electro-osmotic flow), either of which can induce fluid flow in that
opposite direction
and impart viscous forces on the polymer. However, in a low conductivity
solution with
walls appropriately coated to avoid surface charge, opposing viscous forces
have negligible
impact on the electrophoretic driving force, allowing the polymer to proceed
through the
structures and become stretched. In addition, with an appropriately-charged
wall surface,
the electro-osmotic flow can be reversed to provide viscous forces which
assist the
electrophoretic stretching. A field strength of 1000 to 2000 V/m results in
usable polymer
velocities in the 100 micron per second range.
In the cases of electrophoresis and pressure driving forces, the devices
creating the
driving force are generally physically separated from the stretching zone. The
electrodes are
located several millimeters to multiple centimeters away from the stretching
zone, with the
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power supply located even further away. The syringe pump, while advantageous
to be as
close to the stretching zone as possible to minimize the needed pressure drop,
will tend to be
placed outside of the device because of its bulk. In fact, for the sake of
structural flexibility,
it is preferred to place only the stretching and detecting structures
themselves on a small
chip, preferably no larger than 2 cm on a side, and perhaps as small as 1 mm
square, with a
most preferred size (from the standpoint of human handling) of about 1.5 cm by
1 cm, with
a thickness of 0.2 cm. On that substrate, a variety of fluid flow channels are
located. In
such a chip, anywhere between 1 and 160 channels may be comfortably placed on
the
substrate, with 30-40 striking a good balance between having redundancy in the
case of
channel blockage or substrate flaws and having only one channel in a detection
field of view
at one time (with a typical 60x objective).
Substrates. The substrate used is selected for compatibility with both the
solutions
and the conditions to be used in analysis, including but not limited to
extremes of salt
concentrations, acid or base concentration, temperature, electric fields, and
transparence to
wavelengths used for optical excitation or emission. The substrate material
may include
those associated with the semiconductor industry, such as fused silica,
quartz, silicon, or
gallium arsenide, or inert polymers such as polymethylmetacrylate,
polydimethylsiloxane,
polytetrafluoroethylene, polycarbonate, or polyvinylchloride. Because of its
transmissive
properties across a wide range of wavelengths, quartz is a preferred
embodiment.
The use of quartz as a substrate with an aqueous solution means that the
surface in
contact with the solution has a positive charge. When working with charged
molecules,
especially under electrophoresis, it is desirable to have a neutral surface.
In one
embodiment, a coating is applied to the surface to eliminate the interactions
which lead to
the charge. The coating may be obtained commercially (capillary coatings by
Supelco,
Bellafonte PA), or it can be applied by the use of a silane with a functional
group on one
end. The silane end will bond effectively irreversibly with the glass, and the
functional
group can react further to make the desired coating. For DNA, a silane with
polyethyleneoxide effectively prevents interaction between the polymer and the
walls
without further reaction, and a silane with an acrylamide group can
participate in a
polymerization reaction to create a polyacrylamide coating which not only does
not interact
with DNA, but also inhibits electro-osmotic flow during electrophoresis.
The channels may be constructed on the substrate by any number of techniques,
many derived from the semiconductor industry, depending on the substrate
selected. These
3 S techniques include, but are not limited to, photolithography, reactive ion
etching, wet
chemical etching, electron beam writing, laser or air ablation, LIGA, and
injection molding.
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A variety of these techniques applied to polymer-handling chips have been
discussed in the
literature, including Harnson et al. (Analytical Chemistry 1992 (64) 1926-
1932), Seiler et
al. (Analytical Chemistry 1993 (65) 1481-1488), Woolley et al. (Proceedings of
the
National Academy of Sciences November 1994 (91) 11348-11352), and Jacobsen et
al.
(Analytical Chemistry 1995 (67) 2059-2063).
Additional considerations. In preferred embodiments of the invention, the
velocity
in a given planar height of the channel is substantially uniform in a
rectangular channel.
This is true when the height is significantly less than the width of the
channel, such that the
no-slip condition at the wall results in a viscosity-induced parabolic
velocity profile that is
significant in the height axis, leaving only a small boundary region of slower
flow in the
width axis. An aspect (width/height) ratio of approximately 10 or greater is
required for
such embodiments, according to the lubrication theory approximation (Deem
Analysis of
Transport Phenomena, New York: Oxford University Press, 1998. 275-278).
Furthermore,
a small height assists in detection when using a microscope objective in an
optical system.
