Note: Descriptions are shown in the official language in which they were submitted.
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AUTOMATED PARALLEL CAPILLARY ELECTROPHORETIC SYSTEM
TECHNICAL FIELD
This invention relates to an apparatus for performing
electrophoresis. More particularly, it pertains to an
automated electrophoresis system employing capillary
cartridges which are configured for use with commercially
available, microtitre trays of standard size and including a
stacked, dual carousel arrangement, a multi-wavelength beam
generator, a gel delivery system and an off-line reconditioner
to eliminate cross-contamination of samples, improve system
capacity and increase system throughput.
BACKGROUND
Electrophoresis is a well-known technique for separating
macromolecules. In electrophoretic applications, molecules in
a sample to be tested are migrated in a medium across which a
voltage potential is applied. Oftentimes, the sample is '
propagated through a gel which acts as a sieving matrix to
help retard and separate the individual molecules as they
migrate.
One application of gel electrophoresis is in DNA
sequencing. Prior to electrophoresis analysis, the DNA sample
is prepared using well-known methods. The result is a
solution of DNA fragments of all possible lengths
corresponding to the same total sequential order, with each
fragment terminated with a tag label corresponding to the
identity of the given terminal base.
The separation process employs a capillary tube filled
with conductive gel. To introduce the sample, one end of the
tube is placed into the DNA reaction vial. After a small
amount of sample enters the capillary end, both capillary ends
are then placed in separate buffer solutions. A voltage
potential is then applied across the capillary tube. The
voltage drop causes the DNA sample to migrate from one end of
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the capillary to the other. Differences in the migration
rates of the DNA fragments cause the sample to separate into
bands of similar-length fragments. As the bands traverse the
capillary tube, the bands are typically read at some point
along the capillary tube using one of several detection
techniques.
The most popular fluorescent dyes for tag labeling the
DNA samples have absorption maximum wavelength ranging from
490-580 nm. A basic detection technique consists of a CCD
camera with a wide-angle lens, a capillary tube array placed
under the camera lens with its planar surface parallel to the
CCD imaging chip, and a laser beam illuminating across the
capillary array. However, a single laser line provided in the
basic detection technique cannot favor all of the tag labels
at the same time; therefore, either multiple lasers or optical
filters are used to compensate for this shortcoming.
Usually, multiple DNA preparation reactions are performed
in a commercially available microtitre tray having many
separate low-volume wells, each holding on the order of 200-
1000 micro-liters. The microtitre trays come in standard
sizes. In the biotech industry, the currently preferred
microtitre tray has a rectangular array comprising of 8 rows
and 12 columns of wells. The centers of adjacent wells found
in a single row are separated by approximately 0.9 cm,
although this figure may vary by one or two tenths of a
millimeter. The same holds for the spacing between adjacent
wells in a single column. The rectangular array of 96 wells
has a footprint within an area less than 7.5 cm x 11 cm.
Miniaturization has allowed more wells to be accommodated
in a single microtitre tray having the same footprint. New
trays having four times the density of wells within the same
footprint have already been introduced and are fast becoming
the industry standard. Thus, these new trays have 16 rows and
24 columns with an inter-well spacing of approximately 0.45
cm.
It is not uncommon to analyze several thousand DNA
samples for a given DNA sequencing project. Needless, to say,
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it is time consuming to employ a single capillary tube for
several thousand runs.
Prior art devices have suggested means for analyzing DNA
bands in multiple capillaries simultaneously. Such a device
is disclosed in U.S. Patent No. 5,498,324 to Yeung et al.
This reference teaches a means for detecting the DNA
bands as they are separated in multiple capillary
tubes which are positioned parallel to another.
However, in such an arrangement, each capillary tube
is filled with gel and a sample is introduced into
each capillary tube.
The arrangement described above takes a considerable
amount of time to fill each capillary tube with gel. It also
takes considerable effort to introduce a reaction sample into
one end of each of the tubes reproducibly and reliably.
It is also not uncommon that one uses the same capillary
tube for several consecutive sample rugs. This, obviously
risks cross-contamination of samples, which is a further
disadvantage in certain prior art arrangements.
SUMMARY OF T$E INVENTION
One object of the invention is to provide a device which
allows one to simultaneously introduce samples into a
plurality of capillary tubes directly from microtitre trays
having a standard size.
Another object of the invention is to provide a stacked,
dual carousel arrangement to eliminate cross-contamination of
DNA samples without reducing system capacity.
Another object of the invention is to provide a gel
delivery module to uniformly distribute gel through the
capillary tubes quickly.
Another object of the invention is~to provide an off-line
capillary reconditioner to thoroughly clean a capillary
cartridge off-line to improve system throughput with a minimal
increase in cost.
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Another object of the invention is to provide an
apparatus that produces a multi-wavelength beam. This multi-
wavelength beam apparatus allows simultaneous detection of DNA
samples which are tagged with different fluorescent tag
labeling dyes.
These objects are achieved by a disposable capillary
cartridge which can be cleaned between electrophoresis runs,
the cartridge having a plurality of capillary tubes. A first
end of each capillary tube is retained in a mounting plate,
the first ends collectively forming an array in the mounting
plate. The spacing between the first ends corresponds to the
spacing between the centers of the wells of a microtitre tray
having a standard size. Thus, the first ends of the capillary
tubes can simultaneously be dipped into the samples present in
the tray's wells. The cartridge is provided with a second
mounting plate in which the second ends of the capillary tubes
are retained. In another embodiment, instead of the second
mounting plate, the second ends of the capillary tubes are
bundled together and received by a liquid delivery chamber,
preferably a high pressure T-fitting.
Plate holes may be provided in each mounting plate and
the capillary tubes inserted through these plate holes. In
such case, the plate holes are sealed airtight so that the
side of the mounting plate having the exposed capillary ends
can be pressurized. Application of a positive pressure in the
vicinity of the capillary openings in this mounting plate
allows for the introduction of air and fluids during
electrophoretic operations and also can be used to force out
gel and other materials from the capillary tubes during
reconditioning. The capillary tubes may be protected from
damage using a needle comprising a cannula and/or plastic
tubes, and the like when they are placed in these plate holes.
When metallic cannula or the like are used, they can serve as
electrical contacts for current flow during electrophoresis.
In the preferred embodiment, a stacked, dual carousel
arrangement eliminates a cross-contamination problem without
reducing the capacity of the system. The system uses a buffer
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solution with the gel to provide a medium for the migration of
DNA from one end of the capillary tubes to the other end
during electrophoresis. Since the buffer solution also
migrates through the capillary tubes during electrophoresis,
one end of the capillary tubes must be immersed in buffer
solution to continuously replenish the buffer supply in the
capillary tubes. Accordingly, the buffer solution may become
contaminated with the DNA sample during electrophoresis.
Next, the DNA in the buffer solution could migrate into the
capillary tubes during a subsequent execution of
electrophoresis if the same buffer solution is used on
consecutive executions of electrophoresis. The stacked, dual
carousel arrangement eliminates this contamination problem by
providing a buffer tray for each DNA sample tray to avoid
reuse of the same buffer tray. Since the stacked, dual
carousel arrangement has an additional carousel to hold the
buffer trays, the arrangement does not have to displace any
sample trays to provide room for the additional buffer trays.
Thus, the arrangement eliminates the contamination problem
without reducing system capacity.
In another aspect of the preferred embodiment of this
invention, the detection system employs both a multi-
wavelength beam generator and multi-wavelength detector in
order to allow DNA sequencing samples tagged with different
labeling dyes to be detected simultaneously in the same
instrument without switching laser or optical filters.
The multi-wavelength beam generator is provided by an
argon ion laser capable of producing multi-wavelength beam
with wavelengths at 457 nm, 476 nm, 488 nm, 496 nm, 502 nm,
514 nm. The mufti-wavelength beam generator compensates for
the different absorption spectra among the different labeling
dyes, improves the peak detection signal evenness among DNA
fragments and enhances the signal to noise ratio of the
detection signal.
In another aspect of the preferred embodiment, a gel
delivery module quickly and uniformly delivers gel through the
capillary tubes. Since the gel is too viscous to be delivered
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by a pump, the gel delivery module uses a gel syringe to
deliver the gel. The gel delivery module includes a gel
carriage to hold a disposable gel cartridge. A stepper motor
linear actuator has a movable actuator shaft arranged to move
a teflon plunger located at one end of the gel syringe to
cause gel material to quickly flow through a high pressure
fitting at the other end of the gel syringe. Further, the gel
delivery module uses the same components used in
electrophoresis to relax the gel in the capillary tubes to
achieve uniform gel distribution.
In another embodiment of the gel delivery module, a
squeezable gel bag is utilized. In this embodiment, the gel
bag is placed inside a high pressure chamber which includes a
hollow cylinder with an open top and closed bottom and a cap
removably affixed to the top of the cylinder. An outlet
assembly including an inside end removably attached to the gel
bag and an outside end connected to the T-fitting is affixed
to the chamber. The chamber is also connected to a pressure
control assembly capable of increasing or reducing the
pressure inside the chamber. As the pressure increases inside
the chamber, the gel is squeezed out through the outlet
assembly and delivered to the T-fitting.
In another aspect of the preferred embodiment, a
streamlined, off-line capillary reconditioner thoroughly
cleans the capillary tubes off-line to achieve increased
system throughput with a minimal increase in system cost. An
operator can execute electrophoresis while cleaning a
previously used capillary cartridge with the off-line
capillary reconditioner. Since a thorough cleaning typically
takes approximately twenty minutes, the off-line capillary
reconditioner improves system throughput as the system does
not have to wait for a thorough cleaning of the capillary
cartridge 909 between consecutive executions of
electrophoresis.
The off-line capillary reconditioner contains a small
number of low-cost items including solvent containers for
holding the cleaning fluids, manifolds for selection of the
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cleaning fluids and a simple controller for managing the
cleaning. This streamlined nature of the off-line capillary
reconditioner offers the advantage of increasing system
throughput with a minimal increase in system cost.
3
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1 is a side view of an arrangement in accordance
with the present invention;
Figs. 2A and 2B are a top and side view, respectively, of
one embodiment of a cartridge of the present invention;
Figs. 3A and 38 show a tube assembly and mounting
arrangement for a cartridge of the present invention;
Fig. 4 shows a monitor plate which can be used with an
array of needles;
1S Fig. 5 shows the arrangement of an apparatus which can be
used with the cartridge of Figs. 2A & 28;
Figs. 6A and 6B are a side and a top view, respectively,
of a second embodiment of a cartridge of the present
invention;
Fig. 7 shows the valving arrangement for a pressure cell
similar to the one shown in the cartridge of Figs 6A and 6H;
Fig. 8 shows an exploded view of a pressure cell vertical
cross-section;
Fig. 9 shows a top view of a second mounting plate of a
pressure cell having an alternate arrangement of plate holes;
Fig. 10 shows an electrophoretic apparatus in accordance with
the present invention;
Fig. 11 shows a sequencer module which includes the
stacked, dual carousel arrangement;
Figs. 12A and 12B show a detailed view of a carousel
contained in the stacked, dual carousel arrangement;
Fig. 13 shows a flowchart illustrating the operation of
the present invention;
Figs. 14A-C show a front, side and back view of the
3S solvent/gel delivery module.within.the system;
Fig. i5A-C shows a detailed view of a gel syriage
contained the gel delivery module;
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Fig. 16 shows the flow of gel and solvent through the
solvent/gel delivery module to the sequences module;
Fig. 17 shows the off-line capillary reconditioner;
Fig. 18 is a side view of capillary cartridge of the
b present invention;
Fig. 19 is a view of a current supply/monitoring board;
Fig. 20 shows a mufti-wavelength beam generator using one
laser head;
Fig. 21 shows a mufti-wavelength beam generator using two
laser heads;
Fig. 22 shows optical processing functions of a laser
emitter tube;
Fig. 22a illustrates a light beam foot print at the
output of a laser emitter;
Fig. 22b illustrates a light beam foot print after a beam
expander;
Fig. 22c illustrates a light beam foot print after a one-
dimensional focus lens;
Fig. 23 shows the direction in which the light beam from a
laser emitter tube i~inges upon an array of capillary tubes;
Figs. 24 and 25 show structures associated with excitation
and detection.
