Title of Invention

"CIRCUIT TO CLAMP THE VOLTAGES IN THE ELECTRODES OF THE CHEF SYSTEM CHAMBER OF PULSED FIELD GEL ELECTROPHORESIS"

Abstract The invention relates to a circuit for imposing voltages on the electrodes of trays used in the CHEF pulsed field electrophoresis system. Said circuit is formed by two identical application circuits that are connected to a power source by means of an alternator in such a way that only one of the two circuits receives electrical energy each time. Each application circuit consists of several resistors and diodes, which are series connected to form a voltage divider. Voltage repeaters are connected to the nodes formed at the union of two resistors. Each repeater is connected to a pair of electrodes that are to be polarized to the same potential. Diodes are introduced to correct minor errors in the voltage pattern applied to the electrodes. The circuit can maintain the potential in each electrode when conductivity variations occur during electrophoresis. Each application circuit generates a homogenous electrical field having the same value and different direction in a tray used in the CHEF pulsed field electrophoresis system. Trays with different number and arrangement of and separation between electrodes can be polarized with said circuit.
Full Text CIRCUIT TO CLAMP THE VOLTAGES IN THE ELECTRODES OF THE CHEF SYSTEM CHAMBERS OF PULSED FIELD GEL ELECTROPHORESIS.
FIELD OF INVENTION
The present invention is related to electric equipments used in electrophoresis, specifically to the generation of contour clamped electric potentials for generating homogeneous fields that alternate its direction of application.
BACKGROUND OF THE INVENTION AND PRIOR ART
The electrophoresis
The electrophoresis is a technique that separates molecules by their differential migration inside an electric field. The molecules can be placed in a gel and are sieved when the electric field that compels them to migrate is applied. The negative charged molecules migrate toward the anode and the positive charged ones make it toward the cathode. This way the molecules are separated in bands inside the gel, according to their size. For the generation of the electric field, two parallel electrodes connected to a direct current power supply are usually disposed.
DMA molecules are negatively charged when they are dissolved in buffer at neuter or alkaline pH. When the electric field is applied, DNA molecules are elongated and their charge-mass ratio becomes independent of its molecular size. The above mentioned reasons, together to the fact that the DNA molecules migrate through the pores of the gel in a similar way to the movement of a snake, that is to say by means of a reptation mechanism, it determines that the molecules bigger than 20000 base pairs cannot be separated in electrophoresis at constant electric field, even when they are subjected to molecular sieving.
Pulsed field gel electrophoresis
Pulsed field gel electrophoresis (PFGE) was created by Schwartz and Cantor in 1984 (Cell, 37, pp 67-75,1984; US Patent 4,473,452 of September 25th, 1984) and it
increased the range of the DMA molecules that could be separated in electrophoresis. The authors obtained that the large intact DNA molecules, larger than 20000 base pairs, were separated in band patterns inside agarose gels by means of the application of electric pulses of selected duration that periodically alternated their direction of application regarding the separation gel. The changes in the direction of the electric field application cause reorientation of the DNA molecules migration, while the duration of this reorientation depends on the molecular size. The resulting band patterns have been denominated 'electrophoretic patterns', 'molecular kariotypes', 'electrophoretic kariotypes1, etc.
This way, any system of pulsed field gel electrophoresis consists of:
1. The electrophoresis chamber with their accessories
2. The appropriate electronics to alternate the electric fields with the desired intensity and
pulse duration.
3. The method for polarizing the electrodes.
The electric fields that were generated in the initial PFGE equipments, such as those described by Schwartz and Cantor (Cell, 37, pp 67-75, 1984; U.S. Patent No. 4,473,452 of September 25th, 1984) and others as those described by Carle and Olson (Carle G.F., Olson M.V. Nucleic. Acid Res., 12, pp 5647-5664, 1984) they didn't offer homogeneous values of intensity of the electric field along the gel, so the trajectory and the migration velocity of the DNA molecules in this gels depended on the position that they occupied inside the gel.
Generation of homogeneous electric fields in PFGE
In theory, two infinite electrodes placed in parallel and separated to certain distance generate a homogeneous electric field. But the design of such electrophoresis chamber is impracticable. To approach to the obtaining of an electric field of homogeneous intensity along the separation gel using finite electrodes, Chu (Chu G., Vollrath D., Davis R.W. Science, 234, pp 1582-1985,1986) proposed the following:
1. A regular polygon is selected (square, rectangle or hexagon) as a closed contour upon
whose sides an array of electrodes will be placed to generate inside the polygon an
electric field of homogeneous intensity values.
2. The 'X' axis (y = 0) of an imaginary Cartesian plane is made coincide with one of the
sides of the regular polygon.
3. A 0 volts potential is applied to those electrodes placed at y = 0
4. A Vo1 volts potential is applied to the electrodes placed at the opposed side of the
regular polygon that are at a distance y = A from the 'X' axis.
5. In the remaining electrodes, located on the other sides of the regular polygon and at a
distance 'yi' from the 'X' axis, a potential 'V(yj)1 is applied, where V(yi) = V0•Vi/A.
6. This way, the potential generated inside the regular polygon is similar to the one that
would be generated by two infinite and parallel electrodes separated a distance 'A' one to
each other.

