WO2012044193A1 - A method of extraction and purification of nucleic acids from liquid medium and a vessel of plastic for nucleic acids sorption from liquid medium - Google Patents

A method of extraction and purification of nucleic acids from liquid medium and a vessel of plastic for nucleic acids sorption from liquid medium Download PDF

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Publication number
WO2012044193A1
WO2012044193A1 PCT/RU2010/000731 RU2010000731W WO2012044193A1 WO 2012044193 A1 WO2012044193 A1 WO 2012044193A1 RU 2010000731 W RU2010000731 W RU 2010000731W WO 2012044193 A1 WO2012044193 A1 WO 2012044193A1
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Prior art keywords
vessel
nucleic acids
coating
silicon oxide
sorption
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PCT/RU2010/000731
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French (fr)
Inventor
Evgenij Anatol'evich Bogdanov
Aleksandr Jur'evich Gojhman
Maksim Vladimirovich Patrushev
Original Assignee
Federal'noe Gosudarstvennoe Avtonomnoe Obrazovatel'noe Uchrezhdenie Vysshego Professional'nogo Obrazovanija "Baltijskij Federal'nyj Universitet Imeni Immanuila Kanta"
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Priority to EP10836821.8A priority Critical patent/EP2622072A1/en
Publication of WO2012044193A1 publication Critical patent/WO2012044193A1/en

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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
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/02—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material
    • B01J20/10—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material comprising silica or silicate
    • B01J20/103—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material comprising silica or silicate comprising silica
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3202—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the carrier, support or substrate used for impregnation or coating
    • B01J20/3206—Organic carriers, supports or substrates
    • B01J20/3208—Polymeric carriers, supports or substrates
    • B01J20/321—Polymeric carriers, supports or substrates consisting of a polymer obtained by reactions involving only carbon to carbon unsaturated bonds
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3202—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the carrier, support or substrate used for impregnation or coating
    • B01J20/3206—Organic carriers, supports or substrates
    • B01J20/3208—Polymeric carriers, supports or substrates
    • B01J20/3212—Polymeric carriers, supports or substrates consisting of a polymer obtained by reactions otherwise than involving only carbon to carbon unsaturated bonds
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3231—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the coating or impregnating layer
    • B01J20/3234—Inorganic material layers
    • B01J20/3236—Inorganic material layers containing metal, other than zeolites, e.g. oxides, hydroxides, sulphides or salts
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/508—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above
    • B01L3/5082—Test tubes per se
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J7/00—Chemical treatment or coating of shaped articles made of macromolecular substances
    • C08J7/04—Coating
    • C08J7/0427—Coating with only one layer of a composition containing a polymer binder
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J7/00—Chemical treatment or coating of shaped articles made of macromolecular substances
    • C08J7/04—Coating
    • C08J7/043—Improving the adhesiveness of the coatings per se, e.g. forming primers
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J7/00—Chemical treatment or coating of shaped articles made of macromolecular substances
    • C08J7/12—Chemical modification
    • C08J7/16—Chemical modification with polymerisable compounds
    • 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
    • C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
    • C—CHEMISTRY; METALLURGY
    • C23—COATING METALLIC MATERIAL; COATING MATERIAL WITH METALLIC MATERIAL; CHEMICAL SURFACE TREATMENT; DIFFUSION TREATMENT OF METALLIC MATERIAL; COATING BY VACUUM EVAPORATION, BY SPUTTERING, BY ION IMPLANTATION OR BY CHEMICAL VAPOUR DEPOSITION, IN GENERAL; INHIBITING CORROSION OF METALLIC MATERIAL OR INCRUSTATION IN GENERAL
    • C23C—COATING METALLIC MATERIAL; COATING MATERIAL WITH METALLIC MATERIAL; SURFACE TREATMENT OF METALLIC MATERIAL BY DIFFUSION INTO THE SURFACE, BY CHEMICAL CONVERSION OR SUBSTITUTION; COATING BY VACUUM EVAPORATION, BY SPUTTERING, BY ION IMPLANTATION OR BY CHEMICAL VAPOUR DEPOSITION, IN GENERAL
    • C23C14/00—Coating by vacuum evaporation, by sputtering or by ion implantation of the coating forming material
    • C23C14/06—Coating by vacuum evaporation, by sputtering or by ion implantation of the coating forming material characterised by the coating material
    • C23C14/10—Glass or silica
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2220/00—Aspects relating to sorbent materials