Fig. 26 shows a high pressure chamber which supplies high
viscous gel;
Fig. 27 shows a solvent/gel delivery module of the
preferred embodiment;
Fig. 28 shows a back view of the solvent/gel delivery
module;
Fig. 29 shows the flow of gel and solvent through the
solvent/gel delivery module in the preferred embodiment; and
Fig. 30 shows a flowchart illustrating the operation of
the preferred embodiment.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
35' Fig. 1 presents a schematic illustrating.the use of a
device in accordance with the present invention. A cartridge
30 of the present invention comprises a plurality of capillary
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tubes 32 having substantially the same length. The capillary
tubes extend between a sample-side connection array 34 and gel
side connection array 36. The capillary tubes 32 terminate on
the sample-side in an array of first capillary ends 38 and on
the gel side in an array of second capillary ends 40.
Thus, both ends of each of the capillary tubes 32 in Fig.
1 extends through individual plate holes in a base member 42,
which preferably is formed from polycarbonate, or acrylic or
the like. Alternatively, each array of capillary ends may be
retained in a separate mounting plate having the plate holes,
and each of the mounting plates may then be fixed to a base
member. Also, instead of passing each capillary tube through
an individual plate hole, one or more capillary tubes may be
collected together and sent through a common hole, or even no
hole at all.
Between the two arrays, the capillary tubes 32 pass
through a thermoelectric element 44 which is mounted on the
base member 42. The thermoelectric element is arranged on
either side of a window region 46. The thermoelectric element
2o is used to control the temperature of the capillary tubes
within a predetermined range. It should be evident to one
skilled in the art that the thermoelectric element 42 may be
comprised of two or more individual elements. It should also
be evident that alternate temperature control means such as
circulating fluid systems, and air convection may also be used
to control the temperature.
The capillary tubes 32 are arranged parallel to one
another, side by side, in the window region 46. The length of
each capillary tube from its first capillary end to the window
region 46 is substantially the same for all the capillary
tubes 32. This length is determined by the optimization of
(i.e., minimum acceptable) sample run time, and the minimum
acceptable resolution of the separated samples. Nominally,
this length in on the order of 50-70 cm. The window region
46 represents the region allowing access to the parallel
capillary tubes from incoming excitation light. It also
allows access to outgoing fluorescence emission from the
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capillary tubes. Thus, the window region 46 allows the bands
50 in the various capillary tubes to be detected.
As shown in Fig. 1, an excitation light source comprising
a laser 52 and a prism 54 is used to focus a light beam 56
through the window region 46 and onto the capillary tubes 32.
A fluoresced light beam 58 is then sensed by a CCD camera 60,
which captures the bands 50. As is known to those skilled in
the art, other illumination and detection means can also be
used.
The arrangement of Fig. 1 provides for the substantially
simultaneous introduction of samples into the array of first
capillary ends 38 of all the capillary tubes 32. In
particular, the arrangement allows one to introduce the
various samples by simultaneously dipping the array of first
capillary ends 38 into the wells 62 of a sample-side
microtitre tray 64 having a standard size, as described above.
To allow for this, the individual capillary ends are
spaced apart from one another such that they have a spatial
arrangement which is substantially the same as, that of an
array of wells belonging to a microtitre tray of standard
size. Thus, the spacing between adjacent first capillary ends
is approximately 0.9 cm and the entire array of first
capillary ends has a footprint less than 7.5 cm x 11 cm, thus
corresponding to a microtitre tray of standard size.
The array of second capillary ends 40 is inserted into
the wells 66 of a second microtitre tray 68, where they come
into contact with a buffer solution 70, as known to those
skilled in the art. As the wells 66 in the second tray 68 are
separated from one another, the chance of cross-contamination
among the second capillary ends 40 is reduced.
A voltage source 72 is used to provide a voltage
differential between the two arrays of capillary ends. As
shown in Fig. 1, one voltage level is applied through
individual leads 74 to each of the wells 62 of the first
microtitre tray 64 and a second voltage level is applied in
substantially the same manner through leads 76 to the wells 66
of the second tray 68. Thus, current flows through the leads
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74, into the individual samples, through the first capillary
ends 38, through the__capillary tubes 32, through the secoad
capillary ends 40, into the buffer 70 present in the wells 66
of the second microtitre tray 68, and finally through leads
76.
Figs. 2A and 2B shows a top and a side view of one
embodiment of a cartridge 80 in accordance with the present
invention. The cartridge~has a base member 82 formed from
polycarbonate, acrylic or the like. Mounted in the base
member are first and second mounting plates 84, 86,
respectively. Preferably, these plates are formed from an
electrically insulative material.
An array of first capillary ends 88 project from the
bottom surface 90 of the first mounting plate 84 and an array
of second capillary ends 92 project from the bottom surface 94
of the second mounting plate. The capillary tubes 96 pass
through, and are retained in, plate holes formed in the plates
84, 86 and project from the top surfaces 98, 100 of the
plates. Preferably, each of the capillary tubes 96 is
protected by a tube assembly which is secured to a plate
hole in the mounting plate, as it passes through the mounting
plates.
As best seen in Fig. 2A, the tube assemblies, each with
its associated capillary tubes, form a rectangular array of 8
rows and 12 columns as they emerge from the plates 84, 86.
The spacing between adjacent plate holes in which the
assemblies are held, and the spacing of adjacent capillary
ends 88, 92 correspond to the spacing of adjacent wells in a
microtitre tray of standard size. In the preferred
embodiment, adjacent capillary ends are separated by
approximately 0.9 cm and the entire array of capillary ends,
and thus the array of plate holes through which the capillary
tubes 96 pass, form a footprint no larger than about 7.5 cm x
11.0 cm.
The upper surface 98, 100 of each mounting plate 84,. 86
is provided with first and second enclosures designated by
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reference numerals 104, 106, respectively. In the preferred
embodiment, each of the enclosures is provided with an inlet
108 and an outlet 110. The outlet 110 of the first enclosure
is connected to the inlet 108 of the second enclosure by
plastic tubing 112. The inlet 108 of the first enclosure is
connected to a first plastic shut off valve 114 while the
outlet 110 of the second enclosure is connected to a second
plastic shut off valve 116. The plastic shut off valves 114,
116 are connected, in turn, to respective first and second
quick disconnects 118, 120.
During operation, the cartridge 80 can be connected to a
pump assembly 122 which is arranged to circulate a
temperature-controlled liquid coolant through the~~enclosures
104, 106. In such case, the cartridge's first disconnect 118
is connected to the output 124 of the pump assembly 122 while
the second disconnect 120 is connected to the input 126 of the
pump assembly 122. Such an arrangement maintains the
temperature of those portions of the capillary tubes 96
projecting from the upper surfaces 98, 100 of the mounting
plates and present in the enclosures 104, 106. For this to
work, the mounting plates 84, 86 must form a liquid-tight seal
with the base member 82. A liquid-tight seal must also be
formed between the plate holes and the tube assemblies
and/or the capillary tubes 96 themselves.
The capillary tubes 96 pass between the two arrays of
tube assemblies in an area of the cartridge not covered by
the enclosures 104, 106. As explained above, thermoelectric
temperature control means 128, or the equivalent, is arranged
on either side of a window region 130 of the capillary tubes
96 to control the temperature of the capillary tubes when they
are no longer within the enclosures 104, 106.
Within at least a portion of the window region 130, the
capillaries 96 are arranged parallel to one another so that
they may be read by detection means. Preferably, the base
member 82 is provided with an opening 132 above which the
window region 130 is situated. This allows for at least one
of illumination means or detection means to be placed below
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the base member from where they may be in a direct line of
sight to the exposed capillary tubes 96.
Fig. 3A shows a needle 140 used in forming a tube
assembly 160 which can then be directly inserted into a
mounting plate 162, as shown in Fig. 3B. The needle 140
comprises a metallic cannula 142. In the preferred
embodiment, the cannula 142 is formed from stainless steel
having an inner diameter of 0.064 in. and an outer diameter of
0.072 in. The cannula 142 is provided with a bevel 144 at the
end which is dipped into a well.
Within the cannula 142 is a coaxially arranged annular
polyetheretherketone (PEEK) polymer tubing 146 which serves as
a sleeve. The polymer tubing 146 has an inner diameter of
about 0.006 in. and an outer diameter of 0.0625 in. Thus, the
polymer tubing 146 can be comfortably inserted into cannula
142.
Running through the center of the tubing 146, along a
longitudinal axis of the needle 140, is a capillary tube 148
which is associated with the needle 140. The capillary tube
148 is formed from fused silica and has an inner diameter of
about 0.003 in. and an outer diameter of about 0.006 in.
Thus, the capillary tube 148 fits snugly into the polymer
tubing 146. The capillary tube 148 terminates in an end 150
which is substantially across from the end 152 of the cannula
142. Thus, the spacing between the two ends 150, 152 is about
.035 in.
An UV-cured, medical-grade epoxy sealant 154 is used at
both ends of the polymer tubing 146 to secure it and the
capillary tube 148 to the cannula 142. Preferably, the epoxy
sealant 154 forms an air- and liquid-tight seal through the
cannula 142. The epoxy sealant ensures that the polymer
tubing 146 is not exposed to the environment, and also ensures
that the capillary tube 148 does not come into direct contact
with the cannula 142.
It should be noted that a needle may be formed in ways
other than the one depicted in Fig. 3A. For instance, instead
of a tubular cannula, the needle may simply comprise a
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capillary tube encased in a poured or coextruded plastic
material which, in turn, is secured to a metallic strip.
Other arrangements are also possible.
Fig. 3B shows a hollow, high pressure compression fitting
164 formed from nylon into which the needle 140 is inserted to
complete the tube assembly 160. The needle 140 can be
secured to the cylindrical inner walls of the compression
fitting with an epoxy sealant. Each tube assembly 160 is then
inserted into a plate hole 166 tapped in the mounting plate
162 and the plate hole 116, too, can be sealed with epoxy.
When this is done, an air- and liquid-tight seal is provided
between the bottom surface 168 and the top surface 170 of the
mounting plate 162, allowing the mounting plate to withstand a
positive pressure applied on its bottom surface in a region
where the plate holes securing the tube assemblies are
situated.
One may completely do away with the compression fittings
164 and drill plate holes in the mounting plate 162 which
correspond in size to the outer diameter of the needles 140.
In such case, a needle is directly inserted into each plate
hole in the mounting plate 140 and secured thereto by the
epoxy. Such a fitting-less approach can improve the
structural integrity of the mounting plate I62 due to the
reduced size of the plate holes. It may also provide a better
air- and liquid-tight seal since there are fewer interfaces in
which epoxy sealant is used. Moreover, it should also be
understood that one may retain just a capillary tube 148, or
just a capillary tube 148 encased in polymer tubing 146
directly in a plate hole of appropriate size formed in the
mounting plate 162.
