CN103748208A - Filter module in biomolecule isolation - Google Patents
Filter module in biomolecule isolation Download PDFInfo
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- CN103748208A CN103748208A CN201280031951.4A CN201280031951A CN103748208A CN 103748208 A CN103748208 A CN 103748208A CN 201280031951 A CN201280031951 A CN 201280031951A CN 103748208 A CN103748208 A CN 103748208A
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- filter module
- clarification
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- strainer
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
- C12M47/12—Purification
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D39/00—Filtering material for liquid or gaseous fluids
Abstract
The present invention relates to a device for rapid isolation of target molecules from cell lysates and other liquid mixtures comprising particulate material, a method for isolating the target molecules, in particular nucleic acids, using said device and a kit for carrying out said method comprising said device.
Description
The present invention relates to for the device from cell pyrolysis liquid and other liquid mixture sharp separation target molecules that comprise particulate material, use described device to separate the method for target molecules (especially nucleic acid), and for the test kit of the described method that comprises described device.
In biological chemistry and bioprocess, often need to from the liquid mixture that comprises particle or flocculation material or sample, separate and collect the molecule of particular type.Thereby this can be for example realizes by mixture being filtered in solid or flocculation material are trapped on filtering material/filtering material.Circulation liquid can be contacted with the material of selective binding desired molecule, thereby make desired molecule catch from the liquid filtering.Then can be from bond material wash-out collect desired molecule.
For example, from bacterium, plasmid purification is usually directed to produce cell pyrolysis liquid, and described cell pyrolysis liquid comprises solubility plasmid material and insoluble cell debris, albumen and genomic dna particle.
Lysate is by insoluble particle material is removed to clarify, described in remove conventionally and undertaken by centrifugal or filtration unit.Then the lysate of clarification is transferred to nucleic acid in conjunction with on solid material, described nucleic acid in conjunction with solid material in conjunction with needed plasmid DNA.After optional washing step, then wash-out collect target DNA from bond material.
The clarification of cell pyrolysis liquid and the wash-out of plasmid DNA are widely used in the art and describe, and wherein the filtration step in several different embodiments is by the coupling of filter for installation and DNA column is carried out.Such solution is described in, for example WO95/02049A1, KR2004/085927A, DE202005010007U1, WO2008/121121A2, WO2008/150838A1, WO2009/157679A1 or WO2010/075116A2.
Describe other solution, wherein in a post, comprised for precipitating the strainer held back and DNA bond material, therefore can not remove described strainer.Such a embodiment is shown in for example WO2008/150826A1.
In several prior-art devices, contain two kinds of dissimilar, strainers for solid, particle or throw out are held back, for example fore filter (pre-filter) and deep filter.Described fore filter is normally by the coarse filter that for example lysing cell, cell debris and other particles are not held back, and described deep filter is that thinner precipitation or throw out are held back.Described fore filter is normally made by rigid material, and described rigid material for example, as the porous polyethylene of sintering or polypropylene (WO2008/50838A1, WO2008/50826A1, WO2005/12521A1 or US2006252142A), glass (WO2009/58414A1), wire cloth (WO2003/46178A1) or zeolite (WO2009/60847A1).Described deep filter is to be prepared by the filamentary material (comprising paper) of film or layer form the most commonly, wherein said material is selected from polysaccharide as Mierocrystalline cellulose, rhodia, plastics as polyethylene, polypropylene, teflon (Teflon) (PTFE), polyacrylic ester, polymeric amide or poly(vinylidene fluoride) or the polymkeric substance that comprises sulfuryl be as polyphenylsulphine (polyphenylsulfone), polyethersulfone, polysulfones, polyaryl sulfone or polyphenylsulphine.
Described in other, for the material that solid, precipitation and throw out are held back, be pearl or the silk screen of glass, metal or plastics.
In two prior art files, the application (WO2009/157679A1 and WO2009/157680A1) of the hydrogel post for fragment and precipitation are held back has been discussed.The material using is agarose, makes nucleic acid by still most of pollutent of lysate being held back.
The high molecular biomolecules that cell pyrolysis liquid comprises genomic dna representative, it is highstrung for shearing force.The most of filtering materials that use are in the prior art suitable rigidity.Therefore, if genomic dna is applied to high reactive force, for example, during high speed centrifugation, apply high pressure, this rigid material can be sheared this genomic dna.Then the fragment of genomic dna can be passed through strainer, and is attached on DNA binding matrix, produces the pollutent of needed plasmid DNA.
On the other hand, when agarose or another kind of hydrogel are used as filtering material, described material itself is highstrung for applied external force.As discussed in WO2009/157680A1, contain agarose and must prepare soon before use as the post of strainer, but then this post only can be with the centrifugal rotation within the scope of 1000rpm-3000rpm.If use lower rpm, DNA is not through agarose column; If use higher rpm, can destroy agarose.
Therefore, an object of the present invention is to develop someway and device, described method and apparatus will be for comprising the liquid mixture clarification of at least one types of molecules (described molecule is to shearing force sensitivity), then desired molecule and collect the described material through combination in this mixture of selective binding, wherein minimizes the pollutent of desired molecule.
This target by the clarification/coupling apparatus that comprises filter module, described device the application in target separation method and for target, separate, the test kit that comprises this device meets, wherein said filter module comprises elasticity filtering material.
According to clarification/coupling apparatus of the present invention, represented certain device, described device is by the suspension clarification that comprises liquid phase and solid particulate, precipitation and/or throw out, described clarification can separate particle, precipitation and/or throw out and/or hold back to realize by for example filtering from liquid phase, and described device can also be in conjunction with at least one target molecules in for example, liquid composition by clarification device (strainer), thereby from remaining liquid phase, described target is separated.
In a preferred implementation, the invention provides for the clarification/coupling apparatus from sample separating at least one target molecules, described device comprises filter module and target column.Described clarification/coupling apparatus can be single-column clarification/coupling apparatus, and wherein said filter module is contained in post, and described post also comprises target bond material.Preferably, described filter module can be removed from described post.In another embodiment, described clarification/coupling apparatus is twin columns clarification/coupling apparatus, and wherein said filter module is the form that is inserted into the extra-column (further column) in described column.The filter module of the form of the post that comprises described filtering material is preferably formulated for accepts lysate.
Described filter module comprises at least one filtering material, and described filtering material is " filtration of degree of depth bed " material of substantially avoiding filter stoppage.Described material is preferably elastic, and its representation case is as manually lower deformable, and preferably it is soft and flexibility.Described material is preferably selected from and has open bore any foam (foam material) or the sponge in (open duct).The polyethylene that is exemplified as foaming of such material, polypropylene, urethane (comprising the urethane based on polyester), polyester, polyethers, polystyrene, trimeric cyanamide, or other have the plastic polymer in open duct or natural sponge as Porifera, animal fibre sponge or plant fiber sponge.Described material is known in the art, for example, for air filter, for example, for medical facilities, bandage buffering, cleaning applications etc.
