U.S. patent application number 13/751606 was filed with the patent office on 2013-09-19 for multichannel preparative electrophoresis system.
This patent application is currently assigned to SAGE SCIENCE, INC.. The applicant listed for this patent is SAGE SCIENCE, INC.. Invention is credited to Todd J. Barbera, T. Christian Boles, Douglas Grosvenor Sabin, Paul Chandler Sabin.
Application Number | 20130240360 13/751606 |
Document ID | / |
Family ID | 43729427 |
Filed Date | 2013-09-19 |
United States Patent
Application |
20130240360 |
Kind Code |
A1 |
Sabin; Douglas Grosvenor ;
et al. |
September 19, 2013 |
Multichannel Preparative Electrophoresis System
Abstract
The invention provides an electrophoresis cassette, methods for
making the electrophoresis cassette, and method of fractionating
analytes from a sample based upon electrophoretic mobility in a
single application of the sample to an electrophoretic system.
Inventors: |
Sabin; Douglas Grosvenor;
(Marblehead, MA) ; Barbera; Todd J.; (Marblehead,
MA) ; Sabin; Paul Chandler; (Needham, MA) ;
Boles; T. Christian; (Bedford, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
SAGE SCIENCE, INC. |
Beverly |
MA |
US |
|
|
Assignee: |
SAGE SCIENCE, INC.
Beverly
MA
|
Family ID: |
43729427 |
Appl. No.: |
13/751606 |
Filed: |
January 28, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12760548 |
Apr 14, 2010 |
8361299 |
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13751606 |
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12576148 |
Oct 8, 2009 |
8361298 |
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12760548 |
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61195566 |
Oct 8, 2008 |
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61150243 |
Feb 5, 2009 |
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Current U.S.
Class: |
204/627 |
Current CPC
Class: |
G01N 27/44756 20130101;
G01N 27/44791 20130101 |
Class at
Publication: |
204/627 |
International
Class: |
G01N 27/447 20060101
G01N027/447 |
Claims
1. An electrophoresis cassette comprising: a plate including at
least one macrofluidic separation channel, the channel having a
first physically and electrically isolated portion and a second
physically and electrically isolated portion; and an elution
chamber positioned on one or another of the physically and
electrically isolated portions, the chamber comprising at least one
an elution cavity and an analyte-impermeable barrier.
2-85. (canceled)
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application U.S. Ser. No. 12/760,548, filed Apr. 14, 2010 (now U.S.
Pat. No. 8,361,299), which is a continuation-in-part of U.S. Ser.
No. 12/576,148 (now U.S. Pat. No. 8,361,298), filed on Oct. 8,
2009, which claims priority to provisional application U.S. Ser.
No. 61/195,566, filed Oct. 8, 2008, and to provisional application
U.S. Ser. No. 61/150,243, filed Feb. 5, 2009, the contents of which
are each herein incorporated by reference in their entirety.
FIELD OF THE INVENTION
[0002] This invention relates generally to the field of molecular
biology. Systems and methods of the invention are used to prepare
and analyze DNA, RNA, and proteins from biological samples.
BACKGROUND OF THE INVENTION
[0003] Electrophoretic separation of DNA fragments is used for a
number of purposes in molecular and clinical biology and medicine,
including next generation DNA sequencing, medical diagnostics,
forensic science and DNA computing.
[0004] Preparative gel electrophoresis of DNA has good resolution,
adequate capacity and ease of use on a small scale. However, the
manual process is both time and labor intensive. Critically, it is
difficult to remove the desired DNA fraction from the gel. This
removal process routinely entails excising a band or portion from
the gel containing the DNA of interest and then extracting it by a
variety of chemical and physical means including the use of
enzymes, centrifugation, freezing and more. Importantly, these
methods substantially reduce the amount of DNA harvested and dilute
the resultant DNA into large volumes of fluid, therefore, requiring
additional time and expense to re-concentrate it into a smaller,
usable, aliquot. This problem is so significant to the field of
molecular biology, in fact, that the removal of DNA from gels in
small volumes of fluid has spawned a separate industry for making
various types of kits, reagents and devices to accomplish the task.
However, despite a demonstrated need and the efforts of skilled
artisans, a solution has not yet been developed.
SUMMARY OF THE INVENTION
[0005] The invention provides compositions including electrophorsis
cassettes and preparative electrophoresis systems as well as
methods of fractionating analytes from a sample. Electrophoresis
cassettes and preparative electrophoresis systems of the invention
fractionate nucleic acids or polypeptides of a specified or desired
molecular weight or electrophoretic mobility from a biological
sample, and subsequently extract the desired nucleic acids or
polypeptides from the gel matrix or buffer compositions by drawing
them across an analyte and ion permeable barrier and into an
elution chamber.
[0006] Specifically, the invention provides an electrophoresis
cassette containing a plate including at least one macrofluidic
separation channel, the channel having a first physically and
electrically isolated portion and a second physically and
electrically isolated portion; and an elution chamber positioned on
one or another of the physically and electrically isolated
portions, the chamber comprising at least one an elution cavity and
an analyte-impermeable barrier. The elution chamber is attached or
removable. For instance, the elution chamber is generated by
inserting an elution chamber insert into an elution chamber cavity
within the first or second physically and electrically separated
portion of the separation channel, inserting a liquid gel matrix
composition into the separation channel, solidifying the gel matrix
composition, removing the elution chamber insert thereby generating
an elution chamber, placing an analyte-impermeable barrier on the
distal side of the elution chamber, and filling the elution chamber
with an elution buffer composition. The term "solidifying" is meant
to describe a process by which either a liquid matrix organizes
into a solid gel form (which may be temperature-dependent), or
alternatively, a process by which liquid matrix components
polymerize to form a solid gel. Regardless of the gel matrix
composition used, the composition is injected as a liquid, and
subsequently transforms into a solid once inside the cassette.
[0007] Alternatively, or in addition, an elution chamber of the
above electrophoresis cassette further include at least one of an
analyte-permeable barrier, a sample collection chamber including a
sample removal port, and an analyte-impermeable barrier.
[0008] Furthermore, the elution chamber is removable. In certain
embodiments of the elution chamber, the chamber contains, in the
direction of electrophoresis, a first removable side, an
analyte-permeable membrane, a sample collection chamber, an
analyte-impermeable membrane, and a second removable side. The
removable sides are removable portions of the sample collection
chamber with at least one of an opening, protrusion, or recession
for binding either the analyte-permeable or analyte-impermeable
membrane to the sample collection chamber. Alternatively, the
removable sides are O-rings that fit within a first and second side
of the sample collection chamber and bind either the
analyte-permeable or analyte-impermeable membrane to the sample
collection chamber. A removable elution chamber is also used as an
elution chamber insert, as described above. The removable elution
chamber is attached to the elution chamber cavity within the first
or second physically and electrically isolated portion of the
separation channel.
[0009] The analyte-permeable barrier of the elution chamber is a
hydrophilic membrane or filter. In certain embodiments, the
analyte-permeable barrier includes a least one pore having a
diameter range of between 0.4 micron to 50 microns, and preferably,
of between 0.4 micron to 1 micron.
[0010] The analyte-impermeable barrier of the elution chamber is a
membrane, filter, film, or any combination thereof. Preferably, the
analyte-impermeable barrier is an ultrafiltration membrane or a
conductive film. In certain embodiments, the ultrafiltration
membrane contains a least one pore having a diameter range of
between 0.001 micron to 0.1 micron. Alternatively, or in addition,
the ultrafiltration membrane has a molecular weight cutoff of
between 1,000 to 30,000 daltons. Preferably, the ultrafiltration
membrane has a molecular weight cutoff of between 3,000 to 10,000
daltons. In other embodiments, the analyte-impermeable barrier
includes a conductive film having the same charge as the analyte or
a conductive film contacted with negatively-charged sulfate groups.
In certain aspects, the analyte-impermeable barrier is Nafion.
[0011] The electrophoresis cassette also includes a constriction
point provided between the separation channel and at least one of
the first and second physically and electrically isolated
portions.
[0012] In certain embodiments of the cassette, at least one
macrofluidic separation channel is tapered from one end to the
constriction point. Alternatively, or in addition, at least one
macrofluidic separation channel is optically-transparent. In other
embodiments, the separation channel is optically-transparent on at
least one side, on only one side, or on only a portion of one side.
For instance, the separation channel is optically-transparent on
the bottom side, the top side, or both bottom and top sides.
Preferably, optical transparency is maintained along the separation
channel from the distal edge of the sample well cavity to the
division point.
[0013] The electrophoresis cassette contains at least one dam
within at least one separation channel. Preferably, the
electrophoresis cassette contains two dams within at least one
separation channel. The term "dam" is meant to describe a barrier
structure that partitions the separation channel. In one embodiment
of the invention, a dam is positioned in at least one separation
channel distal to the buffer reservoir and proximal to the sample
well cavity. In another embodiment, a dam is positioned in at least
one separation channel distal to a division point and proximal to a
waste reservoir. The dam is formed from a frame onto which is
attached to an ion-permeable barrier. The ion-permeable barrier is
also preferably permeable to the buffer composition. The frame
recapitulates the geometry of the separation channel, i.e. if the
channel is rectangular, then the dam frame is rectangular. The
ion-permeable barrier is composed of a hydrophilic membrane or
filter. In certain embodiments, the hydrophilic membrane or filter
includes a least one pore having a diameter range of between 0.001
micron to 1 micron, and preferably, of between 0.45 micron to 1
micron. The analyte permeable or impermeable membranes described
herein for use in the elution chamber could also be used as a
membrane for a dam. Importantly, the dam structure restrains the
flow of unsolidified gel matrix molecules to the separation channel
during gel casting, e.g. the portion of the separation channel
between the first dam and the second dam. The dam is electrically
conductive, and therefore, does not disrupt or distort electric
fields or currents present in or around the at least one separation
channel. Preferably, dam structures are inserted prior to
attachment of the cover, and, therefore, in these preferred
embodiments, the dam structures are permanent. Alternatively, the
dam is removable from at least one separation channel because the
cover is not permanently attached onto the base of the
electrophoresis cassette. The dam occupies the total
cross-sectional area of the separation channel. Accordingly, a dam
prevents gel matrix molecules from traversing is membrane, that
upon injection of a gel-matrix composition, effectively partitions
the separation channel into at least one buffer- and at least one
gel matrix-filled compartment, respectively.
