RU2632649C2 - Method of obtaining dna from fish ovicell - Google Patents

Abstract

FIELD: veterinary medicine.

SUBSTANCE: method involves sample preparation and DNA purification. In the process of sample preparation, the ovicelles are inseminated with genetically inactivated sperm to produce gynogenetic embryos, from which, after hatching, DNA is exposed. Before the inactivation, the sperm is diluted with 0.9% sodium chloride solution at the ratio of 1:12, inactivation is carried out by UV-radiation at a dose of 300 J/m², and DNA purification is performed by salt extraction method by lysis of the sample in homogenization buffer solution with proteinase K with further shaking with sodium chloride, purification with a chloroform mixture: isoamyl alcohol and settling DNA from the lysate purified with ethyl alcohol.

EFFECT: invention makes it easier to expose and increase the amount of nuclear DNA suitable for molecular genetic analysis, in particular the use of PCR in the analysis of nuclear genetic materials.

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Description

The invention relates to biotechnology and fish farming, in particular to the molecular genetic analysis of fish.

Molecular genetic research in ichthyology and fish farming has been carried out for many years to differentiate natural fish populations, to identify and certify breeds and lines, as well as to identify differences between wild and artificially reproduced individuals.

The successful application of molecular genetic methods of analysis, the most effective of which is polymerase chain reaction (PCR), largely depends on obtaining a sufficient amount of high molecular weight DNA with an undisturbed structure suitable for PCR.

In fish, fragments of fins and blood are the best material for DNA isolation that can be taken intravitally without causing serious damage. However, there are frequent cases when it is necessary to isolate DNA from gametes, including from individual eggs. Such a need arises, for example, when studying the differences between somatic and gametic DNA in the same individual, when developing some special methods of chromosomal manipulations, when searching for sex-determining factors in species with female heterogeneity, and in studying transgene elimination in meiosis. For these purposes, an analysis of nuclear genetic markers is necessary.

DNA extraction methods include 2 main stages - lysis of samples and purification of DNA lysate from impurities (proteins, fats, as well as tissue and cell residues).

For some types of biological samples, as well as for most samples fixed in different ways, preliminary sample preparation is necessary.

The most universal way to obtain high molecular weight DNA from biological materials, which with small modifications can be used for different types of cells and tissues, includes sample preparation and DNA isolation using phenol-chloroform extraction (Guidelines for the use of polymerase chain reaction in animal husbandry. - Dubrovitsy , 1998 .-- S. 5-7).

Preliminary sample preparation consists in sedimenting the desired fractions of cells from liquid biological samples, thawing, washing, etc. DNA isolation consists in first lysing the sample in a lysis buffer with proteinase K, then purifying the lysate with phenol or phenol: chloroform: isoamyl alcohol, then with a mixture of chloroform: isoamyl alcohol. From the purified lysate, total DNA is precipitated with ethyl or isopropyl alcohol.

However, the use of this method for the isolation of DNA from fish eggs has limitations due to prolonged and incomplete lysis, which is due to the complexity of the destruction of dense egg shells.

With incomplete lysis of eggs, as well as a low ratio of nucleus (chromosomes) / cytoplasm (only 1 haploid set of chromosomes per large cell with a high content of proteins and lipids), it becomes necessary to increase the number of stages of lysate purification with organic solvents to remove proteins, fats and non-lysed residues. As a result, the probability of DNA loss and structural damage is increased.

A known method of obtaining high molecular weight DNA from different cells and tissues, including sample preparation of biomaterial and DNA extraction using salt extraction (Guidelines for the use of polymerase chain reaction in animal husbandry. - Dubrovitsy, 1998. - S. 10-11). Its main advantage over the standard phenol-chloroform method is that it does not require extraction with phenol, which is a highly hazardous substance (hazard class 2) and also a strong PCR inhibitor.

In this method, sample preparation is carried out in the same way as in the previous one (washing, sedimentation of cells from liquid samples, thawing, etc.). DNA isolation is carried out by lysing a sample in a homogenization buffer with proteinase K, shaking the lysate with sodium chloride, purification with a mixture of chloroform: isoamyl alcohol, and precipitating the total DNA from ethyl acetate or isopropyl alcohol from the purified lysate.