    • B01J2220/50—Aspects relating to the use of sorbent or filter aid materials
    • B01J2220/64—In a syringe, pipette, e.g. tip or in a tube, e.g. test-tube or u-shape tube
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2220/00—Aspects relating to sorbent materials
    • B01J2220/80—Aspects related to sorbents specially adapted for preparative, analytical or investigative chromatography
    • B01J2220/86—Sorbents applied to inner surfaces of columns or capillaries
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0848—Specific forms of parts of containers
    • B01L2300/0858—Side walls
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/16—Surface properties and coatings
    • B01L2300/161—Control and use of surface tension forces, e.g. hydrophobic, hydrophilic
    • B01L2300/163—Biocompatibility
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J2323/00—Characterised by the use of homopolymers or copolymers of unsaturated aliphatic hydrocarbons having only one carbon-to-carbon double bond; Derivatives of such polymers
    • C08J2323/02—Characterised by the use of homopolymers or copolymers of unsaturated aliphatic hydrocarbons having only one carbon-to-carbon double bond; Derivatives of such polymers not modified by chemical after treatment
    • C08J2323/04—Homopolymers or copolymers of ethene
    • C08J2323/06—Polyethene
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J2323/00—Characterised by the use of homopolymers or copolymers of unsaturated aliphatic hydrocarbons having only one carbon-to-carbon double bond; Derivatives of such polymers
    • C08J2323/02—Characterised by the use of homopolymers or copolymers of unsaturated aliphatic hydrocarbons having only one carbon-to-carbon double bond; Derivatives of such polymers not modified by chemical after treatment
    • C08J2323/10—Homopolymers or copolymers of propene
    • C08J2323/12—Polypropene
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J2369/00—Characterised by the use of polycarbonates; Derivatives of polycarbonates
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J2483/00—Characterised by the use of macromolecular compounds obtained by reactions forming in the main chain of the macromolecule a linkage containing silicon with or without sulfur, nitrogen, oxygen, or carbon only; Derivatives of such polymers
    • C08J2483/02—Polysilicates

Definitions

  • the invention relates to the field of biotechnologies, in particular to a method of extraction and purification of nucleic acids.
  • the drawback of the said invention is a low degree of homogeneity and uniformity of coating as well as corresponding output and processability. High degree of backing material deformation. The particles are not disposed throughout the whole surface of a vessel.
  • a device for DNA isolation and a method Patent WO2004046231 (Al), published 2004-06-03
  • a vessel of plastic for nucleic acids sorption from liquid medium which comprises a coating of silicon oxide on inner surface of the vessel being made of plastic, having a monolayer of particles at least on a part of the surface of the vessel, wherein said particles are selected from silica, silica gel or glass.
  • the method of DNA extraction from crude preparation comprises the sorption thereof on inner walls of the vessel, rinsing out of admixtures and elution in a saline solution.
  • the drawback of the said invention is its low output and processability, low sorption properties, impossibility to uniformly coat the whole surface of the vessel, both on large and limited areas, a possibility of backing material deformation, low optical transparency.
  • the said invention is the closest technical solution to the claimed solution, i.e. the prior art.
  • the technical problem of the present solution is to increase the output and processability of the process of extraction and purification of nucleic acids, increase the process rate, decrease the number of performed operations, increase the purity of obtained nucleic acids, increase the sorption properties of a vessel, in which the process being performed, ensures uniform coating throughout the whole surface, both on large and limited areas, increase the optical transparency of the vessel.
  • a vessel of plastic for nucleic acids sorption from liquid medium comprising a coating of silicon oxide on inner vessel surface representing a nano-coating (defined as coating having a thickness from 1 to 1000 nm), formed by a thin-film synthesis.
  • the plastic is selected from a group comprising poly vinylchloride, polyethylene, polypropylene, polystyrene, polycarbonates, acrylonitrile- butadiene-styrene, polyurethanes, polyamides, thermoelastolayers, polysulfones, polyesteresterketones, as well as mixtures thereof.
  • a vessel may be formed as optically transparent.
  • a vessel may be formed in a form of a vial.
  • the said thin-layer synthesis being performed in an ultrahigh vacuum. Besides, the said thin-layer synthesis by ion-plasmous sputtering.
  • the ion-plasmous sputtering being performed by vacuum sputtering of silica target with Ar+ ion flow.