Whether or not one uses a compression fitting, and
whether or not one uses a cannula and/or polymer tubing, it
should be understood that in the preferred embodiment, each
plate hole has one capillary tube retained therein. The array
of plate holes preferably has a spatial arrangement
corresponding to that of the wells of a microtitre tray of
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standard size. However, it may be possible to form the plate
holes off-center, and then angle the capillary ends.
Furthermore, it should also be understood that it may be
possible to fix an array of capillary ends in the desired
configuration without forming holes in a mounting plate 162.
For instance, this can be done by gluing or clamping the
individual capillaries to a mounting plate so that their ends
are arranged in the desired configuration. Alternatively, the
capillaries may be secured together so that their ends remain
in the desired configuration in a poured acrylic or the like.
What is important is that the spacing of the capillary ends in
the array correspond to the spacing of the wells in the
microtitre tray of standard size.
A conductive plate 172 may be secured to the mounting
plate 162 by screws, adhesives, or other conventional means.
The conductive plate 172 is formed with an array of conductive
holes 174 which corresponds to the plate holes 166 in the
mounting plate. Each of the conductive holes 174 is formed by
an H-shaped slit which forms a pair of tabs 176, 178 between
the legs of the "H". When a needle 140 is inserted in the
conductive hole 174, the tabs 176, 178 give way, and contact
either side of the needle 140.
As the entire plate 172 is conductive, all needles 140 in
the array share a common electrical connection. A voltage
applied to the conductive plate 172 then appears on the
exterior of each needle 140 in the array. During
electrophoretic application, this voltage appears in the
buffer solution found in each well, into which solution the
capillary end 150 is inserted.
As is known to those skilled in the art, the voltage
differential may be delivered to the first capillary ends
through other means as well. For instance, instead of
contacting a common plate to which the needles are connected,
voltage leads may be connected directly to each needle.
Alternatively, individual leads may be dipped into the liquid
in each well. Another alternative is to deliver the voltage
through a metallic coating, such as gold, deposited on the
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exterior of only the terminal portion of each capillary tube,
where it contacts the liquid in the well. Also, the voltage
may be delivered directly to the wells through one or more
leads, as described earlier. One skilled in the art can
readily formulate alternative approaches to delivering a
voltage to the first capillary end.
Fig. 4 shows a monitor plate 190 which can be used with
the cartridge embodiment shown in Figs. 2A and 2B. In a
cartridge of the present invention, the needles of at least
the first mounting plate 84 are provided with a conductive
plate 172 described above. The needles of the second mounting
plate 86 can be provided with a monitor plate 190.
The monitor plate has an array of monitor holes 192. The
array of monitor holes is aligned with the second array of
plate holes formed in the second mounting plate 86. Each
monitor hole 192 is formed with an isolated electrical contact
194 which is electrically connected to a monitor plate
connector 196 by an individual lead 198. Each needle in the
second mounting plate 86 contacts a corresponding electrical
contact 194 in the monitor plate.
The purpose of the monitor plate is to provide a means
for gauging the presence of electrical conductivity between
any needle in the second mounting plate 86 and the needles of
the first mounting plate 84. In this regard, it should be
understood that the monitor plate 190 can be secured to
mounting plate 86 in much the same manner as the conductive
plate 172. What is important is that each of the electrical
contacts 194 connects to only one needle in the second
mounting plate.
Fig. 5 shows a cartridge 200 having a first 202 and a
second 204 array of needles arranged above a first 206 and a
second 208 carousel, each array positioned above a portion of
a respective carousel. A CCD camera 210 is positioned above a
portion of the cartridge between the two to detect bands in
the capillary tubes (not shown in Fig. 5). Each carousel 206,
208, has eight platforms 212, on each of which a microtitre
tray having a standard size is placed.
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The wells in each of these trays hold one or more liquids
such as samples, gels, buffer solutions, acidic solutions,
basic solutions, etc. As configured in Fig. 5, the first
carousel holds 6 sample trays 214, 1 buffer tray and 1 waste
tray, a sample tray being positioned underneath the first
needle array 202. As also shown in Fig. 5, the second
carousel holds a pair of acidic solution trays 220, a pair of
basic solution trays 222, a pair of waste trays 224, one of
which is positioned underneath the second needle array 204, a
buffer solution tray 226, and a gel tray 228. Thus, the first
carousel 206 is the sample-side carousel and the second
carousel 208 is the gel-side carousel.
The cartridge is removably mounted to an automated
electrophoretic apparatus. During operation, a lifting means
raises and lowers the platform 212 which is under either of
the two needle arrays 202, 204. When a microtitre tray is
brought in close proximity to one of the needle arrays 202,
204, the needles in these arrays, and their associated
capillary ends, are dipped into the contents of each well of
that microtitre tray. When the platform under either of the
needle arrays is lowered, the carousel associated with that
platform may be rotated so that a different platform 212
holding a different microtitre tray, can be raised.
When a platform is raised, surfaces around the periphery
of the platform abut opposing surfaces, thus sealing a
pressure chamber beneath the bottom surface of the needle
array. Introducing a pressurized inert gas, such as helium,
into the chamber at a pressure of 30 psi or so, applies a
uniform force to the samples in the wells of the microtitre
tray held on that platform. This causes a portion of each of
samples to enter into the corresponding array of first
capillary ends.
With, or in place of, applying pressurized helium to
introduce samples into the first capillary ends, one may also
apply a high voltage for brief period of time, on the order of
20-40 seconds, to cause the samples to migrate into the first
capillary ends. Using a high voltage for this purpose,
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however, may be size-selective. That is, smaller molecules
are more likely to enter the first capillary ends, potentially
distorting the subsequent electrophoresis analysis.
The operation of the automated electrophoretic apparatus
in accordance with Fig. 5 will now be described. First, the
various microtitre trays are loaded with the designated buffer
solutions, gels, samples, etc. Then, gel tray 228 in carousel
208 is raised and gel is introduced into the capillary tubes
(not shown in Fig. 5) through second capillary ends (hidden in
Fig. 5) associated with the second needle array 204. The gel
tray 228 is then lowered. A sample tray 214 in carousel 206
is then raised, and sample is introduced through the first
capillary ends (hidden in Fig. 5) associated with the first
needle array 202. The sample tray 214 is then lowered.
Carousels 206 and 208 are then rotated to position buffer
trays 216 and 226 under their respective needle arrays 202 and
204. A voltage differential is then applied across the two
needle arrays to perform the electrophoresis run.
Upon completion of the run, the cartridge may be
reconditioned. This is done by flushing out the gel and
samples from the previous run under pressure and cleaning the
capillary tubes using the acidic 220 and/or basic 222
solutions. The cartridge is then ready for re-use, allowing
the samples in another one of the sample trays 214 to be
tested.
It should be obvious that the carousels 204, 206 may be
formed with a different number of platforms. It should also
be obvious that one can use a linear, or rectangular, or other
arrangement of such platforms. All that is required is a
storage and positioning system which allows a first particular
microtitre tray to be brought to the first needle array 206,
and a second particular microtitre tray to be brought under
the second needle array 208.
Figs. 6A and 6B present a side and a top view,
respectively, of a cartridge 280 having a first mounting plate
282 in which the array of plate holes in the first mounting
plate 282 is rotated by 90°. Otherwise, the arrangement for
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connecting the capillary tubes to the first mounting plate is
substantially the same as previously described. The first
capillary ends formed in an array with the desired spacing
project from the bottom surface of the first mounting plate
282, and are retained in plate holes formed in the first
mounting plate.
The second mounting plate 284, however, is not the same
as in the previous cartridge embodiment. In the cartridge
280, the second mounting plate 282 serves as a pressure
containment member of a pressure cell 286 having substantially
cylindrical exterior walls. For the sake of clarity, Fig. 6A
does not show all the capillary tubes on the first mounting
plate, nor any capillary tubes at all on the second mounting
plate 284. It is to be understood, however, that all the
capillary tubes are present.
The second mounting plate has a radially symmetric,
beveled surface 288 in which a plurality of plate holes 290
are formed. Each of these plate holes 290 is fitted with a
section of PEEK polymer tubing 292 in which the capillaries
are encased using an UV-cured epoxy, as described before, to
form an air- and liquid-tight seal in the plate holes 290.
The capillary tubes pass through the PEEK polymer tubing and a
second end of each capillary tube communicates with an
interior cavity of the pressure cell. Although the preferred
embodiment for this cartridge uses just PEEK polymer tubing
and a capillary tube in the second mounting plate, it should
be understood, that needles similar to the ones described
earlier, could also be used. Also, just the capillary tubes
alone, secured by epoxy, can be used as well. What is
important is that each capillary tube 294 is retained in a
plate hole 290 in an air- and liquid-tight manner, and that
the capillary tube's second end communicates with an interior
cavity of the pressure cell 286.
As is the case with cartridge 80 of Figs. 2A and 2B, this
36 cartridge 280 is provided with thermoelectric control means
298, enclosures 300, 302, and its capillary tubes are arranged
in parallel along at least a portion of a window region 304.
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Although not shown in Figs. 6A and 6B, it is understood that
the enclosures 300, 302 can be provided with inlets and
outlets and the like for circulating a coolant, as was the
case with the other cartridge 80.
As shown in Fig. 6A, the second mounting plate 284 has a
truncated cone-shaped upper portion terminating in a flat top
310. The curved, conical surface 288 in which the plate holes
290 are formed, is advantageous for reasons of structural
integrity when a high positive pressure is applied from
underneath the second mounting plate. Also, placing the plate
holes 290 on such a surface allows them to be placed farther
apart, a feature which also enhances the structural integrity
of the pressure cell 286.
The pressure cell 286 is secured to the base member of
the cartridge 280 and projects through the bottom of the base
member. This arrangement allows the pressure cell 286 to be
provided with an inlet 312 and an outlet 314 arranged on
opposite sides of its cylindrical exterior walls. It should
be noted that the inlet could just as easily be formed in the
2o flat top portion 310 of the second plate 284, and the outlet
formed in the bottom surface of a lower portion 316 of the
pressure cell 286. In such case, the pressure cell could be
rest on the base member, rather than project through its
bottom, with a pipe fitting connected to the outlet through a
hole formed in the base member, which hole is then sealed.
Fig. 7 shows a valuing arrangement for a pressure cell
320 which has an inlet 322 at its top surface 324, but
otherwise is substantially similar to the pressure cell 286.
Aside from the inlet 322, the pressure cell 320 is also
provided with an outlet 326, which is connected to a waste
valve 328. The waste valve 328 is opened to expel the
contents of an interior cavity of the pressure cell 320.
Access to the inlet is 322 controlled by a shut-off valve
330. Liquids can be passed through the inlet 322 into the
pressure cell 320 with the use of a pump 332. Preferably, the
pump is a high pressure liquid chromatography (HPLC) pump
having a pumping capacity of 4-40 milliliters per minute, at a
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pressure of about 2000 psi. The pump 332 is connected to a
multi-valve manifold 334 which selectively allows one of four
liquids to be pumped into the pressure cell. The four liquids
are held in separate containers 336, 338, 340, 342, which
respectively hold gel, a buffer solution, an acidic solution,
and a basic solution. Additional containers holding the same
liquids may be held in reserve, or connected in series with
these, so as to increase the total supply.
The waste valve 328, the shut-off valve 330, the pump 332
and the multi-valve manifold 334 are all under the direction
of a controller, preferably a microcomputer, or equivalent.
Thus, the contents of an interior cavity of the pressure cell
are regulated by the controller. Such a controller may also
receive inputs from various pressure and temperature monitors
and other sensors to prevent damage to the pressure cell 320.
