RU81496U1 - DEVICE FOR EXTRACTION OF A GEL WITH DNA FRAGMENTS - Google Patents

Abstract

The invention relates to a technique for DNA sequencing, and in particular to a device for excising an agarose gel with the necessary DNA fragments, followed by its elution. To extract an agarose gel with DNA fragments, a device is used that represents the working part, consisting of one or more elements in the form of segments of a flat hollow tube with a sharp cutting outer edge, and connected to the working part by one or two handles. In the presence of several elements in the form of segments of a flat hollow tube, they are rigidly interconnected by lateral external surfaces. The connection of the working part of the device and the handles can be made rigid. The device for the extraction of the gel allows you to accurately and quickly extract the necessary DNA fragments with a minimum time of exposure to ultraviolet light. The device allows you to extract these fragments with minimal capture of agarose gel, which significantly improves further elution of DNA and provides a high-quality target product. 1 p., 2 cpf, 5 Fig.

Description

The invention relates to a technique for DNA sequencing, and in particular to a device for excising an agarose gel with the necessary DNA fragments, followed by its elution. The scope of the device is genetic engineering, forensics and medicine, scientific research using DNA isolation methods, polymerase chain reaction (PCR) and repeated polymerase chain reaction (rePCR).

There are various methods for extracting DNA from agarose gels, in which the efficiency of extracting fragments larger than 20 kb does not exceed 20% [Southern, 1975; Wu et al., 1976; Smith, 1980.

It is known to use a scalpel to cut a strip of gel in the method of electro-elution of DIC into dialysis tubes or into a bath cut in a gel [Maniatis T. Molecular Cloning / T. Maniatis, E. Fritsch, J. Sembrook. - Per. from English - M .: Mir, 1984. - S.169-171.] [1].

It is known to use a scalpel to extract a strip of gel under ultraviolet radiation after washing it in a method for recovering DNA from agarose gels [PCR Protocols: Methods in Molecular Biology / Ed, by John V. S. Bartlett; David Stirling - Second Edition. - Vol. 266. - Humana Press. - Pp. 71-74] [2].

With various methods of extracting DNA using agarose gels to obtain a satisfactory final result, in addition to meeting the necessary conditions inherent in a particular method, it is very important to quickly and accurately extract the gel with the necessary DNA fragments. Ultraviolet light destroys DNA and irradiates it even for such a short period (about 30 seconds) that can endanger the production of the target product.

In well-known sources, this problem is solved by using for shielding from destructive ultraviolet radiation by bringing under a strip of cardboard gel intended for excavation to protect from light.

The technical result of the proposed solution is to reduce the time of exposure to the removed sample of ultraviolet radiation, reduce the time of extraction of the gel with DNA fragments, improve the quality and reliability of the selected target product.

The technical result is achieved by the use of a device for extraction of agarose gel with DNA fragments, which is a working part, consisting of one or more elements in the form of segments of a flat hollow tube with a sharp cutting outer edge, and connected to the working part by one or two handles.

In the presence of several elements in the form of segments of a flat hollow tube, they are rigidly interconnected by lateral external surfaces.

The connection of the working part of the device and the handles can be made rigid.

A movable connection is a mechanism that rotates a pipe segment and the handle relative to each other in a plane parallel to the central axis of the pipe segment and passing through the connection line of the handle with the pipe segment. The mechanism for providing a movable connection can be made in the form of a flat hinge, etc. The specified mechanism allows you to set the working part relative to the handle in one plane in a convenient position for work. When turning through an angle of more than 90 °, the device is in the idle position.

The device can be made of any durable neutral material, for example, metal, plastic, etc.

The transverse size of one element of the working part is taken close to the size of the hole formed from the teeth of the comb, with which holes are formed for the target product (DNA).

The inventive device is illustrated by drawings.

Figure 1 - image of a device having a working part, consisting of one element in a perspective view.

Figure 2 - a device having a working part from a number of elements.

Figure 3 - position 1 - pockets in agarose gel from the comb; position 2 - pockets formed after cutting the proposed device of tablets from agarose gel with the desired DNA fragments;

Figure 4 - tablets from the gel with DNA fragments cut out by the proposed device.

5 is a gel tablet with a DNA fragment glowing under ultraviolet light.

The device comprises a main working part from an element having the form of a hollow flat tube 1, with a working cutting end 2 and the opposite end 3. The flat tube 1 is connected by one of its external surfaces to the handle 4. The connection of the flat tube 1 and handle 4 is made rigid (Fig. one).

The device may contain a working part made of several elements in the form of hollow flat tubes 1, interconnected by the sides to form a straight row of the required length. In this case, the handles 4 are located on the two end sides of the row.

