RU2651937C1 - Composition for collection and storage of dna or dna-containing biological traces (variants) and their application - Google Patents

Abstract

FIELD: biochemistry.

SUBSTANCE: group of inventions relates to biochemistry. Compositions for the collection and storage of DNA or DNA-containing biological traces (variants), as well as a means for impregnating hygroscopic carrier materials and performing washings of DNA-containing biological traces, overlays or DNA for storage and/or transportation, where the agent is the above compositions. And the compositions include 1–40 mM Tris (hydroxymethyl) aminomethane, 0.05–1 % of Tween 20, 0.05–1 % of Triton X-100, 1–200 mM K₂SO₄, 1–200 mM KCl, 0.01–0.5 mM EDTA, 1–20 % glycerol, 0.01–0.4 % NaN₃ and water.

EFFECT: inventors ensure stability of the DNA of biological samples over a wide temperature range over long periods of time.

26 cl, 6 ex

Description

FIELD OF THE INVENTION

The present invention relates to a buffered liquid composition for collecting and storing DNA or DNA-containing biological traces, including traces of traces, in dry and liquid form. The specified composition ensures the stability of the DNA of biosamples in a wide temperature range over long periods of time.

State of the art

Currently, in medicine and forensics, molecular genetic analysis is one of the most evidence-based methods for the analysis of human biological traces: traces of fat (PZhS), traces of blood, saliva, sperm, etc. Biological traces, such as PZhS, contain a small amount of genetic material, and it is often insufficient for sample identification. The removal of biological overlay at the scene of the incident is often carried out using standard methods, without taking into account the characteristics of the investigated object and the circumstances. The stage of laboratory studies of the obtained samples can be delayed and begin after a long period of time. Often the reason for unsuitability for identification of the selected biological material at the scene is its improper collection, transportation and storage. Therefore, the choice of an effective method for collecting biological traces, ensuring the preservation of the quantity and quality of the selected material, is of great importance.

Storage of biological material, in particular washouts of biological traces, on a solid carrier (for example, on cotton cloth) in a dry form before laboratory tests is the most common and simple way in which the biosamples are at room temperature without the use of expensive equipment. A carrier with a washout of biological traces to prevent the degradation of DNA molecules must be well dried before storage, protected from sunlight, high temperature and humidity.

The most commonly used method for collecting biological traces at the scene of an accident is to use swabs with gauze soaked in deionized water. When working with washouts of biological traces containing a small amount of genetic material, such as PZhS, obtaining a complete genetic profile of the person who left their traces is fraught with significant difficulties. This can be explained both by the initially low content of genetic material in the tracks, and the possible degradation of the active DNA matrix over time in the absence of DNA stabilizing factors (Faleeva T.G., Ivanov I.N., Mishin E.S., Vnukova N. .V., Kornienko I.V. Problems of molecular genetic identification of human fingerprint traces of human fingerprints // Forensic medical examination. - 2016. - No. 2. - P.14-18).

When working in the field of molecular genetic research, one may encounter samples of flushes of biological traces and overlays made using physiological saline. A physiological solution containing high concentration of NaCl (about 150 mM) is able to inhibit the enzymatic amplification reaction, which may make it difficult to conduct a "direct" polymerase chain reaction (PCR), that is, PCR without preliminary isolation and purification of DNA preparations.

TE buffer is widely used in biochemistry and molecular biology to dissolve DNA or RNA and prevent nucleic acid degradation. The TE buffer consists of 10 mM Tris-HCl pH = 8.0, 0.1-1.0 mM EDTA. Despite its protective properties with respect to the genetic material, this buffer has not received widespread use as a storage medium for dry samples washed with its use.

RU 2567145 discloses a composition for storing DNA-containing preparations or DNA and its use for preserving biological samples, ensuring the preservation of nucleic acids. The composition includes 0.5-4% sodium lauroylsarcosine, which is a detergent and inhibitor of PCR, as well as EDTA (ethylenediamine-N, N, N ', N'-tetraacetate) in high concentrations (5-20 mm), which inhibit PCR and significantly make it difficult or impossible to conduct a "direct" PCR without adding various kinds of activators (usually non-ionic detergents).

Patent RU 2164532 provides methods and reagent kits for gene-amplification diagnostics by PCR and RNA-PCR. The proposed methods and kits are intended for gene testing, genetic diagnostics and do not provide long-term storage of genetic material with their use. The reagent kit for isolating the sum of DNA and RNA contains a lysing solution, which contains sodium lauroyl sarcosine, which is a detergent, as well as sodium iodide and / or guanidine thiocyanate, which can inhibit PCR, making it difficult or impossible to perform "direct" PCR.

Patent RU 2258710 proposes a new cytokine zalpha11 ligand. A method for producing an antibody to a zalpha11 ligand polypeptide is described. The described liquid buffer compositions do not provide long-term storage of genetic material with their use. High concentrations of NaCl (100 mM) and SDS (SDS, sodium dodecyl sulfate), which is a powerful detergent, can inhibit PCR in the lysis buffer and make it difficult or impossible to perform “direct” PCR.

Patent RU 2132853 proposes a method for identifying products from a mixture, a product obtained by identification, a method for producing a mixture and a method for co-producing a catalytic nucleic acid and a product. The use of high concentrations of MgCl ₂ (60 mM) for the transcription reaction in the 5x T7 RNA polymerase buffer will lead to complete inhibition of DNA Taq polymerase (which is completely inhibited by MgCl ₂ concentrations above 10 mM), inhibiting or making impossible PCR.

Patent CZ 304780 (B6) proposes a method for storing DNA of histopathological material. The presence of a lysing buffer of proteinase K at a concentration of 20 mg / ml in the composition of K solution is capable (without additional purification) of completely inhibiting PCR due to enzymatic hydrolysis of DNA Taq polymerase.

US Pat. No. 6310045 B1 provides compositions and methods for cancer immunotherapy. High concentrations of NaCl (150 mM) and SDS (sodium dodecyl sulfate), which is a powerful detergent, in the composition of the used lysis buffer for cells can inhibit PCR or make it impossible to conduct "direct" PCR.

Patent KR 20040026519 (A) proposes a DNA isolation method for PCR analysis of transgenic fish. The described method does not provide long-term storage of genetic material. The presence of SDS (sodium dodecyl sulfate), which is a powerful detergent, and a high concentration of NaOH (20 mM) in the buffer solution X, can inhibit PCR and significantly complicate the implementation of “direct” PCR. High concentrations of MgCl ₂ (5 mM) in the composition of buffer solution-Y contribute to the occurrence of nonspecific amplification products during the formulation of “direct” PCR.

Patent KR 20020029476 (A) provides a lysis buffer reagent for one-step gene isolation. The described method does not provide long-term storage of genetic material. The presence of high concentrations of EDTA (ethylenediamine-N, N, N ', N'-tetraacetate) (10 mM) in the lysis buffer is capable of inhibiting PCR (due to the formation of chelate complexes with magnesium (II) ions and, accordingly, turning them off) from the reaction of enzymatic amplification) or significantly complicate the implementation of "direct" PCR.

CN 102796727 (A) proposes a method for extracting nucleic acids of gram-positive bacteria. The described method does not provide long-term storage of genetic material. The presence of detergents SDS (sodium dodecyl sulfate) and sodium lauroyl sarcosine, as well as phenol in extract B of the used reagent in extract A, can inhibit PCR or make it impossible to conduct "direct" PCR.

Patent EA 004880 provides a kit for reverse transcriptase analysis and its use. The described method does not provide long-term storage of genetic material. The use of divalent metal ^ions Mg ²⁺ in a concentration of more than 10 mM as a part of the reverse transcriptase assay kit leads to inhibition of DNA-Taq polymerase, thereby interfering with PCR. The presence of heparin sulfate as an RNase inhibitor in the kit can inhibit PCR or make it impossible to conduct "direct" PCR.

KR 20030006505 proposes a cell lysing buffer for DNA isolation and a method for isolating DNA using it. The described method does not provide long-term storage of genetic material. The presence of such surface-active substances as SDS (sodium dodecyl sulfate) and sarcosyl sulfate, which are detergents, as well as proteases in the lysis buffer, can inhibit PCR or make it impossible to conduct "direct" PCR.

US Pat. No. 6,071,698 teaches a buffer for DNA isolation and a method for using it. The described liquid buffer composition is intended for DNA extraction and does not provide long-term storage of genetic material with its use. The use of high concentrations of NaCl (0.15-2.0 Μ) and EDTA (ethylenediamine-N, N, N ', N'-tetraacetate) (180-500 mM) in DNA isolation buffer can inhibit PCR or make it impossible to direct "PCR.

US 20020115089 A1 proposes a technology for the simultaneous collection of DNA and non-nucleic analytes. The presence of high concentrations of sodium chloride (1-20 mg / cm ² ), calcium chloride (1-20 mg / cm ² ), EDTA (ethylenediamine-Ν, Ν, Ν ', Ν'-tetraacetate) (0.05-0, 15 M), a powerful detergent for sodium dodecyl sulfate (SDS, 0.2-1%), proteinase K (50-150 μg / ml) in the compositions can inhibit PCR or make impossible "direct" PCR.

In laboratory conditions, the washing off of biological traces and overlays on the surface of the studied objects can be carried out using various lyse solutions with a known composition, for example, 10 mM Tris-HCl, pH 7.5, 10 mM Na ₂ EDTA, 50 mM NaCl, 2% SDS ( Kornienko I.V., Kharlamov SG Methods for the study of human DNA: DNA isolation and its quantitative assessment in the aspect of forensic medical research of material evidence of biological origin: a training manual / I.V. Kornienko, S.G. Kharlamov. Rostov n / a: SFU, 2012. - 216 p.) And commercial, the composition of which contains guanidinium salts in high (several mol) concentrations, for example, the lysis buffer (Lysis buffer) of the DNA IQ ™ System reagent kit (Promega, USA) and the lysis buffer (Lysis buffer) of the PrepFiler ™ User reagent kit (Applied Biosystems, USA) . However, this method of obtaining biological material implies the immediate execution of DNA extraction, and "direct" PCR is not possible due to inhibition of the reaction by the components of the solutions.

When isolating DNA from biological traces that contain initially a small amount of genetic material, for example, such as a potassium substance, a method is needed that would allow extraction with minimal loss of genetic material, exclude contamination of the studied objects with extraneous DNA as much as possible, and perform “direct” PCR without preliminary cleaning the drug.

Isolation of DNA using a Chelex-100 ion-exchange resin allows genetic material to be extracted with minimal loss, but it does not always provide the necessary purity of the resulting preparation. When using this method, it is difficult to obtain a concentrated DNA preparation without the threat of inhibition of PCR by particles of the ion-exchange resin Chelex-100.

The phenol-organic extraction method is applicable to an initially large volume of biological material, since it is associated with significant loss of DNA during its extraction. The high toxicity and complexity of the disposal of reagents negatively affected the popularity of this method, and now in most laboratories it has been replaced by methods such as nucleosorption using silicon dioxide (silica), implemented in most commercial kits (Essentials of Nucleic Acid Analysis. A Robust Approach. Ed. Keer JT, Birch L. Published by The Royal Society of Chemistry. Cambridge. - 2008. - 248 p .; Kornienko I.V., Faleeva T.G. An improved method for isolating human DNA from biological samples containing a small amount of genetic material using on reagent boron DNA IQ (PROMEGA) // Clinical and laboratory consultation. - 2013. - No. 4 (47). - S.49-53.). One of these reagent kits is DNA IQ ™ DNA extraction systems (Promega, USA) and PrepFiler ™ User Guide (Applied Biosystems, USA), with which you can obtain highly purified DNA preparations both manually and using robotic technology. Despite this, these reagent kits are quite expensive, and methods using them include a multi-stage DNA extraction process, which leads to its slight loss and the risk of sample contamination. However, even such a small loss of DNA is extremely significant when working with objects containing initially a small amount of genetic material.

Thus, a universal method using a liquid buffer composition for collecting and storing DNA-containing biological traces containing a small amount of genetic material in dry and liquid form, ensuring DNA stability over a wide temperature range and its lossless extraction, has been proposed to date did not have.

Disclosure of invention

The present invention provides:

(1) A composition for collecting and storing DNA or DNA-containing biological traces, containing a buffer solution comprising 1-40 mm Tris (hydroxymethyl) aminomethane, 0.05-1% vol. Tween 20, 1-200 mM K ₂ SO ₄ , 0.01-0.5 mM EDTA (ethylenediamine-Ν, Ν, Ν ', Ν'-tetraacetate), 1-20% glycerol, 0.01-0.4 % NaN ₃ (sodium azide), water - the rest.

(2) Composition (1), where the buffer solution is a mixture of solutions of 12.1 mg Tris (hydroxymethyl) aminomethane, 50 μl Tween 20, 87.1 mg K ₂ SO ₄ , 2 μl 0.5 Μ EDTA (ethylenediamine-Ν , Ν, Ν ', Ν'-tetraacetate), 500 μl of glycerol, 10 mg NaN ₃ (sodium azide), brought to 10 ml with sterile deionized water.

