RU2081919C1 - Method and kit for genome dactyloscopy - Google Patents

Abstract

FIELD: forensic medicine; analytical methods. SUBSTANCE: preliminarily cleaved with restrictases DNA sample is immobilized on filter and hybridized with biotinylized oligonucleotide probe of formula 3'-(CAC)₅ - (C^*T)_n-C*-5' where C* is biotinylized oligonucleotide and n = 2-8. Hybridization filter is then treated with conjugate of streptavidine and alkaline phosphatase, washed, stained, and resultant hybridization picture is analyzed. Kit contains biotinylized oligonucleotide probe in concentration 30 rel.un./ml, above-mentioned conjugate with total protein concentration 1.5 mg/ml, and dyestuffs for alkaline phosphatase (5-bromo-3-indolyl phosphate and nitrotetrazolium blue). One kit ensures accelerated analysis of a great number of DNA samples. EFFECT: accelerated DNA analysis. 2 cl, 1 dwg

Description

 The invention relates to molecular biology and can be used in forensic medicine, forensics, to establish consanguinity in family analysis.

 The main way to detect genetic polymorphism at the level of DNA structure is the standard method of blot hybridization analysis. In this case, homologous DNA sequences of different lengths are detected, which are formed by restriction endonucleases during hydrolysis of genomic DNA. Variations in the structure of genomic DNA are mainly due to point mutations leading to site changes [1] The most highly effective markers of polymorphism are repeating minisatellite DNAs of various types, including the so-called “simple sequences”.

A known method of genomic fingerprinting of human DNA using a 32-P-labeled oligonucleotide probe (CAC) ₅ , which is a repeating simple sequence [2] With this probe, the most informative patterns of blot hybridization on human DNA are obtained, however, the widespread introduction of such a probe in practice difficult due to the instability of the radioactive label and the danger of working with it for laboratory personnel.

 More promising is the non-isotopic variant of genomic fingerprinting, which uses a probe labeled with a non-radioactive tag.

A known method of conducting hybridization analysis of eukaryotic DNA, the closest to the claimed technical solution and the achieved effect (prototype) [3] The known method is based on the use of a series of oligonucleotide probes consisting of repeating simple sequences in length from 2 to 4 bases, namely (GATA) ₄ GA, (GATA) ₅ , (GATA) ₂ GACA (GATA) ₂ , (GATA) ₄ , (GACA) ₄ , (GGAT) ₄ , (GGA) ₅ , (GT) ₈ , (CT) ₈ , and oligonucleotides complementary to them. In the examples used oligonucleotide probes labeled with a radioactive label. The authors of the patent propose the use of the same probes labeled with biotin at the 5'-terminal phosphate, while they note a lower sensitivity of biotinylated oligonucleotides compared to radio-labeled. The authors do not use oligonucleotides (CAC) ₅ as probes.

 The aim of the present invention is to provide a non-radioactive method and kit for genomic fingerprinting of eukaryotic DNA, which has a higher sensitivity and informativeness compared to the isotopic method and allows analysis in a shorter time.

При такой конструкции зонд содержит от 2 до 8 остатков биотина, расположенных вне участка гибридизационного взаимодействия и не препятствующих образованию комплекса зонд-ДНК. Кроме того, остатки биотина в зонде пространственно удалены друг от друга за счет наличия Т-спейсеров, что уменьшает стерические препятствия при выявлении биотиновых групп с конъюгатом стрептавидин-фермент.This goal is achieved by the fact that in the proposed method and in the kit uses a non-radioactive oligonucleotide probe of the following composition:
3'-CACCACCACCACCAC (C ^* T) _n C ^* -5 ',
where n = 2-8,
C ^* modified deoxycytidine containing biotin:

With this design, the probe contains from 2 to 8 biotin residues located outside the site of hybridization interaction and not preventing the formation of the probe-DNA complex. In addition, the residues of biotin in the probe are spatially removed from each other due to the presence of T-spacers, which reduces steric barriers in the identification of biotin groups with streptavidin-enzyme conjugate.

 When conducting a search, no technical solutions were found that have features similar to the distinguishing features of the proposed method, which allows us to conclude that its criteria of the invention are "significant differences".

