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Description  |
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FIELD OF THE INVENTION
This invention pertains to electrophoresis. More specifically, this
invention pertains to improvements in electrophoresis apparatus whereby an
electrical field can be directionally modulated, so as to tend to cause a
molecule being electrophoresed to migrate in any desired direction.
BACKGROUND OF THE INVENTION
It has been known heretofore that electrical parameters governing gel
electrophoresis, i.e., electrophoresis on a flat surface of a gel, in a
buffer solution, include the magnitude of the electric field, the
direction of the electric field, and the evolution of the magnitude and
direction of the electric field as functions of time, as well as the type
and temperature of the buffer solution, the material and configuration of
the electrodes, and other factors. Both homogeneous and nonhomogeneous
electric fields have been used in gel electrophoresis. An electric field
is regarded as homogeneous if it is uniform, in magnitude and direction,
across the flat surface of the gel at any moment in time.
It also has been known heretofore that pulsed modulation of the magnitude
of the electric field, in its direction, or both, enhances the utility of
gel electrophoresis, particularly in the separation of proteins, nucleic
acids, and other such macromolecules. Pulsed modulation refers to
modulation at timed intervals, between which the electric field may be
substantially uniform across the flat surface of the gel.
In recent years, apparatus for gel electrophoresis have advanced from
apparatus employing two parallel driving electrodes, or two parallel
arrays of driving electrodes, which apply electric fields tending to cause
molecules to migrate along straight lines between such electrodes, to
apparatus employing polygonal arrays of driving electrodes, which apply
electric fields that can be directionally modulated so as to tend to cause
molecules to migrate along non-straight paths, e.g., paths having corners
or zig-zag paths.
In one heretofore known type of apparatus for gel electrophoresis, as
mentioned above, a square array of driving electrodes is provided, which
enables electric fields to be alternatively applied in transverse
directions so as to tend to cause samples being electrophoresed to migrate
in paths that change direction at 90.degree. angles. See, e.g., Cantor et
al. U.S. Pat. No. 4,473,452, which discloses that such fields can be
substantially uniform, if applied by and between paired electrodes in like
numbers on opposite sides of the square array, or substantially
fan-shaped, if applied by and between one electrode on one end of a given
side of the square array and plural electrodes on the opposite side of the
square array. This patent also mentions that each electrode can be
selectively maintained at any positive or negative electrical potential
within a selected range.
In another heretofore known type of apparatus for gel electrophoresis, as
mentioned above, a hexagonal array of driving electrodes is provided,
which enables homogeneous electrical fields to be alternatingly applied at
timed intervals so as to cause molecules being electrophoresed to migrate
in zig-zag paths. See, e.g., Biotechnology, December 1986, page 1054,
which refers to electrophoresis in such apparatus as contour-clamped
homogeneous electric field (CHEF) electrophoresis, and which compares CHEF
electrophoresis to other heretofore known techniques including
pulsed-field gel electrophoresis (PFGE) and field-inversion
electrophoresis. See, also, Chu et al., "Separation of Large DNA Molecules
by Contour-Clamped Homogenous Electric Fields," Science, Dec. 19, 1986,
Vol. 234, pp. 1582-5. An apparatus employing parallel driving electrodes,
for field-inversion electrophoresis, is disclosed in Carle et al. U.S.
Pat. No. 4,737,251.
Although some of the heretofore known apparatus and techniques discussed in
the preceding paragraphs have been valuable contributions to the art of
electrophoresis, there has remained a need, to which this invention is
addressed, for greater flexibility in directional modulation of an
electric field in an apparatus for electrophoresis.
SUMMARY OF THE INVENTION
This invention provides an apparatus for conducting electrophoresis in a
gel, which is immersed in a buffer solution, or for conducting
electrophoresis in another suitable medium, which has an array of driving
electrodes and an array of sensing electrodes, each in contact with the
medium.
The driving electrodes are arrayed in spaced relation to one another,
preferably along a closed, curved path. The sensing electrodes are
distinct from the driving electrodes and also are arrayed in spaced
relation to one another, preferably also along a closed, curved path
spaced from, and lying on the concave side of, the path of the driving
electrodes.
Preferably, the driving and sensing electrodes are arrayed respectively
along concentric circles, with the circle along which the driving
electrodes are arrayed enclosing the circle along which the sensing
electrodes are arrayed. Preferably, the driving and sensing electrodes are
arrayed in equal numbers in electrode pairs, and fixed in place, in which
each sensing electrode is radially inward of and paired with a single one
of the driving electrodes. As a highly preferred example, twenty-four
driving electrodes and twenty-four sensing electrodes may be so arrayed,
the driving electrodes in equally spaced intervals along a circle and the
sensing electrodes in like intervals along a circle having a lesser
radius.
