CN101400689A - Methods of extracting RNA - Google Patents
Methods of extracting RNA Download PDFInfo
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- CN101400689A CN101400689A CNA2007800085506A CN200780008550A CN101400689A CN 101400689 A CN101400689 A CN 101400689A CN A2007800085506 A CNA2007800085506 A CN A2007800085506A CN 200780008550 A CN200780008550 A CN 200780008550A CN 101400689 A CN101400689 A CN 101400689A
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Abstract
Methods and materials are disclosed for rapid and simple extraction and isolation of RNA from a biological sample involving the use of an acidic solution and a solid phase binding material that has the ability to liberate nucleic acids from biological samples, including whole blood, without first performing any preliminary lysis to disrupt cells or viruses. No detergents or chaotropic substances for lysing cells or viruses are needed or used. Viral, bacterial and mammalian genomic RNA can be isolated using the method of the invention. RNA isolated by the present method is suitable for use in downstream processes such as RT-PCR.
Description
The application is the common unsettled U.S. Provisional Application No.60/771 that submitted on February 8th, 2006,510 part continuation application.
Technical field
The present invention relates to be used to catch and extract the material of the simplified method of the Yeast Nucleic Acid Yeast Nucleic Acid of biogenetic derivation material (particularly from).
Background technology
The modern molecular biology method in clinical study, clinical diagnosis test and the drug development of being applied to has been utilized the research of Yeast Nucleic Acid (RNA) more and more.RNA exists with the form of messenger RNA(mRNA) (mRNA), transfer RNA (tRNA) and ribosome-RNA(rRNA) (rRNA).Some modern molecular biology techniques for example Northern trace, ribonuclease protection assay and RT-PCR need separate pure undegradable RNA before analyzing.The existence of specific mRNA sequence and the research of mRNA expression level are widely applied.Analysis mRNA (especially using microarray) is the strong instrument in the molecular biology research.Can determine the rise or the downward modulation of individual gene by the level of mRNA sequence in the working sample.The mRNA level can be used as the function of outside stimulus or morbid state and is evaluated.For example, the cancer positive correlation of the variation of p53 mRNA level and various kinds of cell type.
In addition, many have the virus (comprising HIV, HCV, west Nile virus, equine encephalitis virus and Ebola virus) of great effect to have the rna gene group to human health.The ability of extracting viral RNA fast and cleanly from body fluid or tissue is important for virological investigation and transmissible disease diagnosis and treatment.
The method of current extraction RNA from several different methods at the beginning, enter solution and prevent that RNA is by the endogenous RNA enzyme liberating with destruction or lysing cell, release RNA.Cracking discharges RNA with DNA and protein, then must be again isolation of RNA therefrom.Afterwards, handle described RNA with its dissolving or precipitation.Chirgwin etc., Biochem,
18, 5294-5299 (1979) discloses and has used from the liquid guanidinesalt with while lysing cell, dissolving RNA and inhibition RNA enzyme.Other methods by under low pH, extracting dissolved RNA and protein and DNA are separated with phenol/chloroform (D.M.Wallace, Meth.Enzym.,
15, 33-41 (1987)).Single stage method RNA separation commonly used comprises uses 4M guanidinesalt, sodium-acetate (pH4), phenol and chloroform/primary isoamyl alcohol to handle cell successively.Sample is centrifugal, by adding ethanol RNA is precipitated out from the upper strata (P.Chomczynski, Anal.Biochem.,
162, 156-159 (1987)).United States Patent (USP) 4,843,155 have described a kind of method, wherein phenol under the acid pH and guanidinesalt stabilized mixture are joined cell.After the use chloroform is separated, by reclaim the RNA of aqueous phase with ethanol sedimentation.
Other methods comprise hot phenol are joined in the cell suspension, carry out ethanol sedimentation (T.Maniatis etc., Molecular Cloning, A Laboratory Manual, ColdSpring Harbor Laboratory (1982)) then; Use negatively charged ion or nonionogenic tenside are with lysing cell and discharge cytoplasm rna; And use RNA enzyme inhibitors for example vanadyl-ribonucleoside complex (vanadyl riboside complex) and diethylpyrocarbonate (L.G.Davis etc., " Guanidine Isothiocyanate Preparation of Total RNA " and " RNApreparation:Mini Method " in Basic Methods in Molecular Biology, Elsevier, New York, pp.130-138 (1991)).