7. If the polarity of the electrodes placed at two pairs of opposed sides is electronically
exchanged an angle among the lines of force of the resulting electric fields will be form.
This angle is denominated in PFGE Yeorientation angle1.
8. The reorientation angle obtained when the polarity among the electrodes of two different
pairs of sides is electronically exchanged will be 90° in the square and 60° or 120° in the
hexagon.
The configuration of electrodes in a hexagonal array has been the one used in the current systems of PFGE. That system was denominated as Contour Clamped Homogeneous Electric Field or CHEF and it was introduced by Chu in 1986 (Chu G. Science 234, pp 1582-1585, December 16th, 1986).
One of the deficiencies of the current CHEF system is that the closed contour of electrodes is limited to the regular polygons previously described.
Methods to clamp the voltages in the electrodes of the CHEF system and to obtain electric fields of homogeneous intensity inside the gel.
Three methods have been mainly proposed, they were gaining in complexity and electronic components:
1. A simple voltage divider (Chu G., Vollrath D., Davis R.W. Science, 234, pp 1582-
1585,1986).
2. The voltage divider associated to transistor pairs in push-pull configuration (Maule J.,
Green D. K. Anal. Biochem. 191, pp 390-395,1990).
5 3. The use of operational amplifiers to control better the voltages imposed in each electrode of the CHEF system (Clark S.M., Lai E., Birren B.W., Hood L. Science 241, pp 1203-1205,1988).
The simple voltage divider in the PFGE systems.
10
One of the methods to clamp the potential values in the CHEF electrodes is to use a network of resistors that are connected in series. This network forms a voltage divider among the values zero and Vo'. We will name nodes to the place of union between two serial resistors of the voltage divider and at each node is connected an electrode of the
15 hexagon.
The electrodes placed in y = 0 and y = A, that means that in two opposed sides of the hexagon, are connected to the potentials '0' and 'Vo1 respectively. There are two other groups of electrodes, the electrodes of two consecutive sides of the hexagon form each
20 group. Each one of those electrodes is connected to a node of the voltage divider that defines the potential that should be applied in this electrode. The potential value that is imposed is calculated like it was mentioned in the previous paragraph. For that reason, the two electrodes that are in two different sides of the hexagon, but they are at the same distance 'yi' from the more electronegative electrodes (y = 0), they should be at the same
25 voltage value given by V(yi) = Vo•y,/A.
To achieve the change in the application direction of the electric field, which is indispensable in PFGE, the potential difference is applied to other two different groups of electrodes. This is carried out with relays and diodes which connect the electrodes that 30 should be polarized with zero volt and 'V0' to the outputs of the power supply through the system for the electric fields switching.
However, the use of series of resistors to clamp the voltages has an inconvenience. When the network of resistors and the buffer solution came into contact, the latter behaves as a new resistor connected in parallel with the resistors of the network. The currents that are injected from the resistors toward the electrodes and vice versa change the value of the potential in each electrode and affect the electric field homogeneity. The voltage change depends on the amount of current that is injected to or it is extracted from the buffer solution which in turn depends on changes in the concentration, temperature, volume and pH of the buffer solution, among others. This changes affect randomly the conductivity of the buffer and therefore the magnitude of the electric current that is exchanged with the pure resistive circuit (Maule J., Green D. K. Anal. Biochem. 191, pp 390-395, 1990). These random changes in the voltage patterns are uncontrollable and therefore, they affect in a different way the results and the reproducibility of the electrophoretic patterns that are obtained in each experiment.
Those changes can be reduced if the current passing trough the series of resistors is much bigger than the one which circulates by the buffer (Maule, J. and Green, D. K. Anal. Biochem. 191, pp 390-395, 1990). However, that solution has the disadvantage that it causes an unnecessary waste of electric power and forces to use components (especially the resistors) of higher power that are more expensive.
The voltage divider associated to pairs of transistors in push-pull configuration.
To solve the problems outlined for the resistive voltage divider the use of current sources made of semiconductor elements was proposed (Maule J., Green D. K. Anal. Biochem. 191, pp 390-395, 1990). Those current sources separate each electrode from their corresponding node in the series of resistors of the divider. Between each node and their corresponding electrode a pair of transistors is placed in the configuration called 'push-pull'. They inject to and extract electric current from each electrode, then repeating in the electrodes the voltage from the node of the divider without has been affected by the changes of conductivity of the buffer solution. The mentioned system is able to polarize the electrodes appropriately in the two directions of application of the electric field in PFGE. However, it has some limitations:
1. he pairs of electrode that should be polarized with same voltage value, V(yi) = V0«yi/A,
gets its potential from different nodes, therefore, the equality of voltages in all required
electrode pairs is not always achieved.
2. The electrodes nearer the more electropositive electrodes receive the electric current
from the NPN type transistor of the push-pull they are connected to. While the electrodes
nearer the more electronegative electrodes sink electric current toward the PNP type
transistor of the push-pull they are connected to. The fact that transistors of different
polarities are active at the same time introduces errors in the pattern of voltages.
3. The resistors that set the potential pattern in one of the two direction of application of
the field are the same ones that make it in the other direction. For that reason, it is not
possible to make independent adjustment of the potentials pattern in each field. Any
variation wanted to be introduced in one of the two directions necessarily affects the other
direction.
4. The circuit has as many transistor pairs in push-pull configuration as electrodes has the
CHEF chamber. The transistor pairs in push-pull configuration are connected in parallel.
When some of the transistors get broken it is difficult to determine the damaged pair.

