U.S. patent application number 13/862129 was filed with the patent office on 2014-08-21 for device for stretching a polymer in a fluid sample.
This patent application is currently assigned to PathoGenetix, Inc.. The applicant listed for this patent is PathoGenetix, Inc.. Invention is credited to Joshua W. Griffis, Robert H. Meltzer.
Application Number | 20140234985 13/862129 |
Document ID | / |
Family ID | 49515076 |
Filed Date | 2014-08-21 |
United States Patent
Application |
20140234985 |
Kind Code |
A9 |
Meltzer; Robert H. ; et
al. |
August 21, 2014 |
DEVICE FOR STRETCHING A POLYMER IN A FLUID SAMPLE
Abstract
The invention provides structures and methods that allow
polymers of any length, including nucleic acids, to be stretched
into a long, linear conformation for further analysis.
Inventors: |
Meltzer; Robert H.;
(Chelmsford, MA) ; Griffis; Joshua W.; (Gardner,
MA) |
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Applicant: |
Name |
City |
State |
Country |
Type |
PathoGenetix, Inc.; |
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US |
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Assignee: |
PathoGenetix, Inc.
Woburn
MA
|
Prior
Publication: |
|
Document Identifier |
Publication Date |
|
US 20130309780 A1 |
November 21, 2013 |
|
|
Family ID: |
49515076 |
Appl. No.: |
13/862129 |
Filed: |
April 12, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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61625745 |
Apr 18, 2012 |
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61784399 |
Mar 14, 2013 |
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Current U.S.
Class: |
436/501 ;
422/500; 436/174 |
Current CPC
Class: |
G01N 1/286 20130101;
B01L 2300/0867 20130101; B01L 3/502761 20130101; Y10T 436/25
20150115; B01L 2200/0663 20130101; B01L 2300/0816 20130101 |
Class at
Publication: |
436/501 ;
422/500; 436/174 |
International
Class: |
G01N 1/28 20060101
G01N001/28 |
Claims
1. A device for stretching at least one polymer in a fluid sample,
said device 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, said
tapered channel comprising: a first zone having a first tapered
shape; a second zone having a second tapered shape, wherein the
second tapered shape is different than the first tapered shape; and
wherein said at least one polymer, when present, moves along said
tapered channel from said first end to said second end and is
stretched.
2. The device of claim 1, wherein the first tapered shape includes
an increasing strain rate taper.
3. The device of claim 2, wherein the second tapered shape includes
a constant strain rate taper.
4. The device of claim 2, wherein a width w(x) of the tapered
channel in the first zone is defined by the following equations: w
( x ) = 1 ( bx + c ) 2 b = 1 l 1 ( 1 w 2 - 1 w 1 ) c = 1 w 1
##EQU00011## wherein l.sub.1 is the length of the first zone,
w.sub.1 is the width of the tapered channel at the first end of the
tapered channel, and w.sub.2 is the width of the tapered channel at
a transition which separates the first zone from the second
zone.
5. The device of claim 2, wherein a width w(x) of the tapered
channel in the first zone is defined by the following equations: w
( x ) = 2 w i v i ax 2 = F 1 x 2 F 1 = 2 v x w x x . x ##EQU00012##
wherein x is distance along the channel, w.sub.i is channel width
at arbitrary position i, v.sub.i is fluid velocity at arbitrary
position i, F1 is a geometrical taper coefficient for an increasing
strain rate funnel, v.sub.x is fluid velocity at distance x,
w.sub.x is channel width at distance x, and {dot over
(.epsilon.)}.sub.x is strain rate at distance x.
6. The device of claim 3, wherein the width w(x) of the tapered
channel in the second zone is defined by the following equations: w
( x ) = w 2 1 + x a a = l 2 w 2 w 3 - 1 ##EQU00013## wherein
l.sub.2 is the length of the second zone, w.sub.2 is the width of
the tapered channel at a transition which separates the first zone
from the second zone, and w.sub.3 is the width of the tapered
channel at the second end of the tapered channel.
7. The device of claim 3, wherein the width w(x) of the tapered
channel in the second zone is defined by the following equations: w
( x ) = F 2 x F 2 = v x w x . ##EQU00014## wherein x is distance
along the tapered channel, F2 is a constant strain rate taper
coefficient, v.sub.x, is fluid velocity at distance x, w.sub.x, is
channel width at distance x, and .epsilon..sub.x is strain rate at
distance x.
8. The device of claim 1, wherein the elongation structure is
formed on a chip.
9. The device of claim 1, further comprising a delivery region for
delivering said at least one polymer in said fluid sample to said
elongation structure.
10. The device of claim 9, wherein said delivery region comprises a
sample loading port and a delivery channel, said delivery channel
leading into the elongation structure.
11. The device of claim 10, further comprising at least one buffer
channel leading into the elongation structure.
12. The device of claim 11, wherein the at least one buffer channel
comprises at least two opposing buffer channels leading into the
elongation structure.
13. A device for stretching at least one polymer in a fluid sample,
said device comprising: an elongation structure, wherein said
elongation structure comprises a tapered channel, said tapered
channel having a width w(x) which decreases from a first end to a
second end, the tapered channel comprising: a first zone having a
first shape; a second zone having a second shape, wherein the
second shape is different than the first shape; and a transition
which separates the first zone from the second zone; wherein the
width w(x) of the tapered channel in the first zone is defined by
the following equations: w ( x ) = 1 ( bx + c ) 2 b = 1 l 1 ( 1 w 2
- 1 w 1 ) c = 1 w 1 ##EQU00015## wherein l.sub.1 is the length of
the first zone, w.sub.1 is the width of the tapered channel at the
first end of the tapered channel, and w.sub.2 is the width of the
tapered channel at the transition; wherein the width w(x) of the
tapered channel in the second zone is defined by the following
equations: w ( x ) = w 2 1 + x a a = l 2 w 2 w 3 - 1 ##EQU00016##
and wherein l.sub.2 is the length of the second zone, w.sub.2 is
the width of the tapered channel at the transition, and w.sub.3 is
the width of the tapered channel at the second end of the tapered
channel.
14. The device of claim 13, wherein the elongation structure is
formed on a chip.
15. The device of claim 13, further comprising a delivery region
for delivering said at least one polymer in said fluid sample to
said elongation structure.
16. The device of claim 15, wherein said delivery region comprises
a sample loading port and a delivery channel, said delivery channel
leading into the elongation structure.
17. The device of claim 16, further comprising at least one buffer
channel leading into the elongation structure.
18. The device of claim 17, wherein the at least one buffer channel
includes two opposing buffer channels leading into the elongation
structure.
19. A method of stretching at least one polymer in a fluid sample,
the method comprising: delivering a fluid sample into the device
recited in claim 1; stretching the at least one polymer in the
first zone of the tapered channel; and maintaining the tension on
the at least one polymer in the second zone of the tapered
channel.