To use the device, DNA is first extracted from samples according to the method of Doyle and Dickson (Sambrook et al., 1967 [3]; Doyle, Dickson, 1987 [4]), followed by amplification of a specific region of the gene by PCR (polymerase chain reaction) using universal for sample primers. Then, to separate, identify and purify DNA, electrophoresis of the obtained PCR product is carried out on an agarose gel. When separated in gel, you can track

DNA position, since DNA bands are stained with fluorescent or intercalating dye in DNA - ethidium bromide in low concentration. Looking at the gel in ultraviolet light, you can even notice 1 ng of DNA. For horizontal electrophoresis, a gel plate is prepared. To do this, pour a buffer mixture with a certain amount of agarose into a transparent plastic cuvette. A comb is inserted into the heated gel at a distance of 1 mm from the bottom of the cuvette, the teeth of which form holes 5 mm wide (Maniatis, 1984). After the gel has hardened and becomes elastic, the comb is removed. The required volume of PCR product (DNA) is introduced into the formed wells with the bottom lined with the gel and electrophoresis is carried out in a predetermined mode. After electrophoresis, the gel plate with DNA strips is transferred to glass and the desired DNA fragment is found in ultraviolet light.

At this stage, use the proposed device. The device, as if stamped, with a cutting end is immersed vertically in the gel in the region of the marked DNA fragment, then it is quickly removed and again immersed in the gel in the region of the next DNA fragment, etc. Before each new immersion in the gel, the proposed device is disinfected in a weak solution of formic acid or ethanol, or burned in a flame of an alcohol lamp. Thus, in a short time, you can cut the gel with the necessary DNA fragments from samples of different samples.

In the presence of a large number of samples of the same type, a device is used in the form of a series of elements with two handles (Figure 2), which significantly speeds up the process of cutting tablets with DNA fragments from the gel. When immersed vertically in a gel under ultraviolet light in an even shorter time than in the previous version, all samples with DNA fragments of the required length are simultaneously cut out, and the need for re-disinfection of the device is eliminated.

Since the working part of the device coincides in size with the width of the DNA strip (the size of the holes from the teeth of the comb), when cutting such

the method minimizes the capture of agarose. In addition, after removing the cut out tablets of the agarose gel with the necessary DNA fragment with tweezers, a lower agarose layer 0.5-1 mm thick can be removed with a scalpel (the agarose layer is equal in thickness to the distance between the bottom of the well and the cell). Next, the DNA localized in the tablet from a small amount of agarose gel is purified by acceptable methods, primers are added and sequenced. Since agarose contains impurities of sulfated polysaccharides that inhibit certain enzymes (ligases, polymerases, restrictase), the more accurately and quickly the tablet is removed from agarose with the necessary DNA fragment, the better and cleaner the elution of DNA fragments can be carried out to use as matrices or substrates for these enzymes.

The use of the claimed device for the extraction of the gel with the necessary DNA fragments allows you to accurately and quickly carry out the extraction with minimal ultraviolet radiation and with minimal capture of the agarose gel, which significantly improves further elution of the DNA and obtaining a high-quality target product.

The device can be used for fast and high-quality extraction of gel fragments with various high molecular weight bioorganic compounds, for example, such as nucleic acids, lipids, proteins, and others, when isolated by electrophoresis.

Information sources

1. Maniatis T. Molecular Cloning / T. Maniatis, E. Fritsch, J. Sambrook. - Per. from English - M .: Mir, 1984. - S.169-171.

2. PCR Protocols: Methods in Molecular Biology / Ed. by John V.S. Bartlett; David Stirling - Second Edition. - Vol. 266. - Humana Press. - Pp. 71-74.

3. Sambrook, J, Fritsh, E.F., Maniatis T. Molecilar cloning: a laboratory manual // New-York: Cold Spring Harbor. Lab. Press., Pp. 1967. - 1626.

4. Doyle, J.J. and Dickson, E. Preservation of plant samples for DNA restriction endonuclease analysis // Taxon. 1987. - Vol. 36. - Pp. 715-722.

Claims (3)

1. A device for extracting a gel with DNA fragments, characterized in that it has a working part, consisting of one or more elements in the form of segments of a flat hollow tube with a sharp cutting outer edge, and contains one or two handles connected to the working part. 2. A device for extracting a gel with DNA fragments according to claim 1, characterized in that in the presence of several elements in the form of segments of a flat hollow tube, they are rigidly interconnected by lateral external surfaces. 3. A device for extracting a gel with DNA fragments according to claim 1, characterized in that the connection of the working part of the device and the handles can be made rigid.