5. In the transistors pairs configured in push-pull one of the transistors it is always active
while the other one is inactive. This means that in all moment half of the transistors are
inactive. However, those transistors cannot be eliminated from the circuit, because when
the electric field is applied in the other direction, some pairs change the active transistor.
Therefore, the voltage divider network connected to transistors pairs in push-pull
configuration is inefficient, since the total number of transistors inactive in each field is
excessive the same as the total quantity of transistors.
6. All the transistor pairs are connected to the power supply without any element that limits
the current. The failure of a single transistor causes short circuit between the positive and
negative outputs of the power supply. So, it can be concluded that the circuit is not safe.
The use of operational amplifiers to control better the voltages imposed in each electrode of the CHEF system.
Other more complex systems use operational amplifiers to carry out an individual control of the potential imposed in each electrode of the hexagonal array of the chamber (US Patent 5,084,157). Those systems are able to vary the angle between the two directions
of application of the electric field but by means of increasing the electronic complexity of the systems, as much in their construction as their operation. Additionally, the elements that carry out the control of the potentials cannot be properly isolated from the power elements. It is necessary the digital conversion what implies new complexities and the cost of the equipment increases.
On the other hand, Riveron and cols. (Cuban patent, application No.: 2000-306)
demonstrated that for obtaining straight an reproducible band patterns in PFGE is necessary to guarantee electric fields of homogeneous intensity inside the electrophoresis chamber. They determined that the homogeneity of the applied electric field can be only obtained if, besides having a system for the proper polarization of the electrodes in the closed contour, the electric resistance homogeneity of the buffer and the gel is guaranteed. If the electric resistance is desc
ribed as
R = (1 / a) • (d / A)
where: (a) it is the conductivity of the electrolyte, (d) it is the separation among the electrodes of opposed polarities and (A) it is the cross section area to the flow of the electric current.
It is deduced that for the electric resistance was homogeneous in the whole chamber it is necessary that turbulent flow does not exist in the buffer surface neither deformations nor meniscuses in the gel that alter or modify the cross section area to the flow of the electric current.
Therefore, if PFGE systems, still those that has very complex electronic circuits to polarize the electrodes, do not assure the homogeneity of the buffer electric resistance, they cannot guarantee straight band patterns and reproducible experiments. This situation becomes more critic with small chambers.
DETAILED DESCRIPTION OF THE INVENTION
To explain appropriately the circuit proposed in this invention it is necessary to define a reference system.
The reference system
We will consider a closed contour of electrodes (from the PFGE chambers of the CHEF system) to the group of several electrodes placed on the 'm' sides of a regular polygon, where 'm1 is even. In our reference system (figure 1) Li side is defined arbitrarily and it is placed on the 'X' axis of a Cartesian plane. The opposed side (denominated as L(m/2>+i is located at a distance 'A' from the 'X1 axis. This way, the remaining sides of the regular polygon are symmetrically distributed to both sides of the LI and L(m/2)+i sides. Those sides of the regular polygon that are to the left of the sides LI and L(m/2)+i will be denominated as sides 'C' and those that are to the right like sides 'D1.
On each side are placed 'k' electrodes, where 'k' it is a natural number between 1 and 10. There will be 'k1 electrodes placed on the LI side that is on the 'X' axis with ordinate y0 = 0. There will also be 'k' electrodes located on the side L(m/2)+1 at a distance 'A' from the 'X1 axis with ordinate yn+1 = A.
All the electrodes located on the sides 'C' and 'D' will be denominated as EIC, E2c,.., Enc and EID, E20,--, EnD, where 'n' is equal to 'k•(m-2)/2'. The denomination of the electrodes is made in the following order for the sides 'C1 and 'D1, starting from the Li side until arriving to the L(m/2)+i side. Electrodes, E1C, E2c,-., Enc, and electrodes E1D, E2D,.., Eno- The two Eic and EID electrodes are placed at the same distance y, of the 'X1 axis, where T is a natural number between 1 and 'n'. Each one of those ([E1c-E1D], [E2c-E2D],", [Enc-Eno]) will be denominated Pi electrode pairs.
The circuit of this invention to achieve homogeneous electric fields inside a closed contour of electrodes
To explain the circuit, first it will be referred how to achieve a homogeneous field using the previously described reference system. It is assumed that all the electrodes are energized during the electrophoresis with a given voltage among 0 and 'V0' volts that it is obtained from a power supply as follows.
1. To those 'k1 electrodes placed on the LI side are applied 0 volts.
2. To those 'k' electrodes placed on the opposite L(m/2)+1 side are applied 'V0' volts.
owever, when applying a potential difference among the electrodes placed on the LI and L(m/2)+i sides an electric field is set inside the PFGE chamber whose intensity is not homogeneous in all the regions of the chamber. This means that in the Pi electrode pairs a voltage not proportional to the distance yi appears. Therefore, in the remaining electrodes there should be imposed voltage values that homogenize the electric field inside the whole electrophoresis chamber. Then to the Pj electrode pairs is applied a voltage Vj = V0•yi/A.
This way, the electrodes of the closed contour are polarized for generating a homogeneous electric field in a determined direction of application. A similar reasoning is applicable to achieve a homogeneous electric field of same magnitude, but whose lines of force have another direction. It is only necessary to define another side of the regular polygon as LI.In this invention is proposed that both electric fields of the PFGE can be energized with two identical clamping circuits. Those clamping circuits are connected between the positive and negative outputs of the existent circuit to alternate the electric fields or alternator in a way that only one of the clamping circuits receives electric energy at the same time.
Each one of those circuits imposes in the electrodes the voltages that generate an electric field of homogeneous intensity in one of the application direction inside the chambers of the CHEF system. The circuit connections in one of the two directions where the electric field will be applied are carried out in the following way:
I. One of the negative outputs of the alternator is connected through diodes to all 'k'
electrodes of the LI side (side placed on the 'X' axis). The positive output
corresponding to this negative output of the alternator is connected through diodes
to those 'k1 electrodes located on the L(m/2>+i side, that is to the electrodes located
on the side placed at the distance 'A' from the 'X' axis.
II. The connection through diodes between the negative output of the alternator and
the 'k1 electrodes of the LI side is carried out in the following way:
a) each electrode of the LI side of the regular polygon is connected to the anode of
a diode,
b) the cathodes of those diodes, one for each electrode, are all connected together
and to the anode of a second diode,
c) the cathode of that second diode is connected to the negative output of the
alternator.
III. The connection through diodes between the positive output of the alternator and
those 'k' electrodes of the L(m/2)+1 side is carried out in the following way:
a) each electrode of the L(m/2)+i side of the regular polygon is connected to the
cathode of a diode,
b) the anodes of those diodes, one for each electrode, are all connected together
and to the cathode of a second diode,
c) the anode of that second diode is connected to the positive output of the
alternator.

