RU2049821C1 - Method of preparing non-radioactive labelled probe for determination of nucleotide series - Google Patents

Abstract

FIELD: biotechnology. SUBSTANCE: origin nucleic acid is modified by reamination at pH 3.2-4.5 and temperature 60-90 C within 1-12 min. Reagent having formula R₁-S-R₂ where R₁ is derivative of hydroxyl amine which reacts with cytosine of nucleic acid, R₂ is chemical group which reacts with activated derivative being used as labelling agent is used as reamination agent. EFFECT: improves efficiency of method. 2 cl, 2 dwg

Description

 The invention relates to molecular biology, medicine and veterinary medicine and can be used to detect specific nucleic acid (NK) sequences by molecular hybridization for the diagnosis of hereditary and bacterial diseases in humans and animals.

 The synthesis and use of non-radioactive labeled hybridization probes is an urgent task in medicine, veterinary medicine and agriculture. The use of hybridization tests for infection of humans, animals, and plants with various microorganisms in general practice is hampered by the lack of inexpensive, technologically advanced and radionuclide-free methods for preparing probes.

 A known method of producing non-radioactive probes, based on the reaction of expansion of a single-stranded break in DNA using enzymes (nick translation) [1] The method includes the following steps: introducing into the DNA matrix of single-stranded discharges using the enzyme DNAse 1; thermal inactivation of the enzyme DNase 1; polymerization of deoxynucleoside triphosphates on a DNA matrix using DNA polymerase 1, wherein one of the precursors is radiolabeled and the second carries a non-radioactive label; radioactive label control of the polymerization process; purification of labeled DNA from enzymes and unincluded precursors.

 The disadvantages of the method are the use of enzymes and expensive precursors, the need to control the polymerization procedure, a small amount of simultaneously processed DNA (not more than 5 μg), the sensitivity of the enzymes to the quality of the DNA matrix, the possibility of labeling only double-stranded deoxypolinucleotides.

Closest to the proposed method is a method for producing non-radioactive probes, based on the reaction of double-stranded DNA with succinic acid dihydrazide [2] comprising the following stages: thermal denaturation of the DNA at 95 ^about C for 5 min; incubation of denatured DNA with an equal volume of succinic acid dihydrazide pH 5 at 95 ^about C for 15-60 minutes; isolation of modified DNA by gel filtration on a Sephadex G-50; sample concentration; labeling of the modified DNA with N-hydroxysuccinimide ester of D-biotinyl-ε-aminocaproic acid; purification of labeled DNA from non-aligned biotin on a Sephadex G-50.

The disadvantage of this method of DNA modification stringent conditions (prolonged incubation at 95 ° ^C, pH 5) leading to a drop in the quality of the probe. With a sensitivity of 20 pg in hybridization, the scope of such probes is sharply narrowed. The application of this method for labeling only double-stranded deoxypolinucleotides is described.

 An object of the invention is to improve the quality of the target product (increasing sensitivity in detecting a probe, expanding the range of modified NKs (single and double-stranded poly and oligonucleotides, deoxy and ribo derivatives), increasing the stability of the resulting product).

The problem is achieved in that as a transaminating agent, a compound of the general formula R ₁ -SR _{2 is used} , where R ₁ is a hydroxylamine derivative that reacts with cytosine in the composition of NK; S spacer; R ₂ amino or aminooxy group, reacts with activated derivatives of the signaling connections, and transamination process is carried out at pH 3,2-4,5 at 60-90 ^{° C} for 1-12 min. As the transaminating agent, 4-aminooxybutylamine is predominantly used, where the spacer S is a tetramethylene bridge, which eliminates steric hindrances between the DNA duplex and R ₂ .

 The essence of the proposed method is as follows.

A solution of NK 1 mg / ml in water or in a buffer containing no derivatives of amines and carboxylic acids, mixed with an equal volume of an aqueous solution of 4-aminooksibutilamina dihydrochloride, pH is titrated pedvaritelno to 3.75 at 80 ° ^C with a solution of lithium hydroxide;
the components of the mixture are mixed and the mixture is incubated at 80 ^about 5 minutes;
modified NK is isolated by gel filtration on Sephadex G-25 with elution with 50 mM sodium bicarbonate;
NK are precipitated from the mixture by adding sodium chloride to 0.1 M and ethyl alcohol to 67%, followed by centrifugation at 12000 g for 10 min;
the precipitate was dissolved in 0.1 M sodium bicarbonate and then D-biotinyl-ε-aminocaproic acid N-hydroxysuccinimide ester was added to 5 mM. Incubate the mixture at 37 ° C. ^for 30 minutes;
biotinylated NK is purified by gel filtration on Sepjadex G-25 with elution with 50 mM sodium bicarbonate.

The probe can be used immediately or stored at least one year without loss of quality at (-20) ^{° C} in 50 mM sodium bicarbonate.

