EA005714B1 - Method of preserving biological materials - Google Patents

Abstract

The present invention relates to biotechnology, in particular, to the methods of using genetic material of animal or vegetable nature, and can be used, for example, for the identification of humans.

Description

The invention relates to the field of biotechnology, namely methods using the genetic material of animal or vegetable origin, and can be used, in particular, in identifying a person.

In the framework of the present invention, the following concepts and terms will be used.

Biological material - any tissue or fluid of a living organism (muscle, nail, hair, skin, bone, droplet of blood, saliva, other biological fluid, plant tissue, its juice, etc.). This concept summarizes all types of matter of biological origin, from which it is possible to extract any information about a biological creature, the owner of this tissue.

The genetic code is the information on the sequence of amino acids that make up the DNA of a biological substance, written in special symbols. The genetic code, the system for encrypting hereditary information in nucleic acid molecules, which is realized in biological organisms as a sequence of nucleotides.

The genetic material is deoxyribonucleic acid (DNA).

DNA is a biopolymer consisting of many monomers, deoxyribonucleotides, linked through phosphoric acid residues in a specific sequence specific for each individual DNA. The unique sequence of deoxyribonucleotides in this DNA molecule is a code entry.

Today, a significant difficulty is the identification of unidentified human remains resulting from natural disasters, man-made incidents, hostilities, etc. Unfortunately, the remains of biological objects that can be identified as human remains are often found in a state that is practically unsuitable for identification by conventional methods. The remains can be fragments of bodies that do not contain special signs, allowing you to identify a person with confidence.

In this case, the identification can be carried out on the smallest human remains with the use of gene analysis. But for this, it is necessary to have a comparison material, as which it is preferable to have a sample of the human body.

For professions involving an increased risk, it is advisable to place a tissue sample in the data bank in advance, which will be used for gene analysis in identifying unrecognizable remains.

Currently, there are various ways to preserve samples of biological materials.

For preservation, it was suggested to use sample freezing (8ϋ, inventor's certificate 395439), embalming (8I, inventor's certificate 1745765), paraffin embedding (VI, patent 2031114), as well as deposition of a monolayer of tissue cells on a carrier with subsequent storage of the sample in a refrigerator (VI, patent 2014359).

All the above mentioned methods do not ensure the duration of storage of a sample of biological material (for several years) due to the presence of moisture and oxygen in the sample, as well as microorganisms present on the surface of the sample and the container for its storage. The presence of moisture and oxygen in the sample, as well as its contact with oxygen ultimately leads to the oxidation of biological material and its unsuitability for use as a source of body genes from which the sample was taken.

The closest analogue of the proposed method is the method of storing a sample of a biological material, including its isolation with an inert gas (8I, inventor's certificate 1183537), followed by placing the sample in storage.

The disadvantages of this method include the inability to ensure the duration of storage due to the presence in the sample of moisture and oxygen.

The purpose of the invention is to provide the possibility for a long time to use genetic material while minimizing costs.

To achieve this goal, it has been proposed to use a method for storing biological material, including isolating a sample of biological material from the external environment, then keeping it at a low temperature, whereby the sample placed in an open container is sublimated, then the internal surface of the container and the sample are isolated from the external environment and keeping the sample is carried out at a temperature not exceeding + 8 ° C. Usually, sublimation is carried out to a residual moisture content in the sample of not more than 6 wt.%, As well as to a residual oxygen content in the sample of not more than 4 wt.%. Preferably, the sample is isolated from the external environment by sealing the container, or by filling the container with an inert gas, or by filling the container with an inert gas and sealing. Storage can be carried out in thermostats or in waste salt mines. Typically, the sample is stored in glass containers, metal, ceramic or polymer containers. However, it is possible to use other options for the use of long-lived containers.

It was experimentally established that in the process of sublimation of a sample, together with moisture, oxygen is removed from a sample of biological material, and all microorganisms die on the surface of the container. For this reason, the duration of storage of the sample is significantly increased.

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Subsequent isolation of the sample and container from moisture and oxygen of the air contributes to long-term storage. The use of thermostats or developed salt mines for storage with their constant temperature and humidity allows for an unlimited increase in the storage life of a sample of biological material.

