Electrophoresis using contour-clamped electric fields

Abstract: A gel electrophoresis method and apparatus is described in which the shape and orientation of the electric field is controlled by contour clamping. Electrodes are arranged around a closed contour. Two or more electrodes, the driving electrodes, are clamped at a potential difference ₀ to establish the general orientation and strength of the electric field. The remaining electrodes are clamped to intermediate potentials to control the shape of the field established by the driving electrodes. The field is varied in accordance with the purpose of the electrophoresis, depending upon the size of the particles and the information to be determined concerning the particles.

Description: This invention is in the field of gel electrophoresis and pertains to a method and apparatus for controlling the migration of particles by manipulating the shape and orientation of the electric field in the gel. Gel electrophoresis is capable of separating macromolecules such as proteins and nucleic acids on the basis of size, charge, and/or conformation. Most applications involve the use of a single pair of electrodes to generate the electric field. Such a field is necessarily constrained to be uniform and oriented in a single direction, and as a result conventional techniques are limited in many respects. For example, conventional electrophoresis is not capable of resolving DNA fragments much larger than 50 kilobases. The recent introduction of new electrode configurations which generate electric fields in alternating orientations has allowed the separation of large DNA molecules up to 2 megabases in size. For example, US-A-4,473,452 describes an electrophoresis method of separating DNA molecules which involves the use of electric fields deliberately chosen to be nonuniform, rather than the uniform fields sought in previously known electrophoresis methods. The nonuniformity of the electric field, however, causes the DNA molecules to migrate with a mobility and a trajectory that depends on where in the gel the sample is loaded. Thus, comparison of multiple samples across the gel is difficult. For example, conventional electrophoresis may produce distortion, with molecules at the edge of the gel migrating more slowly than those in the center. Consequently electrophoresis is often done inefficiently at voltages lower than necessary to achieve adequate resolution. For example, conventional electrophoresis is limited in resolution by band broadening. Also, conventional electrophoresis is not capable of identifying macromolecules with different secondary structure. Two dimensional electrophoresis has been developed for this purpose, but this method is technically difficult because it involves manipulations of the gel. It is a principal object of the present invention to provide a method for manipulating the shape and orientation of electric fields for the purpose of controlling the migration of particles in a gel. It is another object of the present invention to provide for the high resolution separation of macromolecules, particularly DNA molecules up to or greater than 2 megabases in size, by gel electrophoresis whereby the pattern of separation is independent of position in the gel. It is a further object of this invention to provide a means for separating relatively small macromolecules, such as DNA of less than 50 kilobases, without distortion even at high voltage. Yet another object of the present invention is to minimize band broadening in the electrophoretic separation of macromolecules. A still further object of this invention is to provide a method for identifying secondary structure in macromolecules. These and other advantages were disclosed in our earlier EP-A-256737 which was predicated in part on the discovery that the limitations inherent in the existing electrophoretic separation techniques can be overcome by applying contour-clamped uniform or nonuniform electric fields, with the term "contour-clamped electric field" meaning that the electric field is generated by multiple electrodes arranged along a closed contour clamped to predetermined electric potentials, and with the term "uniform electric field" meaning an electric field having a uniform direction and magnitude within a closed contour. Depending upon the particular application for the method and apparatus, the gel was subjected to a uniform or nonuniform field by clamping a plurality of electrodes at predetermined potentials, where at least two electrodes clamped to different potentials serve as the driving electrodes and at least two more electrodes clamped to intermediate potentials serve to further define the nature of the electrical field. Thus controlled homogeneity or inhomogeneity could be applied, and the orientation of the field affected. In the present invention we go further. We provide for the sequential application of potential to pairs of electrodes at the corners of closed quadrilaterals, with the other electrodes clamped via resistors, with