 The sensitivity of hybridization using probes obtained by the proposed method is 0.3 pg by homologous plasmid, which allows the identification of unique genes during blot hybridization of NK.

Essential difference of the proposed method compared to the prior art is that as pereaminiruyuschego reagent is a compound of the general formula R ₁ -SR ₂ and the process is carried out in the optimal mode, namely, in an environment with pH 3,2-4,5 at ^about 60-90 C for 1-12 minutes, which allows to improve the quality of the probe and expand its functionality i.e. the ability to label the entire spectrum of NK (single and double stranded poly and oligonucleotides, deoxy and ribo derivatives). In addition, the method allows you to enter into the NK a wide range of signal compounds: biotin, various fluorochromes and haptens.

The use of transcendental values of the mode of the transamination process does not allow to obtain high-quality probes. For example, when the pH of the reaction mixture is more than 4.5, a decrease in the rate of modification occurs. When the mixture is incubated for less than 1 min, the necessary degree of modification is not achieved, and for more than 12 minutes the rate of modification decreases. When the mixture is incubated for less than 1 min, the necessary degree of modification is not achieved, and more than 12 min, the by-product of the reaction accumulates, which reduces the quality of the target product. By incubating the mixture at a temperature above 90 ^{° C.} Polymerization probes decreases and accordingly the sensitivity, and at 60 ° ^C falls modification speed.

 Thus, the use of the proposed method allows, in comparison with the prototype, to obtain an additional technical result, which consists in reducing the number of stages of labeling, increasing the sensitivity of hybridization, increasing the stability of the product, and also allows you to expand the range of modified polymers.

PRI me R 1. 50 μl of a solution of NA in a concentration of 1 μg / μl in water or in a buffer that does not contain derivatives of amines and derivatives of carboxylic acids, is mixed with 50 μl of a 2 M aqueous solution of 4-aminooxybutylamine dihydrochloride, the pH of which was previously titrated to 3.75 at 80 ° ^C with a solution of 4.0 M lithium hydroxide. The components of the mixture are mixed and the mixture is incubated at 80 ° C. ^for 5 minutes. Modified NK is isolated by gel filtration on Sephadex G-50 (20x0.5 cm) with elution with 0.2 ml / min of 50 mM sodium bicarbonate. The yield of fractions is monitored by absorbance at 260 nm and 230 nm. NK are precipitated from the first fraction by adding sodium chloride up to 0.1 M and ethyl alcohol up to 67%, followed by centrifugation at 12000 g for 10 minutes. The precipitate was dissolved in 0.1 M sodium bicarbonate and then D-biotinyl-ε-aminocaproic acid N-hydroxysuccinimide ester was added to 5 mM. Incubate the mixture at 37 ° C. ^for 30 minutes. Biotinylated NK was purified by gel filtration on a Sephadex G-50 under similar conditions.

 The quality control of the modified NK is carried out in a hybridization reaction, carried out according to the following procedure.

The target DNA was denatured by heating in 10 mM Tris-HCl, 0,5 mM sodium ethylenediaminotetraacetates (TE) for 5 min at 98 ^{° C,} added to 1 M NaCl and applied to a nitrocellulose membrane. DNA was immobilized by ultraviolet light [3] NK biotinylated probe was denatured by heating in TE for 5 minutes at 98 ^{° C} and applied to a solution containing 0.6 M NaCl, 50% formamide, 0.02% polyvinylpyrrolidone, 0.02% Ficoll 400, 0.02% bovine serum albumin, 0,5% Tween 20, 40 mM sodium phosphate pH 7. Hybridization of the filter with the immobilized DNA is carried out for 16 hours at 37 ^C, after which the filter for 10 min washed 3 times at 37 ^about in 0.5 M NaCl, 50% formamide, 0.4% SDS, 33 mm sodium phosphate, pH 7. Then the filter is incubated with stirring in 0.2% casein, 50 mm Tris- HCl pH 7, 0.15 M NaCl, 5 mM MgCl _2, 0.2% strength Tween 20 for 40 minutes at 23 ° ^C. The conjugate streptavidin-alkaline phosphatase diluted 2000-fold in 50 mM Tris-HCl pH 7, 0, 15 M NaCl, 5 mM MgCl ₂ and incubated filter in this solution for 20 minutes at 23 ° ^C. unbound conjugate is washed from the filter by stirring for 10 minutes at 23 ^{° C} in four changes of 0.15 M NaCl, 50 mM tris-HCl pH 7.5 mM MgCl ₂ , 0.2% Tween 20. Rinse the filter with 0.15 M NaCl, 50 mM Tris-Hcl pH 9.4; 5 mM MgCl ₂ and then develop for 16 hours in a solution of the same composition, but with the addition of 0.33 mg / ml NBT, 0.165 mg / ml BCIP. At the end of the development, the filter is rinsed with water and dried in air.