United States Patent (USP) 5,234 discloses among 809 (Boom etc.) by being incorporated on glass or other solid phase from biogenetic derivation the technology of DNA isolation and RNA simultaneously.By being exposed to the strong (〉 5M among the Tris HCl (pH8.0) that contains EDTA and tensio-active agent Triton X-100) guanidine thiocyanate solution comes the cell in the cracking biogenetic derivation (for example serum or urine).By hatching purify DNA and RNA from biomaterial with diatomite or silica dioxide granule (itself and DNA and RNA formation reversible mixture).
The United States Patent (USP) 5,155,018 of Gillespie provides from biogenetic derivation and to have separated and the method for purifying biological viable rna, and described biogenetic derivation can also comprise DNA, protein, carbohydrate and other cellular material.By in the presence of the binding soln that comprises dense acid chaotropic salt, making biogenetic derivation contact glass fine powder or diatomite come isolation of RNA.Under these conditions, it claims RNA optionally in conjunction with granular silicon materials, but also discloses with ethylate solution-treated solid matter to remove DNA.Other researchist work has afterwards confirmed to have really DNA to pollute.Be attached to described particulate RNA be easy to sample in contained other biological substance be separated.Preferably, cleaning and particle bonded RNA are to remove the material of non-specific adsorption.By discharging bonded RNA, thereby reclaim pure substantially biologically active rna with rare salt buffer wash-out.Also disclose the adding nuclease to remove the DNA in the elutriant, this has brought the further query to the claim of selective binding RNA.The US 5,990,302 of Kuroita etc. has provided the modification of Gillespie method, and it is by merging to come isolation of RNA with sample, chaotropic agent, lithium salts, acid solution and nucleic acid carrier.The US 6,218,531 of Ekenberg provides another kind of improvement, and the solution that wherein will contain RNA and impurity mixes with dilution buffer liquid to form clarifying lysate, then RNA is combined with the silicon-dioxide solid phase.Make it clarification by deposit D NA and protein.Described dilution buffer liquid can be water, but is more preferably the damping fluid SSC for example with neutral pH, wherein contains salt and more preferably contains for example SDS of stain remover.
United States Patent (USP) 5,010,183 and 5,985,572 have described with the monomer cats product lysing cell of a valence charge and ability that RNA and DNA are precipitated from solution simultaneously.In these patents, it is insoluble at first making RNA.In the method for ' 183 patent, the solution of quaternary ammonium surfactant and 40% urea and other additive is added in the cell suspension, and mixture is centrifugal.Precipitation is resuspended in the ethanol, makes nucleic acid from wherein precipitating by adding salt.
The U.S.6 of Michelsen etc., 355,792 disclose the method for following isolating nucleic acid, promptly use pH less than 6.5 damping fluid acidifying liquid sample, institute's acidifying solution is contacted with hydroxyl inorganic oxide material, to be combined with the solid phase material and the liquid separation of nucleic acid, and with pH 7.5 to 11 the basic solution wash-out of (preferred 8-8.5).Acidic solution does not contain ionic detergent, chaotropic agent and any ion＜0.2M.The example of having finished reflects that the use of described method has following prerequisite, and promptly nucleic acid was discharged in the solution before being hunted down.
WO00/66783 and EP1206571B1 disclose the method for free extracellular nucleic acid in the sample separation, promptly by make suspect the sample contain nucleic acid pH less than 7 condition under the water miscible weakly alkaline polymkeric substance of contact make that all nucleic acid form water-insolubles in weakly alkaline polymkeric substance and the sample and precipitate, with water-insoluble precipitation and sample separation, and described precipitation contacted with alkali with the pH that improves solution to being higher than 7, from the weakly alkaline polymkeric substance, discharge nucleic acid thus.But described polymkeric substance contains the protonated amino that can be neutralized by rising pH under acid pH.
The US5 of Backus etc., 582,988 and EP 0707077B1 the method that provides from the nucleic acid of lysate is disclosed, it may further comprise the steps: under less than 7 pH condition, make and suspect that the lysate that contains nucleic acid contacts the nucleic acid that makes in described weakly alkaline polymkeric substance and the described lysate and forms the water-insoluble precipitation with the water-soluble weakly alkaline polymkeric substance of capacity, from described lysate, separate described water-insoluble precipitation, described precipitation is contacted with alkali to improve pH value of solution to greater than 7, from described weakly alkaline polymkeric substance, discharge described nucleic acid thus.