IV. The ends of a voltage divider formed by 'n+1' resistors Ri and a variable quantity of
diodes are also connected to the negative and positive outputs of the alternator.
This way, the total voltage (Vo - 0) is divided in values proportional to the 'yi'
distance that separates each Pj electrode pairs (Eic-Eio) from the 'X1 axis.
V. Each NJ node formed between RI and RM resistors of the voltage divider is
connected to the input of a voltage repeater. Each voltage repeater's output is
connected to one of the Pj electrode pairs. The voltage repeaters have two
functions, one is to repeat in their output (the PI electrode pairs) the voltage at their
input that comes from the Ni node. The other function is to maintain this voltage
constant against the conductivity changes of the buffer during the PFGE.
The voltage repeaters are of two types:
1. When the voltage repeater is connected between a NJ node of the divider and a PI electrode pair where T is a natural number among '[(n/2)+1]' and 'n', this voltage repeater is formed by the following circuit elements:
a NPN type transistor whose base is connected to the NJ node of the voltage divider, its collector to the positive output of the alternator and its emitter to the
anodes of two diodes whose respective cathodes are connected to the electrodes of the already mentioned Pj electrode pair. 2. When the voltage repeater is connected between a N( node of the divider and a Pj
electrode pair where V is natural number between 1 and 'n/2', this voltage repeater is
formed by the following circuit elements:
a PNP type transistor whose base is connected to the Nj node of the voltage divider, its collector to the negative output of the alternator and its emitter to the cathodes of two diodes whose respective anodes are connected to the electrodes of the already mentioned Pi electrode pair.
The value of each Rj resister is chosen to guarantee that the voltage at each Pj electrode pair was proportional to the distance that separates them from the electrodes located on the Li side of the regular polygon.
The other circuit is identical to this, but it is connected to the Ej electrodes in a different way. According to the desired angle between the lines of force of the electric fields that are going to be generated, another side of the polygon is redefined as LI side and the reference system is rotated the necessary angle in order to the new LI side was at the 'X' axis. The 'C1 and 'D' sides, the Ej electrodes and the Pj electrode pairs are redefined starting from the L1 side.
From the previous reasoning it is deduced that Pj electrode pairs from each circuits are different. That is why diodes are required and they cannot be directly connected to the transistor emitters of the voltage repeaters. The diodes allow to join both electrodes from the electrode pairs guaranteeing them to have the same potential when that voltage repeater is active, because the field was applied in that direction. When the electric field is set in the other direction the diodes that join the old Pj electrode pair remain connected in series but with opposite polarities. It is guaranteed this way that the circuit branches between the old electrode pairs that join electrodes at different potential in this moment, have at least a diode inversely polarized. They have a very high electric resistance and the electrodes of that branch become electrically isolated.
The influence of the buffer changes of conductivity upon the potential of each NI node of the divider is decreased by sourcing current into or sinking current away from the electrode until its voltage equals its node voltage. Transistors in "emitter follower" configuration are used as current sources. The electrode pairs located nearer the negative output should always extract current from the buffer because their potential tend to be higher than the one at its corresponding reference node. For this reason a PNP type transistors is used which sink this current toward the negative output of the power supply. The electrode pairs located nearer the positive output should always source current into the buffer because their potential tend to be lower than the one at its corresponding node. For it a NPN type transistor is used which gets current from the positive output. In that way the potential of the reference nodes it is not considerably affected.
Variations of the buffer temperature, concentration, pH, height, etc occur during the electrophoresis. These disturbances tend to alter the voltage pattern at the electrodes. The necessary current to counteract these effects is also managed by the transistors.
Each electrode pair potential resembles to its reference potential but it differs in a certain value. The difference is caused by the transistor base to emitter and diode voltage drops associated to the electrode pairs. This voltage drop is characteristic of the PN junctions of the silicon semiconductor elements and it is approximately similar to 0,7 volt.
The change in the transistors and diodes polarity that occurs in the center of the divider introduces an error in the voltage pattern. This error can be compensated by inserting diodes in series with the central resister of the voltage divider. This way the potential of the reference nodes is modified in the same magnitude but in opposite sense to the effect of the voltage drops in the transistors and diodes of each pair.
Finally it is necessary to insert a diode in series with the diodes that polarize the electrodes located on the L1 and L(m\2)+1 sides and are connected to the negative and positive outputs of the alternator. This is necessary to homogenize the number of voltage drops (caused by forwardly polarized PN junctions) in the path between the outputs of the alternator and each one of the electrodes. These paths have two PN junctions for the electrodes located on the 'C1 and 'D' sides.
Therefore, the circuit proposed in this invention consists of two identical parts that are connected to the power supply through another appropriate electronic circuit to alternate the electric fields with the wanted intensity and pulse duration.
EXAMPLES
The following examples are illustrative of the circuit that it is described but they not limited in any measure the reach of this patent.
Example 1. Circuit to polarize the electrodes of a hexagonal chamber of 18 electrodes. Array of electrodes.
In the figure 2 a group of electrodes 101 to 118 placed on a regular hexagon at three electrodes per each side is shown. In one of the directions of the electric field application (denominated A) the electrodes 101, 102 and 103 (denominated A+ group) are polarized with the maximum potential, close to the potential of the power supply's positive output. The electrodes 110, 111 and 112 (denominated A- group) are polarized with the minimum potential, close to 0 volts. The rest of the electrodes is organized in pairs (table I), each electrode from the same pair will be polarized with the same voltage, the one proportional to the distance from each pair to the electrodes of the A- group.
In the other direction of the electric field application (denominated B) the electrodes 113, 114 and 115 (denominated B+ group) are polarized with the maximum potential, close to the potential of the power supply's positive output. The electrodes 104, 105 and 106 (denominated B- group) are polarized with the minimum potential, close to 0 volt. The rest of the electrodes is organized in pairs (table I), each electrode from the same pair will be polarized with the same voltage, the one proportional to the distance from each pair to the electrodes of the B- group.
In this particular electrode array the 'dist' distance between two consecutive electrodes is the same one. However the distance among the 118-104 electrode pair and the electrodes
of the A+ group is the half, that is 'dist/2'. The same occurs with the 113-109,116-112 and 103-107 pairs with regard to the electrodes of the A-, B+ and B- groups respectively.
Table I. Electrode pairs and transistor type of the voltage repeater they are connected to.(Table Removed)
The lines show the number of the electrodes that should be polarized at the same voltage to generate a homogeneous electric field in the two A and B application directions in a chamber with an electrode disposition similar to one in figure 1.
Seven resistors 201 and 202 connected in series are necessary to generate the reference potentials in this CHEF chamber with three electrodes per side (figure 3). The resistors 201 are of the same value, the resistors 202 have the half of this value. The ladder of resistors 201 and 202 is connected between the positive (+) and negative (-) outputs of a power supply through switches or the alternator. This voltage divider generates the reference potentials that appear in the nodes 203 and 204.
The voltage repeaters 205 and 206 take the voltage from the reference nodes 203 and 204 to appropriately polarize the electrodes in A and B directions.
Voltage repeaters 205 and 206 are detailed shown in figure 4. The base of the NPN type transistor 301 is connected to the node 203. The collector of the transistor 301 are connected to the positive output (+) through switches. The emitter of the transistor 301 are
connected to the anodes of two diodes 302, which in turn are connected by the cathode to the electrodes whose potential corresponds to that particular node 203.
The base of the PNP type transistor 303 is connected to the node 204. The collector of the transistor 303 is connected to the negative output (-) through switches. The emitter of the transistor 303 are connected to the cathodes of two diodes 304 which in turn are connected by the anode to the electrodes whose potential corresponds to that particular node 204.
In table I is pointed out the electrodes polarized with NPN (voltage repeater 204) and PNP (voltage repeater 205) type transistors.
The electrodes of A+ and B+ groups (figure 5) are connected to the cathode of diodes 401 connected together by their anodes. The anode of the diodes 401 is connected to the cathode of another diode 402 which in turn is connected by its anode to the positive output of the power supply through switches.
The electrodes of A- and B- groups are connected to the anode of diodes 403 connected together by their cathode. The cathode of the diodes 403 is connected to the anode of another diode 404 which in turn is connected by its cathode to the positive output of the power supply through switches. The diodes 402 and 404 guarantee that the branches which polarize the electrodes of the A+, A-, B+ and B- groups have the same voltage drops provoked by the PN junctions that the rest of the electrodes of the array.