(3) Composition (1), wherein the DNA-containing trace is a biosample.

(4) Composition (1), wherein the ratio of buffer to bio-sample is 10: 1.

(5) Composition (1), wherein the biosample is a potogens, traces of blood, saliva, vaginal secretions, sperm, cell suspensions, mixed biological traces, DNA preparations.

(6) Composition for collecting and storing DNA or DNA-containing biological traces containing a buffer solution comprising 1-40 mΜ Tris (hydroxymethyl) aminomethane, 0.05-1% vol. Triton X-100, 1-200 mM K ₂ SO ₄ , 0.01-0.5 mM EDTA (ethylenediamine-Ν, Ν, Ν ', Ν'-tetraacetate), 1-20% glycerol, 0.01-0 , 4% NaN ₃ (sodium azide), water - the rest.

(7) Composition (6), where the buffer solution is a mixture of solutions of 12.1 mg Tris (hydroxymethyl) aminomethane, 50 μl Triton X-100, 87.1 mg K ₂ SO ₄ , 2 μl 0.5 Μ EDTA (ethylenediamine -Ν, Ν, Ν ', Ν'-tetraacetate), 500 μl of glycerol, 10 mg NaN ₃ (sodium azide), brought to 10 ml with sterile deionized water.

(8) Composition (7), wherein the DNA-containing trace is a biosample.

(9) Composition (7), wherein the ratio of buffer to bio-sample is 10: 1.

(10) Composition (7), wherein the biosample is a potogens, traces of blood, saliva, vaginal secretions, sperm, cell suspensions, mixed biological traces, DNA preparations.

(11) A composition for collecting and storing DNA or DNA-containing biological traces, containing a buffer solution comprising 1-40 mm Tris (hydroxymethyl) aminomethane, 0.05-1% vol. Tween 20, 1-200 mM KCl, 0.01-0.5 mM EDTA (ethylenediamine-Ν, Ν, Ν ', Ν'-tetraacetate), 1-20% glycerol, 0.01-0.4% NaN ₃ (sodium azide), water - the rest.

(12) Composition (11), where the buffer solution is a mixture of solutions of 12.1 mg Tris (hydroxymethyl) aminomethane, 50 μl Tween 20, 37.3 mg KCl, 2 μl 0.5 Μ EDTA (ethylenediamine-Ν, Ν, Ν ', Ν'-tetraacetate), 500 μl of glycerol, 10 mg NaN ₃ (sodium azide), adjusted to 10 ml with sterile deionized water.

(13) Composition (12), wherein the DNA-containing trace is a biosample.

(14) Composition (12), wherein the ratio of buffer to bio-sample is 10: 1.

(15) Composition (12), wherein the biosample is a potogens, traces of blood, saliva, vaginal secretions, sperm, cell suspensions, mixed biological traces, DNA preparations.

(16) A composition for collecting and storing DNA or DNA-containing biological traces containing a buffer solution comprising 1-40 mM Tris (hydroxymethyl) aminomethane, 0.05-1% vol. Triton X-100, 1-200 mM KCl, 0.01-0.5 mM EDTA (ethylenediamine-Ν, Ν, Ν ', Ν'-tetraacetate), 1-20% glycerol, 0.01-0.4% NaN ₃ (sodium azide), water - the rest.

(17) Composition (16), where the buffer solution is a mixture of solutions of 12.1 mg Tris (hydroxymethyl) aminomethane, 50 μl Triton X-100, 37.3 mg KCl, 2 μl 0.5 Μ EDTA (ethylenediamine-Ν, Ν, Ν ', Ν'-tetraacetate), 500 μl of glycerol, 10 mg NaN ₃ (sodium azide), adjusted to 10 ml with sterile deionized water.

(18) Composition (17), wherein the DNA-containing trace is a biosample.

(19) Composition (17), wherein the ratio of buffer to bio-sample is 10: 1.

(20) Composition (17), wherein the biosample is a potogens, traces of blood, saliva, vaginal secretions, sperm, cell suspensions, mixed biological traces, DNA preparations.

(21) The use of compositions (1) to (20) for the impregnation of hygroscopic carrier materials and for flushing DNA-containing biological traces, overlays or DNA with the aim of removing, storing and / or transporting them.

(22) The use of (21), wherein the hygroscopic carrier material is a cotton cloth, fibrous materials (e.g. a cotton swab), paper media (e.g., chromatographic paper).

(23) The use of (21), wherein the DNA-containing biological trace, overlay or DNA is a dry bio-sample.

(24) The use of (21), wherein the DNA-containing biological trace, overlap or DNA is in liquid form.

I. Objects of the present invention are liquid buffer compositions for the impregnation of porous hygroscopic carriers, for example gauze, cotton swabs, paper, for the purpose of collecting, transporting and storing traces of DNA-containing biological material by washing them off, or by applying onto soaked media buffer solutions that ensure the stability of the DNA of biosamples in a wide temperature range for long periods of time in a dry form.

The sign "%", as used in the present invention, means the percentage by weight of the specified component in the specified composition, buffer.

In the present invention, the terms “biological traces and overlays”, “DNA-containing traces” are used, which means any excretion of the human body containing genetic material left by its owner on various surfaces of environmental objects in the form of overlays and soaks.

One of the constant components of the buffer compositions is 0.01-0.4% NaN ₃ (sodium azide), which has a pronounced antimicrobial and antimycotic effect without inhibition of PCR.

Also, a constant constituent of the compositions is 0.01-0.5 mM EDTA (ethylenediamine-Ν, Ν, Ν ', Ν'-tetraacetate). In animal tissues, Fe ²⁺ and Fe ³⁺ ions are present in large quantities in the composition of hemoglobin and released during its denaturation. The binding constant of EDTA with iron ions is ≈10 ^-4 M ^-1 Fe ²⁺ ) and ≈10 ^-24 M ^-1 (Fe ³⁺ ) (Sillen, LG, Martell, A.E. Stability Constants of Metallon Complexes. The Chemical Society, London. - 1964.). Thus, due to the presence of EDTA in the composition of the buffer, the inhibitory effect of iron ions exerted on PCR is eliminated. At the same time, low concentrations of EDTA in the buffer are not capable of exerting an inhibitory effect on PCR, thereby not interfering with direct PCR.

A slightly alkaline environment is most optimal for storing DNA preparations. Therefore, Tris solution (1-40 mM Tris (hydroxymethyl) aminomethane) is used to create a buffer capacity, maintain a slightly alkaline pH value and stabilize DNA molecules.

In order to stabilize DNA-Taq polymerase, the composition includes activators such as Tween 20 (0.05-1% vol.), Triton X-100 (0.05-1% vol.).

Stabilization of the enzyme DNA-Taq polymerase by increasing the half-life of the enzyme at high temperatures (from + 90 ° C to + 97.5 ° C) at the stage of denaturation of the DNA matrix during PCR is carried out by using activators of enzymatic amplification: K ₂ SO ₄ (1-200 mm), KCl (1-200 mm), glycerol (1-20%), which can significantly increase the reaction efficiency.

The present invention is a combination of the above components in a buffer:

1. The buffer liquid composition (1) is a mixture of solutions (1-40 mm Tris (hydroxymethyl) aminomethane, preferably 10 mm, 0.05-1% vol. Tween 20, preferably 0.5% vol., 1-200 mm K ₂ SO ₄ , preferably 50 mM, 0.01-0.5 mM EDTA (ethylenediamine-Ν, Ν, Ν ', Ν'-tetraacetate), preferably 0.1 mM, 1-20% glycerol, preferably 5%, 0.01-0.4% NaN ₃ (sodium azide), preferably 0.1%). For example, a mixture of 12.1 mg Tris (hydroxymethyl) aminomethane, 50 μl Tween 20, 87.1 mg K ₂ SO ₄ , 2 μl 0.5 Μ EDTA (ethylenediamine-Ν, Ν, Ν ', Ν'-tetraacetate), 500 μl of glycerol, 10 mg NaN ₃ (sodium azide), brought up to 10 ml of sterile deionized water.

2. The buffer liquid composition (2) is a mixture of solutions (1-40 mm Tris (hydroxymethyl) aminomethane, preferably 10 mm, 0.05-1% vol. Triton X-100, preferably 0.5% vol., 1- 200 mM K ₂ SO ₄ , preferably 50 mM, 0.01-0.5 mM EDTA (ethylenediamine-Ν, Ν, Ν ', Ν'-tetraacetate), preferably 0.1 mM, 1-20% glycerol, preferably 5 %, 0.01-0.4% NaN ₃ (sodium azide), preferably 0.1%). For example, a mixture of 12.1 mg Tris (hydroxymethyl) aminomethane, 50 μl Triton X-100, 87.1 mg K ₂ SO ₄ , 2 μl 0.5 Μ EDTA (ethylenediamine-Ν, Ν, Ν ', Ν'-tetraacetate ), 500 μl of glycerol, 10 mg NaN ₃ (sodium azide), brought to 10 ml with sterile deionized water.

3. The buffer liquid composition (3) is a mixture of solutions (1-40 mm Tris (hydroxymethyl) aminomethane, preferably 10 mm, 0.05-1% vol. Tween 20, preferably 0.5% vol., 1-200 mm KCl, preferably 50 mM, 0.01-0.5 mM EDTA (ethylenediamine-Ν, Ν, Ν ', Ν'-tetraacetate), preferably 0.1 mM, 1-20% glycerol, preferably 5%, 0.01 -0.4% NaN ₃ (sodium azide), preferably 0.1%). For example, a mixture of 12.1 mg Tris (hydroxymethyl) aminomethane, 50 μl Tween 20, 37.3 mg KCl, 2 μl 0.5 Μ EDTA (ethylenediamine-Ν, Ν, Ν ', Ν'-tetraacetate), 500 μl of glycerol 10 mg NaN ₃ (sodium azide), adjusted to 10 ml with sterile deionized water.

4. The buffer liquid composition (4) is a mixture of solutions (1-40 mm Tris (hydroxymethyl) aminomethane, preferably 10 mm, 0.05-1% vol. Triton X-100, preferably 0.5% vol., 1- 200 mM KCl, preferably 50 mM, 0.01-0.5 mM EDTA (ethylenediamine-Ν, Ν, Ν ', Ν'-tetraacetate), preferably 0.1 mM, 1-20% glycerol, preferably 5%, 0 , 01-0.4% NaN ₃ (sodium azide), preferably 0.1%). For example, a mixture of 12.1 mg Tris (hydroxymethyl) aminomethane, 50 μl Triton X-100, 37.3 mg KCl, 2 μl 0.5 Μ EDTA (ethylenediamine-Ν, Ν, Ν ', Ν'-tetraacetate), 500 μl of glycerol, 10 mg NaN ₃ (sodium azide), adjusted to 10 ml with sterile deionized water.

The working surface of the hygroscopic carrier object (gauze, cotton swab, etc.) is completely impregnated in one of the four above-mentioned buffer liquid compositions in a ratio of 20-150 μl of solution per 1 cm ² (depending on the capacity of the porous carrier) of the material the carrier immediately before washing off the dry ones or after complete drying to wash off liquid traces of DNA-containing biological material (potogens, traces of blood, saliva, vaginal secretion, sperm, cell suspension, mixed ologicheskie traces DNA preparations), followed by subjecting to drying at room temperature.

When using paper, for example, chromatographic paper, as an object carrier, it is impregnated by a single application of one of the four above-mentioned buffer liquid compositions in an amount of 20-150 μl per 1 cm ² , after which the paper is dried at room temperature. A biological sample is applied in liquid form to the carrier object (washout filtrate, cell suspension, DNA preparation, etc.) in an amount of 20-1000 μl in terms of an area of 1 cm ² to 10 cm ² with a paper thickness of 0 , 15 to 1.0 mm, after which the paper is also dried at room temperature.

Thus, the present invention relates to compositions that maintain stable DNA of traces of biological samples taken by washing, or applying to a hygroscopic carrier object, providing the longest storage of DNA in a wide temperature range.

II. Another object of the present invention is a method for storing DNA preparations in a stable form in a liquid composition for storage, including:

(i) adding a buffer liquid composition of the present invention as a diluent in the final DNA isolation step;

(ii) mixing the purified DNA sample and the buffer solution of the present invention in a ratio of 1:10; and

(iii) storing the DNA preparation.

The objects of the present invention is a liquid buffer composition for storing DNA preparations in liquid form in a wide temperature range (from -70 ° C to + 40 ° C).