During operation, the interior cavity of the pressure
cell 320 is filled by means of the pump 332. This forces the
pumped liquid into the second capillary ends which communicate
with the interior cavity of the pressure cell 320. By filling
the array of first capillary ends and the pressure cell 320
with the appropriate fluids in the appropriate sequence, one
may perform the electrophoresis operations, much as described
above with regard to the apparatus of Fig. 5.
After the run, one may recondition the pressure cell and
the capillary tubes to prepare them for another run. Again,
this is accomplished by flushing the gel and sample from all
the capillary tubes simultaneously. With the pressure cell
320, however, pressures on the order of several thousand psi
can be applied. These increased pressures force the viscous
gel out of the capillary tubes much faster. This reduces the
cycle time between runs, with reconditioning, to about one to
two hours.
Fig. 8 shows an exploded cross-section of a pressure cell
350, similar to the pressure cell 286 in the cartridge 280.
As is the case with the other pressure cells, pressure cell
350 is preferably formed from aluminum or stainless steel.
The pressure cell 350 is provided with a threaded inlet 352
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formed on the top surface 354 of its upper portion 356, which
upper portion comprises the second mounting plate. The
pressure cell 350 is also provided with a threaded outlet 358
on the bottom surface 360 of its lower portion 362. High
pressure pipe fittings can be screwed into the threads of the
inlet 352 and the outlet 358.
The upper portion 356 and the lower portion 362 are held
together by a plurality of bolts (not shown) which are
inserted through bolt holes 368 formed along the periphery of
the bottom surface 360 of the lower portion 362. The bolts
are then screwed into corresponding threaded holes 370 formed
on the bottom surface 372 of the upper portion 356. An O-ring
364 partially fits into a rectangular channel 366 formed in
the second mounting plate 356. The O-ring 364 provides a seal
between the upper portion 356 and the lower portion 362.
Instead of an O-ring, a gasket, or the like may be used to
effect such a seal.
At the center of the pressure cell 350, formed between
the upper 356 and lower 362 portions is an interior cavity
374. A plurality of plate holes 376 are formed in the upper
portion (second mounting plate). For simplicity, in Fig. 8,
the plate holes 376 are only shown on one side of the upper
portion 356. It should be understood, however, that they are
also present on the other side. The plate holes 376 extend
from a beveled surface 378 formed on the upper portion 356 to
the interior cavity 374.
Capillary tubes 380 are retained in these plate holes 376
and their second ends 382 communicate with the interior cavity
374. Preferably, each capillary is encased in a section of
PEEK polymer tubing which extends from a point within each
plate hole 376, proximate to the interior cavity 374, to well
outside the beveled surface 378. For simplicity, however, the
PEEK tubing is not shown in Fig. 8. Nevertheless, it should
be kept in mind that just the capillary tube, or a needle
comprising a capillary tube, PEEK tubing and a cannula, can be
inserted into each plate hole 376, assuming that it is
suitably sized.
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As explained above, an UV-cured epoxy sealant is used to
seal the plate holes 376 at both ends so that they are air-
and liquid-tight. With the pressure cell, the terminal
portion 384 of each plate hole proximate to the interior
cavity 374 is tapped or roughened. This provides a surface to
which the epoxy sealant bonds more readily during assembly.
A liquid held within the interior cavity 374 is in
contact with the material forming the interior cavity. When
the liquid is also in contact with the second capillary end
382, an electrical connection between the interior cavity 374
of the pressure cell 350 and the first capillary end secured
to the first mounting plate, is completed. Thus, grounding
the pressure cell 350 through a contact formed thereon,
applies the ground to the interior cavity 374, completing the
circuit necessary to perform the electrophoresis.
Alternatively, as the pressure cell 350 is electrically
isolated from the base member to which it is mounted, the
potential of the pressure cell 350 may be allowed to float.
This allows one to apply a high voltage to the pressure cell
350, rather than to the needles associated with the first
capillary ends.
Fig. 9 shows an alternate arrangement for the plate holes
390 in a second mounting plate 392 having a top surface 394
and an inlet 396. In this arrangement, each set 398 of three
plate holes is offset at an angle relative to the center of
the top surface 394. This provides for a maximum spacing
between the plate holes. From a structural integrity point of
view, such an arrangement may be preferable to having the
plate holes arranged radially, in a spoke-like fashion, as
shown in Fig. 6B.
Fig. 10 shows an electrophoretic apparatus 400 designed
for use with a capillary cartridge formed in accordance with
Figs. 6-9. The apparatus comprises a user interface 402,
shown as a video display terminal and keyboard, which
communicates with a controller 404, which preferably is a
microprocessor-based computer, or the like. The user
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interface 402 allows a user to enter commands, receive status
information, and view the collected data.
The apparatus 400 also comprises a data processing
computer 406, which receives, stores and processes video
signals from a CCD camera 408. The data processing computer
406 is provided with optical 410 and/or magnetic 412
read/write data storage means. Resident in the data
processing computer 406 are signal and image processing
software to analyze the signal data from the camera 400. The
data processing computer 406 is connected to the controller
404, and responds to requests from the latter, exchanging data
and control information, as needed.
The apparatus 400 is further provided with a high-voltage
power supply 414 which provides the necessary voltage to be
applied across the ends of the capillary tubes. The power
supply's operation is directed by the controller 404.
The controller 404 also directs the operation of a pump
interface 416, which comprises a number of electronic
switches. The pump interface 416 regulates the operation of a
solenoid valve 418. The solenoid valve 418 connects a gas
inlet 420 which is connected to an inert gas source, such as a
pressurized helium tank, to the chamber 422. The pump
interface 436 also regulates the operation of high pressure
liquid chromatography (HPLC) pump 426. The HPLC pump 426,
under the direction of the controller, selectively supplies
liquids in containers 428, 430, 432, 434, gel, buffer
solution, an acid, and a base, to a pressure cell 436 of a
cartridge 438 through a multi-valve manifold 440.
A carousel 442 having a plurality of platforms 448 is
turned by a rotor 444. A lifting means 446, such as a
hydraulic pump or the like, raises and lowers a platform 448
positioned under a first mounting plate. This brings a
microtitre tray 450 on the platform 448 towards and away from
an array of capillary ends, as previously described. Both the
rotor 444 and the lifting means are connected to, and driven
by, the controller 404.
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The apparatus 400 also includes a light source 452,
preferably a laser, which illuminates the capillary tubes 454,
as directed by the controller 404. The light source 452
illuminates the capillary tubes 454 from below, through an
opening in a base member of the cartridge 438, as previously
described. A light shroud 456 covers the camera 408, the
light source 452, and at least the window region of the
capillary tubes 454, as the detection of the capillary bands
is performed in the dark.
l0 During operation, the capillary tubes 454 are first
cleaned and then loaded with gel through the pressure cell 436
by activating pump 452. The pump 452 is then turned off.
Next, a platform 448 carrying a microtitre tray 450 holding
samples is raised by the lifting means 446. This forms a seal
between the platform 448 and the underside of the chamber 422.
It also dips the first capillary ends into the wells of the
microtitre tray 450. With the chamber 422 sealed, the
solenoid valve 418 is opened, allowing pressurized helium gas
to enter through the inlet 420. This puts a uniform positive
pressure on the samples in each of wells of the microtitre
tray 450, on the order of 30 psi, and forces the samples at
least slightly into the first capillary ends. As discussed
above, a high voltage may be applied for a brief period of
time for this purpose, as well. The platform 448 is lowered
and the carousel 442 is rotated, bringing a microtitre tray
filled with buffer solution under the first capillary ends.
Next, the buffer tray is raised so that the first capillary
ends are dipped into the buffer solution, and the pressure
cell 436 is filled with buffer solution so that the second
capillary ends are in contact with buffer solution, as well.
After this, the high voltage source 414 is turned on to
perform the electrophoresis run. The light source 452 and the
camera 408 are used to simultaneously detect the bands in all
the capillary tubes 454. The video signal data from the
camera 408 are processed and stored in the computer 406. The
processed data may then be presented on the user interface
CA 02295227 1999-12-23
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402. After the run, the cartridge 438 may be reconditioned
(i.e., cleaned) and prepared for another run.
The carousel arrangement shown in Fig. 5 has a buffer
contamination problem. As shown in Fig. 5, there are 6 sample
trays 214 and one buffer tray 216 on the first carousel 206.
After a sample has been introduced from a sample tray 214 on
the first carousel 206 through the first capillary ends
(hidden in Fig. 5), the sample tray 214 is lowered, carrousels
206 and 207 are rotated to position buffer trays 216 and 226
l0 under their respective needle arrays 202 and 204, and a
voltage differential is applied across the two needle arrays
202, 204 to perform electrophoresis. While the first
capillary ends (hidden in Fig. 5) are in contact with the
buffer tray 216, some of the DNA sample from the first
capillary ends will diffuse into the buffer tray 216 and
thereby contaminate the buffer tray 216. This contamination
will adversely effect the accuracy of a subsequent
electrophoresis run with a DNA sample from another sample tray
214 because this subsequent electrophoresis run will use the
same buffer tray 216 as the previous run.
This contamination problem could be eliminated by
replacing sample trays 214 with buffer trays 216 until there
is a one to one correspondence between the set of sample trays
214 and the set of buffer trays 216 on the first carousel 206.
With this arrangement, each sample tray 214 has a single,
corresponding buffer tray 216. While this arrangement
eliminates the contamination problem, it adversely effects the
capacity of the first carousel 206. If the first carousel 206
has eight platforms, the first carousel 206 can only have a
maximum of three sample trays 214 in order to achieve the one
to one correspondence between the set of sample trays 214 and
the set of buffer trays 216, necessary to eliminate the
contamination problem.
Fig. 11 illustrates a stacked, dual carousel arrangement
in a sequences module 60o which achieves a one to one
correspondence between the sample trays 214 and buffer trays
216 to eliminate the contamination problem, without reducing
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the capacity of the system. The stacked, dual carousel
arrangement has an upper carousel 601 and a lower carousel 602
which are aligned and spaced apart along a common axis.
In the preferred embodiment, both carrousels 601, 602
have seven sites 618 for accommodating microtitre trays 214,
216 and one large cut-out 620 for enabling passage of
microtitre trays 214, 216. The large cut-out 620 on the upper
carousel 601 allows passage of a tray initially positioned on
a site 618 of the lower carousel 602 through the upper
carousel 601 to the needle array 603.
In one embodiment, the upper carousel 601 holds the
sample trays 214 and the lower carousel 602 holds the buffer
trays 216 corresponding to the sample trays 214. Fig. 12a
shows this tray arrangement on the lower carousel 602. With
this tray arrangement, the lower carousel 602 could hold six
buffer trays 216 and a waste tray 224. The upper carousel 601
could hold six sample trays 214. In the preferred embodiment,
each carrousel includes six sample trays, six buffer trays,
one drain tray, and one water tray. The water tray is
provided for rinsing the first ends of capillaries and
electrodes, However, the number of each type of trays placed
on a carrousel varies as the size of commercially available
microtitre trays changes.
Each of the carrousels 601, 602 in the stacked, dual
carousel arrangement contains a rotor 604 and a motor 608 for
selectively rotating the carrousels 601, 602 to a chosen
angular position. Specifically, the motor 608 selectively
rotates the carrousels 601, 602 to position the appropriate
site 618 accommodating a sample tray 214, buffer tray 216 or
waste tray 224 under the needle array 603. A controller 404
causes the motor 608 to selectively rotate each carrousel 601,
602 to a chosen angular position.
In the preferred embodiment, the stacked, dual carousel
arrangement has a motor 608 for each carousel 601, 602. The
motor 608 is a stepper motor, available from Pacific
Scientific in Wilmington, MA. In another embodiment, the
stacked, dual carousel arrangement has a DC motor 608 and a
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clutching mechanism for selectively engaging and rotating each
carousel 601, 602.