The hole of resilient material (preferred foams or sponge), should be preferably in the scope of 10 μ m-1000 μ m, more preferably in the scope of 25-500 μ m, and most preferably in the scope of 50-200 μ m.Described foam preferably has the quantity in the duct of every centimetre of 5-50, preferably 20-40 duct/cm.
Described foam or sponge be foam or the sponge of " supporting (self-supporting) certainly " preferably.This represents that described foam or sponge are resilient materials, by pressure distortion, but during applied pressure, substantially returns to its original shape when removing.In particularly preferred embodiments, described resilient material is flexible material, and it can be easy to be out of shape by the strength of finger.
Such elasticity also can be measured by being called compressive hardness (or compression dynamics of described foam), for example, by DIN EN ISO3386, measure.Can be by the fixed size sheet of described foam being compressed to predetermined amount (often to 40%) and measuring power (kPa or the N/m that described compression needs
2).The compressive hardness (to 40%) of preferred material in the scope of 0.5-50kPa, preferably in the scope of 1-30kPa, more preferably 1-20kPa and particularly preferably 2-10kPa.
Due to elasticity and the flexible characteristic of filtering material using, compared with the method for not using strainer is for example centrifugal, reduced the shearing of shear sensitive molecule (as genomic dna) and in elutriant the amount of genomic DNA fragment very low.
Filter module also preferably comprises one deck or two-layer the second strainer, preferably under flexible filter.Preferably, described the second strainer also should not prepared by rigid material, and described material is sheared sensitive molecule as genomic dna in high speed centrifugation.Described the second strainer can represent conventional deep filter, and it is preferably prepared by filamentary material.Described the second strainer can be the form of film or layer, and wherein said material can be selected from polysaccharide as Mierocrystalline cellulose, comprises paper, rhodia, plastics as polyethylene, polypropylene,
(PTFE), polyethylene terephthalate (PET), polyacrylic ester, polymeric amide, especially
poly(vinylidene fluoride), the polymkeric substance that comprises sulfuryl is if polyphenylsulphine, polyethersulfone, polysulfones, polyaryl sulfone or polyphenylsulphine or non-DNA are in conjunction with silicon-dioxide.
The hole of the second strainer is less than the hole of flexible filter, and preferably in the scope of 0.1-50 μ m, preferably in the scope of 0.5-30 μ m, most preferably in the scope of 1-10 μ m.As long as overlapping in the scope of the aperture of flexible filter as herein described and the second strainer described in it, be noted that the aperture of described the second strainer should be less than the aperture of described flexible filter.
The thickness of the thickness of described flexible filter and described one or more the second strainers is not limited to the present invention, but the thickness of preferred elastomeric strainer is for example 1-10mm, preferably 2-8mm, more preferably 3-5mm.Because described one or more the second strainer resilience in comparison strainers are held back less material wittingly, the thickness of described one or more the second strainers is preferably lower than the thickness of described flexible filter.Therefore, the thickness of described one or more the second strainers can the scope of each comfortable 0.1-1mm in.
Described one or more strainer allow liquid phase by and substantially hold back whole solid/particle/flocculation materials.Described one or more strainer is arranged in filter module in some way, and described method makes lysate not allow to walk around by described one or more strainers.Therefore, for example, if use Filter column, the sidewall contact of described one or more strainers and described post or be placed in the fixer with described sidewall contact.For example, described flexible filter can customize to meet according to size the inside of described post, and with the sidewall contact of this post, especially when it is moistening, for example, compared with the internal diameter of post, described strainer " size becomes large (oversizing) ".In this case, described strainer " is caught " inside of described post.In addition, described strainer can fix by the ring under described strainer and/or on it.If ring is placed on described strainer, there are like this other advantages, described liquid lysate is directly walked around described strainer by described flexible filter and obstruction lysate by flowing down post jamb.
For filter module of the present invention is provided, flexible filter (for example foam or sponge) is placed in the container with entrance and exit, described container is pipe, post, syringe etc. such as.Described container can be made by plastics, metal, matrix material, glass or its arbitrary combination, or other non-reacted or bio-capacitivity materials are made arbitrarily.Described container can be with manufacturing by injectable plastic material, described can tolerance by power centrifugal or that medium vacuum pressure causes by injectable plastic material.If needed, the second strainer is also placed in the inside of described container, preferably under described flexible filter.At least one in strainer can be supported in container, described supporting for example designs described container by following manner, it represents the such as silk screen of supporting structure that liquid can perviousness in bottom, fence or arbitrarily similar supporting structure are as crosslinked, colyliform etc., or comprise the passage of end face, or described strainer is supported in container by supporting device, such as fixer of described supporting device, ring, silk screen, fence or arbitrarily similar supporting structure are as crosslinked, colyliform etc., or comprise the passage of end face, or strainer can pass through for example glue, glue, sealing or similar tackiness agent stick on described wall of a container.In addition, described supporting member can be the improvement of the internal surface to column, the annular ridge (annular ridge) for example forming on the internal surface of internal holes.In a preferred embodiment, the second strainer is placed under the foam or sponge of post, and wherein said the second strainer can be supported.Described the second strainer itself then can be as the support of flexible filter.So the filter module of the cylindricality formula of preparation can be inserted in extra-column, obtains twin columns device, has the bond material for required target in described extra-column.
Described column is preferably mixed with the filtered sample of accepting from filter module.Described column comprises for the bond material in conjunction with at least one target molecules.Described column can by be known in the art arbitrarily, for the post in conjunction with required target, represent, described target especially the nucleic acid by flexible filter or filter module as plasmid DNA or RNA, or albumen.Except as otherwise noted, the common implication that all technical terms used herein are understood with those skilled in the art with scientific terminology is identical.Conventionally, name used herein is well known and conventional.The term of direction is as "up" and "down", " top " and " bottom ", " more than " and " below " or " top " or " bottom " etc. at device, use the direction of middle finger parts.In the situation that term provides with singulative, also consider the plural form of this term.
Term used herein " preparation in a small amount " or " preparing in a small amount " refer to the purifying scale from the initial volume of culture of about 0.5-5m1.The post using in preparing purifying in a small amount or other devices also can be in the scopes from the about 5ml of about 0.5ml-.Term " middle amount/preparation in a large number " or " middle amount/prepare in a large number " refer to the purifying scale starting from the volume of culture of 5-100ml.The post using in middle amount is prepared purifying or other devices can be in the scopes of the about 15ml of about 5ml-or the about 25ml of about 5ml-, and the post using in preparing purifying in a large number or other devices can be in the scopes of the about 100ml of about 25ml-.