20. A method comprising: moving a polymer through a tapered channel
past at least one detection station, the tapered channel having a
constant strain portion, detecting an object-dependent impulse that
conveys information about structural characteristics of the
polymer; obtaining an observed trace based on the detected impulse,
wherein the observed trace is an intensity versus time trace;
applying an acceleration correction to the observed trace; and
obtaining a corrected intensity versus distance trace from the
application of the acceleration correction to the observed
trace.
21. The method of claim 20, wherein the acceleration correction is
defined by: .DELTA. x c .apprxeq. - L 2 2 x tag .tau. ( 1 - .tau. )
##EQU00017## wherein: .DELTA.x.sub.c is a difference in a distance
a molecule would travel assuming a constant velocity compared to a
distance traveled in an accelerating flow experienced by the
polymer; L is a length of the molecule; x.sub.tag is a distance of
a point of detection from a theoretical asymptotic origin of the
constant strain portion of the channel; and .tau. is a relative
time for the molecule to transit a point of detection.
Description
RELATED APPLICATIONS
[0001] This application claims the benefit under 35 U.S.C.
.sctn.119(e) of U.S. Provisional Application Ser. No. 61/625,745,
entitled "DEVICE FOR STRETCHING A POLYMER IN A FLUID SAMPLE" filed
on Apr. 18, 2012, and U.S. Provisional Application Ser. No.
61/784,399, entitled "DEVICE FOR STRETCHING A POLYMER IN A FLUID
SAMPLE" filed on Mar. 14, 2013, the entire contents of both of
which are incorporated herein by reference.
FIELD
[0002] The present invention is directed to a device for stretching
at least one polymer in a fluid sample, where the device includes a
tapered channel.
SUMMARY
[0003] According to one aspect, a device for stretching at least
one polymer in a fluid sample is provided. The device includes an
elongation structure, where said elongation structure includes a
tapered channel that decreases in width from a first end to a
second end. The tapered channel includes a first zone having a
first tapered shape, and a second zone having a second tapered
shape, where the second tapered shape is different than the first
tapered shape. The at least one polymer, when present, moves along
said tapered channel from said first end to said second end and is
stretched. The first tapered shape may include an increasing strain
rate taper, and the second tapered shape may include a constant
strain rate taper.
[0004] According to another aspect, a method includes moving a
polymer through a tapered channel past at least one detection
station, where the tapered channel has a constant strain portion.
The method also includes detecting an object-dependent impulse that
conveys information about structural characteristics of the
polymer, such as a nucleotide sequence or hybridization of the
polymer to a sequence-specific probe, and obtaining an observed
trace based on the detected impulse, where the observed trace is an
intensity versus time trace. The method also includes applying an
acceleration correction to the observed trace and obtaining a
corrected intensity versus distance trace from the application of
the acceleration correction to the observed trace.
[0005] Various embodiments of the present invention provide certain
advantages. Not all embodiments of the invention share the same
advantages and those that do may not share them under all
circumstances.
[0006] Further features and advantages of the present invention, as
well as the structure of various embodiments that incorporate
aspects of the invention are described in detail below with
reference to the accompanying drawings.
BRIEF DESCRIPTION OF FIGURES
[0007] The foregoing and other objects and advantages of the
invention will be appreciated more fully from the following
figures, wherein like reference characters designate like features,
in which:
[0008] FIG. 1A is a schematic representation of a microfluidic chip
with an elongation structure according to one embodiment;
[0009] FIG. 1B is a schematic representation of a tapered channel
with two zones according to one embodiment;
[0010] FIG. 1C is a schematic representation of a DNA sample being
stretched in a tapered channel according to one embodiment;
[0011] FIG. 1D includes representative fluorescent signals
generated from a single stretched DNA molecule;
[0012] FIG. 2 includes a chart which includes data from prior art
tapered funnels and also data from two embodiments of the present
invention;
[0013] FIG. 3A is a schematic representation of a DNA sample being
stretched in a tapered channel according to one embodiment;
[0014] FIGS. 3B-3F represent single molecule stretching
morphologies;
[0015] FIGS. 4A-4C illustrate molecule extension in increasing
fluid velocity;
[0016] FIGS. 5A-5D illustrate the effect of channel depth on single
molecule morphology
[0017] FIGS. 6A-6B illustrate improved DNA stretching efficiency in
constant strain rate detection funnels;
[0018] FIG. 7A-7C illustrate the accelerated corrected site
specific fluorescence traces; and
[0019] FIGS. 8A-8D illustrates the acceleration correction in
constant tension fluidics.
DETAILED DESCRIPTION OF INVENTION
[0020] The present invention provides devices 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 device. 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, unlimited lengths of DNA molecules can be
monitored, 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, may be achieved.
[0021] An extended labeled polymer may be moved past at least one
detection 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. As used
herein, a "detection station" is a detection arrangement that
detects physical quantities and/or properties of a polymer. Such
properties include emission of energy or light of one or more
wavelengths, wherein such emission is the result of laser
interaction with the polymer or a sequence-specific probe
hybridized to the polymer, including one or more fluorophores
attached thereto. A detection station includes detection
instruments, such as a camera, CCD camera or a silicon-intensified
camera, a laser and optical detector combination, a light and
optical detector combination, confocal fluorescence illumination
and detection, or any other suitable detection instrument. This
process is discussed in greater detail in U.S. Pat. No. 6,696,022
which is herein incorporated by reference in its entirety.
[0022] The devices 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 occurring 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 environment
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.
[0023] 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.
[0024] The genetic information of all cellular and some viral
organisms is encoded in long polymeric chains of DNA. The ultimate
resolution of base-by-base DNA sequence is unique for each
organism. The identity and interrelatedness of organisms however
can be determined by lower resolution detection of repeated
elements in their genetic code. Genomic technology is capable of
optically characterizing the length of individual restriction
endonuclease digested molecules, and identifying the spatial
location of fluorescent labels tagged to repeated sequence motifs
on each molecule of DNA. Genomic technology has wide ranging
applicability in identification of bacterial genomic DNA.
[0025] The ability to optically resolve discrete sites of
fluorescent labeling requires physical manipulation of each
molecule such that it is stretched into a fully elongated,
linearized conformation. Previous studies have demonstrated the
efficacy of stretching long fragments of bacterial genomic DNA in
microfluidic devices with combined shear and elongational flows. In
two-dimensional tapered funnels etched to 1 .mu.m depth, DNA
stretching is strongly affected by the funnel taper, and
confinement of the DNA to the 1 .mu.m deep channel serves to
pre-stretch the DNA, resulting in more uniform stretching. This
ability to elongate single fragments of DNA in continuous flow
conditions is highly advantageous over alternative DNA elongation
methods because of the ease of sample manipulation, high sample
throughput, and simplicity of the microfluidic device.
[0026] The sensitivity of Genomic technology as a detection and
identification method is limited by DNA detection throughput,
resolution of length information, and the range of fragment lengths
that can be fully extended. By careful consideration of the
stretching funnel geometry, Applicants have significantly improved
these factors. First, as set forth below, the relationship between
the effects of fluid velocity, funnel taper, and fragment length
range have been correlated to allow for design of funnel geometries
that stretch a desired range of fragment lengths at any desired
fluid velocity. Second, as set forth below, novel funnel geometries
have been designed that maintain the tension of stretched DNA
molecules during detection. This increases analyzable molecule
throughput by stretching a higher percentage of molecules to the
fully extended state and eliminating relaxation and shear-induced
molecular tumbling during detection. Finally, funnel geometries
that maintain tension in the DNA detection channel dictate
continuous acceleration of the molecule. Correct molecule analysis
requires corrections of this acceleration. Because the acceleration
profile is dictated by the funnel geometry, the acceleration
correction can be determined from the funnel taper.