The angular position of the two carousels are
automatically detected. For this, each carousel 601, 602 also
has an encoder 612 which is operatively engaged to the rotor
604. The encoder 612 senses angular position data of the
rotor 604 and sends this data to the controller 404. In the
preferred embodiment, the encoder 612, available from Stegmann
Corporation in Vandalia, Ohio (Model Number AG612XKRR, 2048)
has an optical sensor and has a 2048 pulse resolution value.
The angular position can also be detected by means of a
linear arrangement of holes 613 placed along the periphery of
the carrousel, adjacent each site. For a carrousel with 8
sites, a leading hole, three coding holes, a trailing hole are
provided. The leading and trailing holes simply serve to
indicate that coded holes maybe present there between. The
three coding holes may each be present, or absent. This
allows one to code for 8 = 2x2x2 sites. The holes are
illuminated by LEDs located above the upper carousel 601; the
LED light passes through the holes and is detected by a
carousel location detector located below the lower carousel
602. The carousel location detector generates the angular
position data from the sequence of holes through which LED is
visible light and sends this data to the controller 404.
The controller 404 determines the rotational position of
each carousel 601, 602 using the angular position data from
either of the above embodiments to cause the motor 608 to
selectively rotate each carousel 601, 602 to a chosen angular
position. Specifically, the controller 404 uses the position
data to compensate for the rotational momentum of the carousel
601, 602 which can cause the carousel 601, 602 to initially go
beyond its chosen angular position.
The stacked, dual carousel arrangement also contains a DC
motor 605 having a movable member to move the chosen tray 214,
216, 224 along the common axis toward or away from the needle
array 603 as needed. The controller 404 causes the motor 605
to move the chosen tray 214, 216, 224 along the common axis.
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The motor 605 also contains a current meter to measure
the current drawn by the DC motor 605. When a DC motor
encounters a load, the current drawn by the motor increases to
permit continued movement of its movable member. Accordingly,
the current increases sharply when a tray 214, 216, 224
reaches the needle array 603 while traveling upward or when a
tray 214, 216, 224 reaches a carousel 601, 602 while traveling
downward. Upon detecting a sharp increase in the current, the
controller 404 causes the DC motor 605 to stop as the tray
l0 214, 216, 224 has reached its proper position.
In the preferred embodiment as shown in Fig. 12a and Fig
12b, the diameter and thickness of each carousel 601, 602 are
23.5 inches and 3/8 inches respectively. Further, each
carousel 601, 602 has a circular hole 610 in its center with a
diameter of 1.375 inches to receive a rotor 604. Each
carousel 601, 602 has four holes 611 with a diameter of 5/16
inches equally spaced along the periphery of a circle centered
at the center of the carousel 601, 602 with a diameter of four
inches. The carrousels 601, 602 are bolted to a bearing
assembly which is fixed on an axle through these four holes
611 in order to balance the carrousels 601, 602 on the bearing
assembly to enable their controlled rotation.
In the preferred embodiment, two rectangular openings
613, 614 form the sites 618 on each carousel 601, 602. The
size of the opening on the top side 613 of the carousel 601,
602 is slightly larger than size of the trays 214, 216, 224.
An exemplary length and width of the top opening 613 are 5.95
inches and 4.187 inches respectively. The size of the opening
on the bottom side 614 of the carousel 601, 602 is slightly
smaller than the size of the trays 214, 216, 224. The
slightly smaller size of the bottom opening 614 allows a lip
of a tray 214, 216, 224 to rest on the site 618. An exemplary
length and width of the bottom opening 614 are 5.45 inches and
3.687 inches respectively. Each site 618 on each carousel
601, 602 also has a recess 615 which matches a tab on the
trays 214, 216, 224 to ensure its proper orientation.
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In the preferred embodiment, the cut-out 620 is an
opening which is entirely bounded by the carousel 601, 602.
In this embodiment, the movable member of the motor 605 must
move within the periphery of the carousel 601, 602 to avoid
hitting the carousel 601, 602 since the opening is entirely
bounded by the carousel 601, 602.
In an alternate embodiment, the cut-out 620 is partially
bounded by the carousel 601, 602 and is unbounded along the
periphery of the carousel 601, 602. In this embodiment, the
movable member of the motor 605 does not need to move within
the periphery of the carousel 601, 602 to avoid hitting the
carousel 601, 602 since the cut-out 620 is unbounded along the
periphery of the carousel 601, 602. Accordingly, the movable
member of the motor 605 can move outside the periphery of the
carousel 601, 602 in this embodiment.
The sequencer module 600 (Fig. 11) also contains elements
discussed previously which work in conjunction with the
stacked, dual carousel arrangement to perform electrophoresis.
These elements include a CCD camera 408, a laser 452, a high
pressure chamber 422, an array of capillary tubes 454, an
optical window region 130, and an enclosure forming a coolant
region 300. The solvent/gel delivery module 800 described
subsequently in Figs. 14A-C delivers solvent or gel to the
sequencer module 600 through the solvent/gel input port 606.
Fig. 13 explains the operation of the stacked, dual
carousel arrangement which was illustrated in Fig. 11, Fig.
12a and Fig. 12b. After the solvent/gel delivery module 800
fills the capillary array 454 with gel in step 705 as
subsequently discussed in detail in Figs. 14A-C, the
controller 404 causes the motor 608 and rotor 504 to rotate
the lower carousel 602 to position the cut-out 620 under the
needle array 603 in step 700.1 to prevent the vertical
movement of any trays 216, 224 on the lower carousel 602 in
subsequent step 700.2. The controller 404 also commands the
motor 608 and rotor 604 to rotate the upper carousel 601 to
position a sample tray 214 under the needle array 603 in step
700.1.
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In step 700.2, the controller 404 causes the motor 605 to
deploy the sample tray 214 of the upper carousel 601 to the
needle array 603 along the common axis. In step 700.3, the
controller 404 increases the gas pressure in the high pressure
region 422 or applies a voltage to transfer the sample from
the sample tray 214 to the capillary array 454. In step
700.4, the controller 404 causes the motor 605 to move the
sample tray 214 away from the needle array 603 along the
common axis.
As shown in Fig. 13, the stacked, dual carousel
arrangement sends feedback to the controller 404 which the
controller 404 will process to ensure that it issues commands
at the proper time. For example, the encoder 612 which is
operatively engaged to the rotor 604 sends feedback indicative
of the carousel 601, 602 position 1 to the controller 404.
The controller 404 will not issue the command to motor 605 to
move the sample tray 214 to the capillary inlets 603 in step
700.2 until the carousel position feedback 1 indicates that
the sample tray 214 on the upper carousel 601 is beneath the
needle array 603.
Similarly, the current meter of the motor 605 sends
feedback indicative of the vertical position of the sample
tray 2 to the controller 404. The controller 404 will not
issue the command to transfer the sample into the capillary
array 454 in step 700.3 until the tray position feedback 2
indicates that the sample tray 214 is deployed at the needle
array 603.
A pressure transducer of the high pressure region 422
sends the pressure value in the region (valve status 3) to the
controller 404. The controller 404 will not issue the command
to cause the motor 605 to move the sample tray 214 away from
the capillary inlets 603 in step 700.4 until the valve status
feedback 3 indicates that the valve of the high pressure
region 422 is in the proper position. Finally, the controller
404 will not issue the command to introduce buffer into the
capillary array 454 in step 701 until the tray position
feedback 2 as determined by the current meter of the motor 605
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indicates that the sample tray 214 has been returned to the
upper carousel 601.
Following the completion of step 700 as indicated by the
tray position feedback 2, the sequencer module 600 introduces
the buffer into the capillary array 454 in step 701. In step
701.5, the controller 404 causes the motor 608 and rotor 604
to rotate the upper carousel 602 to position its cut-out 620
under the needle array 603. In step 701.5, the controller 404
also causes the motor 608 and rotor 604 to rotate the lower
carousel 602 to position a buffer tray 216 under the needle
array 603.
In step 701.6, the controller 404 causes the motor 605 to
move the buffer tray 216 from the lower carousel 602 through
the cut-out 620 of the upper carousel 601 to the needle array
603. In step 701.7, the controller 404 activates an
ultrasonic transducer to rinse the capillary inlets 603.
During step 701, the stacked, dual carousel arrangement
sends feedback to the controller 404 which the controller 404
will process to ensure that it issues commands at the proper
time. The encoder 612 sends feedback indicative of the lower
carousel 602 position 5 and upper carousel 601 position 5 to
the controller 404. The controller 404 will not issue the
command to cause the motor 605 to move the buffer tray 216 to
the capillary inlets 603 in step 701.6 until the carousel
position feedback 5 indicates that the cut-out 620 of the
upper carousel 601 and the buffer tray 216 on the lower
carousel 601 are positioned beneath the needle array 603.
Similarly, the current meter of the motor 605 sends
feedback indicative of the vertical position of the buffer
tray 6 to the controller 404. The controller 404 will not
issue the command to activate the ultrasonic transducer in
step 701.7 until the tray position feedback 6 indicates that
the buffer tray 216 is deployed at the needle array 603.
Finally, the ultrasonic transducer sends feedback indicative
of its status to the controller 404. The controller 404 will
not initiate electrophoresis in step 702 until the transducer
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status 7 indicates that the capillary inlets 603 have been
rinsed.
The stacked, dual carousel arrangement also performs
tasks in step 704 which recondition the capillary array 454 as
further explained below in the description of the solvent/gel
delivery module of Fig. 14A-C. In step 704.15, the controller
404 causes the motor 608 and rotor 604 to rotate the upper
carousel 602 to position its cut-out 620 under the needle
array 603. The controller 404 also causes the motor 608 and
rotor 604 to rotate the lower carousel 604 to position a waste
tray 224 under the needle array 603 in step 704.15. In step
704.16, the controller 404 causes the motor 605 to deploy the
waste tray 224 from the lower carousel 602 through the cut-out
620 of the upper carousel 601 to the needle array 603.
In the preferred embodiment, the controller 404 manages
electrophoresis and DNA analysis by issuing commands to the
components of the electrophoresis apparatus as described in
the discussion of the stacked, dual carousel arrangement
above. Accordingly, the controller 404 manages the tasks
enumerated in the left column of Fig. 13. The data processing
computer 406 is devoted to processing the data obtained from
executing DNA analysis (Step 703) since DNA data processing is
typically computationally intensive. Accordingly, the data
processing computer 406 performs the tasks enumerated in the
right column of Fig. 13. The controller 404 and data
processing computer 406 are connected to a local area network
and communicate via a data processing user-instrument
interface 706.
Figs. 14A-C illustrate the solvent/gel delivery module
800 which is used after DNA analysis to recondition the
capillary array 454 and to refill the capillary array 454 with
gel. Figures 14a, 14b and 14c show a front view, a side view
and a back view respectively of the solvent/gel delivery
module 800. During DNA analysis, the sample travels from the
capillary inlets 603 in Fig. 11 through the capillary array
454 from right to left.
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During capillary reconditioning, the solvent travels from
the solvent containers 801-803 shown in Fig. 14A, through the
solvent/gel input port 606 shown in Fig. 11 and through the
capillary array 454 of the sequencer module 600 of Fig. 11
from left to right. Similarly, during refill of the capillary
array 454 with gel, the gel travels from the gel syringe 804
shown in Fig. 14B, through the solvent/gel input port 606
shown in Fig. 11 and through the capillary array 454 of the
sequencer module 600 of Fig. 11 from left to right.