The diodes 207 (figure 3) compensate the errors caused by the polarity change of the transistors and diodes in the voltage pattern.
fn the table II the theoretical voltages and the ones measured in the electrodes of a CHEF chamber are presented. The electrodes are placed on the sides of a hexagon like that of the figure 2. The separation among the opposed sides is 11,6 cm. The chamber was filled with 225 ml of buffer solution TBE 0,5x (TBE 1x: Tris 89 mM, Boric acid 89 mM, EDTA 2 rnM, pH 8,4) at 20 °C. The voltages were generated with a circuit similar to the one presented. The resistors used were of 470,0 ohm, two resistors were placed in parallel to
achieve half of the value in the resistors 202. MJE340 and MJE350 transistors and 1N4007 diodes were used. The energy was obtained from a 'Macrodrive I' power supply adjusted to a 120,0 volt constant voltage among the positive (+) and negative (-) outputs in the A and B directions.
Table II. Theoretical values and the ones generated by the presented circuit in the electrodes of a CHEF chamber with 18 electrodes located on the sides of a hexagon. (Table Removed)
Electrode numbers, according figure 2, are in bold typeface.
The theoretical voltage was calculated considering a typical voltage drop of 0,7 volt in each PN junction (in the diodes and in the base to emitter of the transistors) of the silicon semiconductors elements. For the calculation transistors were considered like ideal elements with zero base current.
Example 2. Circuit to polarize the punctual electrodes of a hexagonal chamber with 36 electrodes not evenly distributed.
In the figure 6 a group of punctual electrodes 501 to 536 placed upon a regular hexagon at six electrodes per each side is shown. In one of the direction of the electric field application (denominated A) the electrodes 501 to 506 (denominated A+ group) are polarized with the maximum potential, close to the potential of the power supply's positive output. The electrodes 519 to 524 (denominated A- group) are polarized with the minimum potential, close to 0 volts. The rest of the electrodes are organized in pairs (table III), each electrode
from the same pair will be polarized with the same voltage, the one proportional to the distance from each pair to the electrodes of the A- group.
In the other direction of the electric field application (denominated B) the electrodes 525 to 530 (denominated B+ group) are polarized with the maximum potential, close to the potential of the power supply's positive output. The electrodes 507 to 512 (denominated B-group) are polarized with the minimum potential, near to 0 volt. The rest of the electrodes are organized in pairs (table III), each electrode from the same pair will be polarized with the same voltage, the one proportional to the distance from each pair to the electrodes of the B- group.
In this case the distances between two consecutive electrodes are not the same. For example the distance between the electrode 501 and the 502 are different to the distance between the electrode 502 and the electrode 503.
To generate the reference potentials in this CHEF chamber with six electrodes per side thirteen resistors 601, 602 and 603 connected in series are needed (figure 7). The resistance values of resistors 601, 602 and 603 should be chosen in order to the potential at each electrode be proportional to the distance between each electrode and the electrodes of the A- and B- groups for each one of the A and B directions, respectively. In this case the resistors 601 were of 348 ohm the resistors 602 are of 470 ohm and the resistors 603 are of 235 ohm. The chain of resistors 601, 602 and 603 are connected to the positive (+) and negative (-) potentials of a power supply through switches. This voltage divider generates the reference potentials that appear in the nodes 604 and 605. The voltage repeaters 606 and 607 take the voltage from the reference nodes 604 and 605 to properly polarize the electrodes in A and B directions. The voltage repeaters 606 and 607 are identical to the repeaters 205 and 206 (figure 3). Diodes 608 are inserted in series with the resistors to correct the errors in the voltage pattern caused by the polarity change of the active transistors inside the voltage repeaters 606 and 607.
Diodes are used in a similar circuit to the one presented in the example 1 to polarize the electrodes of the A+, A-, B+ and B- groups (figure 8). In this case six diodes 701 and 703 are used to polarize the electrodes of the groups A+ and A- because this particular
electrode array presents six diodes per each side. The function of the diodes 702 and 704 is similar to those of the example 1, to guarantee that the potential of all the electrodes were affected by the same number of voltage drops.
Table III. Theoretical values and the one generated by the presented circuit in the electrodes of a CHEF chamber with 36 electrodes placed on the sides of a hexagon. (Table Removed)
Lines show the number and the voltage of the electrodes that should be polarized to the same potential to generate a homogeneous electric field in the two A and B application direction. The first column shows the theoretical potential that corresponds to each electrode pair. The electrode numbers, according to figure 6, appears in bold typeface. The theoretical voltage was calculated with the same considerations as in the example 1.
In table III the theoretical voltages and the one measured in the electrodes of a CHEF chamber are presented. The electrodes are placed on the sides of a hexagon as the one of the figure 6. The separation among the opposed sides is 11,6 cm. The chamber was filled with 225 ml of buffer solution TBE 0,5x (TBE 1x: Tris 89 mM, Boric acid 89 mM, EDTA 2 mM, pH 8,4) to 20 °C. The voltages were generated with a circuit similar to the
one presented. The energy was obtained from a 'Macrodrive I' power supply whose exit was adjusted to a constant voltage of 120,2 volt between the positive (+) and negative (-) outputs in A and B directions.
The examples that have been presented are illustrative of the present invention and they do not constitute limitations to their scope. Chambers of different size and forms, number and distribution of electrodes can be polarized with similar circuits to the one shown. This is made by varying only the number of circuit elements: transistors, diodes and resistors as well as the resistance value of these last ones and they would be under the scope of the present invention.
BRIEF DESCRIPTION OF DRAWINGS:
Figure 1. Reference system used to describe the distribution of the electrodes in the PFGE chambers of the CHEF system that can be polarized by the circuit of the present invention. The electrodes are placed on a 'm' sides regular polygon, where 'm' it is an even number between 4 and 50. 'k1 electrodes are placed on each side of the polygon, where 'k' it is a natural number between 1 and 10. One of the sides of the polygon (denominated l_i) is made coincide with the 'X1 axis of a Cartesian plane. The opposed side of the polygon (denominated L(m/2)+1 is located at a distance y = A from the 'X' axis. To the left of the LI and L(m/2)+1 sides are the 'C' sides and to the right the 'D1 sides.
Figure 2. Outline of the distribution of the 18 electrodes of a hexagonal CHEF chamber. Symbols A+ and A- indicate the electrodes connected to the positive and negative outputs of a power supply (through an alternator circuit) to establish an electric field in the direction. Symbols B+ and B- indicate the electrodes connected to positive and negative outputs of a power supply (through an alternator circuit) to establish an electric field in the B direction.
Figure 3. Voltage divider formed by diodes and resistors which is connected to the power supply outputs (through an alternator circuit). This circuit generates the voltages that polarize part of the electrodes of a hexagonal CHEF chamber with 18 electrodes. Voltage repeaters are connected to the nodes formed between the resistors.
Figure 4. Voltage repeaters. The base of the transistors is connected to the nodes of the voltage divider and the emitter are connected to two diodes which in turn are connected to a pair of electrodes that should be polarized to the same potential. In the superior part is presented a voltage repeater with a NPN transistor whose collector is connected to the positive output of a power supply (through an alternator circuit). In the inferior part is shown a voltage repeater with a PNP transistor whose collector is connected to the negative output of a power supply (through an alternator circuit).
Figure 5. To the left is shown the circuit that polarizes the electrodes of the A+ or B+ groups in a hexagonal model CHEF chamber with 18 electrodes. The anode of the diode located in the superior part is connected to the positive output of a power supply through switches. To the right is shown the circuit that polarizes the electrodes of the A- or B-groups in a hexagonal model CHEF chamber with 18 electrodes. The cathode of the diode located in the inferior part is connected to the negative output of a power supply through switches.
Figure 6. Outline of the distribution of the 36 electrodes of a hexagonal CHEF chamber. Symbols A+ and A- indicate the electrodes connected to the positive and negative outputs of a power supply (through an alternator circuit) to establish an electric field in the direction. Symbols B+ and B- indicate the electrodes connected to the positive and negative outputs of a power supply (through an alternator circuit) to establish an electric field in the B direction.
Figure 7. Voltage divider formed by diodes and resistors which is connected to the power supply outputs (through an alternator circuit). This circuit generates the voltages that polarize part of the electrodes of a hexagonal CHEF chamber with 36 electrodes. Voltage repeaters are connected to the nodes formed between the resistors
Figure 8. On the top is shown the circuit that polarizes the electrodes of the A+ or B+ groups in a hexagonal model CHEF chamber with 36 electrodes. The anode of the diode located in the superior part of the circuit is connected to the positive output of a power supply through switches. On the bottom is shown the circuit that polarizes the electrodes