  • the inner surface of the vessel being chemically cleansed by alcohol in an ultrasonic bath before sputtering.
  • the nano-coating thickness doesn't exceed 25 nm.
  • the problem is also solved by providing a method of extraction and purification of nucleic acids from liquid medium, comprising sorption thereof on inner walls of a vessel, rinsing out of admixtures and elution in a saline solution, wherein as a sorbent there is used silicon oxide nano-coating formed by a thin-layer synthesis.
  • silicon oxide nano-coating with the size of from 1 to 25 nm.
  • the vessel with Si0 2 thin-film sputtering is further added with chaotropic agent, pH-buffer, the mixture is agitated, then poured out or taken out by automatic pipette, the vessel is added with alcohol, which volume equals to the nominal volume of the vessel, then agitated and poured out.
  • chaotropic agent pH-buffer
  • the vessel is heated up to 95°C to extract DNA, or up to 65°C to extract RNA, thoroughly agitated and the solution of nucleic acids is taken out into another vessel.
  • the problem is solved by providing a method of extraction and purification of nucleic acids from liquid medium comprising sorption thereof on inner walls of a vessel, rinsing out of admixtures and elution in a saline solution, wherein as a sorbent there is used silicon oxide Si0 2 nano-coating formed by a thin-film synthesis in a vessel, wherein the vessel is further added with chaotropic agent, pH-buffer, the mixture is agitated, then poured out or taken out by an automatic pipette, further the vessel is added with alcohol which volume equals to the nominal volume of the vessel, then agitated and poured out.
  • silicon oxide nano-coating with the size of from 1 to 25 nm.
  • the vessel is heated up to 95°C to extract DNA or up to 65°C to extract RNA, thoroughly agitated and the solution of nucleic acids is taken out into another vessel.
  • the problem is solved by providing a method of extraction and purification of nucleic acids from liquid medium, comprising the sorption thereof on inner walls of a vessel, rinsing out of admixtures and elution in a saline solution, heating of a vessel, wherein as a sorbent there is used silicon oxide Si0 2 nano-coating, formed by a thin-film synthesis in a vessel, wherein the vessel is further added with a chaotropic agent, pH-buffer, after adding elution solution the vessel is heated up to 95°C to extract DNA or up to 65°C to extract RNA, thoroughly agitated and the solution of nucleic acids is taken out into another vessel.
  • silicon oxide nano-coating with the size of from 1 to 25 nm.
  • Fig. 1 shows a vessel with coating sputtering 1).
  • the vial 2) is coated with a uniform silicon oxide layer throughout the whole inner surface.
  • a vessel of plastic 1 (Fig. 1) formed of polypropylene, polyethylene or polycarbonate with inner walls coating with Si0 2 .
  • the application of Si0 2 is performed in such a way that vessel 2 stays optically transparent, and the layer thickness decreases with an increase of an interior volume of the vessel. That will make it possible to balance the rate of heating of the liquid being in the vessel.
  • the vessel may be in the form of a vial.
  • the vials without inner coating have the same thickness of the wall throughout the whole volume. Due to the conical shape the liquid heating in vials without inner coating is nonuniform.
  • the synthesis of a thin-film layer of silicon oxide has been performed in a way of sputtering of a target of silicon oxide by Ar+ high-energy ion flow (1000 ⁇ 1500 eV).
  • the vials are subjected to chemical rinsing with alcohol in an ultrasonic bath.
  • the backing holder makes it possible to obtain up to a thousand of samples in a single cycle ( ⁇ 1 hour).
  • the thickness of silicon oxide synthesized on the surface of a vial ( ⁇ 2 ⁇ 400nm) is sufficient for nucleic acids sorption on inner walls and ensures high degree of transparency of obtained samples of plastic vials. Due to optimum ion energy according to the given method the obtained prototypes possess high adhesion and uniform layer of a thin- film coating.
  • the performing of the said process of ultrahigh vacuum Si0 2 thin-film synthesis results in ultrathin silicon oxide nano-coatings on the inner side of the vials with the volume of from 0,2 to 50 ml, used for biochemical, molecular- biological, genetic, cytologic and other researches.
  • the adjustment of synthesis process results in the selection of the most optimum energies of condensable particles making it possible to obtain Si0 2 coatings with high degree of adhesion to the inner vial surface preserving a transparency needed to perform real-time PCR with the use of fluorescent dyes, including the intercalating ones.