Typical objectives may have a depth of focus of 500 nm to several microns, so
while the
depth of channel could be anywhere from 50 nm to 100 pm as long as the aspect
ratio is
kept above 10 to accommodate the polymer being analyzed, the preferred
embodiments
have channel depths of 200 nm to 1 ~m such that all material passing by in a
channel will be
in focus and accurately observed.
The invention also encompasses embodiments where the channels are not planar,
and are fabricated with three dimensional channel fabrication techniques. In
such
embodiments, constant shear is induced not only from side walls, but from a
gradient in
channel height. Similarly, in further embodiments, combinations of structures
have one
force acting on one axis and the other force acting in the other. In some such
embodiments,
an obstacle field spans the width of the channel as its height decreases in a
tapered shape. In
other embodiments, a tortuous, inward-spiral design in a single plane which
also decreases
in channel width is used to impart shear forces which feed at its center into
a vertical exit
from the device through a hole in the bottom of the material, with detection
near the
entrance to the hole. When structures exist in the vertical dimension, gravity
is used in
some embodiments to help create velocity differentials in the fluid. (Notably,
gravity alone
is not adequate to stretch a polymer or move it significantly with a flow
since the force on a
100 kD polymer is barely more than 10-18 N; any effect of gravity will be felt
by the
molecule through viscous forces.)
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6. EXAMPLES
6.1 EXAMPLE 1: FABRICATION OF A CHIP FOR STRETCHING DNA AND ITS
USE IN AN APPARATUS FOR DETECTING FLUORESCENE EMISSION FROM
LABELED DNA
Experimental Apparatus. A sensitive optical apparatus for detection is shown
in FIG. 24.
The apparatus utilizes confocal fluorescence illumination and detection.
Confocal
illumination allows a small optical volume (of the order of femtoliters) to be
illuminated.
Both Rayleigh and Raman scattering are minimized using a small probe volume.
The beam
from a 1 mW argon ion laser is passed through a laser line filter (514 nm),
directed to a
dichroic mirror, through a 100x 1.2 NA oil immersion objective, and to the
sample. The
fluorescent tag on the DNA can be one of several dyes including Cy-3,
tetramethylrhodamine, rhodamine 6G, and Alexa 546. In addition, intercalator
dyes can be
used such as TOTO-3 (Molecular Probes). The fluorescence emission from the
sample is
passed through a dichroic, a narrow bandpass (e.g.. Omega Optical), focused
onto a 100 pm
pinhole, passed through an aspheric lens, and ultimately focused onto an
avalanche
photodiode in photon counting mode (EG&G Canada). The output signal is
collected by a
multichannel scalar (EG&G) and analyzed using a Pentium III type computer. The
confocal
app~'atus is appropriate for quantitative applications involving time-of
flight. Such
applications include measuring distances on the DNA, detecting tagged
sequences, and
determining degrees of stretching in the DNA. Single fluorescent molecules can
be detected
using the apparatus. For applications requiring imaging, an apparatus using an
intensified
CCD (ICCD, Princeton Instruments) mounted on a microscope is appropriate.
Fabrication of the chin. A set of constant-shear channels with a design shear
rate of 0.085 /s
preceded by two rows of 1.5 micron obstacles on a 2 micron pitch were created
in a 0.090
inch thick quartz substrate by photolithography and e-beam methods. The
substrate was
first cleaned by placement in an RCA solution (5 parts deionized water to 1
part 30%
a~onium hydroxide/30 % hydrogen peroxide, the latter two from Sigma Chemical
Co.,
St. Louis, MO) heated to 80°C for twenty minutes, and dried under a
nitrogen stream.
Shipley S 1813 photoresist diluted in a 2:1 ratio with type R thinner
(Shipley, Newton, MA)
was then spun onto the quartz surface at 3250 rpm for 45 seconds in a spin
coater and cured
at 90°C in an oven for 0.5 hours. The coarse constant-shear pattern was
then contact
printed onto the surface by a 12 s exposure to a mercury lamp, e.g., in a
contact aligner from
Carl Zeiss, Germany, followed by a 30 s rinse under 351 developer (Shipley)
diluted in a
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5:1 ratio with deionized water, further rinses in deionized water, and drying
under a nitrogen
stream. After a 10 s UV-ozone cleaning, the substrate was exposed to a 40
minute etch by
CHF3 in a Reactive Ion Etch (RIE) machine. After another wash in RCA solution,
a
solution of polymethylmethacrylate (650 MVO diluted to 3% in chlorobenzene was
spun
$ onto the surface at 2000 rpm for 45 seconds in a spin coater. The coating
was cured for one
hour in an oven at 180°C, and a 60 A layer of chrome was added in an
evaporator. An e-
beam write was performed to make the fine structures, e.g., the rows of
obstacles, followed
by a chrome etch in the REI machine and a deionized water rinse. The substrate
was then
immersed for 90 seconds in a 2:1 v/v solution of isopropyl alcohol:methyl-
isobutyl ketone
heated to 21 °C for developing, followed by another LJV-ozone cleaning.