of the A- or B- groups in a hexagonal model CHEF chamber with 36 electrodes. The cathode of the diode located in the inferior part of the circuit is connected to the negative output of a power supply through switches.
ADVANTAGES OF THE PROPOSED SOLUTIONS.
1) The electrodes of CHEF type chambers with different shape, size, number and
distribution of electrodes are correctly polarized.
2) The circuit is simpler than the previous voltage clamping systems, however the voltage
pattern generated in the electrodes is as accurate as or more exact than the one
generated by the previous systems.
3) Short circuit between the positive and negative outputs of the power supplies used is
nor possible.
4) Equal polarization of the electrode pairs located in the same theoretical equipotencial
line is achieved.
5) The circuits that generate the electric field in the two directions are independent.
6) The number of transistors used is at least three times less that in the previous systems.
7) The circuit is more economic and it is easier to repair and to maintenance.

We Claim
1. Circuit to clamp the voltages in the electrodes of the Contour Clamped Homogeneous Electric Field system chambers of Pulsed Field Gel Electrophoresis and to generate two identical strength but different orientation homogeneous electric fields, which requires a power supply with positive and negative outputs, another circuit for the electric field switching or alternator with two positive and two negative outputs and an electrophoresis chamber filled with buffer solution, chamber which posses an electrode array placed on the 'm' sides of a regular polygon at 'k' electrodes per side, where the L1 side is arbitrarily selected and it is made to coincide with the 'X' axis of a Cartesian plane, the side Lm/2+1 is parallel to the L1 side and the others sides, named 'C and 'D' sides, are located to the left and to the right of the sides L1 and Lm/2+1 respectively being the Eic-EiD electrodes equally spaced at each side of the polygon thus existing 'n' Eic-EiD electrode pairs P1 formed by electrodes located at the same distance from the L1 side, where 'n' is equal to k(m-2)/2 and 'i' is a natural number between 1 and 'n', circuit comprising that
• it is formed by two identical clamping circuits and each circuit set independently one of the two possible electric field orientations by setting potentials at electrodes of the regular polygon that homogenize the electric field, potentials in which zero volts correspond to the electrodes placed on the L1 side, L1 side that is another side of the regular polygon when the electric field is switched
• diodes are provided to connect the electrodes placed on the L1 and the Lm/2+1 sides to the negative and positive outputs of the power supply, respectively in each electric field orientation
• resistors and diodes are provided to generate voltage references inside each clamping circuit to polarize the EiC-EiD electrodes of each Pj electrode pair
• transistors are provided to make stable the potentials at the EiC-EiDelectrodes of each Pi electrode pair against the buffer solution conductivity changes but the two EiC-EiD electrodes from each Pi electrode pair are driven by one single transistor
• pairs of diodes are provided to connect the two EiC-EiDelectrodes of each Pj electrode pair to the same node of the voltage reference through said single transistors and to polarize these two EiC-EiD electrodes at the same potential in one electric field orientation but to electrically isolate said two EiC-EiD electrodes in the other field orientation
• diodes are provided to compensate errors in the voltage applied to the electrodes