Term used herein " post " and " pillar " refer to have entrance and exit and device or container that can receiving fluids.Although post typically refers to device and the container with about cylinder form, be understandable that term used herein " post " can refer to device or the container with arbitrary shape, especially cylindrical, or other shapes, include but not limited to it is mainly spherical, taper, rectangle, irregularly shaped or its combination.
Term " target organisms molecule " or " target molecules " can comprise nucleic acid, albumen, lipid, glycolipid, path product or sugar, and wherein preferred target molecules is nucleic acid or albumen, particularly preferably nucleic acid.
The albumen that term used herein " albumen " or " protein " comprise full-length proteins, protein fragments, native state or the albumen of sex change.The mixture of albumen can be mixture, the mixture of protein fragments or the mixture of full-length proteins and protein fragments of full-length proteins.
Term used herein " nucleic acid " comprises DNA and RNA, irrelevant with molecular weight or source.The four corner of the polymkeric substance that nucleic acid comprises strand or double chain nucleotide, comprises the Nucleotide of chemically modified, and it can form as known in the art base pairing, can be connected and can be cut by endonuclease or exonuclease with other nucleic acid.Nucleic acid can or can be modified from any natural origin.DNA molecular is arbitrary size, from any DNA molecule in any source, comprises from virus, prokaryotic organism and Eukaryotic DNA, and synthetic DNA and variant, derivative and analogue.DNA can be genomic dna or exchromosomal DNA.RNA molecule is arbitrary size, from any RNA molecule in any source, comprises from virus, prokaryotic organism and Eukaryotic RNA, and synthetic RNA and variant, derivative and analogue.RNA and DNA can be strand or two strands, linear or annular or superhelix shape.Preferred nucleic acid is called as " target nucleic acids ".
According to the present invention, " target nucleic acids ", preferably comprises exchromosomal DNA, for example plasmid and fragment thereof, carrier and fragment thereof, phagemid, clay, BAC, PAC, YAC, mitochondrial nucleic acid molecule, chloroplast(id) nucleic acid molecule or its combination.Particularly, preferably carrier and/or plasmid arbitrarily.They can be commercially available or synthetic or engineered or derivatives thereof.This carrier and/or plasmid can be used to clone or the interested nucleic acid molecule of subclone or by clone or the interested nucleic acid molecule of subclone derivative, and therefore also can separate the recombinant vectors that contains inserting paragraph, nucleic acid fragment or gene according to the present invention.The general classification of interested especially carrier comprise protokaryon and/or eukaryotic cloning carrier, expression vector, fusion vector, double cross carrier or oppositely double cross carrier, the shuttle vectors for different hosts, mutational vector, transcription vector, bob folder carrier, for accepting the carrier etc. of large Insert Fragment (yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC) and P1 artificial chromosome (PAC)).Other interested carriers comprise viral source carrier (M13 carrier, bacteriophage carrier, baculovirus vector, adenovirus carrier and retroviral vector), the carrier of high and low or adjustable copy number, have carrier (for example pACYC184 and pBR322) and eucaryon episome replicating vector (for example pCDM8) for the compatible replicon in single host's combination.The carrier that the present invention considers comprises the carrier (for example recombinant vectors) that contains nucleic acid fragment insertion or extra or sequence and derivative or the variant of any carrier as herein described.According to useful expression vector of the present invention, comprise the derivative carrier of the derivative carrier of karyomit(e), episome and the derivative carrier of virus, for example, derived from the carrier of bacterial plasmid or bacteriophage, and derived from the carrier of its combination, as clay and phagemid.
Arbitrary cell, tissue or organism can be used as the source of the target molecules of needs separation, thereby the described target molecules comprising in described cell, tissue or biogenetic derivation (or its part) is discharged from described cell, tissue or organism.Cell can be protokaryon or eucaryon.Organism can be protokaryon, eucaryon or virus etc., and typically refers to the cell that comprises arbitrarily target molecules (for example nucleic acid interested).Term " host " or " host cell " can exchange use in this article.Such host's example is referring to Maniatis etc., " Molecular Cloning:A Laboratory Manual (molecular cloning: laboratory manual) ", cold spring harbor laboratory, cold spring port, New York. and (1982).Preferred prokaryotic hosts includes but not limited to Escherichia (Escherichia) (for example intestinal bacteria (E.coli)), Bacillaceae (Bacillus), Staphylococcus (Staphylococcus), Agrobacterium (Agrobacter) (for example Agrobacterium tumefaciens (A.tumefaciens)), streptomyces (Streptomyces), Rhodopseudomonas (Pseudomonas), Salmonella (Salmonella), serratia (Serratia), the bacterium of Caryophanon (Caryophanon) etc.Most preferred prokaryotic hosts is intestinal bacteria.Interested especially host bacterium comprises coli strain K12, DH10B, DH5 α, HB101, JM109, X11blue, Top10, Top10F and QIAGEN EZ in the present invention.Preferred eucaryon host includes but not limited to that fungi, fish cell, yeast cell, vegetable cell and zooblast, particularly insect cell and mammalian cell comprise people's cell, Chinese hamster ovary celI, VERO cell, Bowes melanoma cell, HepG2 cell etc.Cell can be the cell transforming, clone, cancer cell system or the normal cell of foundation.Exemplary zooblast is that insect cell is as fruit bat (Drosophila) cell, noctuid (Spodoptera) Sf9, Sf21 cell and cabbage looper (Trichoplusa) High-Five cell; Elegans cell is as Caenorhabditis elegans (C.elegans) cell; And mammalian cell is as COS cell, Chinese hamster ovary celI, VERO cell, 293 cells, PERC6 cell, bhk cell and people's cell.According to the present invention, also can use the cell derived of any virus as biomacromolecule (especially nucleic acid molecule).What be also suitable for use as biomacromolecule source is the tissue of blood or mammalian organs, for example, derived from brain, kidney, liver, pancreas, blood, marrow, muscle, nerve, skin, urogenital system, the recycle system, lymph, gi tract and related tissue source and derived from those of the embryo of Mammals (comprising people) or fetus.These cells, tissue and organ can be clone normal, that transform or that set up, or they can be pathologies, as relate to (being caused by bacterium, fungi or yeast, virus (comprising AIDS) or parasite) infectious diseases, heredity or biological chemistry pathology (for example cystic fibrosis, hemophilia, alzheimer's disease, schizophrenia, muscular dystrophy or multiple sclerosis) or cancer and Carcinogenesis those.Other cells, tissue, virus, organ and the organism that those of ordinary skills were familiar with also can be used as the source of biomacromolecule and uses, and the source of described biomacromolecule is for the preparation of according to biomacromolecule of the present invention.According to the present invention, host or host cell can be used as needing the macromolecular cell derived of separation.
