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WO2008150187A1 - Utilisation du trichloracétate d'ammonium en tant que produit de dissociation de complexes naturels d'acides nucléiques et procédé d'extraction d'arn - Google Patents

Utilisation du trichloracétate d'ammonium en tant que produit de dissociation de complexes naturels d'acides nucléiques et procédé d'extraction d'arn Download PDF

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WO2008150187A1
WO2008150187A1 PCT/RU2007/000310 RU2007000310W WO2008150187A1 WO 2008150187 A1 WO2008150187 A1 WO 2008150187A1 RU 2007000310 W RU2007000310 W RU 2007000310W WO 2008150187 A1 WO2008150187 A1 WO 2008150187A1
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rna
complexes
cells
dna
txaa
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PCT/RU2007/000310
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English (en)
Russian (ru)
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Jury Fedorovich Drygin
Iosif Grigorievich Atabekov
Olga Alexandrovna Kondakova
Sergei Nikolaevich Chirkov
Elena Sergeevna Gavryushina
Original Assignee
Jury Fedorovich Drygin
Iosif Grigorievich Atabekov
Olga Alexandrovna Kondakova
Sergei Nikolaevich Chirkov
Elena Sergeevna Gavryushina
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Application filed by Jury Fedorovich Drygin, Iosif Grigorievich Atabekov, Olga Alexandrovna Kondakova, Sergei Nikolaevich Chirkov, Elena Sergeevna Gavryushina filed Critical Jury Fedorovich Drygin
Priority to PCT/RU2007/000310 priority Critical patent/WO2008150187A1/fr
Publication of WO2008150187A1 publication Critical patent/WO2008150187A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C51/00—Preparation of carboxylic acids or their salts, halides or anhydrides

Definitions

  • the invention relates to molecular biology, biotechnology, genetic engineering and medicine, and can be used as a universal method for isolating RNA, including for diagnostic purposes in mass analysis using DNA (PHK) probes, RT-PCR, etc.
  • the invention also relates to the use of ammonium trichloroacetate as an agent that effectively destroys natural complexes of nucleic acids - RNA and DNA.
  • guanidine thiocyanate is used in a mixture with phenol (in the English language literature TRIZOL Reagent (USPattern No.5346994)), hot water-saturated phenol, phenol with 0.5-4% sodium dodecyl sulfate, chloroform with sodium chloride in a concentration of IM and higher (used by the diagnostic company Agdia, USA).
  • phenol in the English language literature TRIZOL Reagent (USPattern No.5346994)
  • hot water-saturated phenol phenol with 0.5-4% sodium dodecyl sulfate
  • chloroform with sodium chloride in a concentration of IM and higher
  • Trichloroacetate ion is a powerful chaotropic agent and ranks first in the series of chaotropes (Hofeister series) used to destroy complex complexes: CCl 3 COO " > SCN " > gyanidine + > C1O 4 " > Br " > NO 3 " [R.M . C. Dawsop, D.S. Elliott, W.N. Elliott, K. M. Jopes, Datog Biochemil Research, Zd ed., CP., Oxford, 1986].
  • Trichloroacetic acid (TCA) is known to be traditionally A 5–20% concentration is used for the quantitative precipitation of proteins and nucleic acids in the cold from solutions at pH ⁇ l (G.
  • RNA purification from proteins is used either by their extraction with phenol (for phenol-detergent deproteinization) or selective sorption-desorption of RNA on amorphous silica gel ( R. Voom, C.J.A. SoI, M.M.M. Salimaps, CL. Japsep, R.M.E. Werthim-vap Dillep, J. vap der Nordaa J. CHn. Misgob.
  • the main objective of the present invention was to develop a method for isolating RNA, and then DNA, in which the toxic chaotropic deproteinizing agents used were replaced by a non-toxic agent to reduce the danger associated with the nucleic acid isolation procedure and drastically reduce environmental pressure on the environment.
  • Another objective of the invention was to develop a method for isolating RNA, in which RNA is purified using cheap and widely available materials based on fibrous cellulose, such as, for example, filter paper and cotton wool.
  • RNA complexes are not limited to ribonucleoprotein complexes (viruses, ribosomes, informosomes, etc.). In nature, RNA can also be in complexes with DNA (see, for example, RF Itzhaki, et al. “Fractiophthalis diffuse hep dilute salt.”, Nuclide Acids Res. (1978) 5 (3): 739-750; cell membranes (L. Dorssers, et al.