diodes are provided in order to connect the two independent circuits to the same array of electrodes.
2. Circuit as claimed in claim 1 comprising that each clamping circuit is connected to the alternator in a way that only one of the clamping circuits receives electric energy at the same time.
3. Circuit as claimed in claim 1 comprising that each electrode located on the L1 side is connected to the anode of a diode, the cathodes of these diodes are joined together and connected to the anode of a second diode and the cathode of this second diode is connected to the negative output of the power supply.
4. Circuit as claimed in claim 1 comprising that each electrode located on the Lm/2+1 side is connected to the cathode of a diode, the anodes of these diodes are joined together and connected to the cathode of a second diode and the anode of this second diode is connected to the positive output of the power supply.
5. Circuit as claimed in claim 1 comprising that those resistors and diodes to generate voltage references form a voltage divider made of'n+1' resistors Ri and up to ten diodes connected across the positive and negative outputs of the power supply, where 'n' is equal to k(m-2)/2.
6. Circuit as claimed in claim 1 comprising that said single transistor is in emitter
follower configuration, its base is connected to one of the nodes of the voltage reference, its
emitter to the corresponding Pi electrode pair throughout a pair of diodes, transistor that is a
PNP type one with its collector connected to the negative output of the power supply when
'i' is a natural number between 1 and n/2 and is a NPN type one with its collector connected
to the positive output of the power supply when 'i' is a natural number between [(n/2)+l]
and n.
7. Circuit as claimed in claim 1 comprising that each pair of diodes are formed
by two diodes whose cathodes are connected to the emitter of the corresponding transistor
and whose anodes are connected to the two electrodes of the corresponding Pj electrode pair,
when T is a natural number between 1 and n/2.