[0027] As set forth in greater detail in U.S. Pat. No. 6,696,022,
it is generally desirable for the polymer sample to be in a
stretched elongated state. However, a polymer sample is typically
in a lower-energy, more coiled conformation. Therefore, aspects of
the present invention are directed to devices with elongation
structures that include tapered channels that are designed to
stretch the polymer sample and then cause the polymer sample to
remain in a stretched conformation.
[0028] As set forth in greater detail below, aspects of the present
invention are directed to devices with tapered channels with at
least a first zone having a first tapered shape and a second zone
having a second tapered shape, where the second tapered shape is
different than the first tapered shape. As discussed below, in one
illustrative embodiment, the first tapered shape includes an
increasing strain rate taper, and the second tapered shape includes
a constant strain rate taper. The increasing strain rate taper may
be configured to stretch, straighten out and/or elongate the
polymer sample. The constant strain rate taper may be configured to
substantially maintain the elongated shape of the polymer sample to
prevent the polymer from returning to a more coiled and/or hairpin
shape.
[0029] By way of background, as discussed in U.S. Pat. No.
6,696,022, a constant shear rate, or change in average velocity
with distance in the channel, is defined as S:
.differential.u/.differential.x=S
[0030] where x is the distance down a substantially rectangular
charnel, and u is the average fluid velocity in the x direction,
which is computed from the overall fluid flow (O) and the cross
sectional area, A, of the channel as follows:
u=Q/A
[0031] In one embodiment where the channel cross-section is
rectangular, the channel may 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:
u=Q/HW
[0032] Applying the boundary condition that the fluid flow must be
continuous (i.e., incompressible), Q is constant. Hence, u 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:
S=.differential.u/.differential.x=Q/H
.differential./.differential.x(1/W)=(-Q/HW.sup.2)(dW/dx)
dW/dx=(-SH/Q)(W.sup.2)
[0033] Integrating this expression, it is found that:
W=(SHx/Q+C).sup.-1
where C is a constant of integration determined by the original
width of the channel (boundary condition). 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 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, t.sub.yz, required to compute
the force is easily expressed in terms of the shear rate:
F=.intg..intg.-tyz,dzdx=.intg..intg.-.mu.(du/dx)dzdx=.intg..intg.-.mu.Sd-
zdx
where .mu. 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=.mu.HLS
where L is the length of the channel wall, approximately the length
of the channel in which 15 the constant shear is maintained.
[0034] 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 a tapered channel
(i.e. a funnel).
[0035] Funnel structures are tapered channels that apply
elongational forces in a regular and continuous manner as the
polymer flows down the channel. The particular elongational forces
are defined by the type of channel structure and shape. The channel
may include a tapered channel that begins at a given width and
continuously decreases to a second width, creating an increasing
elongational force in the funnel portion of the channel defined
by:
du/dx=(-Q/H)(dW/dx)(1/W.sup.2)
[0036] In one embodiment of the invention, for at least a portion
of the channel, the width of the channel 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.degree. and 75.degree., with a most preferred
value of 26.6.degree. for DNA in a low viscosity solution such as
TE (10 mM TRIS, 1 mM EDTA) buffer, pH 8.0. 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, and in one embodiment, values
of 50 microns and 5 microns, respectively, for DNA.
[0037] In one embodiment, at least a portion of the channel may
also be configured such that the width decreases at an increasing
rate as fluid passes down the channel, resulting in an increase in
strain rate as the channel is traversed. Such tapers may 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 elongational forces
large enough to cause the polymer to stretch in the taper and will
not encounter elongational 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. The inventors have
appreciated that, in situations involving pressure driven fluid
flow, increasing strain rate tapers may yield uniform and
reproducible elongation compared to sudden-onset or continuous
strain-rate tapers. The gradual onset of polymer deformation
achieved in increasing strain rate tapers allows for more thorough
sampling of conformational space for each polymer, thus avoiding
trapping of molecules in partially extended states.
[0038] In one embodiment, at least a portion of the tapered channel
is designed such that the strain rate is constant. The value of the
constant strain 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.
[0039] The strain rate of the funnel can be determined by measuring
the distance between two known points on a strand of DNA. For
example, concatamers of .lamda. DNA are used as standards for
elongational 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 .lamda. DNA molecule (48 kilobases). The physical distance
between the probes is determined using video microscopy or
time-of-flight measurements. The physical distance for .lamda. DNA
in native solution is 14.1 This value is compared with the actual
measured physical distance. For instance, if the measured distance
is 15.0 .mu.m, then the strain rate can be calculated from the
amount of stretching that the DNA has experienced in the stretching
structures. The predicted elongational force on the DNA, as
measured by the velocity of the DNA and the dimensions of the
channel, is matched with the elongation of the DNA and its
intrinsic non-linear stiffness.
[0040] Now turning to the figures, as shown in FIG. 1, in one
illustrative embodiment, the device 10 includes an elongation
structure that is formed into a chip. As shown, the device 10 may
include four ports and may include a sample loading port 30, two
sheath buffer channels 40, 50, the elongation structure 60 (may
also be called the DNA stretching funnel), and a waste port 70. As
illustrated, in one embodiment, there is a delivery channel 32
between the sample loading port 30 and the elongation structure,
and there are two opposing buffer channels 40, 50 that also lead
into the elongation structure.
[0041] The DNA stretching funnel 60 geometry was optimized based
upon experimental results. In one illustrative embodiment, the
structure of the funnel 60 is divided into two distinct regions or
zones. The first zone 62 may be defined as the stretching portion
of the funnel, and the second zone 64 may be defined as the
detection region. The first zone 62 may have a first tapered shape,
and the second zone 64 may have a second tapered shape, different
from the first zone. Distinct taper definitions may be required for
each region. The overall funnel geometry may therefore be fully
described by three characteristic widths (w.sub.1=width of first
end of tapered channel, w.sub.2=width of tapered channel at
transition between first zone 62 and second zone 64, and
w.sub.3=width of tapered channel at the second end of the tapered
channel), two characteristic lengths (l.sub.1,=length of first
zone, and l.sub.2=length of second zone), and two taper definition
equations. These geometries are detailed in FIG. 1B.
[0042] Previous studies have demonstrated that DNA stretching is
most uniform when the initial DNA extension occurs gradually in an
increasing-strain rate funnel. In some embodiments, the geometry of
the first zone 62 (the stretching portion) of the funnel may be
described by the following equations:
w ( x ) = 1 ( bx + c ) 2 b = 1 l 1 ( 1 / w 2 - 1 / w 1 ) c = 1 / w
1 Equation 1 ##EQU00001##
Where the width of the channel (w(x)) is a function of the distance
along the funnel (l.sub.1), the initial funnel width (w.sub.1) and
the width at the transition between the stretching and detection
portions of the funnel (w.sub.2).