  • RNA purifier ammonium trichloroacetate
  • the authors preferred the ammonium cation, since the formed ammonium salts of RNA are readily soluble at low concentrations of ammonium salt in solution (important for dissolving RNA from the precipitate) and selectively precipitate at a high concentration of ammonium salt (2-10 M short RNAs precipitate at a higher concentration of ammonium ions), see, for example, the protocol of the company EPICENTRE ® Biotechnology "A Simplicity Method for RNA Purification Technology in Vitro Technology Reactions ”, in which RNA in a pure transcription system is selectively precipitated with high concentrations of ammonium acetate, while the DNA and nucleotides remain in solution.
  • the invention allows to drastically reduce the danger associated with the RNA isolation procedure, drastically reduce environmental pressure on the environment and simplify the method of obtaining the total RNA fraction from healthy, virus or viroid-infected plant, animal or bacterial cells.
  • the present invention provides a method for the isolation and purification of total RNA from healthy or infected with viruses or viroid cells of plants, animals, or bacteria, as well as DNA.
  • high molecular weight chromosomal DNAs can be obtained, since trichloroacetate mixes well with the aqueous phase of the cell or nuclear lysate.
  • This makes it possible to exclude shaking of the DNA solution with a deproteinizing agent, which is necessary in the case of Trizol, phenol, chloroform, etc., which are poorly miscible with water.
  • TXAA is able to effectively exert a dissociating effect on any naturally occurring complexes formed by nucleic acids, in particular nucleoprotein ones.
  • the method can be used both for the isolation of viroid or viral RNA, and for the isolation of RNA and DNA from cells of plants, animals or bacteria.
  • the present invention relates to a method for isolating total RNA from viruses, bacteria, plant cells (including viroid RNA) or animals, comprising the following stages: a) dissociation of nucleoprotein and other natural nucleic acid complexes with ammonium trichloroacetate; and b) purification of RNA by sorption-desorption of RNA on filter paper or cotton wool.
  • the cells of plants, animals, or bacteria are healthy cells.
  • the cells of plants, animals or bacteria are cells infected with viruses, infected or transformed by a viroid.
  • the dissociation of nucleoprotein and other the complexes are carried out using an aqueous solution of ammonium trichloroacetate in a final concentration of 2-4 M in an environment having a pH close to neutral at room temperature.
  • the present invention relates to the use of ammonium trichloroacetate as an agent for the dissociation of natural nucleic acid complexes.
  • RNA complexes are nucleoprotein complexes, more preferably viruses, informosomes, ribosomes.
  • RNA complexes are complexes with DNA or with cell membranes.
  • the natural nucleic acid complexes are DNA complexes.
  • the DNA complexes are nucleoprotein complexes.
  • FIG. IA Electrophoresis of XBK RNA preparations obtained by treating a viral preparation with a 3 M or 6 M TXAA solution at different times.
  • RNA preparations were carried out on a 1% agarose gel, RNA was stained with bromide this day.
  • FIG. 1B Electrophoresis of BTM RNA preparations obtained by treating a viral preparation with a 4 M TXAA solution at different times.
  • RNA preparations were carried out on a 1% agarose gel, RNA was stained with ethidium bromide.
  • FIG. 2 Comparison of the electrophoretic mobility of BTM RNA and XBK RNA preparations isolated from purified viruses using TXAA (lanes 1 and 3) and the phenol-chloroform deproteinization method (lanes 2 and 4).
  • RNA preparations have the same electrophoretic RNA mobility.
  • FIG. 3 TXAA inhibits cell nucleases.
  • Lane 1 A clarified potato leaf lysate (100 mg) of potato was added to an XBK RNA solution in the presence of 3 M TXAA. The mixture was incubated for 30 min at room temperature, passed through a cellulose suspension column and analyzed by RNA gel electrophoresis. Lane 2 - Control. RNA was incubated as described above; water was added to the reaction mixture instead of the cell lysate.
  • FIG. 4 Electrophoresis of total cellular RNA preparations obtained at different final concentrations of TXAA from potato leaf tissue.
  • Lanes 1–4 Total potato RNA isolated using 2.5 M, 3 M, 3.5 M, and 4 M TXAA, respectively. A weighed portion of plant tissue (100 mg) was suspended at room temperature in a different concentration of TXAA solution and kept for 30 min. RNA was purified on a microcolumn with filter paper (see examples). The RNA pellet was dissolved in 50 ⁇ l, 15 ⁇ l of RNA solution was applied to the gel. Lane 5 - XBK RNA (2 ⁇ g) isolated by the phenol-detergent method.
  • FIG. 5 Electrophoresis of RNA preparations obtained using TXAA from ascites carcinoma cells Krebs P.