8. Circuit as claimed in claim 1 comprising that each pair of diodes are formed by two diodes whose anodes are connected to the emitter of the corresponding transistor and whose cathodes are connected to the two electrodes of the corresponding Pi electrode pair, when 7 is a natural number between [(n/2)+l] and n.
9. Circuit as claimed in claims 1 and 5 comprising that said diodes to compensate errors in the voltage applied to the electrodes are the diodes which form a voltage divider to generate voltage references.
10. Circuit as claimed in claims 1 and 3 comprising that said diodes to compensate errors in the voltage applied to the electrodes are the second diode connected between the cathodes of the diodes connected to the electrodes of the L1 side and to the negative output of the power supply.
11. Circuit as claimed in claims 1 and 4 comprising that said diodes to compensate errors in the voltage applied to the electrodes are the second diode connected between the anodes of the diodes connected to the electrodes of the Lm/2+1 side and to the positive output of the power supply.
12. Circuit as claimed in claim 1 comprising that said diodes to connect the two independent circuits to the same array of electrodes are the diodes connected to the electrodes.
13. Circuit as claimed in claim 1 which requires an electrophoresis chamber which posses an electrode array placed on the 'm' sides of a regular polygon at 'k' electrodes per side comprising that 'm' is an even natural number between 4 and 50 and 'k' is a natural number between 1 and 10.


Documents:

2110-DELNP-2003-Abstract-(17-08-2009).pdf

2110-delnp-2003-abstract.pdf

2110-DELNP-2003-Claims-(17-08-2009).pdf

2110-delnp-2003-claims.pdf

2110-DELNP-2003-Correspondence-Others-(17-08-2009).pdf

2110-DELNP-2003-Correspondence-Others-(18-08-2009).pdf

2110-delnp-2003-correspondence-others.pdf

2110-delnp-2003-description (complete).pdf

2110-DELNP-2003-Drawings-(17-08-2009).pdf

2110-delnp-2003-drawings.pdf

2110-DELNP-2003-Form-1-(18-08-2009).pdf

2110-DELNP-2003-Form-1.pdf

2110-delnp-2003-form-13.pdf

2110-delnp-2003-form-18.pdf

2110-delnp-2003-form-2.pdf

2110-DELNP-2003-Form-3-(17-08-2009).pdf

2110-delnp-2003-form-3.pdf

2110-delnp-2003-form-5.pdf

2110-delnp-2003-pct-210.pdf

2110-delnp-2003-pct-304.pdf

2110-delnp-2003-pct-409.pdf

2110-delnp-2003-pct-416.pdf

2110-DELNP-2003-Petition-137-(18-08-2009).pdf


Patent Number 240540
Indian Patent Application Number 2110/DELNP/2003
PG Journal Number 22/2010
Publication Date 28-May-2010
Grant Date 17-May-2010
Date of Filing 05-Dec-2003
Name of Patentee CENTRO NACIONAL DE INVESTIGACIONES CIENTIFICAS (CNIC), a Cuban based Company, at Ave. 25 No. 15202 esq. a 158, Cubanacan, Playa, Ciudad de la Habana 12100, Cuba
Applicant Address AVE. 25 NO. 15202 ESQ. A 158, CUBANACAN, PLAYA, CIUDAD DE LA HABANA 12100, CUBA.
Inventors:
# Inventor's Name Inventor's Address
1 HERRERA ISIDRON, JOSE ALFREDO, a Cuban citizen CALLE 174 EDIF. BBE-2, APTO 14, RTO. FLORES, PLAYA, CIUDAD HABANA 12100, CUBA.
2 RIVERON ROJAS, ANA MARIA, a Cuban citizen CALLE 21 A NO. 20614 ENTRE 206A Y 214, ATABEY, PLAYA CIUDAD HABANA 12100, CUBA.
3 CANINO RAMOS, CARLOS ALBERTO, a Cuban citizen CALLE, 192 NO. 1991 ENTRE 199 Y 201, MARIA DEL CARMEN, BOYEROS, CIUDAD, HABANA 19200, CUBA.
4 LOPEZ CANOVAS, LILIA, a Cuban citizen CALLE 21 A NO. 20614 ENTRE 206A Y 214, ATABEY, PLAYA, CIUDAD HABANA 12100, CUBA.
5 NOA BLANCO, MARIA DOLORES, a Cuban citizen CALLE 10 NO. 125 ENTRE 1RA Y 5TA, PLAYA, CIUDAD HABANA 10400, CUBA.
PCT International Classification Number G01N 27/447
PCT International Application Number PCT/CU02/00004
PCT International Filing date 2002-06-07
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 2001-0133 2001-06-08 Cuba