[0043] In some embodiments, the geometry of the first zone 62 of
the funnel may be described by the following equations:
w ( x ) = 2 w i v i .alpha. x 2 = F 1 x 2 F 1 = 2 v x w x x . x
Equation 2 ##EQU00002##
[0044] Where the width of the channel w(x) is a function of the
distance along the detection channel (x), the width at arbitrary
position i (w.sub.i), and the fluid velocity at arbitrary position
i (v.sub.i). F.sub.1, which describes the geometrical taper
coefficient for an increasing strain rate funnel, is a function of
the distance along the detection channel (x) the fluid velocity at
distance x (v.sub.x), the funnel width at distance x (w.sub.x), and
the strain rate at distance x ({dot over (.epsilon.)}.sub.x). The
funnel taper geometry can be solved to provide a desired strain
rate at any given fluid velocity.
[0045] Previously described DNA stretching funnels utilized a
parallel walled detection channel, which imposed a
constant-velocity fluid profile in this region. For such funnels,
w.sub.3=w.sub.2. This type of second zone 64 configuration is
illustrated in FIG. 1B. In one experimental study, a novel
detection channel geometry was also investigated, in which the
increasing strain rate funnel transitions smoothly into a tapered
detection channel with a constant-strain rate taper. This unique
second zone 64 tapered configuration is also illustrated in FIG.
1B. In some embodiments, the geometry of the second zone 64 (the
detection region) of the funnel may be described by the following
equations:
w ( x ) = w 2 1 + x a Equation 3 a = l 2 w 2 w 3 - 1
##EQU00003##
Where the width of the channel w(x) is a function of the distance
along the detection channel (x), the width at the interface with
the stretching portion of the funnel (w.sub.2), and the final
funnel width (w.sub.3).
[0046] In some embodiments, the geometry of the second zone 64 of
the funnel may be described by the following equations:
w ( x ) = F 2 x F 2 = v x w x . Equation 4 ##EQU00004##
Where the width of the channel w(x) is a function of the distance
along the detection channel (x) and the constant strain rate taper
coefficient F.sub.2. F.sub.2 is a function of the fluid velocity at
distance x (v.sub.x), the funnel width at distance x (w.sub.x), and
the strain rate (.epsilon..sub.x).
[0047] FIG. 1C illustrates a DNA sample passing through the
elongation structure 60 and being stretched in the tapered channel.
Several funnel profiles were investigated to determine the effects
of geometry on DNA extension under multiple operating conditions.
The tested funnel parameters are summarized in FIG. 2.
[0048] In one embodiment, sheathing buffer flows were used to
constrain the DNA stream to the center of the stretching funnel. A
fluidic circuit model was used to design the dimensions of
resistive elements in the microfluidic structure. The driving
constraint was to normalize the width of the DNA stream at the
nominal point of detection to 0.25 .mu.m. This served multiple
purposes; the DNA stream could precisely be targeted to the
projected laser points in the detection channel, and all DNA
molecules were subjected to a uniform flow stream in the center of
the funnel, thus avoiding variations in velocity and fluidic path
length near the outer walls of the funnel. Operational parameters
for tested devices, including predicted driving pressures and bulk
flow through the DNA injector path were derived from the fluidic
models.
[0049] The microfluidic chips were fabricated by Micralyne Inc.
(Edmonton, Alberta, Canada). All channels were etched in 500 .mu.m
thick fused silica to a depth of 1 or 2 .mu.m by reactive ion
etching. Through holes for fluidic access were machined by
ultrasonic drilling and channels were sealed by fusion bonding to a
170 .mu.m thick cover wafer. The finished wafers were then diced to
form individual devices.
[0050] Each chip to be tested was bonded to a custom acrylic
manifold (Connecticut Plastics, Wallingford, Conn.) using
UV-curable adhesive (Dymax 140M, Dymax Corporation, Torrington,
Conn.). To prepare chips for use, the devices were first wetted
with 100 mM NaOH to remove any residual debris or surface
contamination from the fluidic channels. The chips were
subsequently flushed with water and TE buffers prior to use. All
solutions presented to the microfluidic devices were passed through
a 0.2 .mu.m syringe filters (Millipore, Billerica, Mass.) prior to
use.
[0051] A DNA sample, Escherichia coli K12 (MG1655) was purchased
from ATCC (American Type Culture Collection, Manassas, Va.).
Bacterial genomic DNA was prepared in a mini-reactor as described
previously. The purified genomic DNA was restriction digested using
NotI enzyme, and tagged with fluorescent labeled bis-PNA tags. The
prepared DNA sample was eluted at .about.0.5-1.5 ng/.mu.l.
[0052] Immediately prior to stretching on the microfluidic device
10, a 10 .mu.l aliquot of DNA was gently mixed with POPO-1
intercalator (Life Technologies, Grand Island, N.Y.) to uniformly
stain the DNA backbone. Sample concentration was quantified by
ethidium bromide-stained gel prior to intercalation in order to
standardize a ratio of three nucleic acid base pairs per dye. The
extent to which a given fragment of DNA is stretched in the
microfluidic funnel may depend on the ratio of dye to DNA,
therefore for all comparisons presented in one experiment were
performed such that a common stock of intercalated DNA was used for
each experiment set.
[0053] Single-molecule data acquisition was performed as previously
described. A 5.mu.l sample of prepared, intercalated DNA was
pipetted into the sample port 30 on a device 10 to be tested (see
FIG. 1A). The loaded sample concentration typically varied from
0.5-1.5 ng/.mu.l DNA. The chip was then mounted to a custom
confocal fluorescence microscope. Three laser detection points were
projected into the detection channel of the device. Two 455 nm
spots elicited fluorescence from the POPO-1 stained DNA backbone.
These spots were separated by 20 .mu.m along the detection channel.
A third detection point at 532 nm excited ATTO dyes attached to
sequence specific probes. This detection point was fixed at 5 .mu.m
before the first of the 455 nm points. Fluorescence emission was
collected in three discrete channels using a 100.times.
oil-immersion microscope objective. Fluorescence signals were
spectrally separated using a multi-pass dichroic element, and the
individual fluorescence signals were collected through fiber
coupled avalanche photodiodes (APDs, Perkin Elmer). For all
experiments, data acquisition was normalized to 2 bins per micron.
Spatial resolution between neighboring fluorescent events was
therefore maintained regardless of the fluid velocity within the
detection channel (see FIG. 2).
[0054] All data analysis was performed using custom software
developed at PathoGenetix. Collation of DNA backbone and tag
fluorescence signals was performed using a first software
application. Clustering of molecules based on the sequence-specific
tags was performed using a second software application. An example
of a "tag" is a sequence-specific nucleic acid probe. Single
molecule backbone fluorescence morphology was analyzed using a
scripted program. This program was used to detect the incidence of
elevated fluorescence along the POPO-intercalated DNA backbone. An
event was defined by 3 or more consecutive bins with a fluorescence
intensity greater than 1.5 times the average backbone fluorescence.