  • Lanes 1, 6 Total RNA of uninfected Krebs II cells isolated by the phenolic method (4 ⁇ g); lane 2 - total RNA of uninfected Krebs II cells isolated by TXAA (10 ⁇ g); lane 3 — total RNA of Krebs II-infected Mengo virus cells isolated by TXAA (10 ⁇ g); lane 4 — Mengo virus RNA (2 ⁇ g) isolated by the phenolic method; lane 5 - BTM RNA (2 ⁇ g) isolated by the phenolic method.
  • FIG. 6 Optical absorption spectra of total RNA isolated from Krebs II ascites carcinoma cells.
  • FIG. 7 Electrophoretic analysis of the preparation of cellular RNA from the bacterium Escherichia coli obtained using ammonium trichloroacetate.
  • Lane 1 - witness - lbs RNA of E. coli isolated by the phenol-detergent method of deproteinization The preparation contains 5s rRNA and trace amounts of tRNA.
  • Lanes 2-4 An RNA preparation obtained by treating 2.8 M TXAA bacteria. To the holes 12 ⁇ l (2), 5 ⁇ l (3) and 1 ⁇ l (4) were applied from 50 ⁇ l of a cell RNA preparation solution obtained from 20 mg of frozen cells. Less mobile RNA (the zone immediately above the Ibs rRNA in the resulting preparation) corresponds to the mobility of 23 ⁇ rRNA of bacteria.
  • FIG. 8 Detection of the recombinant plasmid pGEM-ZZ (PSTVd) with the cDNA insert of the potato tuber spindle viroid (BBKK) and the viroid itself (BBKK, PSTVd) by MGA ELISA on a nitrocellulose membrane in RNA preparations obtained with TXAA filters and purified paper , from collection varieties of potatoes using a DNA probe (dienPt) pGEM-ZZ (PSTVd).
  • the top row of numbers indicates the amount of recombinant plasmid in picograms (pg) deposited on the membrane.
  • 400 pg of total RNA preparations isolated by TXAA from infected (lane 1) and healthy (lane 2-4) plants were applied to the membrane.
  • Nucleic acid preparations were fixed on a nitrocellulose membrane by irradiation with UV light at 254 nm and were detected by hybridization with a specific DNA probe.
  • a DNA probe is a recombinant plasmid containing an insert of a viroid cDNA that determines the specificity of the probe.
  • the DNA probe contains a label - diene-platinum, which is a hapten and can be detected using specific antibodies (V.I. Kiseleva, M.F. Turchinsky, TB.Kolesnik, A.M. Attorney, Bioorg. Chemistry, T. 20, pp. 14-20 (1994)).
  • the bound primary antibodies were detected using conjugates of secondary antibodies with the enzyme and its chemiluminescent substrate.
  • the luminescence of the enzymatic reaction products illuminates the x-ray film, allowing visualization of BBKK in the analyzed samples.
  • FIG. 9 Diagnostic analysis of the infection of potatoes with the virus X using the MGA-ELISA technology.
  • Lane 1 is a recombinant plasmid containing an XBK cDNA insert.
  • Lanes 2-5 are total RNA preparations obtained from healthy (2, 4) and XBK (3, 5) infected potato plants (Nevsky variety) by the phenolic method (4, 5) and using TXAA (2, 3).
  • FIG. 10 A comparative analysis of the sensitivity of the diagnosis of viroid (BBKK) infection by PCR technology (see Fig. 10A), MGA-ELISA (Fig. 10B) and RT- PCR-MGA-ELISA (Fig. 10B) preparations of total RNA obtained using
  • TXAA from collection potato leaf tissue.
  • Lane d4 positive control — recombinant plasmid pGEM-ZZ (PSTVd); further - RNA preparations from test plants of varieties Udacha (1), Golubizna (2), Ilyinsky (3), Price (4), Lugovsky (5), Bryansk (6), Skoroplodny (7), Zhukovsky early (8), Bryansk novelty (9), Autumn (10), Christmas (11), Orlando (12), Elizabeth (13) and RNA from BBKK-infected tomatoes (14-17).
  • Lane M is a length marker (in bp), the numbers on the right indicate the lengths of DNA fragments of the lambda phage, the products of exhaustive hydrolysis of DNA by PsU restriction endonuclease.
  • RNA preparation from Udacha potato and sample 16 in FIG. 10B is an RNA preparation from a highly infected tomato.
  • FIG. 11 Determination of the specificity (diene-Pt) of a DNA probe (labeled recombinant plasmid with an Mengo virus cDNA insert) using MGA ELISA.
  • RNA preparations were obtained by phenol-detergent deproteinization. The analyzed preparations were applied to the nitrocellulose filter in the indicated amounts.
  • Lane 1 Mengo virus RNA (50 pg); 2 - Mengo virus RNA (5 pg); 3 - encephalomyocarditis virus RNA (VEMK, 50 pg); 4 - VEMK RNA (5 pg); 5 - RNA (BTM, 50 pg); E.