When using this method to isolate DNA from fish eggs, the limitations are the same as in the previous case, i.e., prolonged and incomplete lysis due to the complexity of destruction of dense egg membranes.

The closest in technical essence and the achieved result is a method of obtaining DNA from fish eggs, including sample preparation and further DNA extraction by modification of phenol-chloroform extraction. (Fain S.R. DNA extraction from caviar with organic solvents. - US Department of the Interior, Fish and Wildlife Service, version 5.3.1998).

Sample preparation is carried out as follows: a small amount of caviar is washed in saline to remove fat and separate eggs, intact eggs are selected from them. To isolate DNA, 1 egg is placed in a lysis solution with proteinase K, in which the detergent Triton X-100 is added to increase the lysis efficiency, and incubated in an incubator for 1 hour at 65 ° C. Then, the lysate is purified with a phenol: chloroform mixture, DNA precipitation with 95% ethanol, and DNA dissolution in TE buffer. This method allows to obtain from 1 egg a significant amount of mitochondrial DNA suitable for PCR.

The disadvantage of this method is the inability to obtain from the eggs (eggs) a sufficient amount of nuclear genomic (chromosomal) DNA suitable for molecular genetic analysis (in particular, for use in PCR), and as a result, the inability to analyze nuclear genetic markers.

This is due to the fact that the nuclear DNA in a haploid egg is represented by only one chromosomal copy, and the fish egg itself contains a large amount of proteins and fats (yolk), which make up the supply of nutrients for embryo development. It is difficult to isolate such a small amount of DNA without damaging it, and without loss to clean from a large amount of yolk stored in the egg.

The use of phenol for DNA purification makes the method highly hazardous.

The technical result of the invention consists in facilitating the isolation and increase in the amount of nuclear (chromosomal) DNA extracted from the eggs, suitable for molecular genetic analysis, in particular, for use in PCR when analyzing nuclear genetic markers, as well as to reduce the risk of the process of obtaining DNA.

The indicated technical result is achieved in that in the method for producing DNA from fish eggs, including sample preparation and DNA isolation, the peculiarity is that at the stage of sample preparation, the eggs are seeded with genetically inactivated sperm to obtain gynogenetic embryos, from which DNA is isolated after hatching. In this case, DNA is isolated by salt extraction.

Obtaining gynogenetic embryos at the sample preparation stage is due to the fact that during gynogenesis only the egg nucleus is involved in the development of the embryo. In the process of development of gynogenetic embryos, the chromosome set of initial eggs is repeatedly copied, which allows one to obtain preparations containing a sufficient amount of chromosomal DNA suitable for use in PCR for the analysis of nuclear genetic markers.

During hatching, embryos are released from the dense shells of the egg, as a result of which, when DNA is extracted from the hatched embryos, their complete lysis is achieved, its duration and the number of stages of lysate purification with organic solvents are reduced, and as a result, the probability of DNA loss and structural damage is reduced.

Isolation of DNA from gynogenetic embryos by salt extraction, which does not use highly toxic phenol, reduces the risk of the process of obtaining DNA.

The method is applicable to obtain DNA from the eggs of virtually any species of fish with external reproduction.

The analysis of the prior art made it possible to establish that the source is not detected, characterized by signs identical to all the essential features of the claimed invention, therefore, the proposed invention meets the condition of "novelty."

An additional search for known solutions showed that the claimed invention does not follow explicitly from the prior art for a specialist. The use of the known method of induced gynoginesis in the claimed invention leads to a new technical result - to facilitate isolation and increase the amount of nuclear DNA suitable for molecular genetic analysis, in particular, use in PCR for the analysis of nuclear genetic markers. The set of essential features in the claimed method makes it possible to isolate nuclear DNA from a single egg and conduct molecular genetic analysis for a variety of purposes.

Thus, the claimed invention meets the condition of "inventive step".