[0014] The electrophoresis cassette contains between 1 and 5
macrofluidic separation channels. Alternatively, the cassette
contains between 1 and 9 or between 1 and 13 macrofluidic
separation channels. The maximum number of macrofluidic separation
channels contained in the electrophoresis cassette is determined by
the ability of a detection system to read the cassette, and in
theory, no maximum number exists, however, a practical range of is
between 25-33 macrofluidic channels.
[0015] The electrophoresis cassette also contains a buffer
reservoir for each of the macrofluidic separation channels.
[0016] The electrophoresis cassette includes a cover for the plate.
In one aspect, the cover includes a configuration that corresponds
to the configuration of the top of the plate. In another aspect,
the cover includes at least one of an opening, a protrusion and a
recess that align with at least one of the buffer reservoirs.
Alternatively, or in addition, the cover includes at least one of
an opening, a protrusion and a recess that align with at least one
of the macrofluidic channel, the buffer reservoir, the sample well
cavity, the sample removal port, the elution reservoir, the waste
reservoir, and the first and second physically and electrically
isolated portions. In another embodiment, the cover includes at
least one of an opening, a protrusion and a recess that align with
at least one of the macrofluidic channel, the sample well cavity,
and the sample removal port. The cover may further include at least
one of an electrode port, a vent, and a sample well port. The
electrode port is either negative or positive. In a preferred
embodiment, the at least one negative electrode port is positioned
proximal to the sample well cavity. In another preferred
embodiment, the at least one positive electrode port is positioned
distal to either the elution chamber or the cavity for the second
dam.
[0017] Moreover, the electrophoresis cassette contains at least one
of a cavity for a first dam, sample well cavity, an elution
reservoir, a cavity for a second dam, and a waste reservoir. In
certain embodiments of the invention, the elution reservoir and the
waste reservoir are provided at an end of the first physically and
electrically isolated portions and the second physically and
electrically isolated portions, respectively.
[0018] The electrophoresis cassette further includes a division
point provided between the macrofluidic channel and the elution
reservoir and the waste reservoir. In certain embodiments of the
cassette, the constriction point is the division point.
[0019] The macrofluidic channel of the electrophoresis cassette
includes at least one of a gel matrix composition, a liquid buffer
composition, a solid buffer composition. In aspects, at least one
of a gel matrix composition, a liquid buffer composition, a solid
buffer composition contains at least one of a fluorophore or a
chromophore. The fluorophore is either the analyte or is bound to
the analyte. Similarly, the chromophore is either the analyte or is
bound to the analyte. An exemplary fluorophore is ethidium bromide,
which binds to polynucleic acids and allows detection of the
polynucleic acid analyte. Moreover, a polypeptide analyte is a
chromophore because it can be detected by mere absorption of
ultraviolet light.
[0020] At least one macrofluidic separation channel of the
electrophoresis cassette contains a gel matrix composition. In one
aspect, the gel matrix composition fills a volume of the
macrofluidic separation channel, including at least one of the
first and second physically and electrically isolated portions. In
another aspect, the gel matrix composition defines at least one
sample well within at least one sample well cavity.
[0021] Sample wells have multiple geometries. The geometry of the
sample well reflects the geometry of the sample well insert used to
define the negative space not occupied by the gel matrix
composition. In certain aspects of the invention, a sample well
insert is used in combination with a stripper plate to create a
terraced geometry, the negative space of which will form the sample
well. Critically, the sample wells of the invention have the have a
unique and essential "chimney" shape, forming a "gel chimney," in
which the walls of the sample well extend through the sample well
insert opening and into the sample well port, as depicted in FIGS.
44-48. The cover plate is specifically adapted with walls
surrounding the sample well insert opening to support the sides of
this chimney-shaped sample well. The chimney-shaped sample well
prevents entry of the sample into the seam between the upper
surface of the gel and the bottom surface of the cassette cover
plate. Such entry can occur by capillary flow, or by
electrophoresis. Sample molecules entering the seam travel at a
different rate than that of sample molecules traveling through the
gel. For this reason, undesired sample molecules traveling in the
seam may be drawn into the elution chamber during elution, thereby
contaminating the desired sample components that have been
traveling through the gel. The contamination typically travels
unpredictably, but often faster than the material traveling through
the gel in the separation channel, causing inappropriately large
molecules to enter the elution chamber.
[0022] The macrofluidic separation channel further contains a lens
positioned between the sample well cavity and the division point.
The lens includes a gel matrix composition. In certain embodiments,
the gel matrix composition of the lens contains a higher
concentration of a polymer, thereby making the lens denser. The
lens takes any shape, including a curve that follows the direction
of electrophoresis. Functionally, the lens focuses at least one of
an analyte. The lens is positioned proximal or distal to a
constriction point or to a detection zone within the separation
channel.
[0023] Furthermore, at least one macrofluidic channel of the
electrophoresis cassette contains a buffer composition. In one
aspect, the buffer composition fills a volume of at least one
buffer reservoir, at least one sample well, at least one elution
reservoir, and at least one waste reservoir.
[0024] At least one elution chamber of the electrophoresis cassette
contains an elution buffer composition. In one aspect, the elution
buffer composition fills a volume of at least one elution
chamber.
[0025] The electrophoresis cassette of the invention is meant to be
compatible with a variety of detection systems. As such, certain
embodiments of the cassette contain an integrated electrode array.
In this aspect, the cassette contains: a negative electrode
positioned between the buffer reservoir and a corresponding end of
the separation channel; a positive electrode positioned between an
end of the first physically and electrically isolated portion and
the elution reservoir; and a positive electrode positioned between
an end of the second physically and electrically isolated portion
and the waste reservoir.
[0026] The electrophoresis cassette further includes a removable
seal. Non-limiting examples of seal materials are polymers,
adhesive films, and tapes. The seal encloses at least one of an
opening, a protrusion and a recess of the cover. Alternatively, or
in addition, the seal encloses the entirety of the electrophoresis
cassette. Functionally, the seal prevents spillage and evaporation
of the buffer and gel matrix compositions contained within the
cassette during storage. Moreover, the seal prevents the buffer and
gel matrix compositions contained within the cassette from
contacting or corroding the electrode array during storage.
[0027] Regardless of the features present within the
electrophoresis cassette, the cassette is disposable.
[0028] The invention also provides a method of making an
electrophoresis cassette, including: providing the above
electrophoresis cassette, wherein the cassette further contains at
least one of a buffer reservoir insert, a sample well insert, a
waste reservoir insert, and a cover, wherein the buffer reservoir
insert includes a vent, wherein the buffer reservoir insert
traverses an opening in the cover plate aligned with the buffer
reservoir, wherein the sample well insert traverses an opening in
the cover plate aligned with the sample well cavity, wherein the
waste reservoir insert includes an injection port, and wherein the
waste reservoir insert traverses an opening in the cover plate
aligned with the waste reservoir; inserting a gel matrix
composition through the injection port; solidifying the gel matrix
composition, wherein the gel matrix composition transforms from a
liquid to a solid; removing the buffer reservoir insert, sample
well insert, and waste reservoir insert, wherein a buffer
reservoir, a sample well, an elution reservoir, and a waste
reservoir are generated; filling the buffer reservoir, the elution
reservoir, and the waste reservoir with a buffer composition;
filling the elution chamber with an elution buffer composition; and
sealing the electrophoresis cassette.
[0029] In other embodiments, the method includes providing the
above electrophoresis cassette, wherein the cassette further
contains at least one of a sample well insert and a cover, wherein
the sample well insert traverses an opening in the cover plate
aligned with the sample well cavity; inserting a gel matrix
composition through the injection port; solidifying the gel matrix
composition, wherein the gel matrix composition transforms from a
liquid to a solid; removing the sample well insert, wherein a
sample well is generated; filling the buffer reservoir, the elution
reservoir, and the waste reservoir with a buffer composition;
filling the elution chamber with an elution buffer composition; and
sealing the electrophoresis cassette. In this embodiment, the gel
is cast in the electrophoresis cassette, without the use of a
casting fixture, and the cassette is oriented or placed
horizontally during the inserting and solidifying steps.
[0030] In one aspect the above method further includes the steps
of: providing a casting fixture, wherein the fixture includes a
front plate that contacts the top of the cassette, wherein the
front plate contains at least one opening that aligns with at least
one of the vent of the buffer reservoir insert and the separation
channel, and a back plate that contacts the bottom of the cassette,
wherein the back plate contains at least one opening; attaching the
casting fixture to electrophoresis cassette, wherein the back plate
contacts the bottom of the electrophoresis cassette and the front
plate contacts the top of the electrophoresis cassette, and wherein
the back and front plates are attached to each other; wherein the
casting fixture is provided and attached prior to the injecting
step and, detaching the casting fixture from the electrophoresis
cassette prior to the removing step.
[0031] According to this method, the buffer reservoir insert fills
a volume of the buffer reservoir. Moreover, the sample well insert
fills a volume of the sample well cavity. Furthermore, the waste
reservoir insert fills a volume of the waste reservoir.
[0032] In one aspect of this method the electrophoresis cassette or
casting fixture is horizontal or vertical during the inserting and
solidifying steps.
[0033] The cassette provided for this method contains between 1 and
5 macrofluidic separation channels. Alternatively, the cassette
contains between 1 and 9 or between 1 and 13 macrofluidic
separation channels.