Another CHF3
etch in the REI machine followed by a wash with RCA solution were then
performed.
Cover slips (Fisher Scientific, Pittsburgh, PA) of dimensions 45 mm x 50 mm x
0.1 S
mm were rinsed with deionized water and dried under a nitrogen stream. A 10:1
w/w ,
solution of RTV615A:RTV615B silicone (General Electric, Schenectady, NY) was
spun
onto the cover slips for 60 seconds at 4000 rpm in a spin coater and was then
cured at 80°C
for two hours. A slab of silicone with a hole where the chip is mounted was
placed on a
cover slip, which was then exposed to a 30 W plasma cleaner for 50 seconds in
order to
make the surface hydrophilic. The silicone slab was then removed and the cover
slip was
rinsed in deionized water and dried under nitrogen. The fully-prepared chip
was then
carefully mounted onto the cover slip.
Apparatus for monitoring-object-dependent impulses from stretched DNA. As
shown in
FIG. 25, the delivery system consists of a polymer supply 151, which is driven
by a syringe
pump 150 through a chip 152 (see above) where the polymer is stretched out and
excited by
a laser beam from laser 154 which is detected by optical detector 153 and
analyzed by
computer 155 that also controls the pump 150 and detector 153.
Monitoring fluorescence emission in stretched DNA. Coliphage T4 DNA (Sigma,
St.
Louis, MO) was labeled by the addition of 4040-1 at a S:1 (base-pair:dye)
ratio, incubation
for one hour, and dilution by a factor of 50,000 in O.SX TBE electrophoresis
buffer (45mM
TRIS, 32.3 mM boric acid, and 1.25 mM EDTA at pH 8.3, all from Sigma, St.
Louis, MO).
One microliter of sample was then pipetted onto the cover slip immediately
next to
the chip, where it was loaded into the channels by capillary action. The chip
and cover slip
were placed on the stage of a fluorescence microscope (Microphot series from
Nikon)
equipped with a 60X piano apo lens (from, e.g., Nikon or Carl Zeiss).
Excitation was from
a mercury arc lamp, with a Nikon B2A filter set ensuring adequate excitation
near the 490
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nm peak excitation of YOYO-1. Emission above 520 nm was passed through the B2A
filter
set and captured by a silicon-intensified camera (Hammamatsu's C2400-08) or by
a CCD
camera. The image from the camera was output to a computer through an image
capture
card (such as the PCI-1408 from National Instruments, Austin, TX) and analyzed
with
image processing software, which was a custom-written routine that identified
the DNA on
the screen based on its brightness against background and counted pixels to
determine
polymer length.
Various DNA molecules were observed in this apparatus (FIG. 26). A DNA
molecule of approximately 190 kb (63 microns) is shown stretching out in the
constant-
shear section of the chip in FIG. 27 (a-g). A DNA fully stretched out in the
chip is shown in
FIG. 28. This molecule was measured at 139 microns, or 535 kb.
Data. A small (half microliter) sample of T4 DNA (Sigma) stained with YOYO-1
(Molecular Probes) was loaded into a chip with a rectangular funnel section
incorporating
posts and run under capillary action. The sample was excited with a 100 W Hg
lamp and
observed with a SIT camera (Hammatsu C2400-08). The video signal from the
camera was
fed to a video capture card in a Pentium-class computer running custom LabView
softv~are
that determined the length of a piece of DNA in pixels based on its velocity
and time spent
in the region of interest. Lengths of less than 30 microns were considered to
be fragments
and were discarded automatically, which led to the obtaining of only ten data
points in the
approximately two minute run of sample. Using a known conversion for the level
of
magnification, the DNA were found to be 50.6 pm long, with a range between 42
and 62
pm. A histogram is shown in FIG. 29. The length is somewhat shorter than the
expected
value of 71.1 pm for a stained 164 kbp T4 DNA, implying the stretching in this
design was
not fully complete.