The method is as follows.

Mature fish products are obtained from spawning fish. From eggs (eggs) using induced gynogenesis get gynogenetic embryos. For this, eggs are inseminated with genetically inactivated sperm by UV irradiation. After hatching the gynogenetic embryos, they are selected (and fixed if storage is envisaged), then DNA is extracted from them by salt extraction.

Embryo lysis is carried out in a homogenization buffer with proteinase K, the resulting lysate is shaken with sodium chloride, purified with a mixture of chloroform: isoamyl alcohol and the total DNA is precipitated from the purified lysate with ethyl or isopropyl alcohol.

Examples of the method are illustrated by photographs (Fig. 1-4), which shows electrophoregrams of DNA from eggs and from gynogenetic embryos, as well as electrophoregrams of PCR products obtained with these DNA preparations.

Example 1. From small eggs obtained from female carp, 2 small portions were selected.

One of them was washed in saline, intact eggs were selected for subsequent DNA isolation using the phenol-chloroform method in the modification for eggs (in accordance with the prototype). DNA was isolated from 1, 10, 50, 90, and 270 eggs. The indicated number of eggs was placed in a lysing solution with proteinase K and Triton X-100 detergent and incubated at 65 ° C. The lysis time increased with an increase in the number of lysed eggs (eggs). The lysates were purified with a phenol: chloroform mixture; the number of extractions increased with the number of eggs used to isolate DNA. Then, DNA was precipitated with 95% ethanol. The DNA precipitate was dissolved in bidistilled water.

Another portion of eggs was seeded with genetically inactivated sperm to obtain gynogenetic embryos.

To irradiate sperm, a mercury bactericidal lamp was used as a source of short-wave UV radiation (300 J / m ² ). The distance from the lamp to the irradiated object was 45 cm, the irradiation duration was 11 min. Before irradiation, sperm was diluted in a ratio of 1:12 (6 drops of sperm per 10 ml of 0.9% NaCl solution). 10 ml of a suspension of diluted semen was poured into a 9 cm Petri dish and irradiated with constant stirring on a magnetic stirrer. Gynogenetic embryos (haploid) were incubated in Petri dishes at room temperature. To prevent photoreactivation of UV damage to sperm, the initial incubation period was carried out in a darkened room. Gynogenetic embryos were selected for DNA isolation after hatching.

For the subsequent isolation of DNA from gynogenetic haploid embryos, the salt extraction method was used. 1 haploid embryo was placed for lysis in a homogenization buffer with proteinase K and incubated at 62 ° C for 1-2 hours. The lysate was shaken with sodium chloride, purified with a mixture of chloroform: isoamyl alcohol, and ethanol precipitated from the purified DNA lysate. The DNA precipitate was dissolved in bidistilled water.

The resulting total DNA was separated by horizontal electrophoresis in a 1.0% ethidium bromide agarose gel in TAE buffer (Tris-EDTA acetate system) and visualized under UV light in a dark AutoChemi room on a 2UV transilluminator. Registration of electrophoregrams was carried out on a computer using the AutoChemi image study system (UVP).

The length of DNA fragments is given in base pairs (bp).

The results of electrophoresis of DNA extracts from fish eggs are shown in FIG. 1, from gynogenetic haploid embryos - in FIG. 2. DNA migration on all electrophoregrams - from bottom to top.

In FIG. 1 on lane 1 - DNA marker (100 bp); on lanes 2, 3 - DNA from 1 carp egg, 4, 5 - DNA from 10 carp eggs, 6, 7 - DNA from 50 carp eggs, 8, 9 - DNA from 90 carp eggs, 10, 11 - DNA from 270 eggs carps; 13-15 - DNA from 1 sturgeon egg; 16, 17 - control (extracts from carp fins containing chromosomal DNA).

As seen in FIG. 1, the band corresponding to high molecular weight chromosomal DNA (about 10,000 bp or more in size) is observed only on lanes 16 and 17, on which control DNA preparations were separated from somatic tissues of carp. In lanes 2–11, where DNA extracts from carp eggs (eggs) were separated, the strip corresponding to chromosomal DNA was not visualized. Possible reasons for its absence are the loss or destruction of DNA in the process of isolation.