A comparative analysis of the proposed solution with the prototype shows that the claimed method differs from the known one in that the DNA hybridization analysis uses a non-radioactive oligonucleotide probe (CAC) ₅ containing from 2 to 8 biotin residues located outside the hybridization interaction site, and to detect biotin after hybridization streptavidin conjugate with alkaline phosphatase and dyes are used. Thus, the claimed method meets the criteria of the invention of "novelty." Kits for genomic fingerprinting of such a composition are not produced in our country. Abroad, the production of similar kits by leading companies in the field of genetic engineering and biotechnology has not been identified.

 Using this method and kit in the analysis of human DNA, blot hybridization patterns are obtained that are more informative than those obtained with 32-P-labeled oligonucleotide probes. New features of a technical solution in the total set of features ensure the achievement of the purpose of the invention, therefore, we can conclude that the claimed technical solution meets the criterion of "inventive step".

The invention is illustrated in the drawing, which shows a genomic fingerprinting of human DNA:
and hybridization with a 32-P-labeled oligonucleotide (CAC) ₅ ;
b hybridization with biotinylated (CAC) ₅ ; DNA samples of representatives of two different families are shown (in each triad father, child, mother). DNA is hydrolyzed by restriction enzyme Hinf 1.

 The essence of the technical solution is most fully disclosed in the examples of the specific invention of the method and set.

Example 1. Synthesis of biotinylated (CAC) ₅ .

The synthesis of the modified base is carried out according to the described method [4]. The synthesis of the oligonucleotide probe is carried out on a column of 50 mg of porous glass CP6-300 (Corning) containing 1 μmol of covalently fixed dimethoxytrityl cytosine. The synthesis is carried out automatically on the domestic synthesizer "Victoria-5" according to the following protocol:
1. Detritylation of 3% trichloroacetic acid in dichloromethane, 30 s.

 2. Washing acetonitrile, 15 C.

 3. Condensation of 0.2 ml of 0.1 M phosphamidate dimethoxytrityl-nucleoside + 0.2 ml of 0.5 M tetrazole in acetonitrile, 5 minutes

4. Oxidation of 0.1 M I ₂ in a mixture of dioxane-pyridine-water, 6: 3: 1, 0.5 min.

 5. Washing acetonitrile, 15 C.

 6. Copy 0.5 M acetic anhydrite in a mixture of dioxane-lutidine-N-methylimidazole, 6: 3: 1, 1 min.

 7. Washing acetonitrile, 15 C.

This cycle of operations is repeated until the synthesis is complete, the carrier is treated with 3 ml of 25% aqueous ammonia for 12 hours at 50 ^o C, the solution of the released oligonucleotide is applied to a 250 x 4 mm column with Lichrosorb RP 18 phase (particle size 10 μm) and elute with a gradient of buffer solutions from 100% B (20% acetonitrile, 0.1 M triethylammonium acetate, pH 7.0) to 100% A (20% acetonitrile, 0.1 M triethylammonium acetate) in 30 minutes The synthesized oligonucleotide is collected, the solution is evaporated to dryness, treated with 2 ml of 80% acetic acid for 1 hour at 20 ^o C, evaporated again to dryness and stored at -20 ^o C.

 Example 2. Analysis of DNA samples.

Hydrolysis of DNA by restrictase enzymes, electrophoresis of fragments in an agarose gel and their transfer to filters are carried out as described [5]
1. Prehybridization.

The filter with immobilized DNA is placed in a cuvette with a hybridization solution and prehybridization is carried out for 30 minutes at 40 ^o C.

 Hybridization solution: 6xSSC, 5xDenhardt, 0.5% SDS (20xSSC 3 M NaCl, 0.3 M Na-citrate pH 7.2; 50xDenhardt 1% polyvinylpyrrolidone, 1% ficol, 1% BSA).

 2. Hybridization.

The filter is transferred to a hybridization solution containing a biotinylated oligonucleotide (3 μl of oligonucleotide per 5 ml of solution). A 5 ml mixture is sufficient for a 10x10 cm filter. Hybridize for 1 hour at 40 ^o C.

 3. Washing from the probe.

After hybridization, the hybridization solution is drained (it can be reused), the filter is washed successively for 15 minutes at room temperature, then 15 minutes at a temperature of 40 ^o C in a solution of 4xSSC, 0.1% SDS.

 4. Background blocking.

 After hybridization and washing, the filter is treated with blocking buffer for 30 minutes at room temperature.

Blocking buffer: 0.1 M Tris-HCl pH7.5.0.1 M NaCl, 3 mM MgCl ₂ , 0.5% Tween-20.