In the General case, the invention is implemented as follows. A sample of biological material placed in a container is sublimated using standard equipment, while eliminating microbiological contamination and removing water and oxygen from the sample. The container is closed, after creating a chemically neutral environment around the sample of biological material. In this case, a vacuum is created in the vessel or the air in the vessel is replaced with an inert gas or inert liquid, a liquid polymer, paraffin or another inert medium. After that, the container with the sample is sealed in any known way (sealed, sealed, brewed, screwed, closed with a ground lid). This will ensure that microorganisms, oxidizing agents and moisture cannot get into the container and contact with the sample of the biological material. The container with the sample is placed in storage at a temperature close to 0 ° C, in the conditions of excluding the possibility of sudden changes (temperature). This will provide better storage, because sudden changes in temperature affect the state of the biologically active material. This can be ensured by placing containers for long-term storage in the basement, cave, mine, under water to a depth, where the temperature is constantly equal to 4 ° C, as well as to other storage places, where the specified requirement is met. The storage place should be protected from solar (ultraviolet) radiation, as well as radioactive radiation, except for natural radiation background. Tanks with biological material can be placed in larger containers (containers). This can provide greater protection against external influences on vessels (containers) containing biological material. This additionally ensures greater safety of storage and easier access to specific samples (vessels). Vessels with biological material may be marked, in addition, additional information may be included in them.

Hereinafter the invention will be disclosed using examples of implementation.

A sample of muscle tissue of a person working under increased danger is placed in an open glass container with a ground-in lid. An open container with the sample is placed in the sublimation chamber. After adjusting the residual moisture and oxygen in the tissue to a level of 7 wt.% And 6 wt.%, Respectively, the capacity is removed from the sublimation chamber. The container is poured under a stopper pre-heated to 120 ° C and cooled to room temperature silicone oil. It was established experimentally that, under the sublimation conditions used, no living microorganisms remain on the surface of the sample and the container, just as there is no moisture, oxygen and microorganisms in the silicone oil. Capacity filled with silicone oil is sealed with a stopper. Label the container with an inscription containing information about the donor tissue. The labeled tank is placed a cave in which there are no differences in temperature and humidity, and the average temperature corresponds to the interval from + 2 to + 5 ° C. The shelf life of a sample obtained, as indicated above, under conditions of a similar cave, exceeds 300 years. (The term is determined by extrapolating changes in the sample).

A sample of the nail plate of the test pilot was placed in a plastic container made with the possibility of sealing. The container with the sample is placed in a sublimation chamber, in which the residual moisture of the sample is adjusted to 8 wt.%, And the residual oxygen content to 7 wt.%. The container is filled with dehydrated carbon dioxide and sealed. On the container stick a plate containing information about the donor of the nail plate. The container, along with other similar containers, is placed in a container that is made of vinyl plastic. The container is filled with argon and brewed. In the future, the container is placed on the seabed at a depth of approximately 100 m, where the water temperature is almost always + 4 ° C. The sample storage time according to extrapolated data is at least 280 years.

Sequoia wood sample placed in a ceramic container. The sample container was placed in a sublimation chamber. After processing, the wood moisture content was 7 wt.%, And the oxygen content was 8 wt.%. On the surface of the container there are no microorganisms left. The internal cavity of the tank filled with molten paraffin. A ceramic lid was placed on the hot paraffin, sealing the contents of the container. The container was placed in a spent salt mine with unchanged conditions (temperature + 2 ° C and humidity 27%). The shelf life of the sample according to the extrapolation data exceeds 500 years.

The proposed method of storing biological material allows you to store samples indefinitely long, suitable for both gene analysis and cloning.

Claims (10)

CLAIM 1. A method for long-term storage of biological material, comprising placing the dehydrated sample in a container that does not contain oxygen, followed by isolating the sample of biological material and the inner surface of the container from the external environment and keeping the container with the sample at low temperature, characterized in that the sample is sublimated beforehand, placed in an open container, to a residual moisture content of not more than 6 wt.% and a residual oxygen content of not more than 4 wt.%, and keeping the sample in the container lead at a temperature not exceeding + 4 ° C. 2. The method according to claim 1, characterized in that the isolation from the external environment is carried out by sealing the tank. 3. The method according to claim 1, characterized in that the isolation from the external environment is carried out by subsequent filling of the tank with an inert material. 4. The method of claim 1, characterized in that the isolation from the external environment is carried out by filling the tank with inert gas and sealing. 5. The method according to claim 1, characterized in that the storage is carried out in thermostats. 6. The method according to claim 1, characterized in that the storage is carried out in spent salt mines. 7. The method according to claim 1, characterized in that the storage is carried out in a glass container. 8. The method according to claim 1, characterized in that the storage is carried out in a polymer container. 9. The method of claim 1, characterized in that the container with biological material is placed in a larger container. 10. The method according to claim 1, characterized in that the container with biological material is marked and / or additional information is applied to it.