The solvent containers 801, 802, 803 hold methanol, water
and soap respectively. A feeder tube 806 in each solvent
container 801-803 carries solvent toward a HPLC pump and wash
solvent system 807. As in the previously described
embodiments, the wash solvent system 807 includes a high
pressure cell (HP cell) to create increased pressures for
faster reconditioning of the capillary array 454.
Support rails 808 provide the structure necessary to hold
the components of the solvent/gel delivery module 800
including the solvent containers 801-803, the gel syringe 804,
the HPLC pump and wash solvent system 807 and the controller
404. The controller 404 causes the other components of the
solvent/gel delivery module 800 to recondition the capillary
array 454 and to refill the capillary array 454 with gel
material.
Figs. 15A-C illustrates the gel syringe 804 of Fig. 14B
in more detail. In contrast to the solvent in the solvent
containers 801-803, the gel is too viscous to be delivered by
a pump. Accordingly, the electrophoresis apparatus uses a gel
syringe 804 for gel delivery. The gel syringe 804 contains a
gel tube carriage 891 which holds a gel cartridge containing
gel material 892. Since the gel cartridge is disposable, it
can be removed from the gel tube carriage 891 after refill of
the capillary array 454 with gel material and replaced with a
new gel cartridge for use in a subsequent execution of
electrophoresis and DNA analysis.
A stepper motor linear actuator 893 has a movable
actuator shaft 897 provided with a pushing member 898. The
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pushing member 898 abuts a surface on a teflonTM plunger 894
located at one end of the gel syringe 804, causing the gel
material to flow through a syringe cap 899 and a high pressure
fitting 895 at the other end of the gel syringe 804. The
controller 404 shown in Fig. 14C selectively activates the
stepper motor linear actuator 893 to control gel delivery. A
cylindrical tube 896 forms the outer structure of the gel
syringe 804. 0-rings prevent the gel material 892 from
leaking around the teflon plunger 894 as it moves toward the
high pressure fitting 895.
In the preferred embodiment, the stepper motor linear
actuator, available from A.M.S.I Corporation in Smithtown, NY
can exert 140 lbs of linear force and displaces l0 ml of gel
in 6,000 pulses. The cylindrical tube 896 is composed of
standard acrylic material and the syringe cap 899 is composed
of stainless steel. The high pressure fitting 895 is
available from Swagelock.
Fig. 16 illustrates the integration of the gel syringe
804 and the HPLC wash solvent system 807 into the solvent/gel
delivery module 800. A solvent manifold 850 connects three
inlets from the feeder tubes 806 of the solvent containers
801-803 to an outlet. Feeder tubes 806 from the solvent
containers 801-803 are connected to the inlets of the solvent
manifold 850 by tubing 860. The controller 404 pictured in
Fig. 14C controls the solvent manifold 850 to select solvent
from one of the three solvent containers 801-803.. The inlet
of the HPLC pump B07 is connected to the outlet of the solvent
manifold 850 by tubing 861 and the outlet of the HPLC pump 807
is connected to an inlet of a valve manifold 851 by tubing
862.
The valve manifold 851 connects two inlets and an outlet.
One inlet of the valve manifold 851 is connected to the gel
syringe 804 by tubing 863 and the other inlet of the valve
manifold 851 is connected to the outlet of the HPLC pump 807.
The outlet of the valve manifold 851 is connected to the
solveat/gel input port 606 by tubing 864. The controller 404
pictured in Fig. 14C causes the valve manifold 851 to select
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either the inlet connected to the gel syringe 804 or the inlet
connected to the HPLC pump 807.
In the preferred embodiment, the tubing 860 connecting
the feeder tubes 806 of the solvent containers 801-803 to the
inlets of the solvent manifold 850 is standard teflonTM tubing
with a diameter of 1/8 inches. The tubing 861 connecting the
outlet of the solvent manifold 850 to the inlet of the HPLC
pump 807 is peek tubing with a diameter of 1/16 inches. The
tubing 861 connecting the outlet of the solvent manifold 850
to the inlet of the HPLC pump 807, the tubing 862 connecting
the outlet of the HPLC pump 807 to an inlet of the valve
manifold 851, the tubing 863 connecting the gel syringe 804 to
an inlet of the valve manifold 851 and the tubing~h864
connecting the outlet of the valve manifold 851 to the
solvent/gel input port are peek tubing with a diameter of 1/16
inches.
In the preferred embodiment, the HPLC pump 807, available
from Alltech Corporation (Model Number 301300), has a non-
metal pump head. The valve manifold 851 is a non-metal valve,
available from Alltech Corporation (Model Number 97500).
Fig. 13 explains the operation of the solvent/gel
delivery module 800 which was illustrated in Fig. 14A and Fig.
16. Following DNA analysis in step 703, the proper rotational
positioning of the carrousels 601, 602 in step 704.15 and the
proper deployment of the waste tray in step 704.16, the
controller 404 causes the solvent/gel delivery module 800 to
pump out the used gel from the capillary array 454 via the HP
cell in step 704.17.
To pump out the used gel, the controller 404 causes the
solvent manifold 850 to select the inlet from the water
container 802 and causes the valve manifold 851 to select the
inlet from the HPLC pump 807 as shown in Fig. 16. The HPLC
pump 807 pumps water from the solvent manifold 850 through the
valve manifold 851 to the HP cell to create increased
pressures for faster reconditioning of the capillary array 454
as previously described. The waste tray 852 which was
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properly deployed in step 704.16 collects the used gel from
the capillary array 454.
After the gel has been removed from the capillary array
454, the controller 404 causes the solvent/gel delivery module
800 to pump rinse solutions through the capillary array 454
via the HP cell in step 704.18. To perform this rinsing, the
controller 404 causes the solvent manifold 850 to select the
inlet from the methanol container 801 and causes the valve
manifold 851 to select the inlet from the HPLC pump 807 as
shown in Fig. 16. The HPLC pump 807 pumps methanol from the
solvent manifold 850 through the valve manifold 851 to the HP
cell to create increased pressures for faster rinsing of the
capillary array 454 as previously described. The waste tray
852 which was properly deployed in step 704.16 collects the
used solvent from the capillary array 454.
After rinsing the capillary array 454 with methanol, the
controller 404 causes the solvent/gel delivery module 800 to
perform rinsing of the capillary array 454 with soap from
container 803. To perform this rinsing, the controller 404
causes the solvent manifold 850 to select the inlet from the
soap container 803 and causes the valve manifold 851 to select
the inlet from the HPLC pump 807 as shown ~in Fig. 16. The
HPLC pump 807 pumps soap from the solvent manifold 850 through
the valve manifold 851 to the HP cell to create increased
pressures for faster rinsing of the capillary array 454 as
previously described. The waste tray 852 which was properly
deployed in step 704.16 collects the used solvent from the
capillary array 454. Further, the controller 404 causes the
solvent/gel delivery module to repeat the reconditioning
process with the solvents from the three solvent containers
801-803 until the capillary array 454 is clean.
As shown in Fig. 13, the solvent/gel delivery module
sends feedback to the controller 404 which the controller 404
will process to ensure that it issues commands at the proper
time. For example, the controller 404 will not cause the
gel/solvent delivery module 800 to pump rinse solutions
through the capillary array 454 until the HP pump status
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feedback 17 indicates that the gel has been pumped from the
capillary array 454. Similarly, the controller 404 will not
issue the command to refill the capillary array with gel
material in step 705 until the HP pump status feedback 18
indicates that the capillary array 454 has been rinsed with
methanol and soap.
In the preferred embodiment, the controller 404 will
determine the HP pump status feedback 17. The controller 404
determines the HP pump status feedback 17 by computing the
volume of solution which has passed through the capillary
array 454 from the flow rate of the HP pump specified by the
manufacturer and the amount of time which has elapsed since
the pump was activated.
Following the reconditioning of the capillaries in step
704, the controller 404 causes the solvent/gel delivery module
800 to refill the capillary array 454 with new gel via the HP
cell in step 705.19. To refill the capillary array 454 with
new gel, the controller 404 causes the valve manifold 851 to
select the inlet from the gel syringe 804 as shown in Fig. 16.
The gel syringe 804 delivers gel through the valve manifold
851 to the HP cell to create increased pressures for faster
refilling of the capillary array 454 as previously described.
The waste tray 852 which was properly deployed in step 704.16
collects any solvent or gel which leaves the capillary array
454 during step 705.19.
After filling the HP cell and capillary array 454 with
gel in step 704.18, the process can continue in one of two
ways. In one embodiment, the gel in the HP cell can be used
as the buffer in the subsequent electrophoresis of step 702.
In this embodiment, the process continues with step 705.20.
In an alternate embodiment, the controller 404 causes the
solvent/gel delivery module to pump the gel from the HP cell
and to fill it with buffer using a process similar to step 704
before proceeding to step 705.20.
The dual, stacked carousel participates in the remaining
tasks of step 705. In step 705.20, the controller 404 causes
the rotor 604 to rotate the upper carousel 602 to position its
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cut-out 620 under the needle array 603. The controller 404
also causes the rotor 604 to rotate the lower carousel 604 to
position a buffer tray 216 under the needle array 603 in step
705.20. In step 705.21, the controller 404 causes the motor
605 to deploy the buffer tray 216 from the lower carousel 602
through the large opening of the upper carousel 601 to the
needle array 603.
Step 705 concludes with a step to equilibrate the
capillary array 454 and to circulate buffer through the
capillary array 454 (Step 705.22). Differences in drag
through the capillary array 454 will cause air bubbles and
pressure differences to develop within the gel in the
capillary array 454 during gel delivery in step 705.19. Step
705.22 removes the pressure differences and air bubbles with a
procedure to equilibrate the capillary array 454 and circulate
buffer through the capillary array 454. This procedure is
similar to the electrophoresis executed in step 702 except DNA
is absent from the capillary array 454 during step 795.22
while DNA is obviously present during electrophoresis of step
702. Specifically, a voltage is applied across the capillary
array 454 to induce relaxation of the gel and movement of the
buffer through the capillary array 454. After circulating the
buffer in the capillary array 454 in step 705.22, the process
repeats for the next DNA analysis beginning with sample
introduction in step 700.
As shown in Fig. 13, the solvent/gel delivery module
continues to send feedback to the controller 404 in step 705
as it did in step 704. The controller 404 will process the
feedback to ensure that it issues commands at the proper time.
For example, the controller 404 will not execute any commands
beyond step 705.19 until the gel syringe feedback 19 indicates
that the capillary array 454 has been filled with new gel.
Similarly, the controller 404 will not issue the command to
rotate the upper carousel 601 and lower carousel 602 in step
705.20 until the HP pump status feedback 19 indicates that the
gel has been pumped into the capillary array 454.
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In the preferred embodiment, the controller 404 will
determine the gel syringe feedback 19. The controller 404
determines the gel syringe feedback 19 by computing the volume
of solution which has passed through the capillary array 454
from the displacement rate of the gel syringe and the amount
of time which has elapsed since the gel syringe was activated.
Similarly, the encoder which is operatively engaged to
the rotor 604 sends feedback indicative of the position of the
carrousels 20 to the controller 404 in step 705. The
controller 404 will not issue the command to deploy the buffer
tray 216 in step 705.21 until the carrousel position feedback
indicates that the large opening of the upper carousel 601
and the buffer tray 216 on the lower carousel 601 are
positioned beneath the needle array 603.
15 The current meter of the vertical motor drive 605 also
sends feedback indicative of the vertical position of the
buffer tray 6 to the controller 404. The controller 404 will
not issue the command to circulate the buffer in the capillary
array 454 in step 705.22 until the tray position feedback 21
20 indicates that the buffer tray 216 is deployed at the needle
array 603. Finally, the controller 404 receives feedback on
the high voltage status of the capillary currents 22. The
controller 404 will not proceed to step 700 to introduce the
next DNA sample until the high voltage status 22 indicates
that the buffer has been circulated through the capillary
array 454.