The present invention is a combination of the above components in a buffer:

1. The buffer liquid composition (1) is a mixture of solutions (1-40 mm Tris (hydroxymethyl) aminomethane, preferably 10 mm, 0.05-1% vol. Tween 20, preferably 0.5% vol., 1-200 mm K ₂ SO ₄ , preferably 50 mM, 0.01-0.5 mM EDTA (ethylenediamine-Ν, Ν, Ν ', Ν'-tetraacetate), preferably 0.1 mM, 1-20% glycerol, preferably 5%, 0.01-0.4% NaN ₃ (sodium azide), preferably 0.1%): 12.1 mg Tris (hydroxymethyl) aminomethane, 50 μl Tween 20, 87.1 mg K ₂ SO ₄ , 2 μl 0, 5 Μ EDTA (ethylenediamine-Ν, Ν, Ν ', Ν'-tetraacetate), 500 μl of glycerol, 10 mg NaN ₃ (sodium azide), adjusted to 10 ml of sterile deionized ode.

2. The buffer liquid composition (2) is a mixture of solutions (1-40 mm Tris (hydroxymethyl) aminomethane, preferably 10 mm, 0.05-1% vol. Triton X-100, preferably 0.5% vol., 1- 200 mM K ₂ SO ₄ , preferably 50 mM, 0.01-0.5 mM EDTA (ethylenediamine-Ν, Ν, Ν ', Ν'-tetraacetate), preferably 0.1 mM, 1-20% glycerol, preferably 5 %, 0.01-0.4% NaN ₃ (sodium azide), preferably 0.1%): 12.1 mg Tris (hydroxymethyl) aminomethane, 50 μl Triton X-100, 87.1 mg K ₂ SO ₄ , 0,5 Μ 2 ul EDTA (ethylenediamine-Ν, Ν, Ν ', Ν'- tetraacetate), 500 l of glycerol, 10 mg of NaN ₃ (sodium azide), brought to 10 ml with sterile deioniz bath water.

3. The buffer liquid composition (3) is a mixture of solutions (1-40 mm Tris (hydroxymethyl) aminomethane, preferably 10 mm, 0.05-1% vol. Tween 20, preferably 0.5% vol., 1-200 mm KCl, preferably 50 mM, 0.01-0.5 mM EDTA (ethylenediamine-Ν, Ν, Ν ', Ν'-tetraacetate), preferably 0.1 mM, 1-20% glycerol, preferably 5%, 0.01 -0.4% NaN ₃ (sodium azide), preferably 0.1%): 12.1 mg Tris (hydroxymethyl) aminomethane, 50 μl Tween 20, 37.3 mg KCl, 2 μl 0.5 Μ EDTA (ethylene diamine Ν, Ν, Ν ', Ν'-tetraacetate), 500 μl of glycerol, 10 mg NaN ₃ (sodium azide), brought to 10 ml with sterile deionized water.

4. The buffer liquid composition (4) is a mixture of solutions (1-40 mm Tris (hydroxymethyl) aminomethane, preferably 10 mm, 0.05-1% vol. Triton X-100, preferably 0.5% vol., 1- 200 mM KCl, preferably 50 mM, 0.01-0.5 mM EDTA (ethylenediamine-Ν, Ν, Ν ', Ν'-tetraacetate), preferably 0.1 mM, 1-20% glycerol, preferably 5%, 0 , 01-0.4% NaN ₃ (sodium azide), preferably 0.1%): 12.1 mg Tris (hydroxymethyl) aminomethane, 50 μl Triton X-100, 37.3 mg KCl, 2 μl 0.5 Μ EDTA (ethylenediamine-Ν, Ν, Ν ', Ν'- tetraacetate), 500 l of glycerol, 10 mg of NaN ₃ (sodium azide), brought to 10 ml with sterile deionized water.

Thus, the present invention relates to a liquid composition that maintains stable DNA, containing DNA and one of the four buffer solutions above in a ratio of 1:10, providing the longest storage of DNA preparations in a wide temperature range.

Additional objects and advantages of the invention are indicated in the following section of the description and partially become apparent from the description or can be studied by applying the invention. The objects and advantages of the invention are understood and realized by means of the elements and combinations detailed in the attached claims.

It should be understood that in the framework of the above general description and the following detailed description are only illustrative and explanatory and do not limit the scope of the invention. The complementary figures included and forming part of this description illustrate some of the advantages of the invention and, together with the description, help explain the principles of the invention.

Brief Description of the Drawings

In FIG. 1 shows the results of electrophoresis in 1% agarose gel of DNA preparations isolated using PrepFiler ™ User Guide reagent kits (Applied Biosystems, USA) (sample 1), “DNA IQ ™” (Promega, USA) (sample 2), liquid buffer compositions 1 (sample 3), 2 (sample 4) and control samples (samples 5, 6) in three parallels ((1), (2) and (3)).

In FIG. Figure 2 shows the graphs of the accumulation of PCR products, reflecting the absence of an inhibitory effect on the amplification reaction of FN100 chromatography paper, impregnated with liquid buffer composition 1 (1) or deionized water (2), as well as cotton (3) and (4).

In FIG. 3 ((1), (2)) shows the results of 2% agarose gel electrophoresis of PCR products in the presence of FN100 chromatography paper, impregnated with liquid buffer composition 1 or deionized water, and cotton.

In FIG. Figure 4 shows PCR plots of DNA swabs prepared using liquid buffer compositions 1 ((1), (2)) or 3 ((3), (4)), or deionized water ((5), (6)), which 7 ((1), (3), (5)) and 30 ((2), (4), (6)) were stored at room temperature.

In FIG. Figure 5 shows the results of electrophoresis in a 2% agarose gel of PCR products - DNA preparations obtained from swabs made using liquid buffer compositions 1 or 3, or deionized water, which were stored at room temperature for 0 days (control) (1), 5 days (2) and 7 days (3).

In FIG. Figure 6 shows the graphs of the accumulation of PCR products of saliva samples, where the solvent was deionized water, physiological saline, TE buffer or liquid buffer composition 3 (buffer 3), during incubation + 22 ° C (1) and + 70 ° C (2) for 30 minutes, as well as saliva samples, where the solvent was lysing solutions from the DNA IQ ™ System, PrepFiler ™ User Guide, Prep-n-Go, “A” reagent kits during incubation + 22 ° C (3) and + 70 ° C ( 4) within 30 minutes.

In FIG. Figure 7 shows the graphs of the accumulation of PCR products of the internal positive control (Su5 channel) in the presence of deionized water, physiological saline, TE buffer, liquid buffer composition 3 (buffer 3) (1), as well as lysing solutions from the DNA IQ ™ System, PrepFiler ™ reagent kits User Guide, Prep-n-Go, “A,” inhibiting PCR (2).

In FIG. Figure 8 shows the graphs of the accumulation of PCR products of DNA preparations from swabs of a sweat substance made from the surface of a smooth metal object using liquid buffer composition 3 (1), physiological saline (2), TE buffer (3), or deionized water (4).

The implementation of the invention

Example 1

For a comparative study of methods for extracting DNA from biological traces, we estimated the loss of DNA of known concentration during its extraction using PrepFiler ™ User Guide (Applied Biosystems, USA), “DNA IQ ™” reagents (Promega, USA) and liquid buffer compositions 1, 2 (buffers 1, 2) of the present invention. Research on each of the methods was carried out in seven parallels.

The human DNA was used as genetic material from the XY-Detect test kit (Syntol LLC) at a final concentration of 0.1 ng / μl.

1. DNA isolation using the PrepFiler ™ User Guide reagent kit (Applied Biosystems, USA) was performed according to the basic protocol for the isolation of genomic DNA.

10 μl of 0.5 ng / μl DNA preparations were placed in standard 1.5 ml microcentrifuge tubes. 300 μl of PrepFiler ™ Lysis buffer and 3 μl of 1.0 Μ DTT were added. Vortex was mixed at low speeds for 5 seconds, then briefly centrifuged. Samples were incubated in a thermal shaker at + 70 ° C and 900 rpm for 40 minutes. Microcentrifuge tubes were centrifuged at 12,000 g for 2 minutes. Transfer the lysate to clean 1.5 ml tubes. 15 μl of magnetic particles were added to the tubes containing the lysate. Vortex was mixed at low speeds for 5 seconds, then briefly centrifuged. 180 μl of isopropanol (Binding buffer) was added. Vortex was mixed at low speeds for 5 seconds, then briefly centrifuged. Tubes were placed in a shaker and mixed for 10 minutes at 1000 rpm at room temperature. Vortex samples were mixed. The tubes were placed in a magnetic tripod and held until the sediment ceased to accumulate (2 minutes). Holding the tubes in a magnetic rack, the supernatant was removed and removed. 300 μl of PrepFiler ™ Wash buffer was added to the tubes. Vortex was stirred at maximum speed so that no magnetic particles were visible on the walls of the tubes, then briefly centrifuged. Tubes were placed in a magnetic tripod for 30-60 seconds. Holding the tubes in a magnetic rack, the supernatant was removed and removed. This washing step was repeated three times. Holding the tubes in a magnetic rack, dried the magnetic particles for 7 minutes on the table. 50 μl of PrepFiler ™ Elution Buffer was added to the tubes. Vortex was stirred at a maximum speed of 5 seconds so that no magnetic particles were visible on the walls of the tubes, then briefly centrifuged. Tubes were incubated in a thermal shaker and at + 70 ° C and 900 rpm for 5 minutes. Vortex was stirred at maximum speed so that no magnetic particles were visible on the walls of the tubes, then briefly centrifuged. Put the tubes in a magnetic tripod and hold until the sediment ceases to accumulate (at least 1 minute). The supernatant (which should contain genomic DNA) was transferred to new 1.5 ml storage tubes.

2. DNA isolation using the DNA IQ ™ reagent kit (Promega, USA) was performed according to the basic protocol for the isolation of genomic DNA.

10 μl of 0.5 ng / μl DNA preparations were placed in standard 1.5 ml microcentrifuge tubes. 250 μl of Promega ™ Lysis buffer and 2.5 μl of 1.0 Μ DTT were added. Vortex was mixed at low speeds for 5 seconds, then briefly centrifuged. Samples were incubated in a thermal shaker at + 70 ° C and 900 rpm. within 30 minutes. Microcentrifuge tubes were centrifuged at room temperature at 10,000 g for 2 minutes. Transfer the lysate to clean 1.5 ml tubes. Pre-mixed, 7.0 μl of DNA IQ ™ Resin resin (suspension of magnetic particles) was added to solutions containing DNA. A mixture of the sample in lysis buffer with magnetic particles was shaken on a vortex for 3 seconds. Incubated at room temperature for 5 minutes, vortexing every 30 seconds. Set the tubes in a magnetic tripod. The solution was carefully selected and removed using an automatic pipette, being careful not to disturb the sediment of magnetic particles on the walls of the tubes. 100 μl of Promega ™ Lysis buffer was added to the pellet. The tubes were shaken on a vortex for 2 seconds. Set the tubes in a magnetic tripod and completely remove the Promega ™ Lysis buffer. 100 μl of 1X wash buffer (Promega ™ Wash Buffer) was added to the tubes. The tubes were shaken on a vortex for 2 seconds. Tubes were installed in a magnetic tripod and the wash buffer was removed. Washing with washing buffer was repeated 2 more times. Set the tubes in a magnetic tripod with the lid open. The precipitate of magnetic particles was dried at room temperature for 5 minutes. 50 μl of elution buffer (Promega ™ Elution Buffer) was added. Vortex was shaken for 2 seconds. Tubes were incubated in a microthermostat + 65 ° C for 5 minutes with constant stirring at 900 rpm. After incubation, the samples were mixed at maximum speed for 2 seconds. Put the tubes in a magnetic tripod and hold until the sediment ceases to accumulate (at least 1 minute). The supernatant (which should contain genomic DNA) was transferred to new 1.5 ml storage tubes.

3. Isolation of DNA using liquid buffer compositions 1, 2 (buffers 1, 2), proposed in the present invention.

The composition of the liquid buffer composition 1 (10 mm Tris (hydroxymethyl) aminomethane, 0.5% vol. Tween 20, 50 mm K ₂ SO ₄ , 0.1 mm EDTA (ethylenediamine-Ν, Ν, Ν ', Ν'-tetraacetate) , 5% glycerol, 0.1% NaN ₃ (sodium azide)): a mixture of solutions of 12.1 mg Tris (hydroxymethyl) aminomethane, 50 μl Tween 20, 87.1 mg K ₂ SO ₄ , 2 μl 0.5 Μ EDTA (ethylenediamine-Ν, Ν, Ν ', Ν'-tetraacetate), 500 μl of glycerol, 10 mg NaN ₃ (sodium azide), brought to 10 ml with sterile deionized water.