  • coli 6-16S rRNA (50 pg); 7-12 - recombinant plasmid with the insertion of cDNA of Mengo virus (7 - 1 ng, 8 - 250 pg, 9 - 50 pg, 10 - 12.5 pg, 11 - 2.5 pg, 12 - 0.5 pg).
  • FIG. 12 Determination of viral RNA in Krebs II cells infected Mengo virus, MGA-ELISA
  • the upper row is the total RNA of Krebs II cells infected with the Mengo virus.
  • the bottom row is the total RNA of uninfected Krebs II cells.
  • FIG. 13 Determination of BBKK by a diene-platinum DNA probe in preparations of viroid RNA (rows 2-4) obtained by different methods from leaf tissue of potatoes.
  • Rows 1 and 5 are from healthy plants.
  • the present invention provides a new environmentally friendly method for producing pure RNA preparations from viruses, bacterial, plant and animal cells, and viroid RNA.
  • the proposed method differs from the known methods using a non-toxic chaotropic / dissociating nucleic acid complexes of an agent, ammonium trichloroacetate.
  • Ammonium trichloroacetate is not included in the list of toxic substances (PAN (Restiside ⁇ stiop Netw Albanyrk N Cincinnatirth ⁇ m convincedrisa) Restiside Database, ⁇ mmopium trichloroacetate) in Canada and the USA.
  • PAN Estimatside ⁇ stiop Netw Albanyrk N Cincinnati ⁇ m convincedrisa
  • Restiside Database ⁇ mmopium trichloroacetate
  • Another advantage of the claimed method is its versatility and the possibility of application for the isolation of RNA of various origin.
  • TCA trichloroacetic acid
  • a trichloroacetate ion has a chaotropic (in this case, dissociating complex natural complexes, which include nucleic acids).
  • chaotropic in this case, dissociating complex natural complexes, which include nucleic acids.
  • This chaotropic agent is an alternative to highly toxic substances that are currently widely used in the isolation of RNA, namely, guanidine thiocyanate, phenol and chloroform.
  • RNA preparations obtained by the developed method are suitable for molecular diagnostics of viral infections of plants and animals by the technologies MGA-IFA, PCR (polymerase chain reaction), RT-PCR (reverse transcription - polymerase chain reaction) and DNA chips.
  • the developed method is suitable for isolating a variety of RNA from various sources.
  • the method of the present invention can be used to isolate various RNAs from viruses, as well as from cells of bacteria, plants, and animals, viroid RNA.
  • the method is suitable for isolation of total cellular and viral RNA in the case of plant cells, animals and bacteria infected with viruses or plant cells infected with viruses.
  • RNA preparations obtained from plant tissue and ascites carcinoma cells using the method of the present invention contain typical eukaryotic ribosomal RNAs (18s and 28s rRNAs) with the expected electrophoretic mobility relative to viral RNAs and cell RNAs isolated using the standard phenolic method.
  • the method of the present invention involves the use of filter paper or cotton wool. Without intending to be limited by any scientific theory, the authors suggest that high concentration of TXAA causes the dissociation of both viruses and cellular nucleoprotein complexes, while proteins denature and completely precipitate in the presence of 70% propanol-2, insoluble in water.
  • RNA it is most preferable to purify the isolated RNA on filter paper or cotton wool in a column chromatography format.
  • filter paper is first crushed if necessary, then crushed paper or cotton is loaded into a column, washed and equilibrated with a buffer containing TXAA.
  • the purification procedure includes applying a virus or cell extract preparation treated with TXAA, washing the column with the preparation with buffer containing TXAA, and eluting the RNA with water.
  • RNA sorption-desorption procedure can be carried out by gravity (under the action of gravity), as well as with a small vacuum, which can be created using, for example, a water-jet pump.
  • Columns can be any column that is commercially available from a wide range of manufacturers of chromatographic equipment, as well as conventional medical plastic syringes.
  • the cleaning procedure on columns with fibrous cellulose can be carried out both individually and using a homemade specially made cassette device.
  • TXAA TXAA for the isolation of nucleic acids of various origins, in particular viral RNA from plant and animal viruses. It was found that treatment of potato and tobacco viruses with 3 M TXAA solution (Table 1) for 5 min leads to the dissociation of viral nucleoproteins and the release of RNA molecules, possessing electrophoretic mobility identical to RNA preparations that were obtained from the same viruses by the classical method of phenol-detergent deproteinization, and the dependence of the RNA yield from different plants quantitatively coincided with the theoretical RNA yields (Table 2) and did not yield to RNA yields upon extraction using the phenol chloroform deproteinization.