Means are provided respectively for providing electrical potentials to be
applied to selected ones of the driving electrodes, for applying such
potentials, for sensing electrical potentials at selected ones of the
sensing electrodes, and for adjusting the applied potentials to maintain
the sensed potentials at selected values. If the driving and sensing
electrodes are arrayed in equal numbers in electrode pairs, as discussed
above, the adjusting means adjusts the applied potentials to maintain the
sensed potential at a selected value at the sensing electrode of each
electrode pair.
Since each electrode pair may be independently controlled, the apparatus
may be programmably controllable, as for example by means of a
microprocessor, to enable an electric field to be directionally modulated,
in any direction, e.g., as a function of time.
These and other objects, features, and advantages of this invention are
evident from the following description of a preferred embodiment of this
invention with reference to the accompanying drawing.
BRIEF DESCRIPTION OF THE DRAWING
FIG. 1 is a semi-diagrammatic view, partially in section, of a support for
an electrophoresis gel, a circular array of driving electrodes, a circular
array of sensing electrodes, and an exemplary portion of electrical
circuitry associated with the driving and sensing electrodes, in an
electrophoresis apparatus constituting a preferred embodiment of this
invention;
FIG. 2 is a block diagram of other portions of electrical circuitry of the
electrophoresis apparatus of FIG. 1; and
FIG. 3 is a plot of isopotential contours achieved using the apparatus of
this invention.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENT
As represented in the drawing, an apparatus 10 for electrophoresis
employing a gel matrix G, in a buffer solution, constitutes a preferred
embodiment of this invention.
Such a gel matrix and such a buffer solution are conventional in gel
electrophoresis. In the preferred embodiment, the gel matrix G may be an
aqueous gel of 1% agarose occupying a square area, which measures about 20
cm.times.20 cm. Any conventional buffer solution compatible with the gel
matrix G may be used. The buffer solution must have sufficient resistivity
to avoid short-circuits between driving electrodes described below. Two
preferred formulations for buffer solutions are set forth below in Tables
A and B respectively. Preferably these are used in 0.5X strengths.
TABLE A
TAE (1X formulation)
0.04 Molar Tris-acetate [Tris(hydroxymethyl)aminomethane actetate]
0.001 Molar EDTA [ethylenediamenetetraacetic acid]
balance, water
pH=8.0 approximately
TABLE B
TBE (10X formulation)
0.89 Molar Tris Base [Tris(hydroxymethyl)aminomethane]
0.89 Molar Boric Acid [H.sub.3 BO.sub.3 ]
0.02 Molar EDTA [ethylenediamenetetraacetic acid]
balance, water
pH=8.3 approximately
The apparatus 10 comprises an electrophoresis chamber of a conventional
type including a rectangular, horizontal support 20 for the gel matrix G
and the buffer solution and including outer walls 22, 24, 26, 28, which
contain the buffer solution. The horizontal support 20 and the outer walls
are made of a non-electrically conducting material, e.g., glass or
plexiglass.
In order to maintain the homogeneity of the electric field, the depth and
temperature uniformity of the buffer solution should be controlled, and
the gel matrix G should be immersed entirely within a uniform layer of the
buffer solution, not recessed into a well. For example, it is believed
that a 1% depth variation leads to a localized 1% error in the magnitude
of the electric field; the direction of the electric field is also
affected, but in a more complex way, which is believed to depend upon the
depth variation gradients. The buffer solution should be cooled, in order
to maintain a uniform temperature, by recirculating through cooling means
(not shown) conventional in electrophoresis. Precise temperature control
is important because the conductivity of a buffer solution can vary by as
much as 3% per degree C. variation.
Preferably, the support 20 is levelled, and the buffer solution covers the
gel matrix G at a depth of about 1 cm and is maintained at a uniform
temperature of 5.degree. to 15.degree. C. Maintaining the buffer solution
at such a temperature also suppresses denaturing of the gel matrix G and
of the sample.