The composition of the liquid buffer composition 2 (10 mm Tris (hydroxymethyl) aminomethane, 0.5% vol. Triton X-100, 50 mm K ₂ SO ₄ , 0.1 mm EDTA (ethylenediamine-Ν, Ν, Ν ', Ν'- tetraacetate), 5% glycerol, 0.1% NaN ₃ (sodium azide)): a mixture of solutions of 12.1 mg Tris (hydroxymethyl) aminomethane, 50 μl Triton X-100, 87.1 mg K ₂ SO ₄ , 2 μl 0 , 5 Μ EDTA (ethylenediamine-Ν, Ν, Ν ', Ν'-tetraacetate), 500 μl of glycerol, 10 mg NaN ₃ (sodium azide), adjusted to 10 ml with sterile deionized water.

10 μl of 0.5 ng / μl DNA preparations were placed in standard 1.5 ml microcentrifuge tubes. 40 μl of liquid buffer composition 1 or 2 (buffer 1 or 2) was added. Vortex was mixed at low speeds for 5 seconds, then briefly centrifuged. Samples were incubated in a thermal shaker at + 70 ° C and 900 rpm for 30 minutes. Microcentrifuge tubes were centrifuged at 10,000 g for 30 seconds.

Control samples were prepared that were not incubated. To do this, 10 μl of the preparations of human control DNA from the ΧΥ-Detect test kit (Syntol LLC) with a concentration of 0.5 ng / μl in standard 1.5 ml microcentrifuge tubes were placed and 40 μl of deionized water were added. Vortex was mixed at low speeds for 5 seconds, then briefly centrifuged.

I. The amount of DNA in the preparations was estimated after its isolation using real-time PCR (PCR-RV) in accordance with the manufacturer's instructions. For this purpose, we used a thermal cycler with a multichannel detector for evaluating real-time PCR products iQ5 (USA) Bio-Rad.

Amplicon accumulation and calibration curves were recorded using PCR-RV technology based on hybridization-fluorescence detection using specific probes with fluorescent labels (TaqMan format). Reagent kits were used to determine the number of DNA fragments of the β-actin gene, fragments of the amelogenin gene located on the X (p22.1-22.3) and Υ (p11.2) chromosomes of the human XY-Detect (Syntol LLC). To evaluate the effectiveness of (E) PCR, a calibration curve was constructed for samples containing the following DNA concentrations: 5 ng / μl, 1 ng / μl, 0.2 ng / μl, 0.04 ng / μl and 0.008 ng / μl. For amplification of the β-actin gene, the calculated PCR efficiency was E = 0.971.

In the table. 1 shows the volumes of the components used in the preparation of the reaction mixture per tube.

Before PCR, 20 μl of the prepared reaction mixture and 5 μl of DNA preparations were added to the tubes. As a control, 5 μl of standard DNA preparations with a known concentration of 5 ng / μl, 1 ng / μl, 0.2 ng / μl, 0.04 ng / μl and 0.008 ng / μl were placed in PCR tubes with the reaction mixture.

Amplification was performed in the following mode:

PCR-RV data analysis was performed using the iQ5 Optical System Software (version 2.0, 2006).

Statistical processing of experimental data was performed using the STADIA program (Kulichev AP Methods and means of data analysis in a Windows environment. Stadia 6.0. - Μ .: Computer science and computers. - 1996. -257 p.).

The results of a comparative study of the concentration of DNA isolated using the PrepFiler ™ User Guide, DNA IQ ™ reagent kits and liquid buffer compositions 1, 2 are presented in Table. 2.

When examining the concentration of standard DNA subjected to extraction methods using PrepFiler ™ User Guide reagent kits (Applied Biosystems, USA), “DNA IQ ™” (Promega, USA) and liquid buffer compositions 1, 2 (buffers 1, 2), proposed in the present invention, it was found that the use of all investigated methods allows you to get enough DNA. A smaller amount of active DNA matrix extracted using the proposed liquid buffer compositions 1, 2 (buffers 1, 2) is noted compared with the results obtained using standard DNA isolation protocols of PrepFiler ™ User Guide reagent kits (Applied Biosystems, USA), “DNA IQ ™” (Promega, USA). This may be due to the following reasons: either the rate of DNA degradation in the proposed liquid buffer compositions 1, 2 (buffers 1, 2) is higher than during its extraction using PrepFiler ™ User Guide reagent kits (Applied Biosystems, USA), DNA IQ ™ ”(Promega, USA), or, conversely, the liquid buffer compositions 1, 2 declared in the present invention (buffers 1, 2) stabilize the high molecular weight DNA fraction, which makes it difficult for the Taq DNA polymerase enzyme to access the β-actin locus under study, thereby creating the illusion of a decrease in DNA concentration. To test these hypotheses, electrophoretic studies of the obtained DNA preparations were performed.

II. Qualitative and quantitative comparative evaluations of DNA preparations were performed using PrepFiler ™ User Guide reagent kits (Applied Biosystems, USA), DNA IQ ™ (Promega, USA), liquid buffer compositions 1, 2 (buffers 1, 2) and control samples using electrophoresis in 1% agarose gel.

Additionally, control samples were prepared, which were incubated at + 70 ° C for 30 minutes. To do this, 10 μl of the preparations of human control DNA from the XY-Detect test kit (Syntol LLC) with a concentration of 0.5 ng / μl in standard 1.5 ml microcentrifuge tubes were placed and 40 μl of deionized water were added. Vortex was mixed at low speeds for 5 seconds, then briefly centrifuged and incubated at + 70 ° C for 30 minutes.

A single TBE buffer was prepared in a volume sufficient to fill the electrophoresis chamber and gel preparation. Agarose powder was added to a single TBE buffer in the amount necessary to obtain a 1% solution and heated in a microwave oven until it was completely melted. The resulting liquid solution was cooled to + 50-60 ° C, ethidium bromide was added to a final concentration of 0.5 μg / ml and carefully mixed, avoiding the appearance of air bubbles in the gel. Warm agarose was poured into the appropriate form and made sure that it was evenly distributed over the form. The comb was vertically inserted so that its teeth did not reach the bottom of about 1 mm. An agarose gel cuvette was left for 30 minutes, then the comb was carefully removed. A gel cuvette was placed in an electrophoresis chamber containing the required amount of a single TBE buffer. The studied samples were prepared for electrophoresis by mixing them with six-fold application buffer (5: 1, v / v) containing bromophenol blue and xylene cyanol in a 30% glycerol solution. Gently introduced samples into the corresponding wells of the gel. HindIII restriction enzyme-treated DNA molecular weight marker was added to the wells designated as "M". Electrophoresis was carried out at a constant electric field strength (6.5 V / cm) for 40 minutes (until bromphenol blue reaches the end of the gel). The gel was placed in a UV transilluminator and photographed. The luminescence intensity of the experimental DNA preparations was compared with the luminescence intensity of the samples with control samples, the amount of DNA in the test sample was determined (Kornienko I.V., Kharlamov G.G. evidence of biological origin: textbook. Rostov n / a: SFedU. - 2012. - 216 p.).

Electrophoresis of DNA preparations isolated using PrepFiler ™ User Guide reagent kits (Applied Biosystems, USA), DNA IQ ™ (Promega, USA) and liquid buffer compositions 1, 2 (buffers 1, 2) in 1% agarose gel in three parallels are shown in FIG. one.

Μ - molecular weight marker Lambda DNA / HindIII Markers;

1 - DNA preparation isolated using the PrepFiler ™ User Guide;

2 - DNA preparation isolated using the DNA IQ ™ reagent kit;

3 - DNA preparation isolated using liquid buffer composition 1 (buffer 1);

4 - DNA preparation isolated using liquid buffer composition 2 (buffer 2);

5 - control DNA preparation, not subjected to incubation.

6 - control DNA preparation, incubated at + 70 ° C for 30 minutes.

A marked decrease in the concentration of nuclear DNA in preparations isolated using the PrepFiler ™ User Guide (Applied Biosystems, USA) and “DNA IQ ™” (Promega, USA) reagents was observed compared to a control DNA sample that did not undergo DNA extraction. In these samples (1 and 2 in Fig. 1), pronounced degradation and a significant loss of high molecular weight DNA are noted, which is presented as a "short" of low molecular weight DNA. DNA isolation with liquid buffer compositions 1, 2 (buffers 1, 2) (samples 3 and 4 in Fig. 1) is not accompanied by a loss of high molecular weight DNA: in the region between bands 21226 and 5148, a fraction of mitochondrial DNA (mtDNA) of 16569 size is clearly visible nucleotide pairs. Thanks to the components that make up the liquid buffer compositions 1, 2, the DNA molecules are stabilized and protected from degradation: the high molecular weight bands of nuclear DNA are clearly visible in the wells of the gel, while in the control preparation incubated at + 70 ° C for 30 minutes, DNA is not visualized or poorly visualized. Thus, it was found that the liquid buffer compositions 1, 2 (buffers 1, 2) of the present invention contribute to DNA stabilization.

III. To assess the quality of DNA isolation methods using PrepFiler ™ User Guide reagent kits (Applied Biosystems, USA), “DNA IQ ™” (Promega, USA) and the method using liquid buffer compositions 1, 2 (buffers 1, 2), proposed in of the present invention, DNA loci were genotyped at the STR loci of the COrDIS Plus panel (D3S1358, THO1, D12S391, D1S1656, D10S1248, D22S1045, D2S441, D7S820, D13S317, FGA, TPOX, D18S51, D16S539, D8S115, D8S118, D8S1, D8S118, D8S118 , SE33 and the Amelogenin locus, which determines the gender) of the COrDIS Plus reagent kit for DNA identification of 19 markers and the human amelogenin locus in accordance with tion of the company's products (COrDIS Plus Reagent kit for multiplex analysis of 19 STR-markers and human amelogenin locus User Manual -..., 2014. - 19 p.) "Gordiz" Ltd. (registration certificate №FSR 2012/13548).

For enzymatic amplification, PCR tubes from the kit containing lyophilized PCR components (Taq polymerase, dNTP mixture, reaction buffer, primer mixture) were added with 10 μl of deionized water, then 5 μl of activator solution containing buffer solution and ions were added magnesium Mg ²⁺ . Then, 10 μl of the studied DNA preparations were added. The mixture was stirred and briefly centrifuged. To assess the specificity of the amplification reaction, the positive (test with control DNA MK1 from the COrDIS Plus reagent kit) and negative (test with deionized water instead of DNA preparation) controls were used.

During amplification, the heating rate was 0.3 ° C / s at the stage of increasing from a temperature from 59 ° C to 72 ° C.

Used the following PCR parameters:

94 ° C 3 min 98 ° C 30 sec 59 ° C * 120 sec, 4 cycles 72 ° C 90 sec 94 ° C 30 sec 59 ° C * 120 sec, 6 cycles 72 ° C 90 sec 90 ° C 30 sec 59 ° C 120 sec, 20 cycles 72 ° C * 75 sec 68 ° C 10 min 15 ° C ∞

* Heating rate from 59 ° C to 72 ° C - not more than 0.3 ° C / 1 sec.

Amplification was performed using a GeneAmp PCR System 9700 thermal cycler (Applied Biosystems) with an aluminum block in the GeneAmp 9600 emulation mode.

Alleles were identified according to the S450 size standard and with respect to the allelic leader attached to the COrDIS Plus test system.

To load samples into the device, a mixture of Hi-Di ™ formamide and S450 size standard was prepared in the following ratio: 10 μl of Hi-Di ™ formamide and 1 μl S450 size standard per sample.

The amplification products were identified by the STR loci of the COrDIS Plus panel using an ABI PRISM 3130xl automatic sequencer in accordance with the instructions for use (COrDIS Plus. Reagent kit for multiplex analysis of 19 STR markers and a human amelogenin locus. User manual. - 2014. - 19 pp. And Guidance on the use of the genetic analyzer 3130 / 3130xl. - Applied Biosystems).

In the process of electrophoresis, information about laser gel scanning and fluorescence detection was automatically transferred to the control computer and processed by Run 3130 Data Collection v.3.0.

Processing of the results and identification of alleles was carried out automatically using the GeneMapper ^® ID 3.2 program.

Statistical processing of experimental data was carried out using the STADIA program (Kulachev A.P. Methods and means of data analysis in a Windows environment. Stadia 6.0. - Μ .: Computer science and computers. - 1996. - 257 p.).

The results of genotyping of experimental and control samples are presented in table. 3.

Notes: "+" - indicates the presence of all allelic states in the studied loci; "+/-" - indicates the loss of one of a pair of alleles (the so-called effect of "false homozygous") in the studied loci; “-” - indicates the absence of the amplification product (loss of all alleles) in the studied loci.

When genotyping DNA preparations isolated using the PrepFiler ™ User Guide reagent kit (Applied Biosystems, USA), it is possible to obtain a complete genetic profile for all the studied loci of the COrDIS Plus system (100% of cases), and when using the DNA IQ ™ reagent kit ( Promega, USA) - only 61.7%. When genotyping DNA preparations isolated using the proposed liquid buffer compositions 1, 2 (buffers 1, 2), it is possible to obtain a complete genetic profile for the 18 studied loci of the COrDIS Plus system and the human amelogenin locus, except for the D22S1045 locus.