Molecules with detected elevated events were characterized by the
location of the event along the trace. Events could occur at the
leading edge of the molecule, at the trailing edge of the molecule,
or located within the length of the molecule. These events were
categorized as hairpin, relaxation, or overlap events,
respectively.
[0055] Detection sensitivity in genomic technology devices can be
directly correlated to the detection throughput of well stretched,
analyzable molecules. The first variable addressed to improve
detection throughput was to simply increase the driving fluid
velocity of the detection funnel. The influence of funnel geometry
must be considered when changing the driving velocity in genomic
technology devices.
[0056] The primary attribute affecting the extension of DNA in
genomic technology funnels is the strain rate of the accelerating
fluid that surrounds the DNA. The strain rate (.epsilon.) is
defined by the change in fluid velocity over a given distance along
the axis of the funnel:
.epsilon.=dV/dx Equation 5
Where the velocity scales inversely with the width of the
funnel
V x = V 0 w 0 w x Equation 6 ##EQU00005##
For a given funnel geometry, as the final velocity at the exit of
the funnel is increased, the peak strain rate also increases
proportionally (FIG. 4A). As shown in FIG. 2, a CV 7.5 funnel
(constant velocity funnel) driven at 7.5 .mu.m/ms achieves a peak
strain rate of 5 .mu.s.sup.-1. This value increases to 10 and 20
.mu.s.sup.-1 at 15 and 30 .mu.m/ms respectively. This directly
impacts the peak tension experienced by a given DNA fragment, and
therefore limits the range of well stretched molecules that can be
achieved.
[0057] The peak tension on a single elongated molecule of DNA
(T.sub.max) can be predicted by:
T max = .zeta. .parallel. 8 L mol 2 .parallel. . Equation 7
##EQU00006##
Where .zeta..sub..parallel. is the parallel drag coefficient (also
known as the molecular drag coefficient), L.sub.mol is the extended
length of the molecule in microns, and {dot over
(.epsilon.)}.sub..parallel. is the average strain rate of the
funnel device, defined by:
. = .DELTA. v .DELTA. x = v f - v i L f Equation 8 ##EQU00007##
Where v is the fluid velocity at the entrance (i) or exit (f) of a
funnel of length (L). .zeta..sub.ll has been previously estimated
in computational models to be 0.61 centiPoise (cP). Using this
value, the peak tension was calculated for all molecule lengths up
to 200 um in constant velocity detection funnels (CV7.5). As the
fluid velocity increased from 7.5 um/ms to 15 and 30 um/ms, the
peak tension on a molecule of a given length increased
dramatically. The inventors predict that an 80 .mu.m fragment would
be well stretched at 7.5 .mu.m/ms (35 pN tension), but would
overstretch dramatically at 30 .mu.m/ms (141 pN).
[0058] The relationships between fluid velocity, strain rate,
tension, and molecule extension in the CV7.5 funnel are
demonstrated in FIG. 4. A sample of E. coli genomic DNA digested
with NotI was prepared as described above and was loaded to the
CV7.5 device. Molecule stretching data was acquired at fluid
velocities of 7.5, 15, and 30 .mu.m/ms. At 7.5 .mu.m/ms, DNA
fragments ranging from 40-100 .mu.m in length appear to be well
stretched--fragments appear as discrete bursts with uniform average
backbone fluorescence intensity. Molecules shorter than 40 .mu.m
appear under stretched, with a distribution of higher average
fluorescence intensities. Molecules longer than 100 .mu.m (the 358
and 361 kb fragments predicted in this digest) appear to have
somewhat lower average backbone fluorescence, indicating the onset
of overstretching.
[0059] At 15 and 30 .mu.m/ms fluid velocities, the fragment
distribution appears to shift. Short fragments appear to stretch to
the same observed length, but longer fragments are significantly
overstretched as peak tension overcomes the 60 pN threshold. The
range of well-stretched fragment lengths is obviously compromised
by simply increasing the driving velocity in a fixed funnel
geometry.
[0060] The inventors predicted from equations 7 and 8 that the
average strain rate, and therefore tension on a molecule would
scale inversely with the length of the funnel--At a fixed fluid
velocity, a longer funnel would produce less tension on a molecule.
This effect is demonstrated in computational models in FIG. 4. The
same E. coli digest was run on the CV 30 device (constant velocity
device) at 30 .mu.m/ms, demonstrating a similar DNA stretching
pattern as observed at 7.5 .mu.m/ms in the CV7.5 device. DNA
throughput in stretching funnels can therefore be increased without
loss of long, analyzable fragments if the length of the funnel is
adjusted to achieve a consistent strain rate.
[0061] As demonstrated above, longer stretching funnels may be
required to accommodate high fluid velocities in genomic technology
devices. This poses a technical difficulty in that the fluidic
resistance (R.sub.h) of the microchannel scales with the length of
the funnel. Reducing the geometry to the simple case of a
rectangular straight channel,
R h = 12 L wh 3 ( 1 - 0.630 h w ) Equation 9 ##EQU00008##
Where .mu. is fluid viscosity, and L, w, and h are the length,
width, and height of the channel respectively. Because the
microfluidic device 10 may be driven by applying vacuum at the DNA
waste port 70, the device 10 may be limited to a 1 atmosphere
pressure drop along the channel. In a 1 .mu.m deep device, this may
imposes a constraint on the maximum channel length, fluid strain,
and fluid velocity. To achieve higher fluid velocities in longer
stretching funnels, the inventors explored devices with differing
etch depths.
[0062] The interaction of the channel floor and ceiling may have
complex effects on the stretching of DNA in fluidic devices.
Constraining the coiled, unstretched DNA into the narrow confines
of a 1 .mu.m channel serves to pre-extend the DNA, due to shear
interactions caused by poiseuille flow in the channel. Once
extended, the rate of DNA relaxation back to the condensed state is
also slowed in shallow channels. Conversely, flow in high-shear
structures can lead to decreased stretching efficiency due to
imposed molecular tumbling. Channel etch depth may therefore have
beneficial and adverse effects that must be balanced in the design
of a DNA stretching device.
[0063] To determine the effects of channel etch depth on high fluid
velocity DNA stretching, the inventors compared two
constant-velocity funnels designed for optimal stretching at 30
.mu.m/ms (CV30a, CV30b, FIG. 2). The optimal fluid velocity was
achieved at 25 psi in the 1 .mu.m funnel, but at 7 psi in the 2
.mu.m funnel. Again, the E. coli NotI digest was processed on both
devices. The confocal laser spots were positioned 50 .mu.m from the
onset of the constant velocity zone. Roughly equivalent DNA
stretching was observed on both devices--well resolved comet plots
were achieved without evidence of DNA overstretching (FIG. 4). To
determine subtle effects on a molecule-by-molecule basis, the
inventors utilized the software algorithm described above to
determine if each molecule in a given selection was well stretched,
or if there was evidence of additional fluorescent signal at the
leading edge, trailing edge, or middle of the molecule, thus
indicating hairpinning, relaxation, or molecule overlap
respectively. The inventors individually isolated clusters of
molecules at .about.40, 60, 80, and 100 .mu.m in three discreet
paired experiments for both the 1 .mu.m and 2 .mu.m deep
devices.