The apparatus 10 comprises twenty-four driving electrodes D.sub.n (wherein
n represents integers from 1 to 24 inclusive) in a circular array having a
radius R.sub.D and twenty-four sensing electrodes S.sub.n (wherein n again
represents integers from 1 to 24 inclusive) in a circular array having a
radius R.sub.s and being disposed fixedly and concentrically within the
circular array of driving electrodes. Each of the driving and sensing
electrodes is preferably mounted to make optimal electrical contact with
the liquid buffer in the electroporesis chamber of the apparatus 10, by
penetrating the buffer solution vertically over its full depth. Each of
the driving electrodes may be advantageously made of a platinum foil
covering a cylindrical core, or of coiled or folded lengths of platinum
wire longer than the depth of the buffer solution, or of a porous
conductive ceramic, e.g., a porous titanium suboxide available
commercially from Ebonex Technologies, Inc. under the "Ebonex" trademark.
Each of the sensing electrodes may be advantageously made of a platinum
wire or other conducting media. In the preferred embodiment, the circular
array of the sensing electrodes has a radius R.sub.s of about 16.5 cm, the
circular array of driving electrodes has a radius R.sub.D of about 18 cm,
and the geometric center of the 20 cm.times.20 cm square occupied by the
gel matrix G coincides with the common center of the circular arrays. A
reference radius R.sub.R is indicated on FIG. 1 for a purpose to be later
mentioned. The driving electrodes are arrayed in equally spaced relation
to one another, i.e., at 15.degree. intervals. The sensing electrodes are
arrayed similarly, at like intervals, so that each of the sensing
electrodes is positioned radially inward of one of the driving electrodes.
Thus, the driving electrode D.sub.1 and the sensing electrodes S.sub.1 are
located on a common radius, i.e., the reference radius R.sub.R the driving
electrode D.sub.2 and the sensing electrode S.sub.2 are located on a
common radius, and so on. The driving and sensing electrodes are arrayed,
on the common radii, in electrode pairs. Thus, the driving electrode
D.sub.1 and the sensing electrode S.sub.1 constitute a first electrode
pair, the driving electrode D.sub.2 and the sensing electrode S.sub.2
constitute a second electrode pair, and so on.
Since the electrophoresis chamber of the apparatus 10 contains a bounded,
uniformly electrically conducting layer of the buffer solution, the
potential distribution necessary for a homogeneous field within the
boundary of such layer is the same as would exist if such layer were
embedded in a much larger region exhibiting a homogeneous field. Field
conditions within a region isolated as if removed from a larger whole can
be maintained if boundary conditions of the region can be matched. The
apparatus 10 enables the boundary conditions of the circular array of
sensing electrodes to be matched as if the circular array of sensing
electrodes were within a much larger region exhibiting a uniform field.
At each electrode pair constituted by a driving electrode and a sensing
electrode, an electrical potential is applied directly to the driving
electrode, e.g., between the driving electrode and a point of common
potential usually referred to as ground, of a suitable magnitude to cause
an electrical potential of a desired magnitude to occur at the sensing
electrode of such electrode pair. Moreover, the electrical potential is
sensed at the sensing electrode of such pair. The potential required at
each sensing electrode varies depending on the desired field. In turn, the
potential at the corresponding driving electrode differs from that of its
sensing electrode by varying amounts, as determined by parameters such as
buffer solution formulation, electrical current, electrode polarity, and
spacing.
To establish a uniform field of a desired amplitude E, e.g., between 2
volts per cm and 10 volts per cm, in a desired direction from the center
of the circular arrays, as shown by an arrow representing a vector in FIG.
1, the required voltage at any given one of the sensing electrodes is
given by the equation:
V.sub.s =ER.sub.s cos .theta.
where E is the desired amplitude, as mentioned above, R.sub.S is the radius
of the circular array of sensing electrodes, and .theta. is the angle
between a radius through the given one of the sensing electrodes and a
radius coinciding with the vector arrow, as shown in FIG. 1. The
electrical potential at a central region C of the gel matrix G may be
arbitrarily held at zero potential, and, therefore, the central region C
of the gel matrix G may be said to be grounded. Typically, for an
electrical field of a magnitude between 2 volts per cm and 10 volts per
cm, voltages of up to .+-.165 V are sensed at the sensing electrodes.
Typically, the electrical potentials applied at the driving electrodes are
up to 25 V greater, more commonly up to 15 V greater, than the electrical
potentials sensed at the sensing electrodes. Since the direction of the
electric field can be quite arbitrary, it is not necessary to align the
direction of the electric field with the angular position of any electrode
or electrode pair of the circular arrays.