[0034] The invention also provides a detection system for detecting
a property of an analyte within a sample including: the
above-described electrophoresis cassette; an electrode array
comprising at least one of a negative electrode and a positive
electrode, wherein the negative electrode aligns with a position on
the cassette between the buffer reservoir and a corresponding end
of the separation channel, and wherein the positive electrode
aligns with a physically and electrically isolated portion of the
separation channel; a detector positioned near the separation
channel of the electrophoresis cassette, wherein the detector
detects a property of an analyte; a processor configured to
activate or deactivate power to at least one positive electrode
based upon a signal received from the detector; and a power module
comprising at least one of a power supply and a relay to provide
power to at least one of the processor, the negative electrode and
at least one positive electrode.
[0035] In certain embodiments of this system, the detected property
is an optical property of an analyte. Exemplary optical properties
include, but are not limited to, the emission or absorption of
light. Furthermore, the detected property includes magnetism,
radiation, temperature, color, energy, or changes in any of the
above.
[0036] The sample of this system contains a detectable label, such
as a magnetic, paramagnetic, radioactive, enzymatic, immunological,
or optical label. Non-limiting examples of optical labels are
fluorescent and light-absorbing compounds. In one aspect, the
sample contains a fluorescent compound and the analyte forms a
complex with the fluorescent compound. In another aspect, the
fluorescent compound or the analyte is a fluorophore. In another
embodiment, the sample comprises a light-absorbing compound and the
analyte forms a complex with the light-absorbing compound.
Alternatively, the light-absorbing compound or the analyte is a
chromophore.
[0037] The analyte of this system is a sample or a molecular weight
marker.
[0038] In a preferred embodiment of this system, the detector
detects a property of the molecular weight marker within a first
macrofluidic channel and sends a signal to the processor.
Subsequently, the processor receives the signal from the detector
and applies an algorithm to determine the molecular weight of at
least one of an analyte at the division point of a second
macrofluidic channel.
[0039] The invention provides a method of fractionating analytes
within a sample, including: providing an electrophoresis cassette
described herein, wherein the cassette further comprises at least
one of a buffer reservoir insert, a sample well insert, and a waste
reservoir insert, and a cover, wherein the buffer reservoir insert
includes a vent, wherein the buffer reservoir insert traverses an
opening in the cover plate aligned with the buffer reservoir,
wherein the sample well insert traverses an opening in the cover
plate aligned with the sample well cavity, wherein the waste
reservoir insert includes an injection port, and wherein the waste
reservoir insert traverses an opening in the cover plate aligned
with the waste reservoir; inserting a gel matrix composition
through the injection port; solidifying the gel matrix composition,
wherein the gel matrix composition transforms from a liquid to a
solid; removing the buffer reservoir insert, sample well insert,
and waste reservoir insert, wherein a buffer reservoir, a sample
well, an elution reservoir, and a waste reservoir are generated;
filling the buffer reservoir, an elution reservoir, and a waste
reservoir with a buffer composition; filling the elution chamber
with an elution buffer composition; and inserting the
electrophoresis cassette into a detection system described herein;
programming the processor of the detection system to selectively
activate the positive electrode of the electrode array aligned with
the physically and electrically isolated portion of the separation
channel comprising the elution chamber when the processor
determines that at least one of an analyte of the desired molecular
weight is traversing the division point of the separation channel;
applying the sample to the sample well; applying a voltage across
the electrophoresis cassette; collecting analytes of the sample
having a desired electrophoretic mobility in the elution chamber,
thereby fractionating analytes within a sample. In certain
embodiments, this method includes providing an electrophoresis
cassette described herein, wherein the cassette further comprises
at least one of a sample well insert and a cover, wherein the
sample well insert traverses an opening in the cover plate aligned
with the sample well cavity; inserting a gel matrix composition
through the injection port; solidifying the gel matrix composition,
wherein the gel matrix composition transforms from a liquid to a
solid; removing the sample well insert, wherein a sample well is
generated; filling the buffer reservoir, an elution reservoir, and
a waste reservoir with a buffer composition; filling the elution
chamber with an elution buffer composition; and inserting the
electrophoresis cassette into a detection system described herein.
In this embodiment, the gel is cast in the electrophoresis
cassette, without the use of a casting fixture, and the cassette is
oriented or placed horizontally during the inserting and
solidifying steps.
[0040] This method further includes the steps of: providing a
casting fixture, wherein the fixture includes a front plate that
contacts the top of the cassette, wherein the front plate comprises
at least one opening that aligns with at least one of the vent of
the buffer reservoir insert and the separation channel, and a back
plate that contacts the bottom of the cassette, wherein the back
plate comprises at least one opening; attaching the casting fixture
to electrophoresis cassette, wherein the back plate contacts the
bottom of the electrophoresis cassette and the front plate contacts
the top of the electrophoresis cassette, and wherein the back and
front plates are attached to each other; wherein the casting
fixture is provided and attached prior to the injecting step and,
detaching the casting fixture from the electrophoresis cassette
prior to the removing step.
[0041] In one aspect of this method, the sample contains a
molecular weight marker. In another aspect of this method, the
analyte is a polynucleic acid or a polypeptide. Moreover, the
polynucleic acid contains deoxyribonucleic acid (DNA) or
ribonucleic acid (RNA). Alternatively, or in addition, the
polynucleic acid is double or single stranded. In certain aspects,
the polypeptide is native or denatured.
[0042] In one aspect of this method, the sample contains at
detectable compound. Exemplary detectable compounds are
magnetically-, paramagnetically-, radioactively-, enzymatically-,
immunologically-, or optically-detectable. Optically-detectable
compounds are, for example, fluorescent and light-absorbing
compounds. In certain embodiments, the sample contains at least one
of a complex of an analyte and a fluorescent compound. In one
aspect, the fluorescent compound is a fluorophore. In another
aspect, the analyte is a fluorescent compound or fluorophore.
Alternatively, or in addition, the sample contains at least one of
a complex of an analyte and a light-absorbing compound. In one
embodiment, the light-absorbing compound is a chromophore. In
another embodiment, the analyte is a light-absorbing compound or
chromophore.
[0043] According to this method, at least one of the gel matrix
composition, the buffer composition, or the elution buffer
composition comprises at least one of a fluorophore that complexes
to at least one of an analyte. Moreover, at least one of the gel
matrix composition, the buffer composition, or the elution buffer
composition comprises at least one of a chromophore that complexes
to at least one of an analyte.
[0044] According to this method, the processor of the detection
system selectively activates the positive electrode of the
electrode array aligned with the physically and electrically
isolated portion of the separation channel comprising the elution
chamber when an analyte having a specified electrophoretic mobility
is detected and wherein the specified electrophoretic mobility is
distinct for each macrofluidic channel.
[0045] The invention provides a composition containing an
electrophoresis cassette, the electrophoresis cassette including:
(a) a channel plate including a macrofluidic channel, wherein the
macrofluidic channel comprises, from proximal to distal, a buffer
reservoir, a first end of a separation channel, a sample well
cavity, a constriction point, a division point, an elution chamber
cavity, a second physically and electrically isolated end of the
separation channel, a third physically and electrically isolated
end of the separation channel, an elution reservoir, and a waste
reservoir; (b) an elution chamber including, from proximal to
distal, an analyte-permeable barrier, a sample collection chamber
having a sample removal port, and an analyte-impermeable barrier,
wherein the elution module is attached to the elution chamber
cavity; (c) a cover plate that contacts the top of the channel
plate, wherein the cover plate contains at least one of an opening,
a protrusion, and a recess that align, from proximal to distal,
with the buffer reservoir, the sample well cavity, the elution
chamber, a combination of the second physically and electrically
isolated end of the separation channel and the elution reservoir,
and a combination of the third physically and electrically isolated
end of the separation channel, and the waste reservoir; (d) a gel
matrix composition that fills the macrofluidic separation channel
and defines a sample well within the sample well cavity; (e) a
liquid buffer composition that fills the buffer reservoir, the
sample well, the elution reservoir, and the waste reservoir; (f) an
elution buffer composition that fills the elution chamber; and (g)
a seal that encloses the electrophoresis cassette.
[0046] In other embodiments of this composition, the
electrophoresis cassette includes, (a) a channel plate including a
macrofluidic channel, wherein the macrofluidic channel comprises,
from proximal to distal, a buffer reservoir, a first end of a
separation channel, a cavity for a first dam, a sample well cavity,
a constriction point, a division point, an elution chamber cavity,
a second physically and electrically isolated end of the separation
channel, a third physically and electrically isolated end of the
separation channel, an elution reservoir, a cavity for a second
dam, and a waste reservoir; (b) an elution chamber including, from
proximal to distal, an analyte-permeable barrier, a sample
collection chamber having a sample removal port, and an
analyte-impermeable barrier, wherein the elution module is attached
to the elution chamber cavity; (c) a cover plate that contacts the
top of the channel plate, wherein the cover plate contains at least
one of an opening, a protrusion, and a recess that align, from
proximal to distal, with the sample well cavity and the elution
chamber; (d) a gel matrix composition that fills the macrofluidic
separation channel and defines a sample well within the sample well
cavity; (e) a liquid buffer composition that fills the buffer
reservoir, the sample well, the elution reservoir, and the waste
reservoir; (f) an elution buffer composition that fills the elution
chamber; and (g) a seal that encloses the electrophoresis cassette.
In certain aspects, the cover further includes at least one of an
electrode port, a vent, a sample well port, and an injection port.
The electrode port is either negative or positive. In a preferred
embodiment, the at least one negative electrode port is positioned
proximal to the sample well cavity. In another preferred
embodiment, the at least one positive electrode port is positioned
distal to either the elution chamber or the cavity for the second
dam. In other aspects of the composition, at least one of the
cavity for the first dam and the cavity for the second dam contain
a first dam or a second dam, respectively. Alternatively, or in
addition, the composition contains a sample well that forms a gel
chimney.
BRIEF DESCRIPTION OF THE DRAWINGS
[0047] FIG. 1A is an illustration of the electrophoresis cassette
used for Example 1.
