HU205993B - Appartus for separating macromolecular dns embedded in gel - Google Patents

Abstract

Appts for the electrophoretic sepn of high-molecule DNA in a gel has an electrophoretic chamber with four gps of electrodes which are joined through a switching device to a power source. These four gps of electrodes are designed identical and are mounted along the sides of an equilateral quadrangle which surrounds a block of gel. The electrodes in each gp are positioned equidistantly. - In an alternative version, the four gps (2,3,4,5) in the electrophoretic chamber (1) are arranged in a rhombic shape around a block of gel (6) with the samples (7) of mixts of DNA molecules. The angle ALPHA is e.g. 60 deg. Each gp has additional electrodes (11) along extensions of the major angle with a length of h/tan ALPHA where (h) is the distance between opposite sides of the rhumb. This produces the molecule tracks (12).

Description

The present invention relates to an apparatus for the electrophoresis of gel-embedded high-molecular-weight DNA which is advantageously used in the analysis and separation of biopolymers.

Separation of the various biopolymer mixtures, particularly those consisting of DNA molecules having more than 5 x ^{10 4} pairs of nucleic bases, is accomplished by pulsed electrophoresis, whereby a cross-linked electric field is formed in the gel containing the high molecular weight DNA. Carle GP Frank M. and Olson Μ. V .: "Electrophoretic Separation of High-Molecular DNAs by Periodic Alternating Fields". 236 of Science 1986, pp. 65-68. A device for separating mixtures of DNA molecules, which is provided with a rectangular electrophoresis chamber, which contains two horizontally opposed line electrodes, with a gel unit disposed between these line electrodes. The supply voltage to the electrodes is transmitted through a switching device where a one-way voltage is applied to the gel unit for up to 3/4 of the period and a reverse polarity voltage to the electrodes for 1/4 of the period, and this switching cycle is repeated several times. Thus, in this apparatus, the direction of movement of the DNA molecules is periodically changed by 180 °, but this change of direction, where the mass of the DNA molecules to be separated is very different, is not considered optimal, since it is impossible to separate DNA fractions which Containing more than ⁶ nucleobase pairs. In addition, moving the molecules back and forth increases the detachment process and reduces the overall performance and productivity of the equipment.

Also in the abovementioned issue of Science, No. 234, 1986, pages 1582-1585. describes a device that separates high-molecular-weight DNA from each other by a connected homogeneous electric field. This article was written by G. Ghy, D. Vollart, and R. W. Davis. In this device, there are six groups of point electrodes perpendicular to the surface of the gel unit, which form an electrically enclosed hexagon through the same resistance, and the interior of the gel unit is located substantially therein. With constant polarity, the voltage of the power supply is connected via a switching device to each pair of opposing electrodes. The third pair of electrode groups is passive and serves only to provide a homogeneous electric field between the electrodes in operation. In this arrangement, the movement of the DNA molecules is such that each change of voltage causes a change in direction of 120 If the size of the electrophoresis chamber is the same, the size of the gel unit may be smaller, of course, samples that can be inserted into the gel unit at the same time, and ultimately the performance of the entire device will be reduced. In addition, the power supply of this device should be twice that of the previous device, since the current flowing through the electrodes should not be less than the current flowing through the buffer solution which covers the gel unit.

SU 4,473,452. also discloses an apparatus for separating large molecule DNAs, which practically allows a mixture of DNA molecules having 5x10 ^{4 to} 9x10 ⁶ nucleobase pairs to be separated. In this apparatus, the electrophoresis chamber is rectangular, and the electrode groups are formed by having two adjacent points on each other, and on the other side, point electrodes which are arranged very close to the opposite electrode groups. The apparatus operates by applying a supply voltage at a predetermined interval through a switching device to one of the electrode groups, which is a negative voltage, but at the same time a positive voltage is applied to the opposite point electrode. This is repeated for the second electrode group and the second point electrode. In this way, the gel unit inside the electrophoresis chamber receives a crossing electric space, forcing the DNA molecules to move along a relatively complex path, which varies in direction from 90 to 180 °, depending on how close it is to the point electrode. .

The curvature of the magnetic field created in this way results in curved paths of the individual DNA fractions within the gel unit, but this makes the determination of the molecular weight extremely difficult and requires a special correction program to be applied to the data processing equipment. too. Another disadvantage of this apparatus is that the number of samples to be simultaneously removed is not very large, since the outer paths are formed from the gel unit through the buffer solution.