" cytoclasis " used herein or " lysis " refer to uses the component of composition or composition that cell is opened, the component of described composition or composition is by cell, tissue or organism cracking, fracture or the punching in the biomacromolecule source as required separation, thereby the biomacromolecule comprising in described cell, tissue or biogenetic derivation (or its part) discharges from described cell, tissue or organism.According to the present invention, described cell, tissue or organism do not need complete cracking, fracture or punching, and do not need all interested macromole comprising in derived cell, tissue or organism from wherein discharging.Preferably, cytoclasis or lysis compound or composition generation at least 25%, 50%, 75%, 80%, 85%, 90%, 95%, 97%, 99% or more releasing effect, described release is that the total biomacromolecule interested containing in described cell, tissue or organism is discharged.
According to the present invention, can by by cell with cause or the composition of helper cracking or destruction or compound contacts so that described lysis or destruction, such as, although can use mechanical force or physical force (pressure, ultrasonic, temperature (heating, freezing) and/or freeze thawing etc.) according to the present invention.In addition, can be with destroy/lysing cell of the arbitrary combination of mechanical force, physical force or cleavage composition/compound, as long as described method is not destroyed interested biomacromolecule substantially.
The cytoclasis using or lysis compound or composition do not limit the present invention.The component comprising in cleavage composition is preferably suitable for wanting the required target organisms molecule of isolated or purified.For the required biomolecules of which kind of type, preferably include which kind of component known to those skilled in the art, and do not limit the present invention.Can comprise one or more stain removers, for example sodium lauryl sulphate (SDS), sarcosyl, triton x-100, polysorbas20, NP-40, N alkyl glucoside, N-alkyl maltoside, glucamide, digoxin, deoxycholate salt, 3-[(3-courage amido propyl)-dimethylammonio (dimethylammonio)]-1-propane sulfonic acid ester (CHAPS), cetyl trimethylammonium bromide (CTAB) or Brij35.Concentration can be any appropriate, for example about 0.01%-10% (w/v), more preferably about 0.1%-5%.Can there are one or more chaotropic agents, for example sodium iodide, sodium perchlorate, guanidine or its salt or urea.In addition, can there are one or more enzymes, as N,O-Diacetylmuramidase, lyticase, yeast lyase (zymolyase), neuraminidase, Novozym234, streptolysin, green wooden enzyme (cellulysin), mutanolysin or lysostaphin.One or more inorganic salt that concentration that can about 1mM-5M exists are as sodium-chlor, Repone K, magnesium chloride, lithium chloride or praseodymium chloride.Can there are one or more organic solvents, for example, as toluene, phenol, butanols, Virahol, primary isoamyl alcohol, ethanol, ether (diethyl ether, dme or ethyl-methyl ether) or chloroform, if they for arbitrarily used according to the present invention strainer there is no negative impact.Can there are any other compounds that destroy biomacromolecule (for example PXB) cytolemma of cell derived and/or the integrity of cell walls (for example cracking or cause the formation in hole), or any aforesaid combination.Described composition can comprise other components, for example sequestrant (for example disodium ethylene diamine tetraacetate (Na EDTA), EGTA, CDTA), one or more proteolytic enzyme (Proteinase K, PRONASE A, stomach en-, trypsinase, papoid, subtilisin) or aforesaid arbitrary combination.The desired concn of the activeconstituents of cracking/destroying compositions and combination restriction the present invention, and can use normal experiment easily to measure by those skilled in the art.
Term used herein " clarification " refers to the process of for example, removing unwanted solid, precipitation or flocculation material (for example cell debris and/or large insoluble molecule) from liquid mixture (cell pyrolysis liquid).The common method of clarification cell pyrolysis liquid comprises centrifugal and filters.Preferably, in filter module, substantially hold back unwanted fragment, make the lysate of basic clarification by containing the column of bond material.
Term used herein " wash-out " refers to by the mode of solvent and will be discharged by the required target molecules of binding matrix institute combination.Term " elute soln ", " elution buffer " and " eluting solvent " refer to the solvent for discharge molecule from described material.Term " elutriant " refers to liquor that wash-out obtains and that comprise desired molecule.
" elutriant " can comprise and be considered to " separation " nucleic acid.As described in the term " separation " (as in " biomacromolecule of separation ") using in this article represents material, component or the composition etc. of separation from other materials, pollutent etc. at least partly purifying out, described material, pollutent etc. are not the parts of separated described material, component or composition.For example, " biomacromolecule of separation " is the biomacromolecule of having processed in some way, and described mode is removed macromole and cellular component at least some other, relevant to cell, tissue, organ or organism.Particularly, term " biomacromolecule of separation ", " nucleic acid molecule of separation " or " plasmid of separation " refer to macromole goods or plasmid goods, described macromole goods or plasmid goods are containing the interested biomacromolecule of 10%, 20%, 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% and 93% (% by weight) of having an appointment, preferably exceed 95%, 97.5% and 98%, and most preferably exceed 99%, 99.5% and 99.9%.But as those of ordinary skill will appreciate that, the macromolecular solution that comprises separation can comprise other compositions, for example one or more buffering salts and/or solvent, such as water or organic solvent etc., and with respect to parent material, described macromole is still considered to " separation " macromole.
By device of the present invention, method and test kit, carrying out separate nucleic acid molecule can also be synthetic by amplification, clone, order-checking, mark, transfection, conversion, in-vitro transcription, In Vitro Translation, nucleic acid, endonuclease digestion, other enzyme modifications etc. characterize or operate, and these methods are the conventional methods used of those skilled in the art.
The invention provides certain apparatus and method, described apparatus and method are suitable for clarification or bag filter containing shear sensitive macromolecular liquid mixture or sample, then from through clarification or filtered liquid or sample desired molecule is attached in bond material.The separation of desired molecule can also comprise follow-up from described bond material wash-out desired molecule.One preferred embodiment in, the invention provides the two devices that comprise filter module, described filter module is inserted in column at least partly with the form of clarification/Filter column, wherein said clarification post is held back the solid from liquid mixture, precipitation or flocculation material, and described column is in conjunction with one or more required target molecules of coming in the liquid of inherent filtration.If needed, target molecules that can wash-out institute combination from described bond material.
In yet another embodiment of the present invention, described filter module is inserted in the post identical with described bond material.In this embodiment, described filter module is not post, but for example dish, described dish is sealed above-mentioned flexible filter and the second optional strainer, and it can be placed on the grid in advance (intended lace) of column inside.In this case, described column preferably designs as follows, and described filter module is fixed on its position in advance by above-mentioned mode.According to the present invention, preferably at sample, through after strainer, remove described filter module.Therefore, described filter module is preferably removable from column.