[0048] FIG. 1B is the illustration of FIG. 1, shown from an
alternate perspective.
[0049] FIG. 1C is an illustration of an electrophoresis cassette
providing dimensions of separation channel. Inserts are shown as
transparent outlines. In an exemplary embodiment, the length from
the distal edge of the sample well to the division point is
approximately 53 mm.
[0050] FIG. 1D is a schematic representation of the front view of
channel plate at the division point of the separation channel.
Length of "a" is, for example, 1.75 mm.
[0051] FIG. 2A-L is a series of photographs showing the
fractionation of genomic DNA by size differentiation over time
using the electrophoresis cassette of FIG. 1 and Example 1. The
arrow indicates the direction of electrophoresis at the division
point, i.e. the arrow points in the direction of the positive
electrode that is differentially activated.
[0052] FIG. 2M is a photograph of a 2% agarose gel, in which the
results of the fractionation of FIG. 2A-L were analyzed. The
purified fraction collected in the elution chamber measured
344.+-.20 base pairs (bp).
[0053] FIG. 3A is an illustration of a preparative electrophoresis
cassette with an elution chamber.
[0054] FIG. 3B is an illustration of a preparative elution chamber
from the perspective of the separation channel.
[0055] FIG. 3C is a pair of photographs of a preparative elution
chamber. Left Panel: Exemplary dimensions for the elution chamber
are as follows: diameter of sample collection port=2.5 mm, height
of elution chamber=6 mm, width of elution chamber=8 mm, depth of
elution chamber=6 mm. With respect to the opening in the side of
the elution chamber, exemplary dimensions are as follows: the
width=4 mm, height=3 mm. Right Panel Disassembled view of elution
chamber showing a first removable side, a sample collection chamber
and a second removable side. The sample collection chamber contains
a sample collection port, which is located, for instance, on the
top side of the chamber.
[0056] FIG. 4A is a photograph of an electrophoresis cassette
combined with the elution chamber of FIG. 3 and Example 2.
[0057] FIG. 4B is a photograph of an agarose gel analysis of
fractions collected from the electrophoresis cassette of FIG.
4A.
[0058] FIG. 5A is an illustration of an electrophoresis cassette
with a tapered separation channel combined with an elution chamber
including a spacer, wedge, and O-ring seals. In one embodiment, the
length of the separation channel from the distal edge of the sample
well to the division point is 67 mm.
[0059] FIG. 5B is a series of illustrations of an elution chamber
assembly shown in FIG. 5A. Exemplary dimensions of the elution
chamber are as follows: the height of the sample collection chamber
and the wedge=10 mm, the depth of the sample collection chamber=4
mm, and the diameter of the O-ring is 4 mm.
[0060] FIG. 6A-F is a series of photographs depicting the capture
of a 200 bp fraction, of a DNA ladder over time using the
electrophoresis cassette of FIG. 5 and Example 3. Arrow indicates
the direction of electrophoresis at the division point.
[0061] FIG. 6G is a photograph of an agarose gel analysis of the
experiment of FIG. 6A-F confirming the specific capture of the 200
bp fraction in the elution chamber.
[0062] FIG. 7 is an illustration of a multichannel preparative
electrophoresis cassette.
[0063] FIG. 8A-H is a series of illustrations showing the
fractionation of a sample over time using an exemplary detection
system.
[0064] FIG. 9 is an illustration depicting the collection of a DNA
fraction within an elution chamber having a DNA permeable membrane,
a sample collection chamber for retaining DNA, and a DNA
impermeable membrane, such as Nafion, to prevent DNA escape.
[0065] FIG. 10 is an illustration depicting the collection of a DNA
fraction within an elution chamber having a gel plug, a
buffer-filled sample collection chamber for retaining DNA, and a
DNA impermeable membrane, such as Nafion, to prevent escape.
[0066] FIG. 11 is a schematic representation depicting an
electrophoresis system with a T-shaped elution channel and a gel
lens. This electrophoresis system contains an electrode chamber at
the T-junction. Fractions are collected unilaterally.
[0067] FIG. 12 is a schematic representation depicting an
electrophoresis system with a T-shaped elution channel without a
gel lens. This electrophoresis system contains an electrode chamber
at the T-junction. Fractions are collected unilaterally.
[0068] FIG. 13 is a schematic representation depicting an
electrophoresis system with a T-shaped elution channel and a gel
lens. This electrophoresis system contains an electrode chamber at
the T-junction. Desired fractions are differentiated from the
remaining sample by directing those fractions to the elution
chamber (or selected-band elution well) rather than the waste
reservoir (or waste elution well).
[0069] FIG. 14 is a schematic representation depicting an
electrophoresis system with a T-shaped elution channel without a
gel lens. This electrophoresis system contains an electrode chamber
at the T-junction. Desired fractions are differentiated from the
remaining sample by directing those fractions to the elution
chamber (or selected-band elution well) rather than the waste
reservoir (or waste elution well).
[0070] FIG. 15 is a schematic representation depicting an
electrophoresis system with a T-shaped elution channel and a gel
lens. This electrophoresis system is lacking an electrode module at
the T-j unction depicted in FIGS. 11-14, and contains an optional
electrode in its place. Desired fractions are differentiated from
the remaining sample by directing those fractions to the elution
chamber (or selected-band elution well) rather than the waste
reservoir (or waste elution well).
[0071] FIG. 16 is a schematic representation depicting an
electrophoresis system with a T-shaped elution channel without a
gel lens. This electrophoresis system is lacking an electrode
module at the T-junction depicted in FIGS. 11-14, and contains an
optional electrode in its place. Desired fractions are
differentiated from the remaining sample by directing those
fractions to the elution chamber (or selected-band elution well)
rather than the waste reservoir (or waste elution well).
[0072] FIG. 17 is a schematic representation depicting an
electrophoresis system with asymmetric elution channels and a gel
lens. Fractions are captured by using sample collection chambers
having differentially permeable membranes on either end. Desired
fractions are differentiated from the remaining sample by directing
those fractions to elution Chamber A rather than elution Chamber B,
or vice versa.
[0073] FIG. 18 is a schematic representation depicting an
electrophoresis system with asymmetric elution channels and a gel
lens. Fractions are captured by using sample collection chambers
having differentially permeable membranes on either end. Desired
fractions are differentiated from the remaining sample by directing
those fractions to one or more designated elution chambers (e.g.
Chamber A versus Chamber B or C). Although three elution chambers
are depicted, the illustrated electrophoresis system can contain
multiple channels of any number. Preferred embodiments contain up
to 13 channels for sample or fraction collection.
[0074] FIG. 19 is series of schematics of a multichannel
preparative electrophoresis cassette, having 5 macrofluidic
channels. The channel plate is contacted to the cover plate and the
sample well insert traverses the sample well insert opening of the
cover plate. Three perspectives are shown.
[0075] FIG. 20 is a blow-up schematic of the multichannel
preparative electrophoresis cassette of FIG. 19. The channel plate,
elution chambers, cover plate, and sample well insert are detached
to reveal detail.
[0076] FIG. 21 is a series of schematics of a multichannel
preparative electrophoresis cassette, having 5 macrofluidic
channels. Left Panel: The channel plate shows 5 tapered
macrofluidic separation channels each having an elution chamber.
Right Panel: The cover plate with a configuration of that
corresponds to the channel plate.
[0077] FIG. 22 is a schematic of a multichannel preparative
electrophoresis cassette, having 5 macrofluidic channels. The
channel plate shows 5 tapered macrofluidic separation channels each
having an elution chamber cavity for housing an elution
chamber.
[0078] FIG. 23 is a schematic of the underside of the multichannel
preparative electrophoresis cassette of FIG. 22.
[0079] FIG. 24 is a series of schematics of an elution chamber
having a sample collection chamber and a sample collection port in
two perspectives.
[0080] FIG. 25 is a series of schematics of an elution chamber
having, with respect to the direction of electrophoresis, an
analyte-permeable barrier, a sample-collection chamber, and an
analyte-impermeable barrier. The sample collection chamber further
contains a sample-collection port.
[0081] FIG. 26 is a series of schematics of an electrophoresis
cassette contacted with a cover plate, wherein a buffer reservoir
insert, a sample well insert, and a waste reservoir insert traverse
the cover plate. Exemplary buffer reservoir inserts contain a vent.
Furthermore, exemplary waste reservoir inserts contain at least one
injection port. Three-perspectives are given of these components
assembled and disassembled to show detail. Gels are caste by
inserting a liquid gel matrix into the injection port and allowing
the gel to harden into a solid form. Inserts are then removed and
the resultant buffer reservoir, sample well, elution reservoir, and
waste reservoir are filled with a buffer composition.
[0082] FIG. 27 is a schematic of an "uncovered" detection system,
depicting an electophoresis cassette placed over light-emitting
diodes and an optics housing, surrounded by the processor elements
that signal detection and selective activation/deactivation of the
electrode array.
[0083] FIG. 28 is a schematic of the "uncovered" detection system
of FIG. 27 from an alternative perspective.
[0084] FIG. 29 is a schematic of the "covered" detection system of
FIG. 27.
[0085] FIG. 30 is an illustration of the optical system of an
exemplary electrophoresis system.
[0086] FIG. 31 is a graph of the fluorescence over time of mixed 50
and 100 by ladders detected using the system of FIG. 31,
demonstrating the sensitivity of detection at a concentration of 1
ng per fraction, or band, within the separation channel.
[0087] FIG. 32 is a series of graphs depicting the fluorescence
versus time signal of digested genomic DNA compared to a 100 bp DNA
ladder. These graphs show the real-time optical detection to
control DNA purification.
[0088] FIG. 33 is a schematic diagram of an exemplary basic
electrophoresis system.
[0089] FIG. 34 is a schematic diagram of exemplary shapes of a
constriction point within the separation channel of an
electrophoresis cassette.