  • the coating may be performed on ion-plasmous sputtering plant.
  • a sample of biological material or gel, containing DNA fragments in the amount of 10 - 20000 mg depending on the volume of used vial is put into a vial of polyethylene, polypropylene or polycarbonate with the inner surface being coated with Si0 2. Then from 2 to 10 volumes of solution 1 containing chaotropic agent, lytic agent, salts of univalent cations, pH stabilization agent, agent for chelating, agent for inhibiting fermentative activity are added. An initial addition of the solution with subsequent introduction of biological material is possible. Then the vial containing the components is thoroughly mixed/pipetted/agitated. The solution 2 is taken out of the vial.
  • the implementation of the method is not limited to the given examples. Different combinations of the said agents and salts of univalent elements are possible. For example, there is a possibility to exclude certain agents in case the agent possesses a few said properties. It is possible to exclude an agent with indicated properties if the given property is not vital for biological object which the nucleic acids are extracted or purified from. pH of the final mixture should be within the limits of 7,8 - 8,4 (for DNA extraction and purification), 4,5 - 5,5 (for RNA extraction and purification). The concentration of the salts of univalent cations should be within 250 - 500 mM. It is possible to introduce proteolytic enzymes. In case of proteolytic enzymes introduction, the solution 1 is to be excluded of agents inhibiting the fermentative activity. It is possible to introduce other enzymes which activity is directed at nucleic acids splitting or proteolysis depending on posed objective.
  • the method is performed in the following way.
  • Method 1 10 - 50 mcl of blood is put into a vial containing lytic and/or chaotropic agent (for example, use of thiocyanate, guanidine, sodium dodecylsulphate, lauroyl sarcosine) in the amount of 100 - 180 mcl.
  • the vial contents are thoroughly mixed.
  • sodium chloride is added up to a final concentration of 200-300 mM.
  • the contents are mixed.
  • the contents are poured out. 180 mcl of ethyl alcohol is added to the vial. Mix.
  • the contents are poured out. 180 mcl of ethyl alcohol is added to the vial. Mix.
  • the contents are poured out.
  • the vial is dried up.
  • the mixture-mastermix is added to perform PCR.
  • Method 2 An isolated cell together with a small amount of medium is put into a vial. A buffer containing lOmM of tris-chloride, 2 mM of EDTA, 250 mM of sodium chloride is added. The vial contents are being heated up to 70°C within 5 minutes. Mix. The vial contents are poured out. Mastermix for PCR is added. If the extracted nucleic acids are intended for further fermentative amplification by PCR, reverse transcription, restriction, alloying, modification, then the extracted nucleic acids may be dissolved directly in mixtures to perform the said fermentative reactions. In case of necessity to dissolve the nucleic acids, the vial is introduced with twice-distilled water, water treated by DEPC or a solution preventing nucleic acids degradation. The storage of nucleic acids is effected at minus 20 °C both in dissolved and in sorbed at vial walls states.
  • the percentage (of the total content of DNA or RNA in a sample) of extraction from mammal tissues including human ones - not less than 70 %, bacterial cells - not less than 75 %, of plant tissues - not less than 70 %, of food substances - not less than 60 %, of degraded biological material - not less than 80 %.
  • Residual albumen contents not more than 50 %.
  • a vessel with nano-coating obtained by a thin-film synthesis method preserves all parameters of a vessel (transparency, heat capacity, rigidity) and acquires the new ones - ability for nucleic acids sorption on inner walls of a vial, wherein a high rate of the process, less number of operations, purity of obtained nucleic acids are ensured.
  • a vessel of plastic for nucleic acids sorption from liquid medium may be used to extract and purify nucleic acids from liquid medium, possessing an ability to sorb nucleic acids on inner walls of a vial, providing high process rate, less number of operations, purity of obtained nucleic acids, high reproducibility.