[0064] In both devices, the efficiency of observing well-stretched
molecules increased with the length of the molecule. The 2 .mu.m
deep device may appear to be more efficient in delivering well
stretched DNA, across all lengths. The incidence of overlapping
molecules was essentially equivalent in both devices, indicating
that the molecular occupancy in the detector was uniformly low in
all experiments. The 1 .mu.m deep device caused significantly more
molecules to display elevated fluorescence in the leading edge of
the molecule, indicating that molecular tumbling was enhanced in
the higher-shear 1 .mu.m channel. The 2 .mu.m channel however
showed uniformly higher (although not statistically significant)
incidence of elevated fluorescence in the trailing molecule edge,
suggesting more rapid relaxation of molecules in the deeper
channels.
[0065] In summary, retention of well stretched molecules in the 2
.mu.m channels was no worse than observed in the 1 .mu.m channels.
Because of the benefit of being able to operate devices at lower
vacuum pressures, further experiments with high velocity funnels
were performed in 2 .mu.m deep devices. These observations helped
explain the complex effect of shear flow on the observation of DNA
stretching. The inventors observed that stretching of DNA is
substantially uniform across a long range of molecule lengths as
controlled by the elongational flow established by the stretching
funnel geometry. Many potentially analyzable molecules must be
rejected from further analysis, however due to hairpinned or
relaxed conformational states, both of which are influenced by
shear flow.
[0066] While modeling strain rate as a function of the distance
along the stretching funnel, the inventors note that once a
molecule passes from the increasing strain region (i.e. the first
zone 62) of the funnel into the constant velocity detection zone
(i.e. the second zone 64, or the constant strain rate region) the
fluid strain rate drops to zero, which suggests that tension along
the molecule also decreases. In prior publications, the inventors
have explored different stretching funnel geometries, and
determined that an increasing strain rate profile yielded more
uniform DNA stretching than did a funnel with constant strain rate.
The inventors decided to test whether the combination of an
increasing strain rate stretching funnel with a constant strain
rate detection zone could improve stretching efficiencies by
maintaining tension on each molecule during detection. This may be
known as a compound constant strain rate funnel and data for such a
device is shown in the last two columns in FIG. 2. The funnel taper
profile for this detection region is described above, and the
resulting strain rate profiles for 30 .mu.m/ms constant velocity
and constant strain rate funnels are shown in FIG. 4.
[0067] The relative tension profile along a molecule experiencing
extensional flow may be modeled with a known funnel taper, assuming
the DNA molecule is a rigid rod with a known length. The velocity
of the molecule at any given position within the funnel is the
average of the fluid velocity at every point along the extended
molecule. In accelerating flow, the head of the molecule is
therefore moving more slowly than the surrounding fluid, while the
tail of the molecule moves more quickly than the surrounding fluid.
The drag of the surrounding fluid elements therefore exerts tension
on the molecule. The magnitude of that tension at any point along
the molecule is proportional to the integral of the difference
between the molecule velocity and the fluid velocity along the
length of the molecule.
[0068] The inventors computed the relative tension profile on a 100
.mu.m long molecule positioned with the head of the molecule at the
origin of the constant velocity region, as well as 50 and 100
microns within the channel. At the 0 .mu.m position, the molecule
is fully constrained by the increasing strain rate region of the
funnel, and displays a nearly parabolic tension profile. As the
molecule precedes 50 .mu.m along the detection zone, the inventors
appreciate that the tension profile has shifted towards the tail of
the molecule and the overall tension has dropped significantly.
With the head of the molecule at 100 .mu.m, it is fully within the
constant velocity zone. At this point, the molecule as a whole
travels at the surrounding fluid velocity and tension along the
molecule has reduced to zero. From these observations, the
inventors note that every molecule analyzed in a constant velocity
detection zone is subjected to a complex, dynamic cycle of
stretching and relaxation. The state of stretching and relaxation
is also highly sensitive to the positioning of the detection point
within the constant velocity zone.
[0069] By comparison, a similar model was built for a funnel
comprising the increasing strain rate stretching region (i.e. the
first zone 62) with the constant strain rate detection zone (i.e.
the second zone 64). For this design, the inventors defined the
desired point of detection to be 150 .mu.m from the transition
between the two taper profiles. The overall length of the detection
zone (i.e. the second zone 64) was defined to be approximately 350
.mu.m. Molecules up to 150 .mu.m in length would therefore be fully
constrained within the constant strain rate region (i.e. the second
zone 64) of the funnel during the entire detection process. The
computational model of this funnel design yields a truly parabolic
tension distribution that is identical regardless of position
varying 50 .mu.m in either direction from the nominal detection
point. This suggests that a DNA molecule will experience constant
tension during the entire detection process.
[0070] As an initial observation, the E. coli NotI digest was run
on the CV30 and CS30 devices (FIG. 6). The detection point was set
at the 50 .mu.m position on the CV30 device and the 150 .mu.m
position on CS30. Again, at first inspection of the comet plots,
both devices provide excellent stretching of DNA, and all predicted
fragment lengths were detected without obvious overstretching. Both
data sets were processed through backbone filters, and the
inventors recognize that the percentage of well stretched fragments
was greater through all observed molecule clusters in the constant
strain rate funnel as compared to the constant velocity funnel.
[0071] To further characterize the improvement in DNA stretching in
constant strain rate detection channels, the inventors repeated the
stretching comparisons in CV30 and CS30 devices while varying the
detector spot position up to 100 .mu.m in each device. The CV30
funnel was run at 0, 50, and 100 .mu.m from the onset of the
constant velocity region. The CS30 funnel was run with the
detection point at 100, 150, and 200 .mu.m from the transition from
increasing strain rate to constant strain rate tapers. Both devices
were run at fixed vacuum to achieve 30 .mu.m/ms at 0 .mu.m and 150
.mu.m spot positions respectively. All data sets were again process
through the software to tabulate the different mechanisms by which
individual molecules would fail backbone fluorescence intensity
filtration.
[0072] A significant improvement in the percentage of
well-stretched molecules was detected in CS30 (constant strain
funnel) compared to CV30 (constant velocity funnel). In both
devices, the percentage of good molecules increased with molecule
length, due to increased peak tension on longer molecules. The
percentage of good molecules was uniform regardless of spot
position for each isolated collection of molecule lengths. This is
in stark contrast to what is observed in the CV funnel. There, we
observed that more molecules pass backbone intensity filters when
the spot position is at 0 .mu.m, and decreases with the distance
from the origin. The inventors also observed that the
intra-experimental reproducibility in the percentage of well
stretched molecules was substantially improved in the CS funnel
compared to the CV funnel. The frequency of overlapped molecules
again was uniformly low in all samples, indicating that the sample
concentration was well-controlled for all experiments.