  • Example 1 The selection of RNA from plant viruses using ammonium trichloroacetate on the example of the tobacco mosaic virus RNA (BTM, TMV)
  • the composition of solutions A, E, TE, and C is shown in Table 1.
  • Filter paper (REAHIM) or sterile cotton wool was used. 0.5 g of filter paper was cut with scissors into small squares (2x2 mm). Transferred to a 50 ml centrifuge tube. Pour 10 ml of solution A. Hold for 5 minutes and vigorously shake on a Vortex shaker to grind the bulk of the pulp into a suspension of paper fibers. A suspension of 70-80 ml of water was diluted and filtered under vacuum. The resulting cellulose paste was suspended in TE buffer solution, packaged in suspension (10 mg / ml) and stored at room temperature.
  • Microcolumns for RNA purification were prepared from 1 ml insulin syringes (ejecting the piston) with a removable needle. To do this, put a paper filter on the bottom of the syringes, fill the columns with 1 ml of cellulose paste under the shallow vacuum of the water-jet pump, using the cartridge we designed. The sorbent was washed with 0.5 ml of solution C, and then 2 times with 0.5 ml of 70% ethyl alcohol and dried for 5-10 minutes under vacuum.
  • RNA bound to the sorbent was eluted with deionized water (MiIIiQ). 1 ml of water was layered on the column three times (waiting until the next portion of water was completely absorbed into the cotton wool) and 3 fractions of the eluate were obtained. The volume of the first fraction varied from 250 to 350 ⁇ l; the volume of the second and third fractions was 1 ml.
  • RNA after treatment of viral particles of TXAA were close to theoretical, and the optical characteristics corresponded to highly purified RNA.
  • Example 2 The selection of RNA from plant viruses using ammonium trichloroacetate on the example of potato virus X RNA (XBK, PVX)
  • RNA yield is close to theoretical (Table 2), and the spectral characteristics (A 260 / A 280 'table 2) and electrophoretic mobility (Fig. . 1, 2) correspond to highly purified viral RNA.
  • Example 3 Isolation of cellular RNA by the example of RNA isolation from leaf tissue of potato using TXAA
  • TXAA TXAA to dissociate not only viral, but also cellular nucleoproteins has been studied on plant and animal cells.
  • RNAs Isolation of high molecular weight viral and cellular RNAs from plant and animal cells at room temperature is possible only in the case of inhibition of cellular ribonucleases by the used chaotropic / dissociating agent. Indeed, incubation of viral RNA with a cell lysate in the presence of TXAA does not cause a noticeable hydrolysis of the viral RNA (Fig. 3), and cellular RNAs have an electrophoretic mobility that matches that of whole rRNA and tRNA molecules. At the same time, in the absence of TXAA, all RNAs were completely degraded (not shown).
  • a sample (50-100 mg) of potato plant tissue (test plant) at room temperature was ground in a mortar with 500-1000 ⁇ l of buffer solution E (Table 1) for deproteinization and RNA extraction.
  • the mixture was transferred to a test tube, 50-100 ⁇ l of a suspension of filter paper in a 4 M TXAA solution was added, and to the resulting mixture was added propanol-2 to a final concentration of 70%.
  • the mixture was applied to a microcolumn filled with a suspension (200 ⁇ l) of cellulose or cotton wool (the columns were filled in advance, they can be stored for a long time). As a rule, several (up to a hundred) samples were prepared simultaneously.
  • RNA sorbed on fibrous cellulose was eluted with distilled water (2x500 ⁇ l) and concentrated by precipitation with alcohol. The precipitate was dissolved in 50 ⁇ l of water.
  • Fig. 4 An agarose gel electrophoretic analysis (Fig. 4) of the resulting preparation of total potato cellular RNA showed that the preparation contains a typical set of eukaryotic ribosomal and transport RNAs.
  • the ratio A 260 / A 28 o for preparations of total potato RNA was in the range of 1.85-2.0, which is typical for deproteinized RNA.
  • Example 4 Isolation of cellular RNA from leaf tissue of tobacco Ni restroomtiapa glitiposa using ammonium trichloroacetate A weighed portion of 1 g of fresh tobacco leaf was quickly homogenized in a mortar with 6 ml of buffer solution E with different contents of TXAA. Sample homogenization time during incubation was not included. Aliquots of 1.5 ml were collected in microcentrifuge tubes and incubated for 5, 10, 20, and 30 min at room temperature without stirring. Then, the samples were centrifuged (10,000 x g, 5 min) in a Errepdor 5414 benchtop centrifuge. Centrifugation time was turned on during incubation. 1 ml of the supernatant was taken, mixed with 2.5 ml of isopropanol, and the mixture was layered on a column. The results of the isolation of total cellular tobacco RNA are presented in tables 3.4.