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  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
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  • Chemical Kinetics & Catalysis (AREA)
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  • Life Sciences & Earth Sciences (AREA)
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  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
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  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Polymers & Plastics (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Microbiology (AREA)
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  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
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  • Inorganic Chemistry (AREA)
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  • Materials Engineering (AREA)
  • Hematology (AREA)
  • Metallurgy (AREA)
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  • Apparatus Associated With Microorganisms And Enzymes (AREA)
  • Solid-Sorbent Or Filter-Aiding Compositions (AREA)

Abstract

The invention relates to the field of biotechnologies, in particular to the method of extraction and purification of nucleic acids. The technical problem of the claimed solution is to increase the output and processability of the process of extraction and purification of nucleic acids, increase the process rate, decrease the number of performed operations, increase the purity of obtained nucleic acids, increase the sorption properties of a vessel in which the process being performed, providing uniform coating throughout the whole vessel surface both on large and limited areas, increase the optical transparency of the vessel. The problem is solved by providing a vessel of plastic for nucleic acids sorption from liquid medium comprising silicon oxide coating on inner surface of the vessel, representing a nano-coating (defined as a coating with the thickness of from 1 to 100 nm), formed by a thin- film synthesis. The method of extraction and purification of nucleic acids from liquid medium comprises the sorption thereof on inner walls of a vessel, rinsing out of admixtures and elution in a saline solution, heating the vessel, wherein as a sorbent there is used silicon oxide SiO2 nano-coating formed by a thin-film synthesis in a vessel, wherein the vessel is further added with chaotropic agent, pH-buffer, the vessel after adding the elution solution is heated up to 95°C to extract DNA or up to 65°C to extract RNA, thoroughly agitated and the nucleic acids solution is taken out into another vessel. Besides, there is used silicon oxide nano-coating with the size of from 1 to 25 nm. The embodiments are given.

Description

A METHOD OF EXTRACTION AND PURIFICATION OF NUCLEIC ACIDS FROM LIQUID MEDIUM AND A VESSEL OF PLASTIC FOR NUCLEIC ACIDS
SORPTION FROM LIQUID MEDIUM
Pertinent Art
The invention relates to the field of biotechnologies, in particular to a method of extraction and purification of nucleic acids.
Prior Art
There is known an invention "A METHOD OF EXTRACTION AND PURIFICATION OF NUCLEIC ACIDS" (Patent RU # 2382081, published: 20.02.2010), which discloses a method of D A and RNA extraction and purification by centrifugation which provides nucleic acids sorption on a silicate sorbent being monodisperse spherical silicon dioxide particles with the size of 100-500 run - with subsequent rinsing out of admixtures and elution. The method makes it possible to increase the efficiency of nucleic acids extraction.
The drawback of the said invention is a low degree of homogeneity and uniformity of coating as well as corresponding output and processability. High degree of backing material deformation. The particles are not disposed throughout the whole surface of a vessel.
There is known an invention "A device for DNA isolation and a method" (Patent WO2004046231 (Al), published 2004-06-03), disclosing according to the first embodiment of the invention, a vessel of plastic for nucleic acids sorption from liquid medium which comprises a coating of silicon oxide on inner surface of the vessel being made of plastic, having a monolayer of particles at least on a part of the surface of the vessel, wherein said particles are selected from silica, silica gel or glass. The method of DNA extraction from crude preparation comprises the sorption thereof on inner walls of the vessel, rinsing out of admixtures and elution in a saline solution.
The drawback of the said invention is its low output and processability, low sorption properties, impossibility to uniformly coat the whole surface of the vessel, both on large and limited areas, a possibility of backing material deformation, low optical transparency.
The said invention is the closest technical solution to the claimed solution, i.e. the prior art.
Disclosure of the invention
The technical problem of the present solution is to increase the output and processability of the process of extraction and purification of nucleic acids, increase the process rate, decrease the number of performed operations, increase the purity of obtained nucleic acids, increase the sorption properties of a vessel, in which the process being performed, ensures uniform coating throughout the whole surface, both on large and limited areas, increase the optical transparency of the vessel.
The problem is solved by providing a vessel of plastic for nucleic acids sorption from liquid medium, comprising a coating of silicon oxide on inner vessel surface representing a nano-coating (defined as coating having a thickness from 1 to 1000 nm), formed by a thin-film synthesis.
Besides, the plastic is selected from a group comprising poly vinylchloride, polyethylene, polypropylene, polystyrene, polycarbonates, acrylonitrile- butadiene-styrene, polyurethanes, polyamides, thermoelastolayers, polysulfones, polyesteresterketones, as well as mixtures thereof.
Besides, a vessel may be formed as optically transparent.
Besides, a vessel may be formed in a form of a vial.
Beside, the said thin-layer synthesis being performed in an ultrahigh vacuum. Besides, the said thin-layer synthesis by ion-plasmous sputtering.