SUMMARY OF THE INVENTION It is an object of the present invention to provide an apparatus for electrophoretic separation of high-molecular-weight DNAs embedded in a gel, whereby the electrodes are located in the electrophoresis chamber so as to create a homogeneous electric field within the chamber itself it is also necessary to move the molecules along a linear, parallel path, so that the separated fractions are well separated.

Accordingly, the present invention provides an apparatus for electrophoresis separation of gel-embedded high-molecular-weight DNA comprising an electrophoresis chamber comprising four groups of electrodes disposed therein, which are connected to a switching device on a power supply.

The essence of the device according to the invention is

That the four electrode groups are identical and are arranged on one side of an equilateral rectangle around its gel unit, and that the electrodes in the electrode groups arranged along each side are spaced apart and that each electrode group is at the output of the switchgear connected.

Also preferred is an embodiment of the apparatus according to the invention wherein the four electrode groups are arranged on one side of a regular rhombus and the sides of the rhombus forming a greater inner angle are extended by 1 to h / tga, where h is the opposite sides of the rhombus. and α is the smaller internal angle, and additional extensions are provided on these extensions.

The apparatus of the invention allows the mixture of DNA molecules to be separated by simultaneously utilizing the entire surface of the gel unit, thereby increasing the performance of the apparatus and accurately determining the molecular weight of the separated DNA fractions, even if the separation efficiency is increased. the mass of the molecules varies over a wide range, the latter can be achieved by varying the angle α, and it is also ensured that the result of the separation is automatically evaluated with a gel scanner.

The apparatus according to the invention will now be described in more detail by means of exemplary embodiments in the accompanying drawings. The

Figure 1 is a schematic diagram of an embodiment of the invention, a

Figure 2 shows the pathways of the DNA molecules belonging to the apparatus of Figure 1, a

Fig. 3 shows a further exemplary embodiment of the apparatus according to the invention, while a

Figure 4 illustrates, in the exemplary embodiment of Figure 3, the pathway of DNA molecules in the gel.

1-4. Figures 1 and 2 therefore show two different embodiments of the invention, wherein Figure 1 shows an embodiment comprising a rectangular electrophoresis chamber (1) having four identical electrode groups (2, 3, 4 and 5), and each of the groups of electrodes (2, 3, 4 and 5) is spaced apart and has the same number of point electrodes electrically coupled through diodes, the latter not shown in the figure. The electrode groups (2, 3, 4 and 5) are placed on a support formed as an upper lid of the electrophoresis chamber (1) and not formed in the figure so as to form the sides of a rectangle and this rectangle at the bottom of the electrophoresis chamber surrounding a gel unit (6) provided with patterns along the rim (7).

The gel unit (6) is decapitated with a buffer solution which allows current to flow between the respective electrode groups (2, 3, 4 and 5) by switching on the respective voltage groups. The electrode groups (2 and 4) are connected to one output of the switching device (8), while the electrode groups (3 and 5) are connected to the other output of the switching unit (8) and connected to the output (9) of the switching unit (8).

Figure 2 shows the path (10) through which the DNA molecules of sample (7) move when the electrode groups (2,3, 4 and 5) are formed as in Figure 1.

If the path (10) of the DNA molecules exiting the sample (7) or the angle of each path segment is chosen to be different from 90 °, the electrode groups (2, 3, 4 and 5) should be arranged as shown in Figure 3. , namely along the sides of a rhombus which has a smaller inner angle α = 60 °, for example, but can be preselected. The sides which have a greater internal angle are extended and the additional electrodes (11) are arranged on these elongated portions (1) having a length (1) of 1 = h / tga, where h is the distance between the two opposite sides of the rhombus and α is the smaller inside angle.

Figure 4 shows the path 12 of the DNA molecules leaving the samples 7 located in the gel unit 6, where it is also shown that each path segment of the path 12 has an angle α.

The operation of the apparatus according to the invention is as follows.