[0090] FIG. 35 is a schematic diagram of exemplary edge
characteristics of a constriction point within the separation
channel of an electrophoresis cassette.
[0091] FIG. 36 is a schematic diagram of an exemplary
electrophoresis cassette base, without a cover, containing cavity
for an upper dam (66) (a first dam), located distal to the buffer
reservoir and proximal to the sample well cavity, and a cavity for
a lower dam (67) (a second dam), located distal to the division
point and proximal to the waste reservoir.
[0092] FIG. 37 is a schematic diagram of tilted view of the
electrophoresis cassette shown in FIG. 36.
[0093] FIG. 38 is a schematic diagram of an exemplary
electrophoresis cassette with a first dam, a second dam, and a
solid gel, without a cover plate. The sample wells show a gel
chimney.
[0094] FIG. 39 is a schematic diagram of an exemplary
electrophoresis cassette cover, including at least one electrode
port (68), at least one vent (49), at least one sample well port
(69), at least one sample collection port of an elution chamber
(29), and at least one injection or gel solution input port (50).
The upper dam lies under the cover, positioned between the proximal
electrode port and the vent hole. The lower dam lies under the
cover, positioned between the gel solution input port and the
distal electrode port.
[0095] FIG. 40 is a schematic diagram of a tilted view of the
exemplary electrophoresis cassette and cover shown in FIG. 39.
[0096] FIG. 41 is a schematic diagram of an exemplary
electrophoresis cassette including a first dam positioned within
the cavity for the first dam and a second dam positioned within the
cavity for the second dam.
[0097] FIG. 42 is a schematic diagram of an exemplary
electrophoresis cassette, cross-sectioned in the center of the
separation channel, to demonstrate the relative positions of the
first and second dams, as well as the sample well insert and
stripper plate.
[0098] FIG. 43A is a schematic diagram of a dam with a membrane
attached to its plastic frame.
[0099] FIG. 43B is a schematic diagram of a dam depicted in FIG.
43A, in an exploded view.
[0100] FIG. 44 is a schematic diagram of an exemplary
electrophoresis cassette, cross-sectioned in the vicinity of the
sample well to depict the relative positions of the sample well
insert and the stripper plate, which together, form a
chimney-shaped sample well.
[0101] FIG. 45 is a schematic diagram of a rotated view of the
electrophoresis cassette depicted in FIG. 44.
[0102] FIG. 46 is a schematic diagram of the rotated view of the
electrophoresis cassette depicted in FIG. 45, with the sample well
insert removed to show the resultant chimney sample well.
[0103] FIG. 47 is a schematic diagram of the rotated view of the
electrophoresis cassette depicted in FIG. 46, with sample comb and
the stripper plate removed to depict the chimney sample well ready
for sample loading. Note that the top surface of the gel chimney is
flush with the top of the cassette cover.
[0104] FIG. 48 is a photograph of an exemplary electrophoresis
cassette similar to the one depicted in FIG. 47.
DETAILED DESCRIPTION
[0105] It is a common practice in biological experimentation to
separate macromolecules such as proteins and nucleic acids, e.g.,
DNA or RNA, for analytical and preparative purposes using
electrophoresis. Electrophoresis separates biomolecules by charge
and/or size via mobility through a separating matrix in the
presence of an electric field. Gel separating matrices are
typically prepared from agarose for nucleic acid separation and
polyacrylamide for protein separation. In capillary
electrophoresis, the matrices may be gels or solutions (e.g.,
linear polyacrylamide solution).
[0106] Gel separating matrices are typically made by pouring a
liquid phase material into a mold formed by glass plates or
separating matrix casting molds. In slab gel electrophoresis, for
example, finger shaped outcroppings in plastic material form
"combs" that are embedded in the top of the separating matrix.
Sample loading wells are formed when the combs are removed from the
solidified separating matrix. Loading these wells is typically a
time consuming and technically challenging task. Dense solutions
such as glycerol or polyethylene glycol are often added to samples
prior to electrophoresis to prevent samples from mixing with
electrode buffers and floating out of the wells.
[0107] Samples, generally in an aqueous buffer, are applied to the
separating matrix and electrodes in electrical contact with the
separation matrix are used to apply an electric field. The field
induces charged materials, such as nucleic acids and proteins, to
migrate toward respective anode or cathode positions.
Electrophoresis is usually completed in about 30 minutes to several
hours.
[0108] The migration distances for the separated molecular species
depend on their relative mobility through the separating matrix.
Mobility of each species depends on hydrodynamic size and molecular
charge. Proteins are often electrophoresed under conditions where
each protein is complexed with a detergent or other material that
imparts a negative charge to proteins in the sample. The detergent
causes most or all of the proteins to migrate in the same direction
(toward the electrophoresis anode). Samples are stained prior to,
during, or after a separation run to visualize the nucleic acids or
proteins within the gel. The location of the various components in
the gel is determined using ultraviolet light absorbance,
autoradiography, fluorescence, chemiluminescence, or any other well
known means of detection. To determine the molecular weight and
relative concentration of unknown nucleic acids or proteins, the
band positions and intensities are typically compared to known
molecular standards.
[0109] Electrophoresis cassettes and systems of the invention
separate, condense, detect, analyze, and collect desired fractions
of analytes within a biological sample. As described in the figures
provided, and defined, in part, in Table 1, the cassettes and
systems of the invention includes distinctive features and
corresponding functions.
[0110] Exemplary electrophoresis cassettes are molded from a
plastic, such as polystyrene and its derivatives, or PMMA.
Alternatively, the electrophoresis cassette is molded using any
optically clear polymer. Electrophoresis cassettes are either
molded as one contiguous piece, or they are assembled from multiple
pieces, each molded from plastic or an appropriate optically clear
plastic that are connected to form a contiguous piece.
[0111] Electrophoresis cassettes of the invention include
macrofluidic channels, rather than microfluidic channels or
nanochannels, to direct and fraction samples. The use of
macrofluidic channels is essential to ensure that a sufficient
amount of an analyte or sample is prepared or analyzed within a
single application of the sample to the cassette such that the
collected fraction can be used directly for further manipulation
and analysis. For example, an isolated analyte or fraction is
subsequently sequenced or inserted into a vector or cell.
[0112] Macrofluidic channels of the invention have a minimal
demonstrated width of 2 mm, which occurs at either the constriction
point or division point of the channel (FIG. 1C). The greatest
demonstrated width of the macrofluidic channels of the invention is
7 mm (FIG. 1C), which occurs near the sample well cavity of the
separation channel. In most embodiments the depth of the
macrofluidic channel is uniform, at approximately 6 mm (FIG. 1C).
However, these dimensions increase and decrease within preferred
ranges. The preferred width of a macrofluidic channel ranges from
between 2 mm to 10 mm and the preferred depth of a macrofluidic
channel ranges from between 2 mm to 10 mm.
[0113] Macrofluidic channels of the invention include physically
and electrically isolated portions. The term "physically isolated"
is meant to describe a channel arrangement in which one portion of
the channel is separated from another portion of the channel by a
physical barrier such that the analyte contained in one portion
cannot intermix with the analyte contained in another portion. The
term "electrically isolated" is meant to describe a channel
arrangement in which the electrode positioned at one portion of the
channel is controlled separately from the electrode positioned at
another portion of the channel. The use of electrically and
physically isolated channels both prevents contamination of the
selected fractions, which can occur in gel slab systems that lack
any barriers between lanes, and improves directional elution of
selected fragments.
[0114] Macrofluidic channels also contain cavities and reservoirs.
The term "cavity" is used to describe a portion of the channel that
is reserved for either the attachment of a structure, the insertion
of a structure within its volume, of the generation of a structure.
A structure is formed, for instance by the placement of the sample
well insert into the sample well cavity, the injection and
solidification of a gel matrix composition, and the removal of the
sample well insert. The term "reservoir" is meant to describe a
cavity that is filled with a buffer composition.
[0115] Elution chambers of the invention include analyte-permeable
and analyte-impermeable barriers. The term "analyte-permeable" is
meant to describe any barrier that is permeable to ions,
polynucleic acids, and polypeptides, but not to, any other
component of the gel matrix composition or buffer composition. The
term "analyte-impermeable" is meant to describe any barrier that is
permeable to ions, but impermeable to polynucleic acids,
polypeptides, any other component of the gel matrix composition,
buffer composition, or elution composition.
[0116] One of the superior properties of the electrophoresis
cassette of the invention is the collection analyte, or fraction
thereof, in an elution buffer composition. Other preparative
electrophoresis systems require the user to extract, for example, a
DNA fraction, from a gel or membrane following electrophoresis.
This secondary DNA extraction step is time-consuming and
significantly decreases the overall yield of DNA obtained from that
fraction. In contrast, electrophoresis systems of the invention
integrate the steps of polynucleotide or polypeptide separation and
collection by providing an elution chamber, which simultaneously
fractions and extracts the polynucleotide or polypeptide analyte
into any desired elution buffer.
[0117] Macrofluidic channels also include one or more engineered
constriction points. Constriction points enable and improve the
isolation of an analyte within sample. Physical parameters of the
constriction point vary among exemplary electrophoresis cassettes
and separation channels. Constriction points within existing
preparative electrophoresis systems have been used to hold a
vertical gel in place and reduce the volume of eluting liquid
before capture. In contrast, the physical constriction of the
separation channel within the electrophoresis cassettes of the
invention produces an electric field gradient. In a basic
embodiment, a small bore hole drilled in a plastic block serves as
a constriction point.