Example 2

The extracted effective DNA template was quantified by storage on FN100 chromatography paper impregnated in deionized water or liquid buffer composition 1 (buffer 1) of the present invention, as well as a comparative study of the inhibitory effect of carrier materials (chromatographic paper and cotton fabric).

The composition of the liquid buffer composition 1 (10 mm Tris (hydroxymethyl) aminomethane, 0.5% vol. Tween 20, 50 mm K ₂ SO ₄ , 0.1 mm EDTA (ethylenediamine-Ν, Ν, Ν ', Ν'-tetraacetate) , 5% glycerol, 0.1% NaN ₃ (sodium azide)): a mixture of solutions of 12.1 mg Tris (hydroxymethyl) aminomethane, 50 μl Tween 20, 87.1 mg K ₂ SO ₄ , 2 μl 0.5 Μ EDTA (ethylenediamine-Ν, Ν, Ν ', Ν'-tetraacetate), 500 μl of glycerol, 10 mg NaN ₃ (sodium azide), brought to 10 ml with sterile deionized water.

Chromatographic paper FN100 (series 527, specific gravity 195 g / m ² , thickness 0.35 mm, absorption height 130 mm / 30 min) and cotton fabric (fabric with a tight weave of fibers and gauze) were used as porous carrier objects.

As the genetic material, control human DNA was used from the XY-Detect test kit (Syntol LLC).

To quantify the recovered effective DNA template stored on the carrier, FN100 chromatography paper was impregnated with 0.5 × 0.5 cm 25 μl of buffer 1 or deionized water with the addition of control DNA at a final concentration of 0.1 ng / μl .

To conduct a comparative study of the inhibitory effect on PCR of carrier objects, FN100 chromatography paper of 0.5 × 0.5 cm size was impregnated with 25 μl of buffer 1 or deionized water.

Immediately after the impregnation, each carrier object was transferred to a sterile intact plastic surface and dried for 24 hours at + 22 ° C in a closed PCR box without UV radiation and contact with foreign objects.

The potential inhibitory effect of cotton fabric (fabrics with tight weaving of fibers and gauze) was studied without impregnation in buffer 1 or deionized water.

Two discs with a diameter of 1.2 mm were cut from carrier objects using sterile scissors and a puncher (Harris Uni-Core), which were placed in PCR tubes. Studies of each carrier item were performed in six parallels.

I. The amount of DNA and the inhibitory effect on the PCR of carrier materials by PCR-PB were evaluated in accordance with the manufacturer's instructions. For this purpose, we used a thermal cycler with a multichannel detector for evaluating real-time PCR products iQ5 (USA) Bio-Rad.

Amplicon accumulation and calibration curves were recorded using PCR-RV technology based on hybridization-fluorescence detection using specific probes with fluorescent labels (TaqMan format). Reagent kits were used to determine the number of DNA fragments of the β-actin gene, fragments of the amelogenin gene located on the X (p22.1-22.3) and Υ (p11.2) chromosomes of the human XY-Detect (Syntol LLC). To evaluate the effectiveness of (E) PCR, a calibration curve was constructed for samples containing the following DNA concentrations: 5 ng / μl, 1 ng / μl, 0.2 ng / μl, 0.04 ng / μl and 0.008 ng / μl. For amplification of the β-actin gene, the calculated PCR efficiency was E = 0.971.

In the table. 4 shows the volumes of components used in the preparation of the reaction mixture per tube.

Before PCR, 22.5 μl of the prepared reaction mixture and 2.5 μl of deionized water (in samples where the carrier was impregnated with buffer 1 or deionized water containing DNA) or 2.5 each were added to test tubes with fragments of the carrier object. μl of human control DNA from the XY-Detect test kit (Synthol) at a concentration of 15 ng / μl (in samples where the carrier is impregnated with buffer 1 or deionized water that does not contain DNA or is impregnated with nothing (cotton) . As a control, 2.5 μl of standard DNA preparations with a known concentration of 5 ng / μl, 1 ng / μl, 0.2 ng / μl, 0.04 ng / μl and 0.008 ng / were placed in PCR tubes with the reaction mixture. μl

Amplification was performed in the following mode:

PCR-RV data analysis was performed using the iQ5 Optical System Software (version 2.0, 2006).

The results of a comparative study of the concentration of DNA extracted from a carrier object (FN100 chromatography paper), impregnated with liquid buffer composition 1 (buffer 1) or deionized water, are presented in table. 5.

The results of a comparative study of the concentration of DNA and the inhibitory effect on PCR of carrier objects are presented in table. 6 and in FIG. 2.

Note: C _t (Threshold Cycle) - threshold cycle.

According to the results of a comparative study of the concentration of DNA extracted from a carrier object (FN100 chromatography paper), it was found that more than 1.5 times more it is possible to obtain an active DNA template from paper impregnated with deionized water rather than buffer 1. The result obtained can be explained by a small degree of inhibition of the enzyme DNA-Taq polymerase in the presence of buffer 1.

In FIG. 2, on the graphs of the accumulation of PCR products, starting from 25-26 cycles, one can see a rise in the curves of all experimental DNA samples in the presence of FN100 chromatography paper impregnated with liquid buffer composition 1 (buffer 1) or deionized water, and in the presence of cotton fabric (with dense weaving fibers and gauze). The results obtained indicate the absence of a significant inhibitory effect on PCR of both the components of the proposed liquid buffer composition 1 (buffer 1) and the material of the potential subject carrier of biological samples.

II. A qualitative and quantitative comparative evaluation of PCR-PB products was carried out using electrophoresis in a 2% agarose gel.

A single TBE buffer was prepared in a volume sufficient to fill the electrophoresis chamber and gel preparation. Agarose powder was added to a single TBE buffer in the amount necessary to obtain a 2% solution and heated in a microwave oven until it was completely melted. The resulting liquid solution was cooled to + 50-60 ° C, ethidium bromide was added to a final concentration of 1.0 μg / ml (at 0.8% it was 0.5 μg / ml) and carefully mixed, avoiding the appearance of air bubbles in the gel. Warm agarose was poured into the appropriate form and made sure that it was evenly distributed over the form. The comb was vertically inserted so that its teeth did not reach the bottom of about 1 mm. An agarose gel cuvette was left for 30 minutes, then the comb was carefully removed. A gel cuvette was placed in an electrophoresis chamber containing the required amount of a single TBE buffer. The studied samples were prepared for electrophoresis by mixing them with six-fold application buffer (5: 1, v / v) containing bromophenol blue and xylene cyanol in a 30% glycerol solution. Gently introduced samples into the corresponding wells of the gel. Bench Tor 100 bp DNA Ladder molecular weight marker was added to the wells designated “M”. Electrophoresis was carried out at a constant electric field strength (6.5 V / cm) for 40 minutes (until bromphenol blue reaches the end of the gel). The gel was placed in a UV transilluminator and photographed. The luminescence intensity of the experimental DNA preparations was compared with the luminescence intensity of the samples with control samples, determining the amount of DNA in the test sample (Kornienko I.V., Kharlamov S.G. Methods of investigation of human DNA. DNA isolation and its quantitative assessment in the aspect of forensic research of material evidence of biological origin: textbook. Rostov n / a: SFedU. - 2012. - 216 p.).

The results of electrophoresis of PCR products obtained by PCR-RV in a 2% agarose gel are shown in FIG. 3 in two parallels (1) and (2).

Μ - molecular weight marker Bench Thor 100 bp DNA Ladder;

1, 7 - FN100 chromatography paper, impregnated with buffer 1 with the addition of DNA;

2, 8 - FN100 chromatography paper impregnated with deionized water with the addition of DNA;

3, 9 - FN100 chromatography paper impregnated with buffer 1;

4, 10 - FN100 chromatography paper impregnated with deionized water;

5, 11 - Cotton fabric (with dense weaving of fibers);

6, 12 - Cotton fabric (gauze);

K1 - Control 1 ng / μl;

K2 - Control 0.2 ng / μl;

K3 Control 0.04 ng / μl;

K4 - Control 0.008 ng / μl.

In FIG. 3 ((1), (2)), the strips of the PCR product of the experimental samples are clearly visualized, corresponding to those in the control samples, which indicates the presence of a high-quality PCR product in the presence of components of liquid buffer composition 1 (buffer 1) and potential carrier objects ( chromatographic paper, cotton) biological material. The results obtained indicate the absence of the inhibitory effect exerted on PCR by the components included in buffer 1 and the studied materials, which makes it possible to use them as carriers of biological material and use them for conducting “direct” PCR.

Example 3

A comparative study of DNA degradation in washes performed on cotton fabric soaked in deionized water or liquid buffer compositions 1, 3 (buffers 1, 3) of the present invention.

The composition of the liquid buffer composition 1 (10 mm Tris (hydroxymethyl) aminomethane, 0.5% vol. Tween 20, 50 mm K ₂ SO ₄ , 0.1 mm EDTA (ethylenediamine-Ν, Ν, Ν ', Ν'-tetraacetate) , 5% glycerol, 0.1% NaN ₃ (sodium azide)): a mixture of solutions of 12.1 mg Tris (hydroxymethyl) aminomethane, 50 μl Tween 20, 87.1 mg K ₂ SO ₄ , 2 μl 0.5 Μ EDTA (ethylenediamine-Ν, Ν, Ν ', Ν'-tetraacetate), 500 μl of glycerol, 10 mg NaN ₃ (sodium azide), brought to 10 ml with sterile deionized water.

The composition of the liquid buffer composition 3 (10 mm Tris (hydroxymethyl) aminomethane, 0.5% vol. Tween 20, 50 mm KCl, 0.1 mm EDTA (ethylenediamine-Ν, Ν, Ν ', Ν'-tetraacetate), 5% glycerol, 0.1% NaN ₃ (sodium azide)): a mixture of solutions of 12.1 mg Tris (hydroxymethyl) aminomethane, 50 μl Tween 20, 37.3 mg KCl, 2 μl 0.5 Μ EDTA (ethylenediamine-Ν, Ν , Ν ', Ν'-tetraacetate), 500 μl of glycerol, 10 mg NaN ₃ (sodium azide), brought to 10 ml with sterile deionized water.

The human DNA was used as genetic material from the XY-Detect test kit (Syntol LLC) at a final concentration of 0.25 ng / μl. 20 μl of DNA preparations were applied on a sterile intact plastic surface and dried for two days at + 22 ° C in a closed PCR box without access to UVI. Washings were performed using sterile cotton cloth 0.5 × 0.5 cm in size moistened with 15 μl of deionized water or liquid buffer compositions 1 and 3. The performed washings were stored at + 22 ° C in a closed PCR box without UVI access. At the end of the storage period, 15 μl of deionized water was applied to the tissue to dissolve the deposited and dried DNA.

Groups were formed: control (0 days, that is, the washings were not stored) and washings stored for 7 and 30 days. Each storage period corresponded to 15 parallels in the corresponding medium. Washed tissue was placed in plastic columns with a trellised bottom to squeeze the carrier object, which was inserted into 1.5 ml Eppendorf tubes. The tubes were centrifuged at 13000-14000 g for 5 minutes.

I. Assessed the amount of DNA in the filtrate using PCR-PB in accordance with the manufacturer's instructions. For this purpose, we used a thermal cycler with a multichannel detector for evaluating real-time PCR products iQ5 (USA) Bio-Rad.

Amplicon accumulation and calibration curves were recorded using PCR-RV technology based on hybridization-fluorescence detection using specific probes with fluorescent labels (TaqMan format). Reagent kits were used to determine the number of DNA fragments of the β-actin gene, fragments of the amelogenin gene located on the X (p22.1-22.3) and Υ (p11.2) chromosomes of the human XY-Detect (Syntol LLC). To evaluate the effectiveness of (E) PCR, a calibration curve was constructed for samples containing the following DNA concentrations: 5 ng / μl, 1 ng / μl, 0.2 ng / μl, 0.04 ng / μl and 0.008 ng / μl. For amplification of the β-actin gene, the calculated PCR efficiency was E = 0.971.

In the table. 7 shows the volumes of components used in the preparation of the reaction mixture per tube.

Before PCR, in a test tube with 23 μl of the prepared reaction mixture, 2 μl of the filtrate of the performed washings were added. As a control, 2 μl of standard DNA preparations with a known concentration of 5 ng / μl, 1 ng / μl, 0.2 ng / μl, 0.04 ng / μl and 0.008 ng / μl were placed in PCR tubes with the reaction mixture.

Amplification was performed in the following mode:

Real-time PCR data analysis was performed using the iQ5 Optical System Software (version 2.0, 2006).

Statistical processing of experimental data was performed using the STADIA program (Kulichev AP Methods and means of data analysis in a Windows environment. Stadia 6.0. - Μ .: Computer science and computers. - 1996. -257 p.).