  • the optimal conditions for obtaining a cellular RNA preparation from tobacco should be considered: 3 M TXAA tissue processing, and the incubation time of the cell extract with TXAA is 20 minutes.
  • Example 5 The selection of cellular RNA from cells of ascites carcinoma (mice) Krebs II
  • hypotonic buffer (10 mM Tris-Hcl, 15 mM NaCl, 1 mM MgCl 2 , pH 8.0) was added to 430 mg of Krebs II cells and kept for 20 minutes in ice. Then the cells were homogenized in a downs manual homogenizer (20 tractions) and centrifuged for 10 minutes at 2500 rpm in the cold (nuclear fraction was precipitated). The supernatant was distributed in 12 tubes (0.9 ml each), 100 ⁇ l of 10% SDS, pH 7.8 were added and 3 deproteinizations were performed (equal volumes of phenol / chloroform mixture (9: 1) were added, vigorously shaken for 2-3 min. centrifuged for 2 min at 12,000 rpm).
  • RNAs are poorly soluble already in 2M TXAA and precipitate together with the dissociated denatured protein.
  • hypotonic buffer solution (10 mM Tris-Hcl, pH 8.0, 15 mM NaCl, 1 mM MgCl 2 ) was added, kept for 20 min in ice, homogenized in a Downs hand-held homogenizer (20 tractions) and centrifuged 10 min at 5000 rpm
  • An equal volume of buffer solution E was added to the supernatant (Table 1), stirred and left overnight at + 4 ° C.
  • an equal volume of 100% propanol-2 was added to the mixture to complete RNA deposition, left for 1 h at -2O 0 C and centrifuged for 5 minutes at 12,000 rpm.
  • RNA of uninfected Krebs II cells RNA of the Mengo virus and tobacco mosaic virus isolated by the phenolic method were used.
  • RNA preparations obtained using TXAA are of great importance; nucleic acid preparations obtained by phenol-detergent deproteinization are standard.
  • a comparison of the optical absorption spectra of the total RNA preparations obtained from the cells of ascites carcinoma Kreb II using TXAA and the phenol-detergent method showed their complete identity (Fig. 6).
  • Example 6 Obtaining a preparation of cellular RNA from the bacterium Escherichia coli using ammonium trichloroacetate
  • E. coli cells (20 mg) were suspended in 200 ⁇ l of water for 3 min at room temperature and 800 ⁇ l of E. solution was immediately added. The cell lysate was kept for 5 min at room temperature and centrifuged for 20 min at 12000 rpm on a Errepdor 5414 benchtop centrifuge. 2 volumes of alcohol were added to the supernatant and the volume precipitate was separated by centrifugation. The precipitate was washed twice with 1 ml of 70% alcohol, dried in a vacuum desiccator and suspended for 10 min in 50 ⁇ l of water.
  • the insoluble precipitate was separated by centrifugation and 1, 5 and 12 ⁇ l samples were taken for analysis by agarose gel electrophoresis in the presence of ethidium bromide (Fig. 7). Electrophoretic analysis showed that bacterial cell nucleoproteins dissociate in the presence of TXAA, while ribosomal and transport RNAs are isolated in free form.
  • Example 7 Detection of RNA viroid of the spindle-shaped potato tubers (BBKK) in the total RNA preparation obtained using TXAA from an infected plant
  • the next objective of the invention was to prove the suitability of the obtained RNA preparations for the molecular diagnosis of viroid and viral infections of plants and animals, based on the principle of molecular hybridization of nucleic acids, i.e. for technologies MGA-IFA, RT-PCR, etc.
  • the technology for detecting the target nucleic acid MGA-ELISA is a combination of the Molecular Hybridization Analysis and Immuno-Enzyme Analysis methods.
  • Nucleic acid preparations of the analyzed plant and animal samples are fixed by irradiation with UV light on a nitrocellulose membrane and hybridized with a specific DNA probe.
  • a DNA probe is a recombinant plasmid containing a cDNA insert of a virus or viroid, which determines the specificity of the probe.
  • the DNA probe contains a label - diene-platinum, which is a hapten and can be detected using specific antibodies (V.I. Kiseleva, M.F. Turchinsky, TB.Kolesnik, A.M. Attorney, Bioorg. Chemistry, 20, pp. 14-20 (1994).
  • the bound primary antibodies are detected using commercially available conjugates of secondary antibodies with the enzyme and its chemiluminescent substrate. The luminescence of the enzymatic reaction products illuminates the X-ray film, allowing the pathogen to be visualized in the analyzed samples.