Besides, the ion-plasmous sputtering being performed by vacuum sputtering of silica target with Ar+ ion flow.
Besides, characterized by the inner surface of the vessel being chemically cleansed by alcohol in an ultrasonic bath before sputtering.
Besides, the nano-coating thickness doesn't exceed 25 nm.
The problem is also solved by providing a method of extraction and purification of nucleic acids from liquid medium, comprising sorption thereof on inner walls of a vessel, rinsing out of admixtures and elution in a saline solution, wherein as a sorbent there is used silicon oxide nano-coating formed by a thin-layer synthesis.
Besides, there is used silicon oxide nano-coating with the size of from 1 to 25 nm.
Besides, the vessel with Si02 thin-film sputtering is further added with chaotropic agent, pH-buffer, the mixture is agitated, then poured out or taken out by automatic pipette, the vessel is added with alcohol, which volume equals to the nominal volume of the vessel, then agitated and poured out.
Besides, after adding the elution solution the vessel is heated up to 95°C to extract DNA, or up to 65°C to extract RNA, thoroughly agitated and the solution of nucleic acids is taken out into another vessel.
According to embodiment 2 the problem is solved by providing a method of extraction and purification of nucleic acids from liquid medium comprising sorption thereof on inner walls of a vessel, rinsing out of admixtures and elution in a saline solution, wherein as a sorbent there is used silicon oxide Si02 nano-coating formed by a thin-film synthesis in a vessel, wherein the vessel is further added with chaotropic agent, pH-buffer, the mixture is agitated, then poured out or taken out by an automatic pipette, further the vessel is added with alcohol which volume equals to the nominal volume of the vessel, then agitated and poured out.
Besides, there is used silicon oxide nano-coating with the size of from 1 to 25 nm.
Besides, after adding the elution solution the vessel is heated up to 95°C to extract DNA or up to 65°C to extract RNA, thoroughly agitated and the solution of nucleic acids is taken out into another vessel.
According to embodiment 3, the problem is solved by providing a method of extraction and purification of nucleic acids from liquid medium, comprising the sorption thereof on inner walls of a vessel, rinsing out of admixtures and elution in a saline solution, heating of a vessel, wherein as a sorbent there is used silicon oxide Si02 nano-coating, formed by a thin-film synthesis in a vessel, wherein the vessel is further added with a chaotropic agent, pH-buffer, after adding elution solution the vessel is heated up to 95°C to extract DNA or up to 65°C to extract RNA, thoroughly agitated and the solution of nucleic acids is taken out into another vessel.
Besides, there is used silicon oxide nano-coating with the size of from 1 to 25 nm.
Brief Description of the Drawing
Fig. 1 shows a vessel with coating sputtering 1). The vial 2) is coated with a uniform silicon oxide layer throughout the whole inner surface.
A vessel of plastic 1 (Fig. 1) formed of polypropylene, polyethylene or polycarbonate with inner walls coating with Si02. The application of Si02 is performed in such a way that vessel 2 stays optically transparent, and the layer thickness decreases with an increase of an interior volume of the vessel. That will make it possible to balance the rate of heating of the liquid being in the vessel. The vessel may be in the form of a vial. The vials without inner coating have the same thickness of the wall throughout the whole volume. Due to the conical shape the liquid heating in vials without inner coating is nonuniform.
To form a functional thin-film coating on inner side of vials of polypropylene, polycarbonate or polyethylene with a volume of from 0,2 to 50 ml, used for biochemical, molecular-biological, genetic, cytologic and other researches, there are used the methods of formation (synthesis) of thin films (like thermal precipitation, electronic evaporation, gas-phase deposition, electrolytic deposition, cathode spot deposition, magnetron evaporation, ion-plasmous deposition, impulse laser deposition, etc.). For example, while using ion- plasmous method: - the vials are placed with an open end being in the direction of the atomizated material flow. The synthesis of a thin-film layer of silicon oxide has been performed in a way of sputtering of a target of silicon oxide by Ar+ high-energy ion flow (1000÷1500 eV). Preliminary, the vials are subjected to chemical rinsing with alcohol in an ultrasonic bath. The backing holder makes it possible to obtain up to a thousand of samples in a single cycle (~ 1 hour). The thickness of silicon oxide synthesized on the surface of a vial (~2÷400nm) is sufficient for nucleic acids sorption on inner walls and ensures high degree of transparency of obtained samples of plastic vials. Due to optimum ion energy according to the given method the obtained prototypes possess high adhesion and uniform layer of a thin- film coating.