[0118] Features of the constriction, or constriction point, vary
between electrophoresis cassettes and between separation channels
of a multichannel cassette. For example, the shape of the
constriction by cross-sectional view is either a venturi tube, flow
nozzle, or orifice place, as shown in FIG. 34. The placement of the
constriction point within the separation channel varies. When the
electrophoresis cassette is divided in half horizontally, through
the separation channel, the constriction point is positioned either
within the top or bottom half of the channel. When the
electrophoresis cassette is divided in half vertically, through the
separation channel, the constriction point is positioned either
within the left or right half of the channel. Alternatively, the
constriction point is located in the center of the separation
channel, considered from either above-referenced perspective. From
a perspective directly facing the constriction point, or a head-on
perspective, the shape of the constriction is circular, oval,
square, or rectangular. The cross-sectional area occupied by the
constriction in comparison with the cross-sectional area of the
separation channel, either upstream or downstream of the division
or branch point varies. In certain embodiments the cross-sectional
area occupied by the constriction occupies 90%, 80%, 70%, 60%, 50%,
40%, 30%, 20%, 10%, 5%, or any percentage point in between of the
cross-sectional area of the separation channel. Additional
parameters that vary between separation channels of a multichannel
electrophoresis cassette or between electrophoresis cassettes
include, but are not limited to, the length of the constriction,
the gradient of the taper and/or flare of the constriction, the
symmetry or asymmetry of the constriction, and the material used to
form the constriction, as well as the texture/uniformity of that
material.
[0119] The constriction point of the macrofluidic separation
channel can also be the division point. Alternatively, the
macrofluidic separation channel contains at least one division
point. The term "division point" is meant to describe a point at
which the macrofluidic channel splits or branches into one or more
physically and electrically isolated portions.
[0120] Macrofluidic channels contain at least one of a gel matrix
composition, a liquid buffer composition, or a solid buffer
composition. Gel matrix compositions contain a polymerizing
compound, such as agarose or polyacrylamide, for the separation of
polynucleic acids and polypeptides, respectively. Polymerizing
compounds are provided at percentages ranging from 0.01%-99.9%.
Electrophoresis buffer compositions known in art are used herein.
Buffer solutions are preferably electrolyte solutions.
[0121] Electrophoresis cassettes optionally contain electrodes that
are either disposable or reusable. Disposable electrodes are
integrated into the cassettes and made from epoxy with conductive
particles, inks, or rubber. Reusable electrodes are made of coated
titanium or platinum probes.
[0122] Sample wells have multiple geometries. The geometry of the
sample well reflects the geometry of the sample well insert used to
define the negative space not occupied by the gel matrix
composition. Preferably, the sample wells of the invention have the
have a unique and essential "chimney" shape depicted in FIGS.
44-48. Generally, sample well insert, or sample comb has a simple
rectangular shape, which forms a simple rectangular negative space
within the gel. As such, in such a gel, the top of the sample well
is level with the top of the gel and, if a cover were applied, the
top of the well would be flush or level with the bottom of the
cover. However, under certain circumstances this sample well
geometry, particularly when a cover is attached to the
electrophoresis cassette base, allows for leakage of the sample in
the liquid-filled space between the top of the gel and the cover
plate. This leakage leads to contamination of the desired fractions
within the eluction chamber.
[0123] The chimney geometry was developed in conjunction with an
adaptation of the cover, i.e. the sample well port, to support the
gel chimney and prevent leakage of the sample, and, therefore,
contamination of desired fractions within the elution chamber. The
sample well port of the cover is adapted to support the gel
chimneys of the sample well.
[0124] The chimney well is a superior property of this invention
because the purpose of preparative electrophoresis is the precise
and exact separation of fractions from a sample that differs in a
physical property. In certain circumstances, the differences
between collected and discarded fractions are very subtle.
Contamination of the collected fractions with random analyte from
the sample pulled into the seam by capillary action between the gel
and the bottom of the cover plate significantly distorts the
results. Thus, the prevention of this contamination and the
creation of chimney wells provides a superior and distinguishing
feature of the invention.
Samples, Analytes, and Fractions
[0125] Electrophoresis cassettes and detection systems of the
invention fractionate, analyze, and collect polynucleic acid and
polypeptide analytes or fractions within a sample.
[0126] The term "sample" describes a plurality of molecules that
can be separated using gel electrophoresis. The term "fraction"
describes a subset of the plurality of molecules within a sample. A
fraction is defined or determined by size. Alternatively, a
fraction is defined or determined by any physical property that
causes it to migrate at a faster or slower rate than other
components or fractions of a sample when driven to migrate through
a buffer composition of the invention by the force of an electric
field (i.e., electrophoretic mobility).
[0127] An exemplary sample includes, but is not limited to, a
nucleic acid, an oligonucleotide, a DNA molecule, a RNA molecule,
or any combination thereof. Alternatively, or in addition, a sample
includes, but is not limited to, an amino acid, a peptide, a
protein, or any combination thereof. For example, a sample is a
whole cell lysate, or the DNA or protein fraction of a cell
lysate.
[0128] Nucleic acids are derived from genomic DNA, double-stranded
DNA (dsDNA), single-stranded DNA (ssDNA), coding DNA (or cDNA),
messenger RNA (mRNA), short interfering RNA (siRNA), short-hairpin
RNA (shRNA), microRNA (miRNA), single-stranded RNA, double-stranded
RNA (dsRNA), a morpholino, RNA interference (RNAi) molecule,
mitochondrial nucleic acid, chloroplast nucleic acid, viral DNA,
viral RNA, and other organelles with separate genetic material.
Furthermore, samples include nucleic acid analogs that contain
modified, synthetic, or non-naturally occurring nucleotides or
structural elements or other alternative/modified nucleic acid
chemistries known in the art. Additional examples of nucleic acid
modifications include the use of base analogs such as inosine,
intercalators (U.S. Pat. No. 4,835,263) and minor groove binders
(U.S. Pat. No. 5,801,115). Other examples of nucleic acid analogs
and alternative/modified nucleic acid chemistries known in the art
are described in Current Protocols in Nucleic Acid Chemistry, John
Wiley & Sons, N.Y. (2002).
[0129] PNA oligomers are included in exemplary samples or fractions
of the invention. PNA oligomers are analogs of DNA in which the
phosphate backbone is replaced with a peptide-like backbone
(Lagriffoul et al., Bioorganic & Medicinal Chemistry Letters,
4: 1081-1082 (1994), Petersen et al., Bioorganic & Medicinal
Chemistry Letters, 6: 793-796 (1996), Kumar et al., Organic Letters
3(9): 1269-1272 (2001), WO96/04000).
[0130] Polypeptides or proteins are complex, three-dimensional
structures containing one or more long, folded polypeptide chains.
Polypeptide chains are composed of a plurality of small chemical
units called amino acids. Naturally occurring amino acids have an
L-configuration. Synthetic peptides can be prepared employing
conventional synthetic methods, using L-amino acids, D-amino acids
or various combinations of L- and D-amino acids. The term "peptide"
describes a combination two or more amino acids. Naturally
occurring amino acids have an L-configuration. Peptides having
fewer than ten amino acids are "oligopeptides," whereas peptides
containing a greater number of amino acid units are "polypeptides."
Any reference to a "polypeptide" also includes an oligopeptide.
Further, any reference to a "peptide" includes polypeptides and
oligopeptides. Each different arrangement of amino acids forms a
different polypeptide chain.
[0131] The term "nucleic acid molecule" describes the phosphate
ester polymeric form of ribonucleosides (adenosine, guanosine,
uridine or cytidine; "RNA molecules") or deoxyribonucleosides
(deoxyadenosine, deoxyguanosine, deoxythymidine, or deoxycytidine;
"DNA molecules"), or any phosphoester analogues thereof, such as
phosphorothioates and thioesters, in either single stranded form,
or a double-stranded helix. The term nucleic acid molecule, and in
particular DNA or RNA molecule, refers only to the primary and
secondary structure of the molecule, and does not limit it to any
particular tertiary forms. Thus, this term includes double-stranded
DNA found, in linear or circular DNA molecules (e.g., restriction
fragments), plasmids, and chromosomes. A "recombinant DNA molecule"
is a DNA molecule that has undergone a molecular biological
manipulation. (see Sambrook et al. Molecular Cloning, A Laboratory
Manual, Cold Spring Harbor Laboratory Press).
[0132] Samples are combined with a reagent that imparts a net
negative charge, denatures a peptide or protein, or digests a DNA
or RNA molecule prior to application to an electrophoresis system.
These reagents are known in the art. Furthermore, samples are
combined with agents that impart fluorescent, magnetic, or
radioactive properties to the sample or fractions thereof for the
purpose of detection. In one embodiment of the system, a dsDNA
sample is mixed with ethidium bromide, applied to the
electrophoresis cassette, and fractions of the sample are detected
using an ultrabright green LED.
[0133] All standard and specialty buffers known in the art are used
with samples, and fractions thereof, as well as to make the buffer
compositions the fill the electrophoresis cassettes of the
system.
[0134] Regarding polypeptides, the term "native" is meant to
describe a non-denatured polypeptide. Polypepide analytes of the
invention are native or denatured.
Detection System
[0135] Detection systems of the invention are compact and
automated. These systems are designed and intended for desktop or
bench-top use. Furthermore, electrophoresis cassettes of these
systems are disposable.
[0136] Systems include at least one electrophoresis cassette with
means to fractionate, detect, analyze, and collect a polynucleic
acid or polypeptide analyte or fraction within a sample.
[0137] Systems also include a detection module with means to detect
and analyze, for instance, to quantify, a signal. Exemplary signals
include, but are not limited to, visible light, fluorescent light,
magnetic fields, and radioactivity. Detection modules are
positioned at a detection zone or constriction point of the
separation channel of an electrophoresis cassette. Alternatively,
the position of the detection module is shifted towards the entry
or exit points of the constriction. The constriction point or
detection zone is proximal to the sample well. The detector tracks
a marker and the processor determines, based upon the size, speed,
electrophoretic mobility, and/or timing of the marker, when an
analyte of the desired molecular weight or electrophoretic mobility
will traverse the division point.
[0138] Included in these systems is an illumination source that is
either independently incorporated into the system or incorporated
into the detection module. The illumination source uses ultra
bright light emitting diode (LED) in combination with a filter set
and one or more photodiodes, for instance.