Described clarification/coupling apparatus can be used for the clarification of sample and from described at least one target molecules of sample later separation, wherein make the pollutent of the fragment of shear-sensitive molecule minimize.Described sample can be liquid sample or the dry-eye disease having suspended in liquid.For example, device described herein can be clarified cell pyrolysis liquid sample and isolated nucleic acid molecule from the lysate of clarification.For example, by sample (lysate) filtration and the combined material capture of one or more target molecules after, can remove and abandon internal ramp module (with unwanted fragment).Then can optionally wash and elution of bound nucleic acid or one or more other target molecules to described bond material.
In above-mentioned twin columns embodiment, the corresponding clarification/Filter column of described filter module.Described inner clarification/Filter column and described column both can be by plastics, metal, matrix material, glass or its arbitrary combination, or other suitable non-reacted or biocompatible materialses are made arbitrarily, can tolerate the power producing by using the centrifugal of described device.
In a preferred embodiment, wherein said filter module is the form can be inserted into the post in column, the entrance of described filter module/clarification post or upper, open end are orientated identical direction with entrance or the upper, open end of described column, and the outlet of described filter module/clarification post is orientated identical direction with the outlet of described column.Described filter module/clarification post can pass internal holes along all Directional Extensions, thereby makes the outlet of described filter module/clarification post and the bond material adjacency in described column.Or described filter module/clarification post only can partly be expanded through internal holes, thereby make to have gap between the outlet of described filter module/clarification post and described bond material.
Preferably, when being inserted in column, even, when centrifugal, described filter module comprises for described filter module being fixed on to its any-mode of position in advance.Described mode can be for example encircle, crank, shoulder etc., described mode preferably allows the surrounding of described filter module be greater than column around, described filter module can not be deeper inserted in column, or filter module be placed on the crank or shoulder of its corresponding column inside.
The example that two posts all have the twin columns device of shoulder is shown in Fig. 1 (bond material in described column jacket does not show).In described figure, described column (bond material does not show) shows work (1), and the described filter module being inserted in described column shows work (2).Described flexible filter (3) is placed on the second strainer (4).
Described filter module (for example, to clarify the form of post) can design as follows in addition, be that it can be inserted into (and need not be any for described post being fixed on to the mode of appropriate location (in place)) in described column, but it can be removed after filtration step.
If needed, the internal diameter of described column can customize to hold the size of clarifying post according to size, thereby makes at least a portion outside surface of described clarification post and a part of internal surface of described column contact and provide the tight coupling between described clarification post and column.This tight coupling can be two connections between surface, and it can be realized by the frictional force after contacting with each other between surface.Closely coupling also can, by the shape of the described clarification post of customization and column, realize thereby make one or the other (or two) post slightly depart from dimensionally nominal size, or realize by the cone shape of described two posts.In addition, closely coupling can be by customizing the crank of described post or the shape of shoulder obtains, and described crank or shoulder are fixed on appropriate location by the mode of shoulder or the mutual close contact of crank by described clarification post.Coupling between described clarification post and column can be enough airtight, thereby makes the negative pressure of the outlet that puts on described column in twin columns system, to produce vacuum.Conventionally, when being positioned at described column, described clarification post can be certain size, thereby at least part of outside surface of described clarification post and at least part of internal surface of described column are contacted, and forms and the vacuum-sealing of described column when applying negative pressure.Coupling between described column jacket and clarification post can not hinder when not applying negative pressure, and described clarification post is removed from described column.
Described column comprises for the bond material in conjunction with required target.Described bond material can be the material of any appropriate for catching target molecules, includes but not limited to fiber, matrix, resin, film, dish or strainer or other suitable materials or its combination arbitrarily.Described bond material can be caught the target molecules of at least one type, includes but not limited to nucleic acid.One preferred embodiment in, described bond material is DNA or RNA bond material, especially can be in conjunction with the material of plasmid DNA.Suitable DNA bond material comprises silicon-dioxide and non-silicon-dioxide DNA bond material or its combination.Described DNA bond material can also be the chromatographic material of any appropriate, includes but not limited to silica gel, aluminum oxide, titanium dioxide, sintered glass, polymkeric substance or its arbitrary combination.In addition, described bond material can be electric charge switch membrane (charge switch membrane), comprises glass fibre or nitrocellulose, or anionresin matrix, comprises derivative glass fibre.Described bond material can also be for example combination of the described material in different layers of several types.Described bond material can be positioned near the outlet or outlet of inside of column.Described bond material can be positioned at the position of any appropriate, thereby makes must pass through described bond material by described column and the sample that leaves from outlet opening.
Described bond material preferably comprises the hole of suitable dimension, and to provide enough liquid or the sample of clarification to flow through described device, provide can be by the high surface area of molecule combination simultaneously, thereby and provides the good productive rate at least one target molecules.The size in described hole can be any appropriate, make it possible to the size of at least one target molecules combination, for example, in the scope between approximately 0.5 μ m and approximately 5 μ m.
Described column can comprise the supporting member of bond material described in one or more, or it can adhere on wall of a container by for example glue, glue, sealing or similar tackiness agent.Described supporting member can remain on the position of post inside in connection with material.Described one or more supporting members can be physically to limit the arbitrary structures that described bond material moves.Suitable supporting member includes but not limited to fixer, ring, silk screen or frit.In addition, described supporting member can be the improvement of the internal surface to column, the annular ridge for example forming on the internal surface of internal holes.
The present invention is also provided for clarifying the method for the suspension that comprises at least one target molecules and solid particulate, precipitation and/or throw out, wherein said suspension can also comprise shear sensitive molecule, described method comprise relate to as defined above, for the filtration step of the filter module that described solid particulate, precipitation and/or throw out separated from target molecules, wherein said target molecules is retained in solution.Especially described method is suitable for from this suspension, required target molecules being separated and/or purifying, wherein said suspension can also comprise shear sensitive molecule, as high molecular weight molecules, comprise the filtration step that relates to filter module as defined above.The circulation liquid of gained comprises target molecules required in solution, does not substantially contain particulate material as solid, precipitation and suspended substance.In addition, the solution of gained is not preferably substantially containing the macromolecular fragment of shear-sensitive or the fragment of himself.Described circulation liquid can contact with target bond material, described target is separated/separates from described circulation liquid.
According to the preferred method of the present invention, isolated or purified nucleic acid, the preferred DNA of described nucleic acid, most preferably plasmid DNA.
Preferably, the separation of described nucleic acid and/or purifying comprise described nucleic acid be attached to the step on nucleic acid bond material, and described step is preferably carried out in column.