As a representative example, for an electrical field having a magnitude of
5 V per cm and being directed at an angle of 25.degree. clockwise (as
shown) from the reference radius R.sub.R, the required voltages at the
sensing electrodes are given below in Table C, wherein the number in the
column at the extreme left is the subscript of the designation of the
sensing electrode, and wherein all data are approximate.
TABLE C
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Electrode .THETA.* cos.theta.
Vs(=ER.sub.s cos.theta.)
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1 335.degree. 0.906 74.7 V
2 350.degree. 0.985 81.3 V
3 5.degree. 0.996 82.2 V
4 20.degree. 0.940 77.5 V
5 35.degree. 0.819 67.6 V
6 50.degree. 0.643 53.0 V
7 65.degree. 0.423 34.9 V
8 80.degree. 0.174 14.3 V
9 95.degree. -0.087
-7.2 V
10 110.degree. -0.342
-28.2 V
11 125.degree. -0.574
-47.3 V
12 140.degree. -0.766
-63.2 V
13 155.degree. -0.906
-74.8 V
14 170.degree. -0.985
-82.2 V
15 185.degree. -0.996
-82.2 V
16 200.degree. -0.940
-77.5 V
17 215.degree. -0.819
-67.6 V
18 230.degree. -0.643
-53.0 V
19 245.degree. -0.423
-34.9 V
20 260.degree. -0.714
-14.3 V
21 275.degree. 0.087 7.2 V
22 290.degree. 0.342 28.2 V
23 305.degree. 0.574 47.3 V
24 320.degree. 0.766 63.2 V
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*Measurements are made clockwise from vector E.
A constant positive or negative voltage may be arbitrarily added to all
voltages given in Table C above. The corresponding absolute values of the
electrical potentials applied to the driving electrodes exceed the
absolute values of the electrical potentials sensed at the sensing
electrodes in the respective electrode pairs, typically by up to 25 V,
more commonly up to 15 V, but depend in complex ways on such factors as
the composition of the buffer solution, the polarities, currents drawn by
the respective electrodes, and surface areas of the respective electrodes.
Advantageously, the feedback approach taken by this invention allows such
factors as are mentioned in the preceding paragraph to be largely ignored.
Prudence still dictates, however, that driving electrodes having larger
surface areas are preferred over thin wires. Larger surface areas diminish
the potential drops in the immediate vicinity of the driving electrodes
and lessen the density of gas bubbles formed by electrolysis, thus
reducing excess power dissipation outside the electrophoresis region.
Reduced power loss minimizes the cooling requirements. For example,
driving electrodes with an exposed surface area of about 0.25 cm.sup.2 are
preferred.
Herein, all references to a driving electrode are intended to refer to an
electrode which, at a given potential applied thereto, provides or
receives an electromotive force tending to cause molecules to migrate
toward or away from an electrode of a different potential, e.g., zero
potential or a potential of an opposite polarity. For example, if it were
grounded, the center C of the gel matrix G would define a ground potential
of zero value vis-a-vis each of the driving electrodes to which electrical
potentials are applied. Irrespective of whether the center C of the gel
matrix G is at ground potential, electrical potentials can be thus applied
to a selected two or more, and as many as all of the driving electrodes,
to establish an electric field in any arbitrary direction. Therefore,
herein, all references to selected ones of the driving electrodes are
intended to refer to as few as two of the driving electrodes, and to as
many as all of the driving electrodes. Similarly, herein, all references
to selected ones of the sensing electrodes are intended to refer to as few
as two of the sensing electrodes and as many as all of the sensing
electrodes.
A high gain, error-sensing, differential amplifier arranged for negative
feedback is provided for each of the twenty-four electrode pairs. As
representative examples shown in FIG. 1, such an amplifier 40 is shown, as
provided for the electrode pair constituted by the driving electrode
D.sub.23 and the sensing electrode S.sub.23, and another such amplifier
40' is shown, as provided for the electrode pair constituted by the
driving electrode D.sub.19 and the sensing electrode S.sub.19. Each
amplifier comprises a high gain, integrated circuit operational amplifier
with a discrete component output stage employing conventional high voltage
MOS transistors, to deliver output voltages of up to .+-.250 V, and
capable of switching speeds on the order of 100 .mu.sec. The output stage
of each amplifier is connected directly to the driving electrode of the
electrode pair for which such amplifier is provided. As a representative
example shown in FIG. 1, the output stage of the amplifier 40 is connected
directly to the driving electrode D.sub.23.