 5. Conjugate treatment.

 After the blocking step, the filter is soaked for 5 min in the reaction buffer, then, having soaked with filtered paper, transfer it to a clean sheet of lavsan. Apply a streptavidin-phosphatase conjugate to the filter, previously diluted with the reaction buffer 2,000 times (to a final conjugate concentration of 0.5-1.0 μg / ml).

 A 10x10 cm filter requires 2 ml of diluted conjugate. Cover the filter with a second sheet of lavsan, remove air bubbles between the sheets and leave for 30 minutes.

Reaction buffer: 0.1 M Tris-HCl, pH 7.5, 0.1 M NaCl, 3 mM MgCl ₂ , 0.05% tween-20.

 6. Washing from the conjugate.

 Wash the filter from excess conjugate 3 times for 5 min with blocking buffer and once with AP buffer.

AP buffer: 0.1 M Tris-HCl, pH 9.5, 0.1 M NaCl, 5 mM MgCl ₂
7. Staining.

Phosphatase dyes are dissolved in dimethylformamide (2.5 mg of 5 bromo-3-indolylphosphate in 50 μl of DMF, 4 mg of nitrotetrazole blue in 50 μl of DMF) and transferred to a tube with AP buffer (15 ml). The filter is poured with a dye solution and left in the dark until colored bands appear (usually 1.5-2 hours at room temperature or 30 minutes at 37 ^° C).

 After identifying the bands, the filter is washed with distilled water and dried.

 Example 3. The composition of the kit for genomic fingerprinting.

The kit for genomic fingerprinting includes the following components:
biotinylated oligonucleotide (CAC) ₅ at a concentration of 30 opt.ed./ml
alkaline phosphatase streptavidin conjugate with a protein concentration of 1.5 mg / ml,
dyes for alkaline phosphatase solid-phase substrate 5-bromo-3-indolylphosphate and nitrotetrazole blue.

 The kit is designed for 10 blot hybridization experiments (analysis of about 100 DNA samples).

 The drawing shows that when using a biotinylated probe, the hybridization pattern is clearer, the bands are more discrete, there is practically no background characteristic of hybridization with a radioactive probe. According to the results of the hybridization analysis of the DNA of the two mixtures, we can conclude that in the triad 648-650 there is no blood relationship between the child and the alleged father, while in the triad 639-641 paternity is undoubtedly proved.

 Thus, the proposed method and kit for genomic fingerprinting of eukaryotic DNA are more promising for practical use. The analysis does not require special safety measures for radiation protection. A biotinylated oligonucleotide probe can be stored for a long time for at least a year, while a radioactive probe is unstable (half-life is 14 days). The analysis time is significantly reduced due to the fact that it takes only 2-3 hours to identify a biotin tag after hybridization, while the radio-autography of a filter with a 32-P tag lasts several days.

 List of references.

 1. Jeffreys A.J. Wilson V. Thein S.L. // Nature.-1985-V.136.-P.76-79.

 2. Schafer R. Zischler H. Epplen J.T. // Nucl. Acids Res.-1988.-V.16, N 11. -P.5196.

 3. Epplen J.T. Patent 0266 787 A2 (C 12 Q 1/68) N 871164083 dated 05/11/88.

 4. Urdea M. S. Warner B.D. Running J.A. Stempien M. Clyne J. Horn T. // Nucl. Acids Res.-1988.-V.16, N 11.-P. 4937-4956.

 5. Ryskov A.P. Dzhancharadze A.G. Prosnyak M.I. et al. // Genetics.-1988.-T. 24.-S.227-238.

Claims (2)

1. A method of genomic fingerprinting, comprising blot hybridization of a DNA sample with a biotinylated oligonucleotide probe containing a simple repeating sequence, characterized in that the sequence is used as an oligonucleotide probe
3 '- (САС) ₅ - (С ^* Т) _n С ^* 5,
where C ^* biotinylated deoxycytidine;
n 2 8. 2. A kit for genomic fingerprinting, comprising a biotinylated oligonucleotide probe containing a simple repeating sequence, a streptavidin alkaline phosphatase conjugate, 5-bromo-3-indolylphosphate and nitrotetrazolium blue, characterized in that the probe is a sequence
3 ^' - (CAC) ₅ - (C ^* T) _n C ^* 5,
where C ^* biotinylated deoxycytidine;
n 2 8.