Due to such a combination of parameters there is achieved a required transparency to perform a real time polymerase chain reaction (PCR) with the use of fluorescent dyes, including the intercalating ones.
The performing of the said process of ultrahigh vacuum Si02 thin-film synthesis results in ultrathin silicon oxide nano-coatings on the inner side of the vials with the volume of from 0,2 to 50 ml, used for biochemical, molecular- biological, genetic, cytologic and other researches. The adjustment of synthesis process results in the selection of the most optimum energies of condensable particles making it possible to obtain Si02 coatings with high degree of adhesion to the inner vial surface preserving a transparency needed to perform real-time PCR with the use of fluorescent dyes, including the intercalating ones. The coating may be performed on ion-plasmous sputtering plant.
Thus, there are preserved all physical parameters of a vial (transparency, heat capacity, rigidity) and the new ones are acquired - ability for nucleic acids sorption on inner walls of a vial.
Example of embodiment of the method
A sample of biological material or gel, containing DNA fragments in the amount of 10 - 20000 mg depending on the volume of used vial is put into a vial of polyethylene, polypropylene or polycarbonate with the inner surface being coated with Si02. Then from 2 to 10 volumes of solution 1 containing chaotropic agent, lytic agent, salts of univalent cations, pH stabilization agent, agent for chelating, agent for inhibiting fermentative activity are added. An initial addition of the solution with subsequent introduction of biological material is possible. Then the vial containing the components is thoroughly mixed/pipetted/agitated. The solution 2 is taken out of the vial.
The implementation of the method is not limited to the given examples. Different combinations of the said agents and salts of univalent elements are possible. For example, there is a possibility to exclude certain agents in case the agent possesses a few said properties. It is possible to exclude an agent with indicated properties if the given property is not vital for biological object which the nucleic acids are extracted or purified from. pH of the final mixture should be within the limits of 7,8 - 8,4 (for DNA extraction and purification), 4,5 - 5,5 (for RNA extraction and purification). The concentration of the salts of univalent cations should be within 250 - 500 mM. It is possible to introduce proteolytic enzymes. In case of proteolytic enzymes introduction, the solution 1 is to be excluded of agents inhibiting the fermentative activity. It is possible to introduce other enzymes which activity is directed at nucleic acids splitting or proteolysis depending on posed objective.
The method is performed in the following way.
An example of implementation of the method of extraction and purification of nucleic acids from liquid medium consists in:
Method 1. 10 - 50 mcl of blood is put into a vial containing lytic and/or chaotropic agent (for example, use of thiocyanate, guanidine, sodium dodecylsulphate, lauroyl sarcosine) in the amount of 100 - 180 mcl. The vial contents are thoroughly mixed. Then sodium chloride is added up to a final concentration of 200-300 mM. The contents are mixed. The contents are poured out. 180 mcl of ethyl alcohol is added to the vial. Mix. The contents are poured out. 180 mcl of ethyl alcohol is added to the vial. Mix. The contents are poured out. The vial is dried up. The mixture-mastermix is added to perform PCR.
Method 2. An isolated cell together with a small amount of medium is put into a vial. A buffer containing lOmM of tris-chloride, 2 mM of EDTA, 250 mM of sodium chloride is added. The vial contents are being heated up to 70°C within 5 minutes. Mix. The vial contents are poured out. Mastermix for PCR is added. If the extracted nucleic acids are intended for further fermentative amplification by PCR, reverse transcription, restriction, alloying, modification, then the extracted nucleic acids may be dissolved directly in mixtures to perform the said fermentative reactions. In case of necessity to dissolve the nucleic acids, the vial is introduced with twice-distilled water, water treated by DEPC or a solution preventing nucleic acids degradation. The storage of nucleic acids is effected at minus 20 °C both in dissolved and in sorbed at vial walls states.
Method characteristics
1. The percentage (of the total content of DNA or RNA in a sample) of extraction from mammal tissues, including human ones - not less than 70 %, bacterial cells - not less than 75 %, of plant tissues - not less than 70 %, of food substances - not less than 60 %, of degraded biological material - not less than 80 %.