The gel unit (6), as shown in Figure 1, together with the applied samples (7), which is a mixture of DNA molecules, is placed in the electrophoresis chamber (1) so that it is parallel to the wall of the chamber and then to the buffer solution. which buffer is in contact with all four (2,3,4 and 5) electrode groups. Subsequently, the power supply (9) is turned on, whereby a positive polarity is applied to the electrode group (4) via the switching device (8) and a negative polarity to the electrode group (2). The DNA molecules will be located along the electrical line between the electrode groups (2 and 4) and begin to move toward the electrode group (4), which is at a positive potential. After a predetermined period of time, the switching device (8) switches off the electrode groups (2 and 4) and energizes the electrode group (3 and 5) so that the electrode group (3) receives a positive polarity signal and (5). electrode group with negative polarity. The DNA molecules are rearranged along the altered electric power lines, and depending on the size of the molecules, the rearrangement time will also vary, and then these DNA molecules will move toward the electrode group (3). Thereafter, after a certain time, which is the same as the previous switch-on time, the switching device (8) switches off the electrode groups (3 and 5) and reconnects the voltage to the electrode groups (2 and 4) with the same polarity as as it was first connected to electrode group (2 and 4). After that time

After 205 993 Β, the voltage is applied again to the electrode groups (3 and 5), but now with the polarity opposite to the previous switching cycle. Thus, a full cycle consists essentially of four bars, with a first polarity voltage applied to electrode groups 2 and 4, and a second cycle having a given polarity voltage applied to electrode groups 3 and 5; the voltage is applied to the electrode groups 2 and 4 again, with the same polarity as in the first phase, while the voltage is applied to the electrode groups 3 and 5 from the fourth phase, but this is the opposite of the voltage applied in the second phase. .

This cycle is repeated until the lightest DNA fractions reach the end of the gel unit (6) in relation to the sample (7). Figure 2 shows the path (10) formed with the apparatus shown in Figure 1. Figure 2 shows that during the operation of the switching device B, the movement direction of the molecules changes by 90 ° with each stroke. By changing the direction of the molecules depending on the weight of the molecule, it is possible to efficiently separate molecules containing different numbers of nucleic base pairs and thus, without difficulty, to separate DNA molecules containing 5x10 ⁴ -2x10 ⁶ nucleic base pairs.

As an example in Figure 3! In the embodiment, the electrode groups (2, 3, 4 and 5) are arranged along a rhombic side and are also energized on the electrode groups (2 and 4,) and (3 and 5) at equal intervals, such that a voltage having a positive polarity to the electrode group, a voltage having a negative polarity to the electrode group (2), followed by a positive voltage to the electrode group (3) and a negative polarity to the electrode group (4). This switching cycle is repeated until the isolation is complete.

In addition to the electrode groups (2, 3, 4, and 5), in this embodiment, additional electrodes (11) are provided which, at each end of the diamond, have a larger internal angle, so that the gel unit (6) is sufficiently homogeneous in each case. extensions. Figure 4 shows the path (12) of the embodiment shown in Figure 3 along which the DNA molecules move.

In the case of separating a mixture of DNA molecules where the number of nucleic base pairs in each molecule can be very low, e.g. Can be 4x10 ⁴ or very high, e.g. 5X10 ⁶ , the angle between the sides of the diamond must be varied in order to achieve optimum separation.

Thus, by adjusting that angle, it is possible to separate a mixture of DNA molecules for virtually any size of molecule. The simplest embodiment is shown in Fig. 1, and in practice this is best achieved by placing the electrode groups (2 and 4) on the baseplate of the electrophoresis chamber (1) and the electrode groups (3 and 5) on the electrophoresis (1). chamber cover or a suitable support member for controlling the movement of the lid, if necessary.

The same structure of the electrode groups (2, 3, 4 and 5) and the fact that they are arranged along the sides of an equilateral rectangle and the point electrodes are equally spaced from each other, ensure that homogeneous electric fields are provided towards the gel unit. which are optionally intersected at a predetermined angle, thus providing ideal conditions for all DNA molecules moving on the surface to move.

The device according to the invention can be used primarily in biotechnology, genetics, biochemistry, biophysics for both medical and agricultural purposes.

Claims (2)

Apparatus for separating gel-embedded high-molecular-weight DNA by electrophoresis, comprising an electrophoresis chamber (1) comprising four electrode groups ((2, 3, 4, 5)) connected thereto by means of a switching device (8) connected to a power supply (9). characterized in that the four electrode groups (2, 3,4,5) are formed in the same way and are arranged on one side of an equilateral rectangle around the gel unit (6) and in the electrode groups (2, 3) arranged along each side , 4, 5) are electrically spaced and each electrode group (2, 3, 4, 5) is connected to each output of the switching device (8). Apparatus according to claim 1, characterized in that the four electrode groups (2, 3, 4, 5) are arranged on one side of a regular rhombus and the sides of the rhombus forming a greater inner angle They are extended by a length of 1-h / tga, where h is the distance α from the opposite sides of the rhombus, a smaller internal angle, and on which additional auxiliary electrodes (11) are placed.