The concentration of DNA stored in various media for 0, 7 and 30 days, in dry form at room temperature, is presented in table. 8. The plots of the PCR curves are shown in FIG. four.

Note: “±” - stands for standard deviation.

A comparison of differences in the concentration of DNA stored in various media for 0, 7 and 30 days in dry form at room temperature is presented in table. 9.

A noticeable decrease in the concentration of the active DNA matrix over time in flushes stored at room temperature using deionized water is observed: after 7 days, the DNA concentration decreases by 7.5 times, and after 30 days by more than 18.5 times . In the flushes that were performed using the proposed liquid buffer compositions 1 and 3 (buffers 1 and 3), the concentration of the active DNA matrix decreased after 7 days in 4.9 (buffer 1) and 3.2 (buffer 3) times, and a month later, by 17.5 (buffer 1) and 7.9 (buffer 3) times compared with the control (0 days) (Table 8). It should be noted that after 30 days there is no significant difference in the concentration of DNA stored in washes performed using liquid buffer composition 1 (buffer 1) and deionized water, while liquid buffer composition 3 (buffer 3) continues to exert its stabilizing effect on DNA. That is, for example, storing washings made using the proposed liquid buffer composition 3 (buffer 3) for 30 days is tantamount to storing washings made using deionized water for one week.

In FIG. Figure 4 on the graphs of the accumulation of PCR products shows that after the 35th cycle, an increase in the curves of DNA washout samples performed by deionized water is stored for 7 and 30 days. Moreover, the nature of these curves is unstable and in some samples there is no rise, which reflects an extremely low content or absence of an active DNA matrix in these samples. In samples of DNA washes performed using the proposed liquid buffer compositions 1, 3 (buffers 1, 3), the rise of the curves on the PCR plots always started up to the 35th cycle and was characterized by relative stability of the curves.

In dry form when impregnated with absorbent carrier materials (for example, cotton fabric) of the buffer liquid compositions of the present invention, the shelf life of the biological material DNA at room temperature is significantly longer than in liquid form. This is due to the extremely low content or absence of one of the key components of the hydrolysis of intramolecular phosphodiester bonds - water.

II. A qualitative and quantitative comparative evaluation of PCR-PB products was carried out by electrophoresis in a 2% agarose gel. We studied samples obtained from swabs carried out by the proposed buffer liquid compositions 1, 3 (buffers 1, 3) or deionized water, which were stored for 0 (control), 5 and 7 days.

A single TBE buffer was prepared in a volume sufficient to fill the electrophoresis chamber and gel preparation. Agarose powder was added to a single TBE buffer in the amount necessary to obtain a 2% solution, and heated in a microwave oven until it was completely melted. The resulting liquid solution was cooled to + 50-60 ° C, ethidium bromide was added to a final concentration of 1.0 μg / ml (at 0.8% it was 0.5 μg / ml) and carefully mixed, avoiding the appearance of air bubbles in the gel. Warm agarose was poured into the appropriate form and made sure that it was evenly distributed over the form. The comb was vertically inserted so that its teeth did not reach the bottom of about 1 mm. An agarose gel cuvette was left for 30 minutes, then the comb was carefully removed. A gel cuvette was placed in an electrophoresis chamber containing the required amount of a single TBE buffer. The studied samples were prepared for electrophoresis by mixing them with six-fold application buffer (5: 1, v / v) containing bromophenol blue and xylene cyanol in a 30% glycerol solution. Gently introduced samples into the corresponding wells of the gel. Bench Tor 100 bp DNA Ladder molecular weight marker was added to the wells designated “M”. Electrophoresis was carried out at a constant electric field strength (6.5 V / cm) for 40 minutes (until bromphenol blue reaches the end of the gel). The gel was placed in a UV transilluminator and photographed. The luminescence intensity of the experimental DNA preparations was compared with the luminescence intensity of the samples with control samples, determining the amount of DNA in the test sample (Kornienko I.V., Kharlamov S.G. Methods of investigation of human DNA. DNA isolation and its quantitative assessment in the aspect of forensic research of material evidence of biological origin: textbook. Rostov n / a: SFedU. - 2012. - 216 p.).

The results of electrophoresis in a 2% agarose gel of PCR-PB products of samples stored for 0 days (control) (1), 5 days (2) and 7 days (3) at room temperature are shown in FIG. 5.

Μ - molecular weight marker Bench Thor 100 bp DNA Ladder;

1-5: washings made with deionized water;

6-10: washings made with a liquid buffer composition 1 (buffer 1);

11-15: washings made with liquid buffer composition 3 (buffer 3);

K1 - Control 5 ng / μl;

K2 - Control 1 ng / μl;

K3 - Control 0.2 ng / μl;

K4 - Control 0.04 ng / μl;

K5 - Control 0.008 ng / μl.

In FIG. 5, strips of a high molecular weight PCR product of samples obtained from swabs carried out by the proposed buffer liquid compositions 1, 3 (buffers 1, 3), which were stored for 0 (control) (FIG. 5 (1)), 5 (FIG. 5 (2)) and 7 (Fig. 5 (3)) (excluding samples 6, 9, 13 after 7 days) days, corresponding to those in the control samples, which indicates the presence of a high-quality PCR product in the presence of components of buffers 1 and 3. In this case, strips of a high molecular weight PCR product of samples obtained from washes carried out by deionises storage of water at room temperature for only 5 days (Fig. 5 (2)) is poorly visualized in several samples at room temperature, and on 7 days (Fig. 5 (3)) are absent in all samples.

III. To assess the degradation of DNA in leachates performed on cotton cloth soaked in deionized water and liquid buffer compositions 1 and 3, stored for 0 (control), 7 and 30 days at room temperature, DNA loci were genotyped at STR loci COrDIS Plus panels (D3S1358, THO1, D12S391, D1S1656, D10S1248, D22S1045, D2S441, D7S820, D13S317, FGA, TROX, D18S51, D16S539, D8S1179, local identification, CSF1PO, D5S821 D, locAW, vA, VA, VA, COrDIS Plus reagents for DNA identification of 19 markers and the human amelogenin locus in accordance with the guidelines Twomey company manufacturer (COrDIS Plus Reagent kit for multiplex analysis of 19 STR-markers and human amelogenin locus User Manual -... 2014 -19 pp.) "Gordiz" Ltd. (registration certificate №FSR 2012/13548).

For enzymatic amplification, PCR tubes from the kit containing lyophilized PCR components (Taq polymerase, dNTP mixture, reaction buffer, primer mixture) were added with 10 μl of deionized water, then 5 μl of activator solution containing buffer solution and ions were added magnesium MD ²⁺ . Then, 10 μl of the studied DNA preparations were added. The mixture was stirred and briefly centrifuged. To assess the specificity of the amplification reaction, the positive (test with control DNA MK1 from the COrDIS Plus reagent kit) and negative (test with deionized water instead of DNA preparation) controls were used.

During amplification, the heating rate was 0.3 ° C / s at the stage of increasing from a temperature from 59 ° C to 72 ° C.

Used the following PCR parameters:

94 ° C 3 min 98 ° C 30 sec 59 ° C * 120 sec, 4 cycles 72 ° C 90 sec 94 ° C 30 sec 59 ° C * 120 sec, 6 cycles 72 ° C 90 sec 90 ° C 30 sec 59 ° C 120 sec, 20 cycles 72 ° C * 75 sec 68 ° C 10 min 15 ° C ∞

* Heating rate from 59 ° C to 72 ° C - not more than 0.3 ° C / 1 sec.

Amplification was performed using a GeneAmp PCR System 9700 thermal cycler (Applied Biosystems) with an aluminum block in the GeneAmp 9600 emulation mode.

Alleles were identified according to the S450 size standard and with respect to the allelic leader attached to the COrDIS Plus test system.

To load samples into the device, a mixture of Hi-Di ™ formamide and S450 size standard was prepared in the following ratio: 10 μl of Hi-Di ™ formamide and 1 μl S450 size standard per sample.

The amplification products were identified by the STR loci of the COrDIS Plus panel using an ABI PRISM 3130xl automatic sequencer in accordance with the instructions for use (COrDIS Plus. Reagent kit for multiplex analysis of 19 STR markers and the human amelogenin locus. User manual. - 2014. - 19 p.) And (Guidance on the use of the genetic analyzer 3130 / 3130xl. - Applied Biosystems).

In the process of electrophoresis, information about laser gel scanning and fluorescence detection was automatically transferred to the control computer and processed by Run 3130 Data Collection v.3.0.

Processing of the results and identification of alleles was carried out automatically using the GeneMapper® ID 3.2 program.

Statistical processing of experimental data was performed using the STADIA program (Kulichev AP Methods and means of data analysis in a Windows environment. Stadia 6.0. - Μ .: Computer science and computers. - 1996. -257 p.).

The results of prototyping genotyping are presented in table. 10. The average values presented in Table 10 are calculated from the typing results of the samples after they were stored on a cotton carrier impregnated with the appropriate medium for 7 days (15 parallels) and 30 days (5 parallels).

Notes: "+" - indicates the presence of all allelic states in the studied loci; "+/-" - indicates the loss of one of a pair of alleles (the so-called effect of "false homozygous") in the studied loci; “-” - indicates the absence of the amplification product (loss of all alleles) in the studied loci.

A noticeable decrease in PCR products over time was observed in washes stored at room temperature using deionized water: after 7 days, alleles were identified in 8.3% of the studied loci, and after 30 days only 2%. In the flushes that were performed using the proposed liquid buffer composition 1 (buffer 1), after 7 days it was possible to identify alleles in 40.4%, and after 30 days in 21% of the studied loci. In the flushes performed using the proposed liquid buffer composition 3 (buffer 3), after 7 days it is possible to identify alleles in 37.5%, and after 30 days in 43% of the studied loci.

Thus, when storing traces of biological origin in washes using deionized water, there is a significant degradation of DNA over time. The use of the proposed liquid buffer compositions 1, 3 (buffers 1 and 3) as a medium for carrying out washings of traces of biological origin and further storage of the performed washings at room temperature helps to stabilize and increases the safety of DNA on the carrier. It should be noted that the use of liquid buffer composition 3 (buffer 3) helps to obtain more genetic information about the stored object.

Example 4

We studied the efficiency of DNA extraction from cells, as well as the possibility of simultaneous "direct" PCR of the obtained samples by evaluating the inhibitory potential of the compared solvents, including the liquid buffer composition 3 (buffer 3), proposed in the present invention. The following solutions were used:

1. The composition of the liquid buffer composition 3 (10 mm Tris (hydroxymethyl) aminomethane, 0.5% vol. Tween 20, 50 mm KCl, 0.1 mm EDTA (ethylenediamine-Ν, Ν, Ν ', Ν'-tetraacetate), 5% glycerol, 0.1% NaN ₃ (sodium azide)): a mixture of solutions of 12.1 mg Tris (hydroxymethyl) aminomethane, 50 μl Tween 20, 37.3 mg KCl, 2 μl 0.5 Μ EDTA (ethylenediamine-Ν , Ν, Ν ', Ν'-tetraacetate), 500 μl of glycerol, 10 mg NaN ₃ (sodium azide), brought to 10 ml with sterile deionized water.

2. Saline solution containing NaCl at a concentration of 150 mm.

3. Lysing solution, conventionally referred to in this example as “A”, containing: 10 mM Tris-HCl, pH 7.5, 10 mM Na ₂ EDTA, 50 mM NaCl, 2% SDS and deionized water.

4. Commercial Lysis buffer of the DNA IQ ™ System reagent kit (Promega, USA).

5. Commercial Lysis buffer of the PrepFiler ™ User Guide reagent kit (Applied Biosystems, USA).

6. Commercial lysing solution Prep-n-Go (Applied Biosystems, USA).

7. TE buffer containing 10 mM Tris-HCl pH = 8.0, 0.1-1.0 mM EDTA and deionized water.

8. Deionized water.

To assess the efficiency of cell lysis using the compared solutions, the buccal epithelium in the saliva of eight volunteers was used as genetic material. Saliva was taken on an empty stomach 30 minutes after rinsing the oral cavity with water.

290 μl of each solution was placed in 0.5 ml tubes in two parallel. 10 μl of saliva samples were added. Vortex was mixed at low speeds for 10 seconds, then briefly centrifuged. The first parallel of the samples was left at room temperature (+ 22 ° C) for 30 minutes. Tubes of the second parallel were placed in a thermal shaker and incubated at + 70 ° C for 30 minutes. After incubation, the samples were mixed on a vortex at low speed for 5 seconds, then briefly centrifuged.

The inhibitory effect on the PCR of the test solutions was evaluated using PCR-RV in accordance with the manufacturer's instructions. A thermal cycler with a multichannel detector was used to evaluate the real-time PCR products of iQ5 (USA) Bio-Rad.