  • RNA preparation obtained from infected cells can be determined using a specific DNA probe.
  • RNA preparation from 100 mg of potato leaf tissue was dissolved in 60 ⁇ l of sterile water. Prior to analysis, the experimental and control samples were denatured in a boiling water bath (3 min), quickly cooled in ice water, and 2 ⁇ l of the analyzed RNA solution was applied to nitrocellulose filters Protap BA-85 or BA-83 (Schleicher & Shuell).
  • the filters were baked at 80 ° C in vacuum (on a standard dryer for polyacrylamide gels) for two hours or irradiated with UV light (Drygin Yu.F., Buzmakov AB, Teterina H.L., Morozov SJ. Molec. Biol . - 1992. - T. 26. - ML. - p. 59-69).
  • RNA fraction from infected potato enriched with viroid RNA by differential precipitation of BBKK with LiCl solutions was separated by electrophoresis in a 7% polyacrylamide gel.
  • Four zones close in mobility to a polynucleotide with a length of 459 nucleotides were excised from the gel.
  • coli cells and isolation of pGEM-ZZ was performed according to the protocols (Mapiatis, T ., Fritsh, E.F. apd Sambrook, J. 1989. Molesuloplopg, A Laboru Mapual (2 nd ed.), CoId Sprig Nagor Laborogo, CoId Sprig Nagog, NY). Additionally, the plasmid was purified by equilibrium centrifugation (see A Laboru Mapul, mentioned above) in the density gradient of cesium chloride with ethidium bromide and was hydrolyzed with WatHl restrictase. Phenol deproteinized in the presence of sodium dodecyl sulfate plasmid was used to synthesize a DNA probe.
  • RNA preparations for molecular diagnostic analysis were determined visually.
  • a plasmid with experimental samples was subjected to serial dilutions of denatured pGEM-ZZ (PSTV in a series of 200 - 0.1 pg of plasmid, which served as an internal indicator of the sensitivity of pathogen determination in this experiment.
  • Samples applied onto a nitrocellulose membrane were hybridized with labeled pGEM-ZZ recombinant DNA diene-platinum (PSTV).
  • PSTV labeled pGEM-ZZ recombinant DNA diene-platinum
  • the bound probe was determined by indirect enzyme-linked immunosorbent assay on membranes. Hybridization conditions of the BBKK-specific DNA probe and visual determination of Tests of the analyzed samples (see Fig.
  • a DNA probe i.e. about Zl O "20 g mole [Drygin Yu.F., Musin SM., Kondakova OA, Savenkov E.I., Solomatin CB, Mozheva KA, academician PACXH Atabekov I.G. ((Molecular diagnosis of infection of healthy potato varieties Of the Russian Federation with viroid spindle-shaped spiders ”, PACXH Reports, 6, 24-25 (1996); Kondakova OA, Drygin Yu.F. Diagnosis of potato viroid disease with probes (diene) Pt-DNK, Biotechnology, JY, pp. 83- 90 (1999)).
  • Example 8 The suitability of RNA preparations obtained from plants infected with viruses for diagnostic MGA-ELISA technology
  • RNA The detection of viral (BTM and XBK) RNA in infected plants was carried out similarly as described in Example 7, except for the use of specific for each virus DNA probes.
  • cDNAs of each virus were obtained by cloning ip vitro using RT-PCR and appropriate primers.
  • the length of the cloned DNA fragments was in the range of 450-1600 nucleotide pairs.
  • an XBK potato infection is reliably visible in a series of concentrations of total RNA of 200-2000 ng / ⁇ l, moreover, viral RNA is determined in the same way both in preparations obtained by classical phenol-detergent deproteinization and in a preparation obtained using TXAA ( Fig. 9).
  • Example 9 The suitability of RNA preparations obtained from plants infected with viruses for the diagnosis of RT-PCR and RT-PCR-MGA-ELISA technologies
  • TXAA-derived RNA preparations were amplified by RT-PCR and the products were analyzed by gel fluorescence (Fig. 10A). Similar samples were applied to a nitrocellulose filter (Fig. 10B) and analyzed by MGA-ELISA (see Example 7) or by RT-PCR- MGA-ELISA (Fig. 10B).
  • MGA-IFA ⁇ OT-PCR MGA-IFA-OT-PCR
  • RNA preparations obtained from plant materials are suitable for diagnostic analysis using RT-PCR and RT-PCR-MGA-ELISA.
  • Example 10 Determination of viral RNA by MGA-ELISA technology in a total RNA preparation obtained from Krebs II carcinoma cells infected with Mengo virus
  • RNA preparations from uninfected and infected cells were obtained as described in Example 5.