Each amplifier compares an electrical potential corresponding to the
electrical potential sensed at the sensing electrode of the electrode pair
for which such amplifier is provided, to a program voltage supplied to
such amplifier for such electrode pair in a manner to be later described.
The electrical potential sensed at the sensing electrode of such electrode
pair may be and, as shown, is scaled down by a factor of 100, before being
fed back to such amplifier, by a voltage divider comprising a resistor
connected to the sensing electrode of such electrode pair and a resistor
connected between the resistor and ground. As a representative example
shown in FIG. 1, a voltage divider scaling down the electrical potential
sensed at the sensing electrode S.sub.23 comprises a resistor 42 having a
resistance of 1 Meg ohms connected to the sensing electrode S.sub.23 and a
resistor 44 having a resistance of 10k ohms connected between the resistor
42 and ground. Such resistor values produce negligible current flow
through the sensing electrodes and allow them to sense the buffer solution
potential at their locations without errors due to electrochemical
electrode activity. Similarly, as shown, a voltage divider scaling down
the electrical potential sensed at the sensing electrode S.sub.19
comprises a resistor 42' connected to the sensing electrode S.sub.19 and a
resistor 44'. Each amplifier applies an electrical potential having a
value equal to a program voltage plus or minus a feedback voltage, which
is provided by such amplifier in response to the scaled down, sensed
voltage fed back to such amplifier, the applied potential being applied to
the driving electrode of the electrode pair for which such amplifier is
provided. Consequently, an electrical potential of the desired magnitude
tends to occur at the sensing electrode of such electrode pair.
As shown in FIG. 2, the program voltage for each electrode pair is
provided, as a digital signal, from a data source 50, which generates or
retrieves such program voltage. Further, the data source 50 supplies the
digital signal through a digital-to-analog converter 52, which converts
the digital signal to an analog voltage, through a buffer 54, which
buffers the analog voltage, and through a multiplexer 60, to the amplifier
provided for such electrode pair. The multiplexer 60, which has a timed
cycle that may be controlled by the data source 50 through a lead 62, or
independently, has one input stage, which is connected to the
digital-to-analog converter 52 through the buffer 54, and twenty-four
output stages, one for each electrode pair. The output stage of the
multiplexer 60 for each electrode pair includes an output terminal, which
is connected to the amplifier for such electrode pair, and a capacitor for
storing a voltage, i.e., the program voltage supplied by the multiplexer
60 to the amplifier for such electrode pair. Thus, as shown in FIG. 2, the
multiplexer 60 has an output stage including such an output terminal
V.sub.1 and such a capacitor C.sub.1 for the electrode pair constituted by
the driving electrode D.sub.1 and the sensing electrode S.sub.1, and such
an output terminal V.sub.2 and such a capacitor C.sub.2 for the electrode
pair constituted by the driving electrode D.sub.2 and the sensing
electrode S.sub.2. Also as shown in FIG. 2, the multiplexer 60 has such an
output terminal V.sub.23 and such a capacitor C.sub.23 for the electrode
pair constituted by the driving electrode D.sub.23 and the sensing
electrode S.sub.23 and such an output terminal V.sub.24 and such a
capacitor C.sub.24 for the electrode pair constituted by the driving
electrode D.sub.24 and the sensing electrode S.sub.24. The output terminal
V.sub.23 is shown also in FIG. 1.
In place of the multiplexer 60, a plural number of multiplexers may be
used, each addressing a different group of the electrode pairs, e.g., six
multiplexers, each addressing a different group of four electrode pairs,
whereupon, in place of the digital-to-analog converter 52 and the buffer
54, as a subcombination, a like number of subcombinations of
digital-to-analog converters and buffers may be used, each subcombination
comprising a digital-to-analog converter supplying an analog signal
through a buffer, and each subcombination addressing a different one of
the multiplexers. Although shown with a mechanical symbol, the multiplexer
60 preferably is electronic.
The data source 50 may be any suitable source of digital signals
representing the programming voltages for the driving electrodes. Thus,
the data source 50 may comprise a computer, microprocessor, or
programmable controller, which generates such signals pursuant to a stored
program. The stored program may reside in an erasable, programmable,
read-only memory (EPROM) which is used as a "look up" table, and from
which data are retrieved, as digital signals, pursuant to external control
through external leads 56, 58, shown in FIG. 2. External control of such a
memory may be provided by an array of switches. In a simpler form, the
data source 50 may comprise a resistor-divider string, which is adapted to
be selectively switched. Details of the data source 50 are outside the
scope of this invention.