Amplicon accumulation and calibration curves were recorded using PCR-RV technology based on hybridization-fluorescence detection using specific probes with fluorescent labels (TaqMan format). Reagent kits were used to determine the number of DNA fragments of the β-actin gene, fragments of the amelogenin gene located on the X (p22.1-22.3) and Υ (p11.2) chromosomes of the human XY-Detect (Syntol LLC).

In the table. 11 shows the volumes of the components used in the preparation of the reaction mixture per tube.

Before PCR, 5 μl of the test samples were added to tubes with 20 μl of the prepared reaction mixture. To control the inhibitory effect on PCR, 4 μl of test solutions of 1-7 or deionized water without adding saliva samples and 1 μl of human control DNA from the XY-Detect test kit (Synthol LLC) were placed in test tubes with the reaction mixture. concentration of 5 ng / μl.

Amplification was performed in the following mode:

Real-time PCR data analysis was performed using the iQ5 Optical System Software (version 2.0, 2006).

Statistical processing of experimental data was performed using the STADIA program (Kulichev AP Methods and means of data analysis in a Windows environment. Stadia 6.0. - Μ .: Computer science and computers. - 1996. -257 p.).

The results of a comparative study of the inhibitory effect on PCR of the studied solutions in the composition of DNA preparations obtained from saliva samples that were incubated at + 22 ° C for 30 minutes by inhibiting the PCR of the β-actin gene (FAM channel) are presented in Table. 12 and in FIG. 6 (graphs (1) and (3)).

Notes: C _t (Threshold Cycle) - threshold cycle _; ΔC _t is the difference between the control (drug 8) and the experimental (drugs 1-7) threshold cycles; N / A - No PCR product.

The results of a comparative study of the inhibitory effect on PCR of the studied solutions in the composition of DNA preparations obtained from saliva samples, incubated at + 70 ° C for 30 minutes, inhibition of β-actin gene PCR inhibition (FAM channel) are presented in Table. 13 and in FIG. 6 (graphs (2) and (4))

Notes: C _t (Threshold Cycle) - threshold cycle; ΔC _t is the difference between the control (drug 8) and the experimental (drugs 1-7) threshold cycles; N / A - No PCR product.

The results of a comparative study of the inhibitory effect on PCR of the studied solutions, including liquid buffer composition 3 (buffer 3), on the inhibition of PCR of the internal positive control (channel Su5) are presented in table. 14 and in FIG. 7 (graphs (1) and (2)).

Notes: C _t (Threshold Cycle) - threshold cycle _; ΔC _t is the difference between the control (drug 8) and the experimental (drugs 1-7) threshold cycles; N / A - No PCR product.

In FIG. 6, the graphs of the accumulation of PCR products show that, starting from the 27th cycle, the curves of DNA samples rise, incubated at + 22 ° C for 30 minutes, in which the solvent is deionized water, saline and TE buffer, and from 28 - samples with a liquid buffer composition 3 (buffer 3) (Fig. 6 (1)). Incubation of saliva samples (buccal epithelium) at + 70 ° C for 30 minutes contributes to the destruction of cell structures, which facilitates access of the DNA Taq polymerase enzyme and primers to the DNA matrix. Therefore, the rise in the curves of saliva samples incubated at + 70 ° C for 30 minutes in deionized water, saline or liquid buffer composition 3 (buffer 3) begins already in the 26th cycle, and in the TE buffer - from the 27th cycle. The nature of the curves is more uniform and smooth (Fig. 6 (2)). Samples in which the lysing solutions from the DNA IQ ™ System reagent kits, PrepFiler ™ User Guide and Prep-n-Go lysing solutions, “A” were the solvent, the curves did not rise as at + 22 ° C (Fig. 6 (3)) , and at + 70 ° C (Fig. 6 (4)) incubation, which reflects the complete inhibition of PCR by the components included in their composition, and the inability to conduct a “direct” PCR in contrast to other solvents, including the proposed liquid buffer composition 3 ( buffer 3).

In FIG. 7 shows the absence of the inhibitory effect of deionized water, physiological saline, TE buffer, liquid buffer composition 3 (buffer 3) (1), and the inhibitory effect on PCR of the internal positive control (Su5 channel) of lysing solutions from DNA IQ ™ System reagents, PrepFiler ™ User Guide, Prep-n-Go, “A” (2).

Example 5

A comparative study of biological overlaps containing a small amount of genetic material was carried out, using the example of human potogira substances left on the surface of artificial leather made using the liquid buffer compositions 1, 3 (buffers 1, 3) of the present invention or deionized water. The influence of the incubation modes of the studied samples on the amount of DNA obtained and the efficiency of its genotyping were evaluated.

1. The composition of the liquid buffer composition 1 (10 mm Tris (hydroxymethyl) aminomethane, 0.5% vol. Tween 20, 50 mm K ₂ SO ₄ , 0.1 mm EDTA (ethylenediamine-Ν, Ν, Ν ', Ν'- tetraacetate), 5% glycerol, 0.1% NaN ₃ (sodium azide)): a mixture of solutions of 12.1 mg Tris (hydroxymethyl) aminomethane, 50 μl Tween 20, 87.1 mg K ₂ SO ₄ , 2 μl 0.5 Μ EDTA (ethylenediamine-Ν, Ν, Ν ', Ν'-tetraacetate), 500 μl of glycerol, 10 mg NaN ₃ (sodium azide), adjusted to 10 ml with sterile deionized water.

2. The composition of the liquid buffer composition 3 (10 mm Tris (hydroxymethyl) aminomethane, 0.5% vol. Tween 20, 50 mm KCl, 0.1 mm EDTA (ethylenediamine-Ν, Ν, Ν ', Ν'-tetraacetate), 5% glycerol, 0.1% NaN ₃ (sodium azide)): a mixture of solutions of 12.1 mg Tris (hydroxymethyl) aminomethane, 50 μl Tween 20, 37.3 mg KCl, 2 μl 0.5 Μ EDTA (ethylenediamine-Ν , Ν, Ν ', Ν'-tetraacetate), 500 μl of glycerol, 10 mg NaN ₃ (sodium azide), brought to 10 ml with sterile deionized water.

As the genetic material, we used the endocrine discharges of the fingerpads of 20 people (10 men and 10 women) aged 18 to 48 years.

Before the study, the hands of the subjects were not exposed to water and cleaned for 1-2 hours. The application of the fingertips of the fingertips of both hands on a pre-sterile surface of artificial leather tissue cleaned from the possible presence of extraneous DNA (composition: 62% polyamide, 34% polyethylene fiber, 4% elastane) was carried out by their single contact with moderate pressing force and rolling for 5 seconds. The contact field for each finger was 6 cm ² . Biological material was removed 20-30 minutes after printing by swabbing on sterile cotton cloth 0.5 × 0.5 cm in size, soaked in 25 μl of liquid buffer compositions (buffer 1 or 3), or deionized water. During the experiment, the studied surface of the artificial skin was at room temperature, without direct exposure to sunlight and contact with foreign objects. Each wash on cotton fabric obtained from one fingerprint was placed in a separate lattice-bottomed filtration column, which was inserted into a 1.5 ml Eppendorf type tube. The tubes were centrifuged at 14000-15000 rpm for 5 minutes. The column and carrier were removed from the tube. The filtrate of the performed washings, located in 1.5 ml Eppendorf tubes, was incubated in a thermal shaker at + 70 ° C for 15 minutes. After incubation, the samples were mixed at low speeds and briefly centrifuged.

In addition, the filtrate was studied for swabs of potogiric substances carried out using a liquid buffer composition (buffer 3). The filtrates were incubated in three modes: at room temperature for 30 minutes (control samples), at + 70 ° C for 30 minutes, and at + 99 ° C for 8 minutes.

After incubation, the samples were mixed on a vortex at low speed for 5 seconds, then briefly centrifuged.

For subsequent comparative genotyping, subjects were selected samples of buccal epithelium. Saliva was taken with cotton swabs on an empty stomach 30 minutes after rinsing the oral cavity with water.

DNA was extracted from samples of buccal epithelium using a Chelex 100 ion exchange resin in accordance with the methodology described in the training manual (Kornienko I.V., Kharlamov S.G. Methods for the study of human DNA. DNA isolation and its quantitative assessment in the forensic aspect -medical research of material evidence of biological origin: teaching aid / I.V. Kornienko, S.G. Kharlamov. Rostov n / A: SFedU, 2012. - 216 p.).

Clippings of cotton swabs with saliva samples were placed in separate sterile microcentrifuge 1.5 ml Eppendorf tubes. 1 ml of sterile deionized water was added to the tubes. Samples were incubated at room temperature for 30 minutes, stirring occasionally. Samples were centrifuged for 3 minutes at 13,000 rpm. 970 μl of supernatant were removed from the tubes, leaving approximately 30 μl of the carrier object in the tubes. 190 μl of a 5% suspension of Chelex and 10 μl of proteinase K (10 mg / ml) were added. Incubated at + 56 ° C in a thermal shaker for 20 minutes. Vortexed for 10 seconds. Then the samples were incubated at a temperature of + 99 ° C in a thermal shaker for 8 minutes. Vortexed for 10 seconds. Centrifuged for 3 minutes at 13,000 rpm. For PCR, the supernatant was used.

I. Assessed the amount of DNA in the studied drugs using PCR-RV in accordance with the manufacturer's instructions. A thermal cycler with a multichannel detector was used to evaluate the real-time PCR products of iQ5 (USA) Bio-Rad.

Amplicon accumulation and calibration curves were recorded using PCR-RV technology based on hybridization-fluorescence detection using specific probes with fluorescent labels (TaqMan format).

Reagent kits were used to determine the number of DNA fragments of the β-actin gene, fragments of the amelogenin gene located on the X (p22.1-22.3) and Υ (p11.2) chromosomes of the human XY-Detect (Syntol LLC). To evaluate the effectiveness of (E) PCR, a calibration curve was constructed for samples containing the following DNA concentrations: 5 ng / μl, 1 ng / μl, 0.2 ng / μl, 0.04 ng / μl and 0.008 ng / μl. For amplification of the β-actin gene, the calculated PCR efficiency was E = 0.971.

In the table. 15 shows the volumes of components used in the preparation of the reaction mixture per tube.

Before PCR, 2 μl of DNA preparations or standard DNA preparations with a known concentration of 0.008, 0.04, 0.2, 1.0 and 5.0 ng / μl were added to tubes with 23 μl of the prepared reaction mixture.

Amplification was performed in the following mode:

Real-time PCR data analysis was performed using the iQ5 Optical System Software (version 2.0, 2006).

Statistical processing of experimental data was performed using the STADIA program (Kulichev AP Methods and means of data analysis in a Windows environment. Stadia 6.0. - Μ .: Computer science and computers. - 1996. -257 p.).

According to the results of statistical analysis of the data, it was found that the distribution was different from the normal one, therefore, as a quantitative estimate, we did not use the average values, but the medians of the samples.

The DNA concentration and threshold cycles of the PCR curves in the washes performed using liquid buffer compositions 1, 3 (buffer 1, 3), as well as deionized water, incubated for 15 minutes at + 70 ° C, are presented in table. 16.

The concentration of DNA in washes performed using liquid buffer composition 3 (buffer 3), subjected to various incubation modes, are presented in table. 17.

According to the results of a quantitative assessment in swabs of a potogir substance made using the proposed liquid buffer compositions 1 and 3 (buffers 1 and 3), the DNA concentration is more than 1.6 times higher compared to the amount of DNA in swabs made with deionized water.

Using the liquid buffer composition 3 (buffer 3) as an example, due to its large stabilizing properties exerted on DNA (proved in Example 3), it is shown that using various incubation regimes of the filtrates of the washes, it is possible to obtain high-quality DNA preparations.

II. To evaluate the solvents used as a liquid medium for washing off biological traces (potogirous substances), genotypes of DNA filtrate loci incubated for 15 minutes at + 70 ° C were genotyped using STR loci of the COrDIS Plus panel (D3S1358, THO1, D12S391, D1S1656, D10S1248, D22S1045, D2S441, D7S820, D13S317, FGA, TROX, D18S51, D16S539, D8S1179, CSF1PO, D5S818, vWA, D21S11, SE33 and the Amelogenin locus, which determines the gender of the human DNA locus of the COrDIS Plus locus and DNA marker Plus in accordance with the manufacturer’s manual (COrDIS Plus. Reagent kit A multiplex analysis of 19 STR-markers and human amelogenin locus User Manual -.. 2014 - 19) "Gordiz" Ltd. (registration certificate №FSR 2012/13548)..

For enzymatic amplification, PCR tubes from the kit containing lyophilized PCR components (Taq polymerase, dNTP mixture, reaction buffer, primer mixture) were added with 10 μl of deionized water, then 5 μl of activator solution containing buffer solution and ions were added magnesium Mg ²⁺ . Then, 10 μl of the studied DNA preparations were added. The mixture was stirred and briefly centrifuged. To assess the specificity of the amplification reaction, the positive (test with control DNA MK1 from the COrDIS Plus reagent kit) and negative (test with deionized water instead of DNA preparation) controls were used.