Fig. 17 illustrates an off-line capillary reconditioner
900 which is used periodically to thoroughly clean a capillary
cartridge 909. To thoroughly clean the capillary cartridge
909, the operator removes it from the automatic
electrophoresis system and installs it in the off-line
capillary reconditioner 900. Accordingly, the operator can
install another clean capillary cartridge 909 in the automatic
electrophoresis system and execute DNA analysis with that
capillary cartridge while the off-line capillary reconditioner
900 is thoroughly cleaning the previously used capillary
cartridge 909. Since a through cleaning typically takes
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twenty to thirty minutes, the off-line capillary reconditioner
improves the throughput of the automatic electrophoresis
system as the system does not have to wait for a thorough
cleaning of the capillary cartridge 909 between consecutive
executions of DNA analysis.
The off-line capillary reconditioner 900 is a low-cost
streamlined version of the solvent/gel delivery module 600
previously explained in Fig. 11 as it does not contain all of
the items included in the solvent/gel delivery module 600.
For example, the off-line capillary reconditioner 900 does not
have a camera, a laser, or a gel syringe. The off-line
capillary reconditioner 900 does not include a gel syringe for
gel delivery because undesirable hardening of the gel could
occur at the ends of the capillary array 454 during movement
of the capillary cartridge from the off-line capillary
reconditioner 900 to the automatic electrophoresis~system if
the gel were delivered off-line prior to movement of the
capillary cartridge. The streamlined nature of the off-line
capillary reconditioner 90o gives it the advantage of
increasing the throughput of the system with a low cost.
The solvent containers 901, 902, 903 hold methanol, water
and soap respectively. A feeder tube 908 in each solvent
container 901-903 carries solvent toward a solvent manifold
905. The solvent manifold 905 connects three inlets to one
outlet. The three inlets of the solvent manifold are
connected to the feeder tubes 906 of the solvent containers
901-903 to establish a one to one correspondence between the
set of inlets and the set of feeder tubes 906.
A HPLC pump 906 has one inlet, which is connected to the
outlet of the solvent manifold, and one outlet, which is
connected to the solvent input port 907 at one end of the
capillary cartridge 909. The off-line capillary reconditioner
900, like the solvent/gel delivery module 600 described in
Fig. 14A, also has a HP cell to create increased pressures for
faster reconditioning of the capillary cartridge 909. A
controller manages the operation of the solvent manifold 905
and HPLC pump 906. A waste container 904 collects waste
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during capillary reconditioning at the other end of the
capillary cartridge 909.
In the preferred embodiment, the controller is a simple,
low-cost digital signal processing system which receives
status feedback and issues commands to the HPLC pump 906 and
the solvent manifold 905 in a predetermined order as explained
below. Alternatively, a general purpose computer like a
personal computer could be used to execute a simple control
program to manage the off-line capillary reconditioner.
l0 After the operator installs the capillary cartridge 909
in the off-line capillary reconditioner 900, the internal
controller causes the solvent manifold to select the inlet
from the water container 901 and activates the HPLC pump 906.
The HPLC pump 807 pumps water from the solvent manifold 850 to
the HP cell to create increased pressures for faster
reconditioning of the capillary cartridge 909 as previously
described. The waste container 904 which was properly
deployed previously collects the used gel from the capillary
cartridge 909.
After the gel has been removed from the capillary
cartridge 909, the off-line capillary reconditioner 900 rinses
the capillary cartridge 909 via the HP cell with methanol.
First, the controller causes the solvent manifold 905 to
select the inlet from the methanol container 902. The HPLC
pump 906 pumps methanol from the solvent manifold 905 to the
HP cell to create increased pressures for faster rinsing of
the capillary cartridge 909. The waste container 904 which
was properly deployed previously collects the used solvent
from the capillary cartridge 909.
Next, the off-line capillary reconditioner 900 rinses the
capillary cartridge 909 via the HP cell with soap from
container 903. First, the controller causes the solvent
manifold 905 to select the inlet from the soap container 903.
The HPLC pump 906 pumps the soap from the solvent manifold 905
to the HP cell to create increased pressures for faster
rinsing of the capillary cartridge 909. The waste container
904 which was properly deployed previously collects the used
42
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solvent from the capillary cartridge 909. Further, the
controller 404 causes the off-line capillary reconditioner 900
to repeat the reconditioning process with the solvents from
the three solvent containers 801-803 until the capillary array
454 is clean.
Fig. 18 illustrates another preferred embodiment of the
capillary cartridge 1180. In this embodiment, the capillary
tubes run from their first ends 1188 disposed in an
electrode/capillary array 1181. The capillary tubes, then,
run inside multilumen tubing 1183. The multilumen tubing is
taught in detail in U.S. Patent Number 6,063,251. The multilumen
tubing 1183 is held firmly in place by tubing holders 1185.
The capillary tubes, without the protection the multilumen
tubing, pass through an optical detection region 1187. Beyond
the optical detection region 1187, the capillary tubes have a
common termination and are bundled together and cemented into
a high pressure T-shaped fitting 1182 made from electrically
conductive material, which, during electrophoresis, is
connected to electrical ground.
The tubing holders 1185 and the T-fitting 1182 are fixed
to a cartridge base 1186. The cartridge base 1186 is made
from polycarbonate plastic for its dielectric characteristic.
The base 1186 in turn is removably attached to a shuttle 1179
which includes a set of rail couplings 1184 protruding from
its bottom. These rail couplings 1184 are arranged so that
they fit on to a railing system (not shown in Fig. IS) of the
sequences module 400 in Fig. 10 or 600 in Fig. 11. The
railing system allows the shuttle 1184 to move between an in
position and out position. The base 1186 is detached from the
shuttle 1179 so that the cartridge 1180 is disposed (or
cleaned) and a new (or cleaned) capillary cartridge is
attached when the shuttle 1179 is in its out position. The
combination of the railing system and the shuttle 1179 allows
the newly attached capillary cartridge to be repeatedly
located at the same position as that of the disposed capillary
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cartridge in relation to a camera and a laser (not shown in
Fig. 18) when the shuttle 1179 is in its in position.
In a preferred embodiment, the shuttle 1179 extends the
length of the base 1186 with an opening to accommodate the
electrode/capillary array 1181; the shuttle 1179 is attached
to the base 1186 by a plurality of removable fasteners 1178.
The electrode/capillary array 1181 is held in place by a
current supply/monitoring board 1190 depicted in Fig. 19. The
board 1190 is preferably a printed circuit board for supplying
l0 high voltage.
The board 1190 preferably includes a plurality of large
holes 1193 so that a set of fasteners can be used to attach
the board 1190 to the base 1186. However, any other means,
e.g., gluing, can be utilized as well to attach the board 1190
to the base 1186 .
The board 1190 also includes a plurality of tube
holes 1194 arranged to be co-located with holes in the base
1186 to allow the first ends of the capillary tubes to
protrude through the tube holes 1194 when the board 1190 is
attached to the base 1186. The plurality of pins 1195,
preferably gold plated, are also disposed on the board 1190.
At least one pin is placed proximate to each tube hole forming
a pin-hole pair 1192. Each pin-hole pair is dipped into one
sample well of the sample microtitre tray.
The board 1190 further includes high voltage electrical
wire leads 1198. The wire leads 1198 electrically connect
each pin 1195 to corresponding connector ends 1196 formed on
the periphery of the board 1190. Each connector end 1196 is
shaped to receive a high voltage connector which preferably
includes about 50 electrical connections. The high voltage
connectors then connect the wire leads 1198 to power supply
lines from a high voltage power supply, preferably
manufactured by Bertan (not shown in Fig. 19). This
establishes a closed electrical circuit from the pins 1195 to
a second electrode connected to the high pressure T-fitting
1182 when the capillary tubes are filled with gel. The second
electrode is preferably connected to the system ground.
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In addition, the high voltage connectors are also
connected to an electronic current monitor which monitors the
electric currents. In the current monitor, the current
flowing in each power supply line is preferably monitored in
multiple power supply lines at a time and in sequence. This
allows the current monitor to make integrated current
measurements.
Fig. 20 illustrates a multi-wavelength beam generator.
The beam generator includes a laser head 1200, preferably an
argon ion laser capable of producing multi-wavelength laser
beams in the wavelengths of 457 nm, 476 nm, 488 nm, 496 nm,
502 nm, 514 nm. The beam generator also includes a laser
emitter tube 1207, which is connected to the argon ion laser
1200 by an optical coupling assembly 1202.
The optical coupling assembly 1202 include a fiber
coupler 1201, which is connected to the laser head 1200, and
an optical fiber cable 1203, which connects the fiber coupler
1201 with the laser emitter tube 1207. The fiber coupler 1201
optically aligns the laser head 1200 with the fiber cable 1203
which is an achromic optical fiber cable. This optical
coupling assembly 1202 permits the laser emitter tube 1207 to
be remotely located from the laser head 1200 and still produce
laser beams, coherent light, at the laser emitter tube 1207.
This also allows the laser head 1200, which generates heat, to
be located in a less sensitive area of the sequencer.
The laser emitter tube 1207 includes a one dimensional
focuser 1208, preferably a positive cylindrical optical lens
with 10 cm focal length, located at an output end 1204 of the
laser emitter tube 1207. The laser emitter tube 1207 also
includes a fiber emitter tube 1205 receiving the optical fiber
cable 1203 and a beam expander 1209, preferably a negative
cylindrical optical lens with 1.9 cm focal length, placed
between the fiber emitter tube 1205 and the one dimensional
focuser 1208. The laser emitter tube 1207 is preferably
housed in a 1" dia. x 6" long tube.
Fig. 22 schematically depicts the optical processes
performed by the laser emitter tube 1207. The laser light
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emitted by the fiber emitter tube 1230 is expanded by the beam
expander 1231. The laser beam then is focused in only one
direction by the one dimensional focuser 1233. The resulting
beam is directed toward the capillary array 1235. The
resulting laser beam is narrowed in one direction and
elongated in the other direction. Figs. 22a-c illustrates the
foot prints of the laser beams at each processing step.
Fig. 21 illustrates another embodiment of the multi-
wavelength beam generator in which two laser heads 1211, 1215
are provided. The first laser head 1211 is identical with the
laser head 1200; the second laser head 1217, however,
generates different wavelength laser beam. The second laser
head 1217 is preferably a solid state laser which produces
laser beam with the wavelength longer than 532 nm. In an
alternative embodiment, the second laser head 1217 produces
multi-wavelength laser beams with the wavelengths different
from the beam generated by the first laser head 1211.
In this embodiment, two optical coupling assemblies with
fiber couplers 1213, 1217 and optical fiber cable 1225, 1221
are provided; the two optical coupling assemblies function
identically with the optical coupling assembly 1202 of Fig.
20. The laser beams generated by the two laser heads 1211,
1215 and delivered by the optical coupling assemblies are
combined in a laser emitter tube 1224 designed to receive
laser beams from two laser sources.
The laser emitter tube 1224 has two input ends 1210, 1212
and one output end: a first fiber emitter tube 1227, receiving
laser beams from the first laser head 1211, is located at the
first input end 1210; a second fiber emitter tube 1223,
receiving laser beams from the second laser 1215, is located
at the second input end 1212; a one dimensional beam facuser
1226, preferably a positive cylindrical optical lens with to
cm focal length and outputing the combined laser beams, is
located at the output end 1214.