The amplifiers noted above and the voltage dividers associated therewith
apply electrical potentials, as provided by the digital-to-analog
converter 52 through the buffer 54 and the multiplexer 60, to selected
ones of the driving electrodes. As the voltage dividers sense electrical
potentials at selected ones of the sensing electrodes, the amplifiers
adjust the applied potentials so as to tend to maintain the sensed
potentials at selected values. The driving electrodes to which electrical
potentials are applied and the sensing electrodes at which electrical
potentials are sensed are selected from the same electrode pairs.
Moreover, the capacitors noted above store the provided potentials, and
the multiplexer 60 restores the potentials stored by such capacitors to
the provided potentials periodically, e.g., every 200 to 600 microseconds.
By appropriate selection and timed control of the programming voltages to
be thus applied to selected ones of the driving electrodes, the apparatus
10 may be readily adapted to practice a wide range of different techniques
of gel electrophoresis, as exemplified by but not limited to such
techniques as are discussed in the Cantor et al. and Carle et al. patents
noted above and in the Biotechnology and Science references noted above.
A uniform field that is constant as a function of time can be thus used to
separate smaller molecules of a sample. Forcing the molecules of a sample
to change direction of movement at an angle, however, causes the smaller
molecules to proceed faster than the larger molecules.
To practice gel electrophoresis according to a technique similar to the
technique discussed in the Cantor et al. patent noted above, the electric
field discussed in the above example as having a magnitude of 5 V per cm
and as being directed at an angle of 25.degree. (clockwise as shown) from
the reference radius R.sub.R can be initially applied for a specified
period of time, e.g., from 1 sec. to 120 sec., or more, whereupon the
electric field can be angularly rotated 90.degree. (i.e., new electrical
potentials are applied to the driving electrodes so that the voltage
sensed initially at the sensing electrode S.sub.1, is sensed at the
sensing electrode S.sub.1, so that the voltage sensed initially at the
sensing electrode S.sub.2 is sensed at the sensing electrode S.sub.8, and
so on) and there applied for the same period of time, or for a different
period of time, whereupon the electric field can be similarly rotated back
(to where the electric field was applied initially) and there applied for
the same period of time, or for a different period of time, and so on for
a desired number of such alternations of the electric field.
To practice gel electrophoresis according to a technique similar to the
technique disclosed in the Carle et al. patent noted above, the electric
field discussed above as having a magnitude of 5 V per cm and as being
directed at an angle of 25.degree. (clockwise as shown) from the reference
radius R.sub.R can be initially applied for a specified period of time,
e.g., about 120 sec., whereupon the electric field can be angularly
rotated 180.degree. (i.e., new electrical potentials are applied so that
the voltage sensed initially at the sensing electrode S.sub.1, is sensed
at the sensing electrode S.sub.13, so that the voltage sensed initially at
the sensing voltage S.sub.2 is sensed at the sensing electrode S.sub.14,
and so on) and there applied at a selected, lower magnitude, e.g., 5/3 V
per cm, for the same period of time, or 5 V per cm for a shorter (e.g., 40
sec.) period of time, whereupon the electric field can be similarly
rotated back (to where the electric field was applied initially) and there
applied at its original magnitude of 5 V per cm for the same period of
time, and so on for a desired number of such alternations of the electric
field.
To practice gel electrophoresis according to a technique similar to the
technique discussed in the Biotechnology reference noted above, an
electric field can be analogously switched back and forth 120.degree., at
timed intervals.
The electric field also may be so controlled as to undergo many small step
changes in amplitude and/or direction in a rapid sequence, so as to
approximate a smooth evolution of angle and amplitude, over time. As will
be apparent from the earlier discussion, any angle of electric field can
be programmed, and within the .+-.250 Volt constraints of amplifiers 40,
any amplitude between 0 and 10 V/cm can be programmed. The field can also
be controlled to cause a funneling effect tending to narrow a band of
molecules of a sample being electrophoresed as the band progresses along
the gel matrix G.
FIG. 3 illustrates the kind of uniform field that can be generated in
matrix G, as actually measured at isopotentials using the arrangement of
FIG. 1. In this experiment, vector E was directed towards electrodes
D.sub.22 and S.sub.22. The buffer was 1 cm deep (0.5X TBE formulation), at
about 28.degree. C., with a voltage gradient of about 5 V/cm and power
dissipation in the buffer of about 60 W.
Various modifications may be made in the apparatus 10 without departing
from the scope and spirit of this invention.
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