The US 5,973,137 of Heath discloses the method for separating undegradable substantially RNA from biological sample, promptly uses the cell cracking agent of being made up of less than 6 buffered soln anionic detergent, sequestrant and pH to handle sample.The effect of described anionic detergent it is believed that to be lysing cell and/or solubilising protein and lipid and to make protein denaturation.When being used for, at first with containing NH from the whole blood isolation of RNA
4Cl, NaHCO
3With the reagent splitting erythrocyte of EDTA, separate white corpuscle and cracking white corpuscle independently in the presence of protein-DNA precipitation reagent.Described precipitation reagent is the sodium salt or the sylvite (for example acetate or hydrochloride) of high density normally.Last step is to make the supernatant precipitation that contains RNA by adding lower alcohol.Thereby isolation of RNA needs to use in addition lyase, glycerine and calcium chloride to discharge nucleic acid with the cell in the digestion prepared product from yeast and gram-positive microorganism.
The US 5,973,138 of Collis discloses and has been used for making nucleic acid to combine with acidic solution paramagnetic particle suspension reversibility.Disclosed particle is ferriferous oxide, iron sulfide or the iron chloride that exposes in this method.Described acidic solution is considered to strengthen the positive polarity of described particle iron part, promotes the combination of the negative electricity phosphate group of nucleic acid thus.Related U.S. patent 6,433,160 disclose similar methods, and wherein said acidic solution contains Glycocoll hydrochloride.
The U.S.6 of Bhikhabhai, 410,274 disclose by separate to come the method for plasmid DNA purification on insoluble matrix, and it comprises a) lysing cell; B) precipitate most of DNA and RNA with divalent-metal ion; C) remove described precipitation; D) with the described lysate of anionite-exchange resin purifying (use the acidic buffer of pH4-6, use more alkaline damping fluid then); And e) further uses the described plasmid of second ion-exchange resin purification.
The US 6,737,235 of Cros etc. discloses to use and has comprised or the particle of hydrophilic crosslinked polyacrylamide polymers of the cation group method of coming isolating nucleic acid of wrap quilt.By the protonated formation cation group of amido on the described polymkeric substance under low pH.Bind nucleic acid under the low pH condition is more discharging in the high ionic strength buffers liquid in low ionic strength buffer liquid.Described polymkeric substance must have 25-45 ℃ lower critical solution temperature.Alkaline pH and comparatively high temps also promote desorption.
The U.S.6 of Simms, 875,857 disclose the method and the reagent of isolation of RNA from vegetable material, and it has used the reagent composition that comprises nonionogenic tenside IGEPAL, EDTA, anion surfactant SDS and high density 2 mercapto ethanol.
The US 7 of Sprenger-Haussels, 005,266 disclose the method that is used for from sample (for example ight soil) purifying that contains nucleic acid processive enzyme inhibitor, stable or isolating nucleic acid, it is by the homogenate sample, then with pH2-7, salt concn〉100mM and contain in the phenol and material (for example polyvinylpyrrolidone) and the solution-treated institute homogenate sample that randomly contains stain remover and sequestrant to form lysate.On based on the conventional solid phase material of silicon-dioxide, handle described lysate then.
For example US 6,270,970,6,310,199, US5,652,348, US 5,945,520, WO96/09116, WO99/029703, EP1234832A3, EP1036082B1, U.S. patent application disclose some patents of 2001/0018513,2003/0008320 and 2003/0054395 etc. and application and disclose nucleic acid is reversibly captured on the bond material, and it is to change the protonation state that changes amido on the bond material by pH between combination and the elution buffer to mediate.Similarly, the US 6 of Bayer etc., 447,764 disclose the method for separating negatively charged ion organic substance (comprising nucleic acid) from aqueous system, this is by reversibly in conjunction with the non-cross-linked polymer nano particle of positively charged ion protonated form, they is separated and improve pH to make described particle deprotonation described negatively charged ion organic substance realizes to discharge from medium.
The U.S.5 of Kausch etc., 665,582 disclose the method that reversibly biomaterial is anchored on the solid support, it comprises invertible polymer is placed on the solid support, reversible joint is connected to described polymkeric substance, and uses bonding composition that biomaterial is connected to reversible joint, described bonding composition comprises nucleic acid, antibody, antiidiotypic antibody or albumin A, so that described biomaterial reversibly anchors on the solid support, wherein said biomaterial can be a nucleic acid.
The US 5,756,126 of Burgoyne discloses the drying solid medium that is used to preserve the genetic stocks sample, and described medium comprises solid substrate and is adsorbed onto the composition of described matrix, and described composition comprises weak base, sequestrant and anionic detergent.