In a particularly preferred embodiment, the invention provides for separating of and/or the method for purification of nucleic acid, said method comprising the steps of:
(i) by the sample that comprises nucleic acid with comprise at least one by as the strainer made of above-mentioned resilient material contact
(ii) to described sample, apply external force to make described sample by described filter module
Described method is preferably further comprising the steps of:
(iii) the circulation liquid of step (ii) is contacted with nucleic acid bond material
(iv) optionally wash the nucleic acid of described combination
(v) from described bond material by described nucleic acid wash-out.
Preferably cell pyrolysis liquid of described sample, particularly comprises the cell pyrolysis liquid of genomic dna and target organisms molecule.Preferred target organisms molecule is target nucleic acids or albumen, especially plasmid DNA.
Can in the post that comprises described filter module, add the sample that contains desired molecule.If described filter module is the form with above-mentioned clarification post, described sample joins in clarification post by the opening end of described clarification post.
Preferably, applying centrifugal force or negative pressure (vacuum) makes sample pass through described filter module/clarification post.
Preferably, described circulation liquid is through entering column.Because described sample is by clarification post, the strainer comprising can prevent that large insoluble molecule is by the outlet of described clarification post.Due to the elastic property of described flexible filter (preferred foams or sponge), even in the situation that applying high centrifugal speed to described sample, also substantially shear sensitive molecule can not sheared as genomic dna.Therefore, the amount of the fragment of shear-sensitive molecule is minimized.
The sample of described filtration preferably contacts with the bond material that is arranged in described column.Described bond material can be in conjunction with one or more desired molecules, and centrifugal force or vacuum make remaining liq leave described device by the outlet of described column simultaneously.Then can remove described filter module/clarification post.Optionally can add one or more washing solns, and make it by described bond material and leave described device by centrifugal or vacuum.Described washing increases the purity of desired molecule by removing unconjugated molecule, impurity or other fragments of self bonded material.Then can to add in described column elutriant with from described bond material by desired molecule wash-out.Preferably collect elutriant.Can use the elute soln of many equal portions.
In the preferred implementation of described method, described suspension or liquid mixture are cell pyrolysis liquids, and described filter module is with the formal operations of clarification post.The lysate of clarification is by described bond material, and described bond material is in conjunction with interested nucleic acid, preferred plasmid DNA.Then can remove the plasmid DNA of described clarification post and wash-out and the described combination of collection.Twin columns device of the present invention makes it possible to clarification and combination in or vacuum step centrifugal at single short (1-3 minute).
In a particularly preferred embodiment, the invention provides for separating of and/or the method for plasmid DNA purification, said method comprising the steps of:
(i) cell pyrolysis liquid that comprises genomic dna and plasmid DNA is contacted with filter module, described filter module comprises at least one strainer of being made by the foam certainly supporting or sponge
(ii) to described sample, apply external force to make described sample by described filter module
(iii) the circulation liquid of step (ii) is contacted with DNA bond material
(iv) optionally wash the DNA of described combination
(v) from described bond material by described DNA wash-out.
Test kit for interested target molecules being separated from sample is also provided herein.For the test kit that interested target molecules is separated, comprise at least one filter module as described herein.
Preferably, described test kit comprises the clarification/coupling apparatus at least one target molecules being separated from sample, described device comprises: be mixed with the filter module/clarification post for accepting sample, described clarification post comprises at least one strainer of being made by flexible filter material described above (preferably from foam or the sponge of supporting), it is formulated as and from described sample, filters at least one non-target molecules, and be disposed for accepting the column from the sample after filtration of filter module/clarification post, described column comprises for the bond material in conjunction with at least one target molecules.
Another preferred embodiment in, described test kit comprises the post that contains filter module and bond material, described filter module comprises at least one strainer of being made by resilient material described above (preferred foams or sponge),, wherein preferred described filter module can be removed from post.
Described test kit also comprises that at least one is selected from other following compositions: at least one lysis buffer; At least one RNA enzyme liquid storage; At least one resuspended damping fluid; At least one neutralization buffer; At least one lavation buffer solution; At least one elution buffer; Carry out the specification sheets of described target separation/purification method.
Accompanying drawing:
Fig. 1 is presented in centrifugal column for filtering and in conjunction with the filter module of coupling, wherein the bond material in centrifugal column does not show.(1) centrifugal column (bond material does not show), (2) filter module, (3) are soft, elastic top filtering material, (4) compacting the second strainer.
May designing of Fig. 2 display filter module, the bottom of its center pillar represents support pattern (filtering material does not show).
Fig. 3 has shown the plasmid preparation separating according to embodiment 2 on sepharose.
1 strainer, described strainer has non-binding silicon-dioxide as bottom filtering material, and sintered glass material, PE, 10 μ m are as top filtering material
2 strainers, described strainer has sintered glass material PE7-12 μ m as bottom filtering material, and Gaze PET, and 20 μ m are as top filtering material
3 strainers, described strainer has non-binding silicon-dioxide as bottom filtering material, and sintered glass material, PE, 20-60 μ m is as top filtering material
4 strainers, described strainer has non-binding silicon-dioxide as bottom filtering material, and sintered glass material, PE, 10-20 μ m is as top filtering material
(5) reference: replace filter within 10 minutes, centrifugally will precipitate pelletizing
Fig. 4 has shown on sepharose a small amount of preparation according to embodiment 3, felt (needle punched felt) and (2) reference of comprising (1) acupuncture: 10 minutes centrifugal will precipitate pelletizing.
The sepharose that Fig. 5 shows represents that the plasmid that uses the filter module that comprises foam to carry out according to embodiment 1 prepares result (1), and according to the preparation result (2) of reference (10 minutes centrifugal will precipitate pelletizing).
Fig. 6 shows according to the type of the strainer for lysate clarification, with the content of comparing genomic dna according to the separation plasmid of embodiment 5.Shown in being prepared as follows in a small amount.(1) reference: 10 minutes plasmids centrifugal will precipitate pelletizing, (2) are used foam to separate as filtering material that replacement is filtered, and (3) are used the plasmid of needle punched felt as filtering material separation.
Fig. 7 shows according to the type of the strainer for lysate clarification, with the content of comparing genomic dna according to the separation plasmid of embodiment 5.Two kinds of preparations have in a small amount been shown.(1) reference: 10 minutes centrifugal will precipitate pelletizing, the plasmid that (2) are used foam to separate as filtering material, and (3) plasmid of using needle punched felt to separate as filtering material.
Embodiment
Embodiment 1:
According to following scheme isolated plasmid dna from bacterial cell.By by cell pellets centrifugal in 1.5ml Eppendorf pipe from bacterial cell substratum harvested cell.The damping fluid, solution and the DNA column that use are from commercially available QIAprep test kit (the Qia Gen company (Qiagen) of Heerden, Germany), and it is designed for the separation of plasmid DNA.
centrifugal scheme
All centrifugation step are carried out under 13.000rpm.