[0139] The detection module of the system, which optionally
includes an illumination source, is coupled with a microprocessor
control system. The microprocessor control system includes a
microprocessor, software, and a set of relays with means to control
a voltage switching scheme that differentially activates a
combination of the negative and at least one positive electrode in
order to divert a sample or fraction thereof to an intended
collection point at the end of the separation channel. In another
aspect of the invention, a laptop is substituted for the use of an
incorporated microprocessor. Exemplary software for controlling
these systems is developed for use on a laptop or with the
incorporated microprocessor.
[0140] Systems further include an integrated or separate power
source.
[0141] Systems of the invention are designed to such that the
separation channels of the incorporated electrophoresis cassettes
are positioned horizontally with respect to a table- or desktop.
Alternatively, the system is configured such that the separation
channels of the incorporated electrophoresis cassettes are
positioned vertically with respect to a table- or desktop.
TABLE-US-00001 TABLE 1 FIGURE Reference Numbers Reference Number
Structure 1 Base plate 2 Channel plate 3 Waste reservoir 4 Elution
reservoir 5 First physically and electrically isolated portion of
separation channel 6 Second physically and electrically isolated
portion of separation channel 7 Division Point 8 Constriction Point
9 Separation channel 10 Sample well cavity 11 Buffer reservoir 12
Alignment excision 13 Elution reservoir insert opening 14 Waste
reservoir insert opening 15 Constriction and division point opening
16 Sample well insert opening 17 Buffer reservoir insert opening 18
Buffer reservoir insert 19 Sample well insert 20 Elution reservoir
insert 21 Waste reservoir insert 22 End of separation channel 23
Cover plate 24 DNA sample 25 Desired analyte or fraction 26 Groove
27 First removable end of elution chamber 28 Sample collection
chamber of elution chamber 29 Sample collection port of elution
chamber 30 Second removable end of elution chamber 31 Gasket 32
Analyte-permeable barrier (e.g. Durapore Membrane) 33
Analyte-impermeable barrier (e.g. Nafion Membrane) 34 Elution
chamber spacer 35 Elution chamber wedge 36 Sample collection
channel 37 O-ring 38 DNA Marker 39 Negative Electrode 40 Sample
well 41 Positive Electrode 42 Connector 43 Elution chamber opening
in cover 44 Alignment protrusion 45 Elution chamber cavity 46
Elution chamber 47 Elution reservoir and waste reservoir cavity 48
Waste reservoir insert 49 Vent 50 Injection Port 51 Electrophoresis
cassette 52 Processor board for power module 53 Input/Output
connector 54 Single computer board 55 Optics housing 56
Photodetector 57 Photodetector processor board 58 Hard disk drive
59 Light-emitting diode 60 Casing of detector system 61
Light-emitting diode 62 Emission filter 63 Light-focusing filter 64
Dichronic mirror 65 Excitation filter 66 Cavity for upper dam
(first dam) 67 Cavity for lower dam (second dam) 68 Electrode Port
69 Sample Well Port 70 Dam Frame 71 Dam Membrane 72 Gel Chimney 73
Cover Alignment Pins 74 Negative (-) Electrode Port 75 Positive (+)
Electrode Port 76 Upper/First Dam 77 Lower/Second Dam 78 Top of Gel
79 Gel 80 Stripper Plate
EXAMPLES
Example 1
Size Fractionation of Genomic DNA by Agarose Gel Electrophoresis in
Y-Shaped Cassette
[0142] The Y-shaped cassette used for this experiment is
illustrated in FIG. 1. The channel plate and cover were machined
from polycarbonate, the base was fused silica, and the molding
inserts used to form buffer and sample wells were machined from
Teflon. To cast the gel cassette, the channel plate was coated
front and back with a thin coating of a dielectric silicone sealant
to prevent leakage of buffer and electrical current. The base and
cover were pressed against the channel plate and held together with
binder clips throughout casting and electrophoresis.
[0143] The electrophoresis gel used was 2% agarose (SeaKem LE
agarose, Lonza) in 0.5.times.KBB buffer (1.times.KBB buffer is 12.4
g/liter Tris base, 14 g/liter TAPS acid, 0.048 g/liter EDTA free
acid). The gel and liquid buffer contained 1.5 ug/ml ethidium
bromide, to enable visualization of the DNA by fluorescence under
UV transillumination. The agarose was heated in water until
dissolved and then cooled to approximately 60.degree. C. Buffer and
ethidium were added and the solution was thoroughly mixed. The
cassette was filled in a horizontal position, with all well-forming
inserts removed, until the cassette was slightly overfilled. The
well-forming inserts were immediately installed. The triangular gap
in the cassette cover which is located over the channel
constriction was covered with a glass coverslip. Care was taken to
avoid introduction of bubbles or silicone sealant into the
channel.
[0144] The cassette was allowed to solidify for approximately 1
hour at room temperature. The well-forming inserts were removed
from the cassette and all wells were filled with electrophoresis
buffer (0.5.times.KBB buffer with 1.5 ug/ml ethidium bromide). The
cassette was placed in a horizontal position on a UV
transilluminator (Fotodyne, 300 nm peak output). A high voltage
electrophoresis power supply (E-C apparatus) was connected to
platinum electrodes in the buffer reservoirs of the cassette.
[0145] A sample of calf thymus DNA (Sigma Chemical) was digested to
completion with BfuCI (New England Biolabs). Two ug of digested DNA
was dissolved in 40 ul of 40% sucrose, 10 mM Tris-HCl, pH 8.0, 1 mM
EDTA, and loaded in the sample well of the cassette.
[0146] Electrophoresis was carried out at a constant voltage of 100
V. The negative electrode was connected to the single buffer
reservoir upstream from the sample well. The positive electrode was
connected to buffer reservoir of the waste leg of the cassette
initially. The electrophoresis buffer reservoirs were exchanged
with fresh buffer every 10 minutes during the run.
[0147] Electrophoresis was carried out using the waste leg
electrode for 57 minutes (see images FIG. 2A-C). At that point,
waste leg electrode was disconnected, and the purification leg
electrode was connected to the power supply for 2 minutes (see
images FIGS. 2D-G). After 2 minutes, the purification leg electrode
was disconnected and the waste leg electrode was reconnected.
Electrophoresis was continued into the waste leg for approximately
3-4 more minutes (see images FIGS. 2H-L). Then power was turned
off. The cassette was unclamped and the cover was removed. The gel
in the separation channel was cut away from the gel in the
purification and waste legs of the cassette near the narrowest
point of the legs. The separation channel gel was discarded and the
separation channel was refilled with electrophoresis buffer.
[0148] DNA in the waste and purification channels was isolated by
electrophoresing the DNA onto strips of DEAE ion-exchange membrane
(Sartorius Stedim) were inserted into thin horizontal slits in the
gel just downstream of the desired DNA fractions (see FIG. 2L).
Electrophoretic capture of the DNA on the membranes was carried out
at 100 V for approximately seven minutes. Purification leg DNA was
isolated first and sample membrane was removed to prior to
isolation of DNA from the waste leg.
[0149] DNA was recovered from ion-exchange membranes, by immersing
membranes in 0.4 ml 1.times.KBB and 1M NaCl at 65.degree. C. for 30
minutes. After removal of ion-exchange strips from tubes, 10 ul of
0.25% linear polyacrylamide was added to each tube and vortexed to
mix. DNA was precipitated with 1 ml ethanol to each tube. DNA
pellets were rinsed in 100% ethanol, and air dried. DNA samples
were resuspended in 15 ul TE buffer (10 mM Tris-HCl, pH 8.0, 1 mM
EDTA), and mixed with 15 ul of 40% sucrose in TE buffer. Entire
amount was loaded onto 2% analytical slab agarose gel in
0.5.times.KBB with 1.5 ug/ml ethidium bromide. The image of the
analytical gel is shown in FIG. 2M. The DNA recovered from the
purification leg measures approximately 344 bp with an edge to edge
band width of approximately 40 bp. The DNA from the waste leg shows
almost complete absence of DNA in the region of 340 bp, indicating
good purification efficiency into the purification leg.
Example 2
Size Fractionation of Genomic DNA by Agarose Gel Electrophoresis in
Y-Shaped Cassette with Liquid Filled Elution Chamber
[0150] In order to demonstrate recovery of fractionated
size-selected DNA in a liquid filled buffer chamber, the device of
FIG. 1 was modified to include a membrane-bounded chamber in the
purification channel. The cassette with elution chamber is shown in
FIGS. 3A-C. The dimensions of the separation channel and waste
channel were similar to the cassette of FIG. 1.
[0151] The elution chamber was a rectangular plastic channel
(polycarbonate) that was bounded on the front side (the side
proximal to the separation channel, see FIG. 4A, 3B) by a membrane
that is porous to DNA, and low in nonspecific DNA binding (Durapore
SVLP, Millipore, 5 um pore size). On the back side of the chamber
(the side proximal to the (+) electrode, see FIG. 4A, 3B), a
membrane that is non-porous to DNA and low in nonspecific DNA
binding was installed (Nafion 117, Ion Power). The membranes were
tightly sealed over the faces of the elution chamber by rectangular
plastic frames that snap over the protruding lip of the chamber
(see FIG. 3C). The top surface of the elution chamber has a small
circular hole which was used for filling and emptying the channel
with a standard handheld micropipettor (FIGS. 3C and 4A). The
volume of the fully assembled elution chamber was approximately 90
ul.
[0152] The cassette was assembled as described in Example 1, with
the exceptions that dielectric silicone sealant was not used
between the channel plate and the top cover. In this example, the
top surface of the channel plate was sealed with a cast silicone
gasket, which is labeled in FIGS. 3C and 4A.