The US6 of Fomovskaia etc., 746,841 disclose the method for purification of nucleic acid, and wherein a part comprises provides the dry substrate that comprises by the solid substrate of the quilt that anion surfactant wraps that is used for lysis, and sample is applied to described substrate and capture nucleic acid.The purposes of catching RNA specifically discloses or illustrates.
GB2419594A1 discloses with the amino surface promoting agent and has randomly stablized nucleic acid with nonionogenic tenside.
The United States Patent (USP) 6,602,718,6 of Augello, 617,170 and 6,821,789 and U.S. Patent Application Publication 2005/0153292 disclose by the method that suppresses or blocking gene is induced or nucleolysis is preserved biological sample (for example whole blood) and preservation RNA and/or DNA.Described gene induced blocker can comprise stablizer and acidic substance.Cationic detergent is preferred stablizer.Latter's lysing cell also causes nucleic acid as precipitating with the mixture of described stain remover.
US 6,916, and 608B2 discloses the method and composition of stable nucleic acid, and it comprises and methyl-sulphoxide blended alcohol and/or ketone mutually.
United States Patent (USP) 6,204,375 and 6,528,641 disclose the method for stabilized cell RNA composition, and it joins cell by for example ammonium sulfate solution of pH4-8 and carries out.Described salts solution makes cell permeabilization, causes RNA and cell protein coprecipitation, makes that RNA can not be near the nuclease that may make its degraded.
The rapid feature of multistep of the aforesaid method trouble of isolation of RNA makes that the application of RNA in clinical practice is complicated.Method must overcome at RNA by the difficulty of before nuclease (for example RNA enzyme) degraded protein in RNA and the cell and DNA being separated.These nucleases are present in the blood, present in an amount at least sufficient to the rapid damage unprotected RNAs.Therefore, must be able to stop the degraded of RNA enzyme from the successful methods of cellular segregation RNA.Still need from biological sample, to extract quickly and easily the method for RNA in this area.These methods will make hydrolysis and the minimum degradation of RNA, make it can be used for multiple analysis and downstream process.
The U.S. Patent Application Publication of owning together 2005/0106576,2005/0106577,2005/0106589,2005/0106602,2005/0136477 and 2006/0234251 discloses material and the method that is used for extracting from biomaterial nucleic acid (comprising RNA).Described method depends on the solid material of the unique types that is used to destroy cell or virus, and does not need to carry out chemical cracking and handle.
Summary of the invention
In one aspect, the invention provides the novel method of extraction and isolation of RNA from biological sample quickly and easily, it comprises use acidic solution and solid phase bond material.Implementing used solid phase bond material among the present invention has at first not carrying out any pre-cracking discharge the ability of nucleic acid under with the situation of destroying cell or virus from biological sample.Described solid phase bond material can comprise quaternary ammonium group, quaternary phosphine group or uncle's sulfonium group.
In yet another aspect, the invention provides from biological sample and to extract and/or the method for purifying RNA, it comprises the use acidic solution and has the matrix part and base and the other solid phase bond material that is connected the matrix part and the cut joint of base that also comprises that described base is selected from quaternary ammonium group, quaternary phosphine group or uncle's sulfonium group.
Detailed Description Of The Invention
Definition
Alkyl: contain side chain, straight chain or the cyclic alkyl of 1-20 carbon, described alkyl can be replaced by 1 or more a plurality of substituents other than hydrogen.Low alkyl group used herein refers to contain the alkyl that can reach 8 carbon.
Aralkyl: with the alkyl of aryl replacement.
Aryl: contain the aromatic group that contains of 1-5 carbocyclic ring aromatic nucleus, it can be replaced by 1 or more a plurality of substituents other than hydrogen.
Biomaterial: comprise whole blood, anticoagulated whole blood, blood plasma, serum, tissue, cell, cellular constituent and virus.
Cell material: intact cell or comprise the material (comprising tissue) of the intact cell of animal, plant or bacterial origin.Cell can be complete active metabolism cell, apoptotic cell or dead cell.
The nucleus composition: the nucleic acid in the phalangeal cell material, it can be genomic dna and RNA and other nucleic acid, for example from the nucleic acid of infectious substance (for example virus and plasmid).
Magnetic-particle: the particle, microparticle or the pearl that the external magnetic field are had response.Self can have magnetic, paramagnetism or superparamagnetism described particle.It can be attracted by external magnets or the magnetic field that applies when using super paramagnetic or ferromagnetic material.Particle can have the solid core part, described core be magnetic response and surrounded by one or more non-magnetic response layers.As an alternative, described magnetic