the result that the electric field in the gel can have any desired orientation. In the drawings Figure 1a is a schematic vector diagram structure and Figure 1b a schematic diagram of one embodiment of a circuit suitable for driving the electrodes of the structure; Figure 2a is a vector diagram and Figure 2b is a circuit model depicting the action at the corners of the contour of the embodiment of Figure 1a; Figure 3 is a schematic diagram depicting the use of solid state relays in a switching circuit; and Figure 4 is a schematic diagram depicting one embodiment of a circuit suitable for driving an electrode of a structure embodying this invention. DETAILED DESCRIPTION While the invention will be described in connection with certain embodiments, it is not intended to limit the invention to those particular embodiments. On the contrary, it is intended to cover all alternatives, modifications, and equivalent arrangements as may be included within the scope of the invention. For general background, please see EP-A-0256737 and the embodiments described therein. While those embodiments allow specific reorientation angles based upon the shape of the closed contour as defined by placement of the electrodes, in an embodiment of the present invention a structure is provided such that an arbitrary reorientation angle is capable of being provided within a single closed contour. Thus, in accordance with this embodiment, a single apparatus defining a single closed contour can be used to provide any desired reorientation angle. One embodiment of such a structure is depicted in Figure 1a, in which the reorientation angle is controlled by clamping the corner electrodes to appropriate potentials, for example using the circuit of Fig. 1b. As shown in Fig. 1b, there are two pairs of resistors R31,R32 (with resistance value R i ) and R33,R34 (with resistance value R j ). In one embodiment of Fig. 1b, R 1 - R 20 2K 1 / 2 W R 31 , R 33 10K 2W R 32 , R 34 up to 20K 2W  = 2tan -1 ( R j R i ); where  is the reorientation angle. Resistors R31 through R34 act as voltage dividers so that the corner electrodes 1, 6, 11, and 16 are clamped to potentials appropriate for a given electric field orientation. When electrodes 1 and 11 are the driving electrodes (A- and A+), electrodes 6 and 16 are clamped to potentials which fix the orientation angle of the electric field, as shown in Figs. 2a and 2b. For a field orientation at an angle 1 / 2  with respect to the vertical (i.e., a reorientation angle of  between alternating fields), V' should reflect the relative perpendicular distances from the driving electrodes. Those distances are x and y in Fig. 2a, x = d sin ( 1 2 ) y = d cos ( 1 2 ) V' = [ x x+y ] V o = [ 1 1+ y x ] V o = [ 1 1+ cos( 1 2 ) sin( 1 2 ) ] V o = [ 1 1+cot( 1 2 ) ] V o A similar calculation yields V'' = [ 1 1+tan( 1 2 ) ] V o The resistive voltage bridge in Fig. 2b produces potentials V' and V'' with the correct relationship: V' = [ R j R i +R j ] V o = [ 1 1+ R i R j ] V o V' = [ R i R i +R j ] V o = [ 1 1+ R j R i ] V o Thus, if the angle  is defined by the relationship, R i R j = cot ( 1 2 ) then R j R i = tan( 1 2 ) and substitution of Equations 8 and 9 into Equations 6 and 7 yields expressions for V' and V'' in the form of Equations 4 and 5. Thus, V' and V'' have assumed values which are appropriate to an electric field with orientation angle 1 / 2 , proving the validity of Equation 1. In another embodiment, as shown in the schematic diagram of Fig. 1b, a variable resistor (typically approximately 20K, 5 watts) is used as resistor R 35 in the B portion of the electrical circuitry in order to cause a lower potential to be used in the B direction than in the A direction, while using a single voltage source V 0 . This has the advantage of allowing the structure to be used to, for example, separate DNA on the basis of secondary structure In one embodiment, as shown in the schematic diagram of Fig. 3, solid state relays (such as the Crydon PVR3301) are used as the switching mechanisms, rather than the relay mentioned above. The use of a solid state relay permits more rapid switching, if desired, and provides increased reliability and less audible noise. In another embodiment, transistor buffer circuits are used between each electrode and the driving circuitry. As shown in FIG. 1b for example, buffer circuits B1 through B24 are used in order to clamp the electrodes to the appropriate potentials more precisely since the resistance paths between electrodes through the chemical buffer are electrically isolated from the resistive voltage divider, thereby allowing the resistive voltage divider to operate more precisely. Such buffer circuits have relatively high input impedance, so as not to load down the resistor voltage divider, and a relatively low output impedance, so as to provide adequate clamping of the electrodes. It is to be understood that buffer circuits such as buffer circuits B1 through B20 can be used to advantage in any embodiment of this invention. Similarly, it is to be understood that buffer circuits such as buffer circuits B21 through B24 can be used to advantage in any embodiment of this invention. In one embodiment of this invention, such buffers are formed as shown in the schematic diagram of Fig. 4 in which emitter follower pull-up and pull-down transistors T98 and T99 are used, respectively. The gain provided by the transistors allows the voltage dividers formed by the series of resistors to operate more precisely, without being loaded down by large amounts of current flowing through the resistors. In one such embodiment, the following component values are used: T98 2N3439 T99 2N5416 R98 150K 1/4 Watt R99 150K 1/4 Watt D98 1N4004 D99 1N4004