1. the bacterial cell of resuspended pelletizing and being transferred in Eppendorf tube in the damping fluid P1 of 250 μ l.
2. add 250 μ l damping fluid P2 and thoroughly mix by pipe being put upside down to 4-6 time.
3. add 350 μ l damping fluid N3 and by pipe being put upside down to 4-6 time, thoroughly mix immediately.
4. by decant or move liquid and be added to filter module by comprising the whole sample volume precipitating from step 4, described filter module is to insert in the form of the post in QIAprep centrifugal column.Described filter module comprises polyurethane foam and uncombined silicon dioxide film (for example not the silicon dioxide film of bind nucleic acid).
5. centrifugal 1 minute of whole assembly, discards circulation liquid.
6. from QIAprep centrifugal column, remove and abandon described filter module.
7. by adding 0.5ml damping fluid PB and centrifugal 30-60 to wash described QIAprep centrifugal column second.Discard circulation liquid.
8. by adding 0.75ml damping fluid PE and centrifugal 30-60 to wash described QIAprep centrifugal column second.
9. discard circulation liquid, and by post more centrifugal 1 minute to remove remaining lavation buffer solution.
10.QIAprep post is placed in clean 1.5ml Eppendorf tube.For eluted dna, to the center of each QIAprep centrifugal column, add 50 μ l damping fluid EB (10mM TrisCl, pH8.5), place 1 minute, and centrifugal 1 minute.Collect elutriant.
vacuum scheme
1. according to the detailed content in QIAprep Miniprep handbook, prepare vacuum manifold and QIAprep centrifugal column.
2. the bacterial cell of resuspended pelletizing in the damping fluid P1 of 250 μ l, and be transferred in Eppendorf tube.
3. add 250 μ l damping fluid P2 and thoroughly mix by pipe being put upside down to 4-6 time.
4. add 350 μ l damping fluid N3 and by pipe being put upside down to 4-6 time, thoroughly mix immediately.
5. by decant or move liquid and be added to filter module by comprising the whole sample volume precipitating from step 4, described filter module is the form of the post in the QIAprep centrifugal column being inserted on vacuum manifold.Described filter module comprises polyurethane foam and uncombined silicon dioxide film.
6. open vacuum source so that solution suction is passed through to described filter module and QIAprep centrifugal column, then close vacuum source.
7. from QIAprep centrifugal column, remove and abandon described filter module.
8. by adding 0.5ml damping fluid PB to wash described QIAprep centrifugal column.Open vacuum source so that washing soln suction is passed through to described post, then close vacuum source.
9. by adding 0.75ml damping fluid PE to wash described QIAprep centrifugal column.Open vacuum source so that washing soln suction is passed through to described post, then close vacuum source.
10. described in, QIAprep centrifugal column is placed in the collection tube of 2ml and is transferred to Eppendorf tube.Under 13.000rpm, carry out 1 minute centrifugal to remove residual lavation buffer solution.
Described in 11., QIAprep post is placed in clean 1.5ml Eppendorf tube.For eluted dna, to the center of each QIAprep centrifugal column, add 50 μ l damping fluid EB (10mM TrisCl, pH8.5), place 1 minute, and under 13.000rpm centrifugal 1 minute.Collect elutriant.
as the conduct as described at QIAprep Miniprep handbook (in June, 2005) 22-23 page is joined
the scheme of ratio
1. the bacterial cell of resuspended pelletizing and being transferred in Eppendorf tube in the damping fluid P1 of 250 μ l.
2. add 250 μ l damping fluid P2 and thoroughly mix by pipe being put upside down to 4-6 time.
3. add 350 μ l damping fluid N3 and by pipe being put upside down to 4-6 time, thoroughly mix immediately.
4. by sample in benchtop microcentrifuge under 13,000rpm centrifugal 10 minutes.
5. from the supernatant of step 4, be transferred to QIAprep centrifugal column.
6. by centrifugal described QIAprep centrifugal column 60 seconds.Discard circulation liquid.
7. by adding 0.5ml damping fluid PB and centrifugal 30-60 to wash described QIAprep centrifugal column second.Discard circulation liquid.
8. by adding 0.75ml damping fluid PE and centrifugal 30-60 to wash described QIAprep centrifugal column second.
9. discard circulation liquid, and by post more centrifugal 1 minute to remove remaining lavation buffer solution.
10. described in, QIAprep post is placed in clean 1.5ml Eppendorf tube.For eluted dna, to the center of each QIAprep centrifugal column, add 50 μ l damping fluid EB (10mM TrisCl, pH8.5) or water, place 1 minute, and centrifugal 1 minute.Collect elutriant.
As " reference ", respectively by identical method processing sample, except replacing filtration step, by centrifugal (10 minutes, 13.000rpm) pelletizing is removed solid, precipitation and throw out, and described supernatant is transferred in new pipe and further processing.
The plasmid comparison of preparation in a small amount of using the plasmid of implementing for the filtration (filter module that comprises rigidity top filtering material) of lysate clarification to prepare in a small amount or implement as centrifugal (pelletizing) of reference:
According to the separation quality grain pUC19 from the 5ml LB substratum of intestinal bacteria TOP10F cell of the centrifugal scheme described in embodiment 1.In filter module, use four kinds of dissimilar filtering materials as upper filter.
1 strainer, described strainer has non-binding silicon-dioxide as bottom filtering material, and sintered glass material, PE, 10 μ m are as top filtering material,
2 strainers, described strainer has sintered glass material PE, and 7-12 μ m is as bottom filtering material, and Gaze PET, and 20 μ m are as top filtering material,
3 strainers, described strainer has non-binding silicon-dioxide as bottom filtering material, and sintered glass material, PE, 20-60 μ m is as top filtering material,
4 strainers, described strainer has non-binding silicon-dioxide as bottom filtering material, and sintered glass material, PE, 10-20 μ m is as top filtering material.
All samples are analyzed on sepharose.Described glue is shown in Fig. 3.On sepharose, operation is according to the OD of each elutriant
260150ng DNA, described elutriant comprises plasmid DNA separately.Can see, and by by compared with preparation centrifugal thick lysate (5), the material (1-4) of all sintering has produced the obviously gDNA pollutent of higher required plasmid DNA.
Comparison with the filtration of rigidity top filtering material with the pelletizing as reference: Fig. 4
According to the centrifugal scheme described in embodiment 1, respectively from the 5mlLB substratum of intestinal bacteria DH10B cell separation quality grain pUC19 or from the 5ml LB substratum of e.colidh5αcell separation quality grain pCMV β.As the top filtering material in filter module, use needle punched felt (1) (rigid filter material) and reference (2) to compare.All samples is all analyzed in triplicate.On sepharose, operation is according to the OD of each elutriant
260150ng DNA, described elutriant comprises plasmid DNA separately.Described gel shows compared with the sample of being clarified by pelletizing, comprises obviously more (fragmentation) genomic dna with the sample that needle punched felt filters.