[0073] The percentage of molecules exhibiting hair pinned or
relaxed morphologies dramatically demonstrated the difference in
stretching in CS (constant strain) and CV (constant velocity)
funnels. In the CS30 funnel, molecules tended to have fewer
observed hairpins with increasing molecule length. The percentage
of observed hair pins was independent of spot position. In the CV
funnel, however, there was a significant dependence of the number
of observed hair pins on the detection point in the funnel. For
example, for an 80 .mu.m fragment, only .about.30% had hairpinned
conformations when the detection point was at the origin of the
constant velocity channel, but this increased to nearly 60% when
the detection point was shifted 100 .mu.m down the channel. This
indicates that as molecules are allowed longer time in the constant
velocity, no-tension portion of the channel, there is increased
opportunity for shear-induced tumbling of the leading end of the
molecule. This effect was eliminated in the constant strain rate
funnel shown in FIG. 1B. The remaining hairpinned molecules likely
represent the percentage of molecules that are not provided
sufficient cumulative strain to resolve complex initial
conformations--these molecules are likely never fully extended in
the first place.
[0074] The CV30 funnel (constant strain funnel) also produced a
uniformly high incidence of molecules exhibiting high fluorescence
on the trailing edge. The number of observed relaxed molecules was
highly variable form run to run, but appeared more pronounced with
the spot position 100 .mu.m from the onset of the constant velocity
zone. Relaxed molecules was nearly eliminated in the CS funnel, as
would be expected if the molecules were held under constant
tension.
[0075] In total, use of the CS funnel (shown in FIG. 1B) as opposed
to the prior art CV funnel at a given fluid velocity results in
nearly two-fold improvement in the recovery of well-stretched,
analyzable molecules by eliminating shear-induced molecular
tumbling and relaxation.
Acceleration Correction in Constant Strain Rate Detection
Channels
[0076] One ramification of detecting stretched DNA molecules in
constant-tension conditions is that the molecules are continuously
accelerating during the time of observation. In genomic analysis,
DNA molecules are stretched to reveal the locations of
sequence-specific fluorescent probes. For accelerating molecules,
the spatial resolution between closely located probes will be
greater at the leading end than the trailing end, due to the
decreasing residence time for each segment of DNA at the detection
spot. This imposes an acceleration-dependent bias on the optical
signal generated from each molecule.
[0077] The detection channel may include multiple detection points.
In some embodiments, the detection channel includes two backbone
spots that are spaced from one another along the detection channel.
The backbone spots detect fluorescence from the intercalator
fluorophore. The detection channel also includes two tag spots that
are spaced from one another along the detection channel. The tag
spots detect fluorescence from the fluorescently labeled tags. The
spatial distance between the two backbone spots is fixed and known,
as is the spatial distance between the two tag spots. The position
of the first backbone spot determines the position of the remaining
spots within the detection channel. Each spot could be a laser spot
or any other suitable detection arrangement. It should be
appreciated that any number of backbone spots and/or tag spots may
be used, as this aspect is not so limited.
[0078] The constant-strain detection channel geometry retains
molecules under constant tension with a well-defined acceleration.
The form of this acceleration can be derived analytically from the
funnel geometry. Obtaining this acceleration may be critical to two
related stages of data processing. In the first stage (position
acceleration correction), the time and position dependence of the
acceleration can be used to determine the entrance and exit time of
the molecule in the tag spot, based on the entrance and exit time
of the molecule in the backbone spots. In the subsequent stage
(trace acceleration correction), the time dependence of the
acceleration can also be used to convert the fluorescence pattern
from the time domain in which it is acquired to the positional
domain along the molecule. The "time domain" is also referred to
herein as the intensity versus time trace or the observed trace.
The "positional domain along the molecule" is also referred to
herein as the distance domain or the intensity versus distance
trace. The acceleration correction is used to properly convert the
intensity versus time trace that was obtained during detection into
an intensity versus distance trace by shifting each data point in
the observed trace by an acceleration correction.
[0079] An approximation of the exact acceleration correction
.DELTA.x.sub.c is given by:
.DELTA. x c .apprxeq. - L 2 2 x tag .tau. ( 1 - .tau. ) Equation 10
##EQU00009##
[0080] Where .DELTA.x.sub.c is the difference in the distance a
molecule would travel assuming a constant velocity compared to the
distance traveled in the accelerating flow in the tapered channel.
L is the length of the molecule. x.sub.tag is a funnel-geometry
derived parameter, and is the distance of the point of detection
from the theoretical asymptotic origin of the constant strain
portion of the channel. The length of the molecule L is measured
from observations of the intercalator backbone signal. The velocity
of the molecule is estimated from the time-of-flight of the
intercalator signal from the first backbone spot to the second
backbone spot. This estimated velocity of the molecule, along with
a measured dwell time in the first backbone spot, is then used to
calculate the length of the molecule L. This calculated length L
approximates the actual length of the molecule, as it corrects any
acceleration-dependent bias. x.sub.tag values for the CS30 and CS50
devices are presented in FIG. 2.
[0081] .tau. is the time for the passage of the molecule through a
detection spot. In some embodiments, the detection spot may be one
of the tag spots. .tau.=0 corresponds to the time at which the
leading end of the molecule enters the detection spot. .tau.=1
corresponds to the time at which the trailing end of the molecule
leaves the detection spot. Values of .tau. between 0 and 1
correspond to the times at which the remaining portions of the
molecule between the leading end and the trailing end enter the
detection spot. For example, a value of .tau.=0.1 may correspond to
the time at which a portion of the molecule that is located behind
the leading end of the molecule enters the detection spot, and a
value of .tau.=0.2 corresponds to the time at which another portion
of the molecule that is located even further behind the leading end
of the molecule enters the detection spot.
[0082] As such, each intensity data point plotted in the observed
intensity versus time trace receives a unique .tau. value ranging
from 0 to 1, inclusive. Because the acceleration correction is a
function of .tau., the value of the acceleration correction will
differ among the data points in the observed trace. Thus, when the
acceleration correction is applied to the observed trace, each data
point in the observed trace will shift by an amount that depends on
the acceleration correction value that is associated with that
specific data point. For example, the intensity point associated
with .tau.=0 will not shift at all, since the value of the
acceleration correction at .tau.=0 is 0. Similarly, the intensity
point associated with .tau.=1 also will not shift, since the value
of the acceleration correction at .tau.=1 is also 0. Finally, the
maximum value (amplitude) of the acceleration correction
.DELTA.x.sub.cmax occurs at .tau.=1/2, as shown in Equation 11
below.
.DELTA. x cmax .apprxeq. - L 2 8 x tag Equation 11 ##EQU00010##
Thus, the intensity point associated with .tau.=1/2 will shift by
the greatest amount during the application of the acceleration
correction.