6.2 EXAMPLE 2: STRETCHING OF PHAGE LAMBDA DNA USING
APPARATUSES OF THE INVENTION
Two different apparatuses were used to obtain the data shown in FIG. 30 (a)
and 30
(b). The apparatus shown in FIG. 20 was used to obtain the data shown in FIG.
30 (b). The
apparatus used to obtain the data in Fig. 30 (a) had the same channel
boundaries as the
apparatus used to obtain the data shown in FIG. 30 (b) (i.e., the ratio of the
sizes of the two
tapered regions of the two-funnel apparatus were identical), except that there
were no posts
present in the structure.
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A fused silica wafer (Hoya Corp., San Jose, CA) was etched with the pattern in
FIG.
20 by a contractor using photolithographic methods described above. The wafer
was diced
into 1 cm by 2 cm chips using a dicing saw (e.g. from Disco Corp., Santa
Clara, CA), and a
fused silica cover slip (e.g. from Esco, Oak Ridge, NJ) was attached by
thermal bonding.
Double stranded lambda DNA (Promega, Madison, WI) having a uniform length of
48.5 kilobases (i.e., an anticipated stretched length of 16-17 microns), was
labeled by
addition of a like amount of 3 ~M TOTO-3 iodide (Molecular Probes, Eugene OR)
intercalating dye and then diluted by a factor of approximately 50,000 in 1X
TE buffer (10
mM TRIS, and 1 mM EDTA at pH 8.0, all from Sigma, St. Louis, MO). The
anticipated
stretch length of lambda DNA stained with an intercalating dye is 21 pm
(approximately
30% longer than unstained DNA) for the double stranded 48.5 kilobase DNA
sample used
here.
The chip and cover slip were placed on the microscope stage of a fluorescence
microscope (e.g., Microphot series from Nikon) equipped with a 100 X plano apo
lens (e.g.,
from Nikon, Carl Zeiss) and a filter set optimized for use with TOTO-3 (e.g.,
XF-47 from
Omega Optical, Brattleboro, VT). Excitation was from a 633 nm HeNe laser
(e.g., from
Melles Griot) focused on two spots aligned on the same flow line within the
microchannel.
The sample was loaded at the entrance of the channels by capillary action and
the flow
sustained using a vacuum at the other end of the chip (created by a vacuum
pump from, e.g.,
welch Vaccum, Skokie, IL). As DNA molecules passed through the laser spots,
emission
above 650 nm was passed through the filter set and captured by a pair of
confocal detectors
aligned above the spot. Time of flight between the detectors was used to
determine
velocity, which was used along with residence time in a laser spot to
calculate the lengths of
the molecules.
The results of these experiments indicate that the two-funnel apparatus
comprising
posts stretches 48.5 kilobases of double-stranded, dye-stained lambda DNA to a
length of
approximately 19.5 pm (FIG. 30 (b)), whereas the two-funnel apparatus without
posts only
stretches the DNA to a length of about 10 pm (FIG. 30 (a)). Thus, although
there is
stretching of the DNA in the tapered channel without posts, on average, the
DNA is
stretched only to somewhat more than half of its full length and very few
individual
molecules are fully stretched, as is evidenced by the wide distribution of the
histogram in
FIG. 30 (a). By contrast, in the structure having a post field combined with a
downstream
tapered channel, the molecules are, on average, stretched to close to full
length and the
majority of molecules are within 20% of their anticipated fully-stretched
length. Therefore,
the two-funnel apparatus with posts stretches DNA better than the same
apparatus without
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WO 01/13088 PCT/US00/22253
posts. Furthermore, this apparatus stretches the polymers more uniformly and
efficiently
than the two-funnel structure without posts.
7. REFERENCES CITED
All references cited herein are incorporated herein by reference in their
entirety and
for all purposes to the same extent as if each individual publication or
patent or patent
application was specifically and individually indicated to be incorporated by
reference in its
entirety for all purposes.
Many modifications and variations of this invention can be made without
departing
fTOm its spirit and scope, as will be apparent to those skilled in the art.
The specific
embodiments described herein are offered by way of example only, and the
invention is to
be limited only by the terms of the appended claims, along with the full scope
of equivalents
to which such claims are entitled.
20
30
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