The band corresponding to chromosomal DNA is clearly visible in lanes 1-6 (Fig. 2), on which DNA preparations were separated from gynogenetic haploid carp embryos. Thus, it can be concluded that the presence of high molecular weight chromosomal DNA in all these preparations. Each DNA preparation is obtained from 1 gynogenetic haploid embryo.

Example 2. To assess the suitability of DNA preparations obtained from egg cells and gynogenetic haploid carp embryos for PCR, they were used in amplification of the target 540 bp DNA fragment (recombinant ptMTa-scGH construct integrated into the nuclear genome) with specific primers AL4 and G35 .

PCR was performed in a final volume of 25 μl with 1 × PCR buffer, 2 mM MgCl ₂ , 0.2 mM dinucleotide triphosphates, 10 pmol of each primer and 1.5 U Taq polymerase using 2.5 μl of DNA extract.

PCR mode: preliminary DNA denaturation - 95 ° C - 13 min; synthesis of PCR products: 5 cycles - 94 ° C - 40 sec, 62 ° C - 40 sec, 72 ° C - 40 sec; 5 cycles - 94 ° С - 40 sec, 60 ° С - 40 sec, 72 ° С - 40 sec; 35 cycles - 94 ° С - 30 sec, 58 ° С - 15 sec, 72 ° С - 15 sec; final completion of chains - 72 ° С - 10 min.

PCR products were separated by electrophoresis on a 1.0% ethidium bromide agarose gel in TAE buffer (Tris-EDTA acetate system) and visualized under UV light in a dark AutoChemi room on a 2UV transilluminator. Registration of electrophoregrams was carried out on a computer using the AutoChemi image study system (UVP).

The results of electrophoresis of PCR products obtained with DNA from carp eggs are shown in FIG. 3, with DNA from gynogenetic haploid embryos, FIG. 4. DNA migration on both electrophoregrams - from bottom to top.

In PCR products obtained with DNA preparations from carp eggs, the amplified target fragment of 540 bp in length is absent (lanes 3-12 in Fig. 3). This may be due to an insufficient amount of chromosomal DNA for PCR, as well as a violation of its structure during extraction.

This target fragment was detected in PCR products obtained with 10 DNA preparations from individual gynogenetic haploid carp embryos (lanes 2-4, 6, 10, 12-15, 17 in Fig. 4). Its absence in the remaining 9 amplitudes (lanes 1, 5, 7-9, 11, 16, 18, 19 in Fig. 4) is associated with the hemizygosity of the females from whom the eggs were obtained for this DNA fragment (ptMTa-scGH construct).

The above examples illustrate that the proposed method allows to obtain high molecular weight chromosomal DNA, suitable for research using PCR, in sufficient quantities, as well as reduce the danger and the complexity of the DNA isolation procedure.

Thus, the above information indicates that when using the claimed invention the following combination of conditions:

- a method for producing DNA from fish eggs according to the claimed invention is intended for use in fish farming, in particular when obtaining DNA for molecular genetic analysis of fish;

- for the claimed method in the form described in the independent clause of the claims, the possibility of its implementation using the means and methods described in the application is confirmed.

Therefore, the claimed invention meets the condition of "industrial applicability".

Claims (1)

A method of obtaining DNA from fish eggs, which involves sample preparation and DNA isolation, characterized in that the eggs are seeded with genetically inactivated sperm during sample preparation to produce gynecogenetic embryos, from which DNA is isolated after hatching, while the sperm is diluted with a 0.9% solution before inactivation sodium chloride in a ratio of 1:12, inactivation is carried out by UV radiation at a dose of 300 J / m ² , and DNA is isolated by salt extraction by lysis of the sample in a homogenization buffer with a oteinase K with further shaking with sodium chloride, purification with a mixture of chloroform: isoamyl alcohol and precipitation of DNA from purified lysate with ethyl alcohol.

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