  • the DNA probe was a recombinant plasmid containing a Mengo virus cDNA insert labeled with dien-platinum.
  • the labeling of specific recombinant DNA with dien-platinum and the detection of the target viral RNA were performed as described in Example 7.
  • the specificity of the DNA probe was determined on control (positive and negative) RNA preparations. Determination of the specificity of the (diene-Pt) -DNA probe (labeled recombinant plasmid with the addition of Mengo virus cDNA) by MGA-ELISA is shown in FIG. 11.
  • the used DNA probe labeled with diene-platinum has a high specificity.
  • DNA probe is not recognizes not related RNAs, namely, BTM RNA and E. coli 16S rRNA.
  • the DNA probe to the Mengo virus is significantly better (compare samples 2 and 4) than VEMK RNA recognizes the Mengo virus RNA.
  • FIG. Figure 12 shows the result of a diagnostic analysis of ascites carcinoma cells in total RNA preparations obtained with TXAA after a 5-hour infection with their Mengo virus. Diagnostic analysis was carried out similarly to that described in Example 7. As can be seen, 10 ng of total cellular RNA isolated from infected cells 5 hours after infection is sufficient to determine viral infection in the early stages of infection, while “diene-platinum” The DNA probe does not bind to 1 ⁇ g of cellular RNA.
  • RNA preparations obtained from test plants or leaf potato tissue or from animal cells using TXAA are suitable for specific and sensitive diagnosis of viroid and viral infections of potato, plum and animal cells. It is important that animals can be diagnosed with picornavirus infection as early as 5 hours after infection, i.e. in its early stages, moreover, 10 ng of total cellular RNA isolated from infected cells is sufficient to detect viral RNA. It is important that the “wild” diene-platinum DNA probe is highly specific and does not bind to a huge excess (1 ⁇ g) of cellular RNA (Fig. 12).
  • Example 11 Comparative analysis of the detection efficiency of viroid RNA preparations obtained by different methods from leaf tissue of potato

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Abstract

L'invention concerne les domaines de la biologie, de la biotechnologie, du génie génétique et de la médecine et permet d'assurer un procédé universel d'extraction et de purification d'ARN, y compris à des fins diagnostiques, lors des analyses de masse par procédés de sondes ARN (ADN), OT-PCR, etc. L'extraction d'ARN et d'ADN s'effectue grâce à l'utilisation de trichloracétate d'ammonium dans un milieu aqueux neutre et une suspension de papier filtrant ou de coton pour la sorption d'ARN et le nettoyage de celui-ci des protéines, des substances à faible masse moléculaire, etc. Le procédé de la présente invention permet d'extraire l'ARN à partir de virus de plantes et d'animaux, à partir de cellules végétales, animales ou bactériennes, le rendement et la pureté des préparations à base d'ARN étant identiques au rendement et à la pureté obtenus par des procédés traditionnels au moyen d'agents hautement toxiques et dangereux. En outre, la présente invention prévoit l'utilisation du trichloracétate d'ammonium en tant que produit universel pour dissocier des complexes naturels formés par des acides nucléiques. Le trichloracétate d'ammonium est une composition facilement disponibles, hydrosoluble, non toxique et sans danger pour l'environnement.
PCT/RU2007/000310 2007-06-08 2007-06-08 Utilisation du trichloracétate d'ammonium en tant que produit de dissociation de complexes naturels d'acides nucléiques et procédé d'extraction d'arn WO2008150187A1 (fr)

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Cited By (2)

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Publication number Priority date Publication date Assignee Title
EP2345719A1 (fr) * 2010-01-18 2011-07-20 Qiagen GmbH Procédé d'isolation de petit ARN
CN102725407A (zh) * 2010-01-07 2012-10-10 比格科技私人有限公司 分离核酸的方法及其试剂盒

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WO2003040687A2 (fr) * 2001-11-06 2003-05-15 Cortex Biochem, Inc. Isolement et purification d'acides nucleiques
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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102725407A (zh) * 2010-01-07 2012-10-10 比格科技私人有限公司 分离核酸的方法及其试剂盒
JP2013516186A (ja) * 2010-01-07 2013-05-13 ビッグテック プライベート リミテッド 核酸の単離方法およびそのキット
EP2521780A4 (fr) * 2010-01-07 2013-07-31 Bigtec Private Ltd Procédé pour isoler des acides nucléiques, et kit à cet effet
EP2345719A1 (fr) * 2010-01-18 2011-07-20 Qiagen GmbH Procédé d'isolation de petit ARN
WO2011086195A1 (fr) * 2010-01-18 2011-07-21 Qiagen Gmbh Procédé d'isolement de petit arn

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