During amplification, the heating rate was 0.3 ° C / s at the stage of increasing from a temperature from 59 ° C to 72 ° C.

Used the following PCR parameters:

94 ° C 3 min 98 ° C 30 sec 59 ° C * 120 sec, 4 cycles 72 ° C 90 sec 94 ° C 30 sec 59 ° C * 120 sec, 6 cycles 72 ° C 90 sec 90 ° C 30 sec 59 ° C 120 sec, 20 cycles 72 ° C * 75 sec 68 ° C 10 min 15 ° C ∞

* Heating rate from 59 ° C to 72 ° C - not more than 0.3 ° C / 1 sec.

Amplification was performed using a GeneAmp PCR System 9700 thermal cycler (Applied Biosystems) with an aluminum block in the GeneAmp 9600 emulation mode.

Alleles were identified according to the S450 size standard and with respect to the allelic leader attached to the COrDIS Plus test system.

To load samples into the device, a mixture of Hi-Di ™ formamide and S450 size standard was prepared in the following ratio: 10 μl of Hi-Di ™ formamide and 1 μl S450 size standard per sample.

The amplification products were identified by the STR loci of the COrDIS Plus panel using an ABI PRISM 3130xl automatic sequencer in accordance with the instructions for use (COrDIS Plus. Reagent kit for multiplex analysis of 19 STR markers and the human amelogenin locus. User manual. - 2014. - 19 p.) And (Guidance on the use of the genetic analyzer 3130 / 3130xl. - Applied Biosystems).

In the process of electrophoresis, information about laser gel scanning and fluorescence detection was automatically transferred to the control computer and processed by Run 3130 Data Collection v.3.0.

Processing of the results and identification of alleles was carried out automatically using the GeneMapper ^® ID 3.2 program.

Statistical processing of experimental data was carried out using the STADIA program (Kulachev A.P. Methods and means of data analysis in a Windows environment. Stadia 6.0. - Μ .: Computer science and computers. - 1996. - 257 p.).

The results of prototyping genotyping are presented in table. eighteen.

The average values presented in table 18 were calculated by typing samples of human potogir substances taken from the surface of artificial skin using washes with liquid buffer compositions 1, 3 (buffers 1, 3), proposed in the present invention, or with deionized water in seven parallels.

Notes: "+" - indicates the presence of all allelic states in the studied loci; "+/-" - indicates the loss of one of a pair of alleles (the so-called effect of "false homozygous") in the studied loci; “-” - indicates the absence of the amplification product (loss of all alleles) in the studied loci.

When genotyping swabs of potogiric matter, as the most difficult object of identification due to the extremely low amount of genetic material contained in it, made using the proposed liquid buffer compositions 1 and 3 (buffers 1 and 3), it is possible to identify alleles in the studied loci, on average, 13 times more and more than 1.7 times less, the absence of an amplification product is noted in comparison with washings made with deionized water.

The results are explained by the presence in the proposed liquid buffer compositions 1, 3 (buffers 1, 3) of chaotropic agents Tween 20 and Triton X-100, under the influence of which, in the process of flushing, in the flush and its filtrate, cell structures are destroyed, and This facilitates the access of the DNA Taq polymerase enzyme and primers to the DNA template. Thanks to the PCR activators (Tween 20, Triton X-100, KCl, K ₂ SO ₄ , glycerol) included in the composition of the proposed liquid buffer compositions 1, 3 (buffers 1, 3), the half-life of the DNA Taq polymerase enzyme is increased at + 95 ° C at the stage of DNA denaturation.

Example 6

Investigated the efficiency of DNA extraction from washouts of biological overlays (potogirova substances), made using various solvents, including liquid buffer composition 3 (buffer 3), proposed in the present invention. Used deionized water and solutions, which according to the results presented in example 4, did not have an inhibitory effect on PCR:

1. The composition of the liquid buffer composition 3 (10 mm Tris (hydroxymethyl) aminomethane, 0.5% vol. Tween 20, 50 mm KCl, 0.1 mm EDTA (ethylenediamine-Ν, Ν, Ν ', Ν'-tetraacetate), 5% glycerol, 0.1% NaN ₃ (sodium azide)): a mixture of solutions of 12.1 mg Tris (hydroxymethyl) aminomethane, 50 μl Tween 20, 37.3 mg KCl, 2 μl 0.5 Μ EDTA (ethylenediamine-Ν , Ν, Ν ', Ν'-tetraacetate), 500 μl of glycerol, 10 mg NaN ₃ (sodium azide), brought to 10 ml with sterile deionized water.

2. Saline solution containing NaCl at a concentration of 150 mm.

3. The TE buffer containing 10 mm Tris-HCl pH = 8.0, 0.1-1.0 mm EDTA and deionized water.

4. Deionized water.

As an analogue of biological traces containing a small amount of genetic material, we used a potogir substance left by the fingertips of eight volunteers on a smooth metal object 9 months after their application. Washings were performed using sterile cotton swabs dipped in 50 μl of liquid buffer composition 3, saline, TE buffer or deionized water.

Cotton swabs with flushes were placed in plastic columns with a trellised bottom, which were inserted into 1.5 ml Eppendorf tubes. Tubes were centrifuged at 13000-14000 g for 5 minutes. The filtrate of the performed washings, located in 1.5 ml Eppendorf tubes, was placed in a thermal shaker and incubated at + 70 ° C for 30 minutes in a mixing mode of 1150 rpm. After incubation, the samples were mixed on a vortex at low speed for 5 seconds, then briefly centrifuged.

Evaluated the effectiveness of DNA extraction from washes of biological overlays (potogirova substances) using PCR-RV in accordance with the manufacturer's instructions. A thermal cycler with a multichannel detector was used to evaluate the real-time PCR products of iQ5 (USA) Bio-Rad.

Amplicon accumulation and calibration curves were recorded using PCR-RV technology based on hybridization-fluorescence detection using specific probes with fluorescent labels (TaqMan format). Reagent kits were used to determine the number of DNA fragments of the β-actin gene, fragments of the amelogenin gene located on the X (p22.1-22.3) and Υ (p11.2) chromosomes of the human XY-Detect (Syntol LLC).

In the table. 19 shows the volumes of components used in the preparation of the reaction mixture per tube.

Before PCR, 5 μl of the test samples were added to tubes with 20 μl of the prepared reaction mixture.

Amplification was performed in the following mode:

Real-time PCR data analysis was performed using the iQ5 Optical System Software (version 2.0, 2006).

Statistical processing of experimental data was carried out using the STADIA program (Kulachev A.P. Methods and means of data analysis in a Windows environment. Stadia 6.0. - Μ .: Computer science and computers. - 1996. - 257 p.).

The results of a comparative study of the efficiency of DNA extraction from washes of biological overlays (potogirova substances) 9 months old are presented in table. 20 and in FIG. 8.

Notes: ΔC _t is the difference between the control (preparation 4) and the experimental (preparations 1-3) threshold cycles.

In FIG. Figure 8 on the graph of the accumulation of PCR products shows that, on average, starting from the 32nd cycle, the curves of all DNA samples of a potogaster substance are lifted for 9 months, in which the solvent is liquid buffer composition 3 (1), physiological saline (2), buffer TE (3) or deionized water (4). The results show the efficiency of DNA extraction from washes of biological overlays containing an extremely low amount of genetic material using the example of a long-stored potogirous substance on the surface of a smooth metal object.

Claims (26)

1. Composition for collecting and storing DNA or DNA-containing biological traces containing a buffer solution comprising 1-40 mm Tris (hydroxymethyl) aminomethane, 0.05-1% vol. Tween 20, 1-200 mM K ₂ SO ₄ , 0.01-0.5 mM EDTA (ethylenediamine-N, N, N ', N'-tetraacetate), 1-20% glycerol, 0.01-0.4 % NaN ₃ (sodium azide), water - the rest. 2. The composition of claim 1, wherein the buffer solution is a mixture of solutions of 12.1 mg Tris (hydroxymethyl) aminomethane, 50 μl Tween 20, 87.1 mg K ₂ SO ₄ , 2 μl 0.5 M EDTA (ethylenediamine-N , N, N ', N'-tetraacetate), 500 μl of glycerol, 10 mg NaN ₃ (sodium azide), adjusted to 10 ml with sterile deionized water. 3. The composition of claim 1, wherein the DNA-containing trace is a biosample. 4. The composition according to p. 3, where the ratio of buffer to bio-sample is 10: 1. 5. The composition according to p. 3, where the biosample is a substance, traces of blood, saliva, vaginal secretions, sperm, cell suspensions, mixed biological traces, DNA preparations. 6. Composition for collecting and storing DNA or DNA-containing biological traces containing a buffer solution comprising 1-40 mm Tris (hydroxymethyl) aminomethane, 0.05-1% vol. Triton X-100, 1-200 mM K ₂ SO ₄ , 0.01-0.5 mM EDTA (ethylenediamine-N, N, N ', N'-tetraacetate), 1-20% glycerol, 0.01-0 , 4% NaN ₃ (sodium azide), water - the rest. 7. The composition according to claim 6, where the buffer solution is a mixture of solutions of 12.1 mg Tris (hydroxymethyl) aminomethane, 50 μl Triton X-100, 87.1 mg K ₂ SO ₄ , 2 μl 0.5 M EDTA (ethylenediamine -N, N, N ', N'-tetraacetate), 500 μl of glycerol, 10 mg NaN ₃ (sodium azide), adjusted to 10 ml with sterile deionized water. 8. The composition of claim 7, wherein the DNA-containing trace is a biosample. 9. The composition of claim 8, wherein the ratio of buffer to bio-sample is 10: 1. 10. The composition according to p. 8, where the biosample is a potogo substance, traces of blood, saliva, vaginal secretions, sperm, cell suspensions, mixed biological traces, DNA preparations. 11. Composition for collecting and storing DNA or DNA-containing biological traces containing a buffer solution comprising 1-40 mm Tris (hydroxymethyl) aminomethane, 0.05-1% vol. Tween 20, 1-200 mM KCl, 0.01-0.5 mM EDTA (ethylenediamine-N, N, N ', N'-tetraacetate), 1-20% glycerol, 0.01-0.4% NaN ₃ (sodium azide), water - the rest. 12. The composition of claim 11, wherein the buffer solution is a mixture of solutions of 12.1 mg Tris (hydroxymethyl) aminomethane, 50 μl Tween 20, 37.3 mg KCl, 2 μl 0.5 M EDTA (ethylenediamine-N, N, N ', N'-tetraacetate), 500 μl of glycerol, 10 mg NaN ₃ (sodium azide), adjusted to 10 ml with sterile deionized water. 13. The composition of claim 12, wherein the DNA-containing trace is a biosample. 14. The composition according to p. 13, where the ratio of buffer to bio-sample is 10: 1. 15. The composition according to p. 13, where the biosample is a substance, traces of blood, saliva, vaginal secretions, sperm, cell suspensions, mixed biological traces, DNA preparations. 16. Composition for collecting and storing DNA or DNA-containing biological traces containing a buffer solution comprising 1-40 mm Tris (hydroxymethyl) aminomethane, 0.05-1% vol. Triton X-100, 1-200 mM KCl, 0.01-0.5 mM EDTA (ethylenediamine-N, N, N ', N'-tetraacetate), 1-20% glycerol, 0.01-0.4% NaN ₃ (sodium azide), water - the rest. 17. The composition according to p. 16, where the buffer solution is a mixture of solutions of 12.1 mg Tris (hydroxymethyl) aminomethane, 50 μl Triton X-100, 37.3 mg KCl, 2 μl 0.5 M EDTA (ethylenediamine-N, N, N ', N'-tetraacetate), 500 μl of glycerol, 10 mg NaN ₃ (sodium azide), adjusted to 10 ml with sterile deionized water. 18. The composition according to p. 17, where the DNA-containing trace is a biosample. 19. The composition according to p. 18, where the ratio of buffer to bio-sample is 10: 1. 20. The composition according to p. 18, where the biosample is a substance, traces of blood, saliva, vaginal secretions, sperm, cell suspensions, mixed biological traces, DNA preparations. 21. The use of a composition according to any one of paragraphs. 1-20 for the impregnation of hygroscopic carrier materials and flushing of DNA-containing biological traces, overlays or DNA for the purpose of their storage and / or transportation. 22. The application of claim 21, where the hygroscopic carrier material is a cotton fabric, fibrous substances, paper carrier. 23. The use of claim 22, wherein the fibrous substance is a cotton swab. 24. The use of claim 22, wherein the paper carrier is chromatographic paper. 25. The application of claim 21, wherein the DNA-containing biological trace, overlay, or DNA is a dry bio-sample. 26. The application of claim 21, wherein the DNA-containing biological trace, overlap, or DNA is in liquid form.

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