[0083] As an example of the application of the acceleration
correction to experimental data, a sample of E. coli NotI digest
was prepared and run on the CS 30 device. The average tag
fluorescence traces were plotted for groups of molecules with
similar observed length (FIG. 7A, top traces). As a DNA molecule
has equal probability of entering the stretching funnel in either
orientation, the observed average tag fluorescence traces are
expected to appear symmetrical about the center of the average
molecule. To exemplify the acceleration induced asymmetry of the
raw traces, the fluorescence trace was also inverted and
superimposed on the original. This highlights significant asymmetry
in each of the selected examples. The amplitude of that asymmetry
increases with the length of the selected fragment. For each
average trace, acceleration correction was applied using Equation
10. The resulting corrected forward and reverse oriented traces are
plotted in FIG. 7A (bottom traces). This demonstrates that the
matched traces are superimposable.
[0084] FIG. 7B shows the acceleration correction amplitude along
the length of each molecule.
[0085] Acceleration correction depends only on the tag spot
position x.sub.tag, and the length of the molecule, L. The
amplitude of the acceleration correction is, to a good
approximation, proportional to the squared fragment length. This
dependency is shown in FIG. 7C. The maximum error to this
approximation in the tag spot positions with 120 .mu.m molecules is
less than 0.05 .mu.m.
[0086] The effect of acceleration dependent signal bias can be
readily observed in the average fluorescent signal generated from a
cluster of similarly sized molecules. FIG. 8A shows the average tag
signal observed in an 85 .mu.m cluster of molecules. Individual
molecules generated from a single restriction digest fragment can
enter the detection funnel in either a "head first" or "tail first"
orientation, with equal probability. Any average trace signal is
therefore expected to be symmetrical assuming sufficient detected
molecules. When the average signal trace, however, is superimposed
on its reverse, the inventors observe that discrete peaks are not
directly matched, but appear to be somewhat phase shifted. The
amount of this phase shift correction can be computed directly from
observed data on a fragment by fragment basis in automated
software. Once molecules are corrected for acceleration, the
average traces become directly superimposable with its reverse
(FIG. 8B).
[0087] The acceleration correction can be calculated for all
molecules within the working fragment range for samples processed
on either the CS30 (closed circles) or CV30 devices (open circles,
FIG. 8C). The detection spot position was set at the 150 .mu.m
position in the CS chip and 50 .mu.m in the CV chip, as described
previously. All molecules in the CS device required correction for
acceleration, but that correction scaled predictably with fragment
length. More strikingly, all analyzed molecules also required
acceleration correction in the CV30 device. The slope of the
required acceleration correction was comparable to what was
required in the CS funnel, but the onset of acceleration correction
was shifted towards longer molecules. In the CV funnels, molecules
longer than the distance from the onset of the parallel walled
channel to the detection point will project into the accelerating
strain portion of the funnel. Therefore shorter molecules would be
expected to travel at constant velocity but longer molecules would
still experience acceleration, thus requiring correction. These
observations were repeated using three paired comparisons of CS and
CV funnels, and yielded reproducible fits for the acceleration
correction in the slope (FIG. 8D) and intercept (FIG. 8E).
[0088] Assuming that acceleration correction is required regardless
of funnel type, there are significant advantages to applying such
corrections in the constant strain rate geometry over the constant
velocity geometry. First, the requirement of acceleration
correction is uniform for all analyzable molecule lengths. In the
constant velocity funnel, the need for acceleration correction is
dependent on the precise location of the detection spot in the
detection channel. This causes different acceleration correction
regimes depending on individual molecule length. Second, because
molecules in constant velocity channels experience relaxation of
the trailing end, the acceleration correction must accommodate for
this distortion as the tail accelerates towards the head of the
molecule. Finally, because molecules are observed under constant
tension conditions and competing effects of shear-induced tumbling
and relaxation are minimized in the CS funnels, the required
acceleration correction can be predicted directly for all lengths
of DNA from the funnel taper profile.
[0089] The inventors have uncovered several developments in the
understanding of the behavior of DNA extension in mixed flow
microfluidic funnels. There are several key observations. First,
DNA extension is dictated by the tension applied to each molecule.
This allows for the taper of a stretching funnel to be tailored to
any desired fluid velocity and range of molecule lengths. Second,
the etched depth of the funnel has competing effects on the
efficiency of DNA stretching. The benefits of pre-extension of DNA
and reduced rate of molecular relaxation in shallow channels,
however may be outweighed by the benefits of reduced fluidic
resistance and shear-induced molecular tumbling experienced in
deeper channels. Furthermore, application of constant tension
detection channels, as the third significant improvement,
eliminates any disadvantage incurred by complex shear flow and
permits observation of single molecules under constant tension
conditions.
[0090] Detection under constant tension conditions clearly provides
a better physical basis for understanding the mechanics of DNA
stretching in extensional flow. Similarly, stretching DNA in
constant tension provides a uniform, geometry based framework for
the prediction of molecular acceleration. Acceleration correction
in constant velocity funnels is much more complicated, as it
depends on the length of a DNA molecule, the distance of the
detection point from the onset of the constant velocity channel,
and also must accommodate any acceleration of the trailing end of
the molecule towards the leading end and the molecule begins to
relax.
[0091] All of the modifications to funnel design serve to
significantly improve the throughput of analyzable, well-stretched
molecules. According to one embodiment, the ultimate evolution of
the funnel design in 2 .mu.m etch depth is the CS50 device. This
permits uniform DNA stretching at 50 .mu.m/ms at .about.10 psi
vacuum. The length range of this device at 50 .mu.m/ms is
comparable to that of the CV7.5 at 7.5 .mu.m/ms, representing a
6.66 fold improvement in throughput due to velocity. Changing from
a 1 .mu.m to 2 .mu.M etch depth served to double the bulk flow
through the DNA injector onto the device, contributing an
additional 2 fold improvement. Transitioning from the constant
velocity detection channel to the constant strain rate channel also
contributes about 2 fold improvement in retention of stretched
molecules due to reduced relaxation and hairpinning. In all,
through better understanding of DNA stretching in fluidic
two-dimensional funnels, the work presented here demonstrates
nearly 25 fold improvement in molecule throughput in a simple
fluidic device. The throughput improvements demonstrated here are
also compatible with microfluidic devices designed to improve
molecule throughput by electrokinetic stacking of DNA onto
semi-permeable polymer gels and elimination of molecule overlap by
fractionation of short, information poor molecules. Improvements in
throughput in genomic technology enhances its applicability of
detection of rare pathogens in complex bacterial mixtures and in
detection from low starting masses of bacterial isolates.
[0092] It should be appreciated that various embodiments of the
present invention may be formed with one or more of the
above-described features. The above aspects and features of the
invention may be employed in any suitable combination as the
present invention is not limited in this respect. It should also be
appreciated that the drawings illustrate various components and
features which may be incorporated into various embodiments of the
present invention. For simplification, some of the drawings may
illustrate more than one optional feature or component. However,
the present invention is not limited to the specific embodiments
disclosed in the drawings. It should be recognized that the present
invention encompasses embodiments which may include only a portion
of the components illustrated in any one drawing figure, and/or may
also encompass embodiments combining components illustrated in
multiple different drawing figures.
[0093] It should be understood that the foregoing description of
various embodiments of the invention are intended merely to be
illustrative thereof and that other embodiments, modifications, and
equivalents of the invention are within the scope of the invention
recited in the claims appended hereto.
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