[0153] The agarose gel (same composition as in Example 1) was cast
with an empty, membrane-free elution chamber inserted into the
cassette. After the gel solidified, the top plate was removed and
the gel column was sliced across the front and back openings of the
gel elution chamber. The elution module was removed and the gel was
cleaned from the inside of the module. Nafion and Durapore
membranes were assembled onto the chamber, and it was reinserted
into the cassette. Dielectric silicone sealant was used on the side
and bottom exterior surfaces of the elution chamber to prevent
electrical leaks around the elution chamber. The assembled cassette
was clamped with binder clips as in Example 1.
[0154] The sample was 2 ug BfuCI-digested calf thymus genomic DNA.
Electrophoresis was carried out at constant voltage of 100 V. The
waste channel electrode was used for 1 hr and 9 minutes, and then
voltage was switched to the purification channel for 5 minutes.
Following this, voltage was switched back to the waste channel for
an additional 3 minutes before terminating electrophoresis.
[0155] After the run, 90 ul of buffer was removed from the elution
chamber with a hand-held micropipette. Eluted sample was ethanol
precipitated as described in example 1. To estimate the efficiency
of DNA recovery by the process, DNA in the gel of the waste leg was
isolated in the vicinity of the "gap" in the DNA pattern (caused by
the removal of DNA into the purification channel). DNA was
extracted from the gel slice using a commercial kit (Qiagen
Minelute Gel extraction kit).
[0156] An analytical 2% agarose gel of the products (see FIG. 4B)
shows efficient purification of a DNA band of approximately 300 bp,
similar to the results shown in Example 1. In this example,
however, the selected DNA product was obtained in liquid buffer
directly from the elution chamber, without the need to perform gel
extraction. The fractionation process was efficient, as judged by
the absence of similar-sized DNA in the sample recovered from the
waste channel.
Example 3
Purification of Specific DNA Band in Cassette with Tapered Channel
and Simplified Elution Chamber Design
[0157] An alternative cassetted design is shown in FIGS. 5A-B. The
cassette features a tapered separation channel. As seen in FIGS.
6D-F, DNA bands are compacted from their original thin and wide
shape near the sample well into square (or compact rectangular)
shapes as they arrive at the three-way channel junction. For this
reason, a tapered separation channel should provide improved size
resolution in purification when compared with separation channels
with rectangular profile like those described in Examples 1 and 2
above.
[0158] The elution chamber of this cassette is constructed from
three plastic parts shown in FIG. 5B. Compressible O-rings are used
to position and seal membranes on either side of the elution
chamber, as illustrated in FIG. 5B. The volume of the elution
chamber is approximately 50 ul.
[0159] To cast the gel used for this example (same gel and buffer
formulation as used in Example 1 above), the top and bottom surface
of the channel plate was sealed with clear packaging tape (Scotch
brand packaging tape, 3M). The purification channel and
electrophoresis buffer compartments were left uncovered on the top
side of the channel plate. The elution chamber was assembled with a
non-porous sheet of PETG sealing the chamber entrance from the
separation channel side. The gel was cast through the buffer
reservoir of the waste channel, thereby filling the waste and
separation channels only. The purification channel contained no
gel, except at the entrance to the elution chamber. After the gel
solidified, the elution chamber was disassembled and the PETG
membrane was discarded. The purification channel was filled with
electrophoresis buffer. The elution chamber was reassembled in the
buffer-filled purification channel with porous membrane (Durapore
BVPP, 1 um pore size, Millipore) on the separation channel side of
the chamber, and non-porous membrane (Nafion 117) on the electrode
side of the chamber. Care was taken to ensure that no bubbles were
trapped in the channel through the elution chamber and spacer.
[0160] The sample consisted of 1 ug of a 100 bp DNA marker ladder
(100 bp ladder, New England Biolabs). Electrophoresis was carried
out a constant voltage of 100 V. The waste channel electrode was
used (see FIGS. 6A-D) until the 200 bp marker arrived at the three
way junction between separation, purification, and waste channels
(approximately 71 minutes into the run, see FIG. 6E). At this
point, voltage was switched to the purification channel and the 200
bp band was driven into the elution chamber for 6 minutes (see
FIGS. 6E-F). The voltage was switched back to the waste channel for
an additional 15 minutes after which the run was terminated.
[0161] The purified sample (50 ul) was withdrawn from the elution
chamber with a handheld micropipette DNA from the gel of the waste
channel was extracted using a commercial kit (Qiagen QIAquick Gel
Extraction kit) and eluted in 50 ul of 10 mM Tris-HCl buffer. Input
DNA (1 ug of 100 bp DNA ladder, NEB) was diluted to 50 ul in TE
buffer. All three samples were mixed with 10 ul of 40% sucrose in
TE buffer containing a small amount of bromophenol blue loading dye
and loaded on a 5% acrylamide gel (29:1, mon:bis, 0.5.times.KBB
buffer) for analysis. The image of the ethidium-stained gel is
shown in FIG. 6G. There is some distortion of the bands due to the
extremely large sample volume (60 ul for all samples), and
differences in salt: waste channel DNA and input ladder DNA was
dissolved in 10 mM Tris-HCl, whereas purified DNA was loaded in
electrophoresis buffer from elution chamber. However, the results
show that the targeted 200 by band was efficiently removed from the
input sample (see absence of 200 bp band in waste channel DNA) and
efficiently recovered from the elution chamber.
Example 4
Multichannel Cassettes for Automated Preparative
Electrophoresis
[0162] In some embodiments of the invention, multichannel cassettes
are used. Exemplary multichannel cassettes are shown in FIGS. 7,
19, 20, 21, 22, 23, 26, 28, 29, 30, and 31. Multichannel cassettes
rapidly process multiple samples. Moreover, multichannel cassettes
provide a means by which the molecular weight of an uncharacterized
sample in a first macrofluidic channel of the cassette can be
estimated by comparison with molecular weight markers run in a
second macrofluidic channel of the same cassette.
Example 5
Vertical Casting of Multichannel Cassettes
[0163] The macrofluidic separation channel, including the first and
second physically and electrically separated ends, to the proximal
sides of the elution chamber (up to the face of the permeable
membrane) and the waste reservoir, respectively, are filled with
agarose gel. To cast the gel, in accordance with FIG. 26, the
channel plate is contacted with the cover plate, and a waste
reservoir insert is inserted into the corresponding opening in the
cover plate, a sample well insert is inserted into the
corresponding opening in the cover, and a buffer reservoir insert
is inserted into the corresponding opening in the cover plate. The
buffer insert contains a vent and the waste insert contains an
injection port. The inserts are designed to seal tightly against
the cover plate to prevent leakage of the molten agarose solution.
Molten agarose is injected into each channel through ports that
extend through the waste reservoir insert and open into the bottom
end of the second physically and electrically isolated portion of
the separation channel. Molten agarose mixture is injected from
syringes or automated liquid dispensing instruments through the
injection port into the second physically and electrically isolated
portion of the separation channel. During casting of the gel, the
cassette is held in a vertical position (proximal end up), thereby
filling the separation channel and the proximal regions of the
first and second physically and electrically isolated portions from
the bottom up. In the first physically and electrically isolated
portion of the separation channel, the molten gel fills the space
extending from the division point to the proximal side of the
elution chamber. Care is maintained to avoid trapping air bubbles
at any point. The injection and vent ports completely occupy the
volume of the waste and buffer reservoirs, thereby precisely
determining the boundaries of the gel column on either end, where
the gel meets the ports.
Example 6
Horizontal Casting of Multichannel Cassettes
[0164] The macrofluidic separation channel, including the first and
second physically and electrically separated ends, to the proximal
sides of the elution chamber (up to the face of the permeable
membrane) and the waste reservoir, respectively, are filled with
agarose gel. To cast the gel, in accordance with FIGS. 36 and 37,
the first and second dams are inserted into the electrophoresis
base plate, which, subsequently, is contacted with the cover plate
(as shown in FIG. 39). A sample well insert is inserted into the
sample well port. The sample well port contains a raised edge
around the opening in the cover plate through which the samples
well insert(s) traverses. With the aid of the stripper plate, the
teeth of the sample well insert are held centrally within the
opening in the cover place such that a space is preserved on all
sides of the teeth and the resultant sample well containing a deep
central portion with high walls (FIG. 44), however, the walls or
"gel chimneys" do not extend past the bottom of the stripper plate.
Molten agarose is injected into the injection port of the cover
corresponding to each channel. The sample port is located proximal
to the second dam. Molten agarose mixture is injected from
syringes, pipettes, or automated liquid dispensing instruments
through the injection port. During casting of the gel, the cassette
maintained in a horizontal position, thereby allowing the molten
agarose to spread through the separation channel until it reaches
the first dam at the proximal end of the sample well cavity, in
which the sample well insert resides. Air is permitted to escape
through the vents in the cover while the molten agarose is being
inserted (FIG. 39). At the completion of the casting process, the
cover is removed and the buffer reservoir, elution chamber, elution
reservoir, and waste reservoirs are filled with a buffer
composition. The elution chamber is filled with an elution buffer.
The portion of the separation channel extending from the sample
well cavity through the proximal side of the elution chamber and
the proximal side of the waste reservoir is filled with solid
agarose. The cover is replaced and the cassette is sealed.
Other Embodiments
[0165] While the invention has been described in conjunction with
the detailed description thereof, the foregoing description is
intended to illustrate and not limit the scope of the invention,
which is defined by the scope of the appended claims. Other
aspects, advantages, and modifications are within the scope of the
following claims.
[0166] The patent and scientific literature referred to herein
establishes the knowledge that is available to those with skill in
the art. All United States patents and published or unpublished
United States patent applications cited herein are incorporated by
reference. All published foreign patents and patent applications
cited herein are hereby incorporated by reference. All other
published references, documents, manuscripts and scientific
literature cited herein are hereby incorporated by reference.
[0167] While this invention has been particularly shown and
described with references to preferred embodiments thereof, it will
be understood by those skilled in the art that various changes in
form and details may be made therein without departing from the
scope of the invention encompassed by the appended claims.
* * * * *