Comparison with the filtration of elasticity/soft top filtering material with the pelletizing as reference: Fig. 5
According to the centrifugal scheme described in embodiment 1, respectively from the 5mlLB substratum of intestinal bacteria DH10B cell separation quality grain pUC19 or from the 5ml LB substratum of e.colidh5αcell separation quality grain pCMV β.Upper filter in fast as strainer mould, urethane, polyethylene or polystyrene foam are for comparing with the reference pelletizing of precipitation.All samples is all analyzed in triplicate.On sepharose, operation is according to the OD of each elutriant
260150ng DNA, described elutriant comprises plasmid DNA separately.According to the gel analysis shown in Fig. 5, in the sample of two types, all there is no visible genomic dna.
Genomic dna pollutent quantitatively
For the quantitative difference of genomic dna pollutent in sample again, according to the scheme separation quality grain of embodiment 1, described plasmid is from the pUC19 of the 5ml LB substratum of TOP10F cell with from the pBRCMV β of the 5ml LB substratum of DH5 α cell.Use as the polyurethane foam in embodiment 4 or if the needle punched felt in embodiment 3 is as top filtering material.After the wash-out of plasmid DNA, use the plasmid DNA of 125ng as the masterplate of PCR in real time.In order to measure the amount of genomic dna pollutent, use primer and DNA probe for the annealing of karyomit(e) pyruvate kinase gene.The sequence of described primer and probe is as follows:
Primer A Tcg taa gcg ttc tga cgt tat c
Primer B Cat gat gcc gtc aga ggc ttc gag
Probe FAM-acc tga aag cgc acg gcg gcg aa
By thering is the amount of the genomic dna that the standard series mode of genomic dna of known quantity quantitatively pollutes.
As shown in Figure 6 and Figure 7, in the sample filtering with rigid material the pollutent of genomic dna far above using the sample filtering according to foam of the present invention.As reference, by plasmid preparation, as masterplate, described plasmid preparation is to use the precipitation in lysate is carried out to pelletizing to prepare.Respectively in Fig. 6 and Fig. 7, sample (1) represents reference: 10 minutes centrifugal precipitates with pelletizingization, sample (2) represents the plasmid that uses filter module to separate, described filter module contain foam as top filtering material and non-binding silicon-dioxide as lower filter, and sample (3) represents the plasmid that uses filter module to separate, described filter module comprise needle punched felt (more rigid material) as top filtering material and non-binding silicon-dioxide as lower filter.
Claims (15)
1. comprise clarification/coupling apparatus of filter module, wherein said filter module comprises elasticity filtering material.
2. the application of the filter module that comprises elasticity filtering material in the method for separating of nucleic acid.
3. clarification/coupling apparatus as claimed in claim 1 or application as claimed in claim 2, is characterized in that, foam or sponge that described elasticity filtering material is perforate.
4. clarification/coupling apparatus as claimed in claim 3 or application, it is characterized in that, described strainer is to be made by the polyethylene foaming, polypropylene, urethane, polyester, polyethers, polystyrene, trimeric cyanamide, natural sponge, animal fibre sponge or plant fiber sponge.
5. clarification/coupling apparatus or the application as described in any one in claim 1-4, is characterized in that, described filter module also comprises the second strainer of being made by filamentary material.
6. clarification/coupling apparatus or the application as described in any one in claim 1-5, it is characterized in that, described the second strainer is placed under the flexible filter on flow direction or afterwards, and the aperture of described the second strainer is less than the aperture of described flexible filter.
7. clarification/coupling apparatus or the application as described in any one in claim 1-6, it is characterized in that, described device is single-column clarification/coupling apparatus, post in described device comprises described filter module, also comprise target bond material, or described device is twin columns clarification/coupling apparatus, wherein said filter module is the form that is inserted into the extra-column in described column.
8. clarification/coupling apparatus or the application as described in any one in claim 1-7, is characterized in that, described filter module can be removed from described column.
9. the method for clarified suspension, described suspension comprises at least one target molecules and solid particulate, precipitation and/or throw out, wherein said suspension also optionally can comprise shear sensitive molecule, described method comprises the filtration step relating to as the defined filter module of any one in claim 1-8, described filter module is for separating described solid particulate, precipitation and/or throw out from described target molecules, and wherein said target molecules is retained in solution.
As claimed in claim 9 for separating of and/or the method for purification of nucleic acid, described nucleic acid, as target molecules, said method comprising the steps of:
(i) sample suspension that comprises nucleic acid is contacted with filter module as defined in any one in claim 1-8
(ii) to described sample, apply external force to make described sample by described filter module.
11. methods as claimed in claim 10, described method is further comprising the steps of:
(iii) the described circulation liquid of step (ii) is contacted with nucleic acid bond material
(iv) optionally wash the nucleic acid of described combination
(v) from described bond material by described nucleic acid wash-out.
12. methods as described in any one in claim 9-11, is characterized in that, described target molecules is plasmid DNA or RNA.
13. methods as described in any one in claim 9-12, is characterized in that, externally applied forces is centrifugal force or vacuum in step (ii).
14. methods as described in any one in claim 11-13, is characterized in that, by using twin columns device that the described circulation liquid in step (iii) is contacted with nucleic acid bond material.
15. 1 kinds for carrying out the test kit of method as described in claim 9-14 any one, described test kit comprises filter module or the clarification/coupling apparatus as described in any one in claim 1-8, and optionally comprise other components arbitrarily, described component is selected from: at least one lysis buffer; At least one RNA enzyme liquid storage; At least one resuspended damping fluid; At least one neutralization buffer; At least one lavation buffer solution; At least one elution buffer; And/or carry out the specification sheets of described target separation/purification method.
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| EP11005418.6 | 2011-07-01 | ||
| EP11005418 | 2011-07-01 | ||
| PCT/EP2012/002759 WO2013004366A1 (en) | 2011-07-01 | 2012-06-29 | Filter module in biomolecule isolation |
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| CN103748208A true CN103748208A (en) | 2014-04-23 |
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| CN (1) | CN103748208A (en) |
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| CN112358943A (en) * | 2015-01-21 | 2021-02-12 | 新加坡科技研究局 | Column-based apparatus and method for size-based recovery of rare cells, and uses thereof |
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Also Published As
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| WO2013004366A1 (en) | 2013-01-10 |
| JP2014518074A (en) | 2014-07-28 |
| EP2726595A1 (en) | 2014-05-07 |
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