Claims: An apparatus for electrophoretic separation and detection of particles of a sample using an electric field, said apparatus comprising: a support for a gel medium in which one or more particles of a sample can be placed; a plurality of electrodes (1-20) arranged in a quadrilateral closed contour around said gel medium; and a clamping device simultaneously clamping all of the electrodes arranged in the closed contour at predetermined electrical potentials, thereby establishing an electric field in the gel medium of a desired magnitude and orientation for performing the electrophoretic particle separation and detection; characterised in that said electrodes includes one (1,6,11,16) at each corner of the quadrilateral and said clamping device further includes four resistors (R i , R j , R 31-34 ) connected to form an input voltage divider circuit, the resistors being connected respectively across adjacent corner electrodes, said input voltage divider circuit being responsive to potentials applied thereto to apply potentials to the four corner electrodes in pairs, whereby by selection of the resistances of the four resistors (R i , R j , R 31-34 ) in the input voltage divider circuit the electric field in the gel medium can have any orientation. An apparatus according to claim 1 wherein the resistances of at least some (R 33 , R 34 ) the resistors (R 31-34 ) are adjustable so as to adjust the reorientation angle . An apparatus according to claim 1 or claim 2, wherein the quadrilateral is a square or rectangle, and wherein one of the four resistors connected across a pair of adjacent corner electrodes and the resistor connected across the remaining two corner electrodes form a first pair of resistors (R i , R 31 , R 33 ) having substantially the same first resistance value, the remaining two of the four resistors forming a second pair of resistors (R j , R 32 , R 34 ) having substantially the same second resistance value. An apparatus according to claim 3, wherein the resistors (R 32 , R 34 ) in one of the two pairs of resistors are adjustable in tandem in resistance value. An apparatus according to claim 1, said clamping device further comprising: intraset resistors (R 1 ) connecting adjacent pairs of electrodes in each set; interset resistors each connecting a corner electrode (1,6,11,16) to a resistor in each set of intraset resistors; wherein the potentials applied by the input voltage divider circuit to two of the corner electrodes cause electrical potentials to be applied to the remaining electrodes of said plurality of electrodes; and said clamping device further including at least a buffer device (B 21 -B 24 ) connected to one or more electrodes for driving said one or more electrodes. An apparatus according to claim 5, wherein said at least one buffer device includes a plurality of buffer circuits (B 21 -B 24 ), each connected to a corresponding electrode for driving such electrode. An apparatus according to claim 6, wherein each of said buffer circuits includes one or more transistors (T98,T99). A method for electrophoretic separation and detection of particles of a sample using an electric field, said method comprising: providing a support for a gel medium in which one or more particles of a sample can be placed; providing a plurality of electrodes arranged in a quadrilateral closed contour around said gel medium; and simultaneously clamping in a clamping step all of the electrodes arranged in the closed contour at predetermined electrical potentials, thereby establishing an electric field in the gel medium of desired magnitudes and orientations for performing the electrophoretic particle separation and detection, characterized in that the clamping step clamps the electrodes (1,6,11,16) in pairs at respectively different potentials at different times such that by applying potentials to an input voltage divider circuit (R i , R j , R 31-34 ) connected respectively across adjacent corner electrodes, whereby by selection of resistances in the input voltage divider circuit the electric field in the gel medium has any desired orientation.

Inventors: CHU GILBERT, DAVIS RON, VOLLRATH DOUGLAS, CHU, GILBERT, DAVIS, RON, VOLLRATH, DOUGLAS
Applicants: UNIV LELAND STANFORD JUNIOR, THE BOARD OF TRUSTEES OF
Application year: 1989
Main classification: G01N27
IPC codes: B01D57, G01N27