2. Reproducibility of quantitative and qualitative indices - 90 %.
3. Residual albumen contents - not more than 50 %.
Thus, a vessel with nano-coating obtained by a thin-film synthesis method preserves all parameters of a vessel (transparency, heat capacity, rigidity) and acquires the new ones - ability for nucleic acids sorption on inner walls of a vial, wherein a high rate of the process, less number of operations, purity of obtained nucleic acids are ensured.
Industrial applicability
A vessel of plastic for nucleic acids sorption from liquid medium may be used to extract and purify nucleic acids from liquid medium, possessing an ability to sorb nucleic acids on inner walls of a vial, providing high process rate, less number of operations, purity of obtained nucleic acids, high reproducibility.

Claims

1. The vessel of plastic for nucleic acids sorption from liquid medium, comprising a coating of silicon oxide on the inner surface of the vessel characterized by the coating being a nano-coating formed by a thin-film synthesis.
2. The vessel according to p. 1 characterized by a plastic being selected out of the group consisting of polyvinylchloride, polyethylene, polypropylene, polysterene, polycarbonates, acrylonitrile-butadiene-styrene, polyurethanes, polyamides, thermoelastolayers, polysulfones, polyesteresterketones, as well as mixtures thereof.
3. The vessel according to p. 1, characterized by the vessel being formed as optically transparent.
4. The vessel according to p. 1 characterized by being formed in the form of a vial.
5. The vessel according to p. 1, characterized by a thin-film synthesis being performed in ultrahigh vacuum.
6. The vessel according to p. 5 characterized by the said thin-film synthesis by ion-plasmous sputtering.
7. The vessel according to p. 6, characterized by ion-plasmous sputtering being performed by vacuum sputtering of silica target with Ar+ ion flow.
8. The vessel according to p. 5, characterized by the inner surface of the vessel being chemically cleansed by alcohol in an ultrasonic bath before sputtering.
9. The vessel according to p. 1, characterized by the nano-coating thickness not exceeding 25 nm.
10. The method of extraction and purification of nucleic acids from liquid medium comprising the sorption thereof on inner walls of the vessel, rinsing out of admixtures and elution in a saline solution, characterized by the use as a sorbent of silicon oxide nano-coating formed by a thin-film synthesis.
11. The method according to p. 10 characterized by the use of silicon oxide nano-coating with the size of from 1 to 25 nm.
12. The method according to p.10 characterized by the vessel with a thin-film Si02 sputtering is further added with chaotropic agent, pH-buffer, the mixture is agitated, then poured out or taken out by an automatic pipette, the vessel is then added with an alcohol which volume equals to the nominal volume of the vessel, agitated and poured out.
13. The method according to p. 10 characterized by the vessel after adding an elution solution is heated up to 95°C to extract DNA or up to 65°C to extract RNA, thoroughly agitated and the nucleic acids solution is taken out into another vessel.
14. The method of extraction and purification of nucleic acids from liquid medium comprising the sorption thereof on inner walls of the vessel, rinsing out of admixtures and elution in a saline solution, characterized by the use as a sorbent of silicon oxide Si02 nano-coating formed by a thin-film synthesis in a vessel, wherein the vessel is further added with chaotropic agent, pH-buffer, the mixture is agitated, then poured out or taken out by an automatic pipette, the vessel is further added with an alcohol which volume equals to the nominal volume of the vessel, then it is agitated and poured out.
15. The method according to p. 14 characterized by the use of silicon oxide nano-coating with the size of from 1 to 25 nm.
16. The method according to p. 14 characterized by a vessel after adding an elution solution is heated up to 95°C to extract DNA or up to 65°C to extract R A, thoroughly agitated and the nucleic acids solution is taken out into another vessel.
17. The method of extraction of nucleic acids from liquid medium comprising sorption thereof on inner walls of a vessel, rinsing out of admixtures and elution in a saline solution, heating a vessel, characterized by the use as a sorbent of silicon oxide Si02 nano-coating formed by a thin-film synthesis in a vessel wherein the vessel is further added with chaotropic agent, pH-buffer, the vessel after adding the elution solution is heated up to 95°C to extract DNA or up to 65°C to extract RNA, thoroughly agitated and the nucleic acids solution is taken out into another vessel.
18. The method according to p. 17 characterized by the use of silicon oxide nano-coating with the size of from 1 to 25 nm.
PCT/RU2010/000731 2010-09-30 2010-12-06 A method of extraction and purification of nucleic acids from liquid medium and a vessel of plastic for nucleic acids sorption from liquid medium WO2012044193A1 (en)

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