 The method is illustrated in figures 1 and 2. Hybridization of a plasmid DNA probe biotinylated in accordance with example 1 does not give a signal with 1 ng of control eukaryotic DNA, and the hybridization sensitivity is 0.3 mg of a homologous plasmid.

 The resulting sensitivity allows the identification of a unique gene during blot hybridization.

 Indirect quality criteria of the resulting probe are also the form of the kinetics of NK modification and the product polymerization determined by electrophoresis under denaturing conditions.

PRI me R 2. The method is carried out analogously to example 1 except that the pH of the reagent is adjusted to 5.0. Figure 1 shows a graph of the rate of modification of plasmid DNA at a concentration of 0.5 μg / μl from the pH of the medium. The concentration of 4-aminooxybutylamine 0.5 M, the modification temperature of 80 ^about C. On the vertical axis, the percentage of modified bases, on the horizontal axis of the pH of the reaction medium at 80 ^about C.

PRI me R 3. The method is carried out analogously to example 1 except that the mixture of NK and the reagent is incubated at 98 ^about C. In this case, a decrease in the polymerization of DNA probes labeled 12 minutes in accordance with example 3 depending on the modification temperature (the polymerization of the probes was checked by electrophoresis in a 2% alkaline agarose gel).

 PRI me R 4. The method is carried out analogously to example 1 except that the mixture of NK and the reagent is incubated for 15 minutes Figure 2 shows the kinematics of DNA modification with 4-aminooxybutylamine in accordance with example 1. On the vertical axis, the percentage of modified DNA bases, on the horizontal axis, the reaction time in minutes. One can see a change in the linear nature of the kinetics of modification due to the disappearance of the target reaction product at a modification time of 6 minutes and the accumulation of a reaction by-product at a modification time of more than 12 minutes.

PRI me R 5. 50 μl of a DNA solution at a concentration of 1 μg / μl in water or in a buffer that does not contain derivatives of amines and derivatives of carboxylic acids, mixed with 50 μl of a 0.8 M aqueous solution of adipic acid dihydrochloride pH 3, 9 (R ₁ -R ₂ -carbonyl hydrazide, S-pentamethylene). The components of the mixture are mixed and the mixture is incubated at 80 ° C. ^for 10 minutes. Modified NK is isolated by gel filtration on Sephadex G-25 (20x0.5 cm) with elution with 0.2 ml / min of water. The yield of fractions is monitored by absorbance at 260 and 230 nm. The eluate was concentrated with butan-1-ol, the alcohol was extracted with sulfuric ether, the ether was removed in vacuo. Sodium phosphate pH 7 to 0.1 M and D-biotinyl-ε-aminocaproic acid N-hydroxysuccinimide ester up to 5 mM are added. The mixture was incubated at 60 ° ^C for 5 hours. To attach fluorescein dye is added to 1 g / l and 0.1 M sodium bicarbonate and incubated at ³⁷ for Sephafex G-25 gel filtration purified C. for 3 hours. The biotinylated and / or DNA fluorohromirovannuyu as described earlier.

 The quality control of the modified NK is carried out in a hybridization reaction carried out according to the procedure described in Example 1. Hybridization of a plasmid DNA probe biotinylated in accordance with Example 5 gives a sensitivity of about 20 pg by homologous plasmid.

 Adipic acid dihydrazide binds at a rate comparable to that of 4-aminooxybutylamine. The degree of modification is determined by the ratio of absorbances at 485 nm and 260 nm of the fluorescent derivative of DNA. Product polymerization, determined by 2% agarose gel electrophoresis, also shows the suitability of the modified DNA for hybridization.

 PRI me R 6. The method is carried out analogously to example 1 except that 1,4-di (aminooxy) butane is used for the transamination in the first stage. Probes modified with both 4-aminooxybutylamine and 1,4-di (aminooxy) -butane have the same sensitivity. DNA was applied on the filters in an amount of: 1 100 pg; 2 31.6 pg; 3 10 pg; 4 3.16 pg; 5 1 pg; 6 0.316 pg.

Claims (2)

1. METHOD FOR PRODUCING A NON-RADIOACTIVE TEST PROBE FOR DETERMINING NUCLEOTIDE SEQUENCES, Including incubation of a nucleic acid in a buffer solution containing a transaminating reagent, isolation and concentration of a modified nucleic acid, labeling it with a suitable signal compound, and purifying it from the impurity general formula
R ₁ SR ₂ ,
where R ₁ is a hydroxylamine derivative that reacts with cytosine in a nucleic acid;
S spacer;
R _{2 is a} chemical moiety that reacts with an activated derivative of a signal compound,
and the process of transamination is carried out at a pH of 3.2 to 4.5 and a temperature of 60 - 90 ^o C for 1 to 12 minutes  2. The method according to claim 1, characterized in that 4-aminooxybutylamine is used as the transaminating agent.

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