The laser beams received by the first and second fiber
emitter tubes 1227, 1223 are combined by a dichroic filter
1229 which transmits laser beams received from the first fiber
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emitter tube 1227 and reflects the laser beams received from
the second fiber emitter tube 1223, thereby combining the
laser beams from the first and second fiber emitter tubes
1227, 1223.
. 5 A beam expander 1222, preferably a negative cylindrical
optical lens 1222 with 1.5 cm focal length, is provided
between the dichroic filter 1229 and the one-dimensional
focuses 1226. The combination of the beam expaader 1222 aad
the focuses 1226 optically function substantially identical to
l0 the optical processes described in Fig. 22 and Figs. 22a-c.
In the preferred embodiment, polarization optics in the
laser heads 1200, 1211, 1215 are removed to maximize the laser
output power. The laser output power is increased by more
than four times in the embodiment depicted in Fig. 21 with the
15 combination of multi-line and unpolarized emission compared to
a polarized single-line laser.
Even though Fig. 22 depicts the resulting laser beam
impinging upon the capillary array 1235 without any angle, a
small angle entrance excitation configuration, as shown in
20 Fig. 23, improves the excitation laser light coupling
efficiency at the detection window. This also reduces the
required laser power to excite a 96 (or more) capillary array
and allows the use of a portable air-cooled argon ioa laser in
the DNA sequencez instrument. In other words, the multi-
25 wavelength laser emitter tubes described in Figs. 20 or 21 is
aligned to illuminate a larger number of capillary array (such
as 1000-. capillaries) while preserving focusability of laser
beams across a wide range of capillary array.
Fig. 24 illustrates the integration of the CCD camera 1257
30 with the laser emitter tube 1243. Fig. 25 illustrates a
structure 1251 associated with the detection region. In the
preferred embodiment, the laser emitter tube 1243 is positioned
so that the laser beam from the laser emitter tube 1243 impinges
at an angle upon capillary array 1200 as shown in Fig. 23.
35 The laser emitter tube 1207 is positioned on a level with
the capillary detection window with the beam emitting end of
the tube 1207 facing away from the instrument operator. In
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the preferred embodiment, the laser emitter tube 1207 is
secured to a bottom end of a tube control arm 1267 which is
rotatably connected to an arm mount 1265. The arm mount 1265
is attached to the bottom ends of flexible rotators 1261,
1263. The top ends of the rotators in turn are connected to
dials accessible from the exterior of the sequencer. The arm
mount 1265 is also mounted on laser emitter positioning rails
1255 and moved by an arm mount position controller 1253. With
the dials and the controller 1253 described above, the laser
emitter tube 1207 is optimally positioned to deliver its
output laser beam to the capillary array.
The CCD camera 1257 is mounted a camera mount (not shown
in Fig. 25) which in turn is movably mounted on the laser
emitter positioning rails 1255. A camera rotating cable (not
shown in Fig. 25) moves the CCD camera plate in the horizontal
direction on the rails 1255. A camera focus gear 1271 is
connected to a gear control cable 1254 which controls the
movements of the CCD camera lens assembly 1269. The
preceding camera controllers allow the CCD camera 1257 to
focus on the portion of capillary array impinged by the laser
beam from the laser emitter tube 1243.
Another preferred embodiment of the high viscosity liquid
delivery system is illustrated in Fig. 26. The liquid
delivery system has a high pressure chamber 1401 which holds
low viscosity liquid 1413 such as water and a squeezable and
disposable bag 1411 which contains a high viscosity liquid
such as gel.
The chamber 1401 includes a cylinder 1402, preferably
made from metallic material such as aluminum or stainless
steel for sustaining interior pressures up to 2000 psi, has a
substantially hollow body with a closed bottom and an open
top. A cap 1404 is removably affixed to the top of the
cylinder 1402, whereby the cylinder 1402 and the cap 1404 form
the high pressure chamber 1401 to hold the liquid 1413 and the
disposable bag 1411.
The gel container 1411 is removably attached to an outlet
assembly 1410, preferably by a Swagelock 1409. The viscous
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liquid is forced out through the outlet assembly 1410 when the
pressure inside the chamber increases. The outlet assembly
1410 is fitted to the cap 1404 with a water tight fitting
1407, available from Swagelock. The outlet assembly 1410 also
includes a gel delivery tubing 1403. Because the pressure
around the squeezable gel bag 1411 is uniformly applied by
the liquid 1413, pressure rating requirement for the gel
container is minimum. The gel bag 1411 is, therefore,
economically made from a disposable bag with a large gel
holding capacity sufficient for multiple gel runs.
The pressure inside the chamber 1401 is increased or
decreased by a pressure control assembly 1406. When more
liquid is supplied to the chamber 1401 by the pressure control
assembly 1406, the pressure inside the chamber 1401 increases.
When the pressure is increased by an excessive amount or when
the cap 2404 is to be opened to replace the bag 1411, the
liquid inside the chamber is released by the pressure control
assembly to reduce the pressure.
The pressure control assembly includes a high pressure
pump 1423 controlled by a controller 1404. In the preferred
embodiment, the high pressure pump 1423 is another HPLC pump.
The pressure control assembly 1406 also includes an inlet
tubing 1421 connected to the pump 1423 and fitted to the
bottom of the cylinder by a water tight fitting 1419.
An outlet tubing 1427 is also provided to the pressure
control assembly 1406. The outlet tubing 1427 is fitted,
preferably, to the bottom of the cylinder 1402 by a second
water tight fitting 1417. In turn, the outlet tubing includes
a release valve 1425 and a pressure transducer 1429 for
generating a feedback signal, preferably less than 6 volts,
which is communicated to the controller 1404. The release
valve 1425 is controlled by the controller 1404.
The controller 1404 is preferably a part of the central
computer controller 404 in Fig. 10 in order to save space;
however, in alternative embodiments, the central computer
controller 404 are replaced with other types of controllers
such as another computer, microprocessor, or any other
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electronic device capable of controlling a water pump and a
valve.
By monitoring the feedback signal, which indicates the
pressure inside of the chamber 1401, the controller 1404
performs the following functions: (1) when the pressure inside
the chamber 1401 needs to be increased by a certain amount,
the controller 1404 activates the HPLC pump 1423 to pump more
liquid into the chamber 1401 via the inlet tubing 1421; or (2)
when the pressure inside the chamber 1401 needs to be
decreased by a certain amount, the controller 1404 opens the
releasing valve 1425 to release the liquid from the chamber
1401. Once a sufficient amount of gel is pushed out, the pump
stops which caused the pressure inside the chamber to decrease
and return the pressure to a previous equilibrium. The
preferred gel filling pressure is 500 psi.
Fig. 27 illustrates the preferred embodiment of a
solvent/gel delivery module 1800 which is used after a DNA
analysis to recondition the capillary array and to refill the
capillary array with gel. Figure 28 shows a back view of the
solvent/gel delivery module 1800. The solvent/gel delivery
module 1800 is preferably placed next to the sequencer. The
purpose of the solvent/gel delivery module 1800 is to provide
sequential, automated gel delivery and capillary
reconditioning.
Fig. 29 illustrates the integration of the high pressure
gel delivery system 1805, either the gel delivery syringe 804
in Fig. 16 or the high pressure chamber 1401 in Fig. 26, into
the solvent/gel delivery module 1800 of Fig. 27. In this
preferred embodiment, a solvent manifold 1850 connects four
inlets from the feeder tubes 1806 of solvent containers 1801-
1804 to an outlet. Feeder tubes 1806 from solvent containers
1801-1804, two bottles with methanol, a bottle with poly vinyl
pyrrolidone (PVP), preferably 2 % of PVP, and a bottle with a
buffer solution, are connected to the inlets of the solvent
manifold 1850. The inlet of the HPLC pump 1807 is connected
to the outlet of the solvent manifold 1850 by a pump
connecting tube 1861 and the outlet of the HPLC pump 1807 is
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connected to an inlet of a valve manifold 1851 by pump outlet
tube 1862. An outlet from the HPLC pump 1807 (not shown in
Fig. 29) is connected to the high pressure chamber 1805 as
discussed above.
A valve manifold 1851 connects two inlets and an outlet.
One inlet of the valve manifold 1851 is connected to the gel
delivery system 1805 by a gel outlet tube 1863 and the other
inlet of the valve manifold 1851 is connected to the outlet of
the HPLC pump 1807. The outlet of the valve manifold 1851 is
l0 connected to a liquid delivery chamber 1810, preferably the
high pressure T-fitting 1182 of Fig. 18, by a manifold outlet
tube 1864. The liquid delivery chamber 1810 includes a purge
valve 1867 for draining waste in the liquid delivery chamber
1810 to a waste container 1865.
The controller 404 illustrated in Fig. 10 includes
connections to the solvent manifold 1850, the HPLC pump 1807,
the pressure control assembly for the gel delivery system
1805, the valve manifold 1851 and the drain valve 1867 for
controlling the connected components.
For example, the controller 404 controls the solvent
manifold 1850 to a select solvent from the four solvent
containers 1801-1804 and causes the valve manifold 1851 to
select either the inlet connected to the chamber 1804 to
receive the gel or the inlet connected to the HPLC pump 1807
to receive the solvent.
In the preferred embodiment, the lengths of the pump
connecting tube 1861, the pump outlet tube 1862 and the
manifold outlet tube 1864 are minimized to reduce wasting gel
and solvents. In particular, the pump connecting and outlet
tubes 1861, 1862 hold old solvent when new solvent is needed
to be supplied to the valve manifold 1851, thereby requiring
the old solvent to be wasted. The similar waste also occurs
between the solvents and the gel in the manifold outlet tube
1864, which preferably is less than 50 cm and is more
preferably less than 25 cm and is most preferably less than l0
cm.
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As illustrated in Fig. 28, the solvent/gel module 1800
includes a blower 1801 for the laser head located is the
sequences. The laser head is housed in the bottom level of
the sequences module. The cooling blower is configured to
essentially suck air out of the sequences module and blow it
out to the exhaust in the washing machine module 1800. The
result is cold air moving across the laser, without creating
huge amounts of turbulence in the sequences module. A 5"
diameter flexible hose is connected from the rear of the laser
to the blower intake. The hot exhaust is carried out through
another 5" diameter hose that is then connected to ceiling
ductwork and expelled (not shown in Fig. 28).
Similar to the steps 700-703 of Fig. 13, the steps 1901,
1903 and 1905 of Fig. 3o explain the operation of the stacked,
dual carousel arrangement which was illustrated in Fig. 11,
Fig. 12a. Since the steps are substantially identical to each
other and the differences between them are self explanatory,
no detailed explanations of the steps 1901, 1903 and 1905 is
provided.
Furthermore, the steps 1907, 1909, 1911 and 1912 of Fig.
31 describe the operation of the solvent/gel delivery module
1800 which was illustrated in Figs. 28~30. Since the
operations of the solvent/gel delivery module 1800 is
substantially identical to the'operations of the solvent/gent
module 800, duplicative discussions will be skipped. However,
the different steps are discussed below.
In step 1907.17, the rinse step includes rinsing the
capillary tubes with methanol for 24 minutes and, then,
rinsing with PVP for 8 minutes. In step 1911.23, the current
monitor attached between the current/monitoring board and the
power supply is activated. In steps 1912.25 and 1912.26, a
rinse tray is utilized for rinsing the first ends of the
capillary tubes and the pins protruding from the current
supply/monitoring board.
While the above invention has been described with
reference to certain preferred embodiments, it should be kept
in mind that the scope of the present invention is not limited
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to these. One skilled in the art may find variations of these
preferred embodiments which, nevertheless, fall within the
spirit of the present invention, whose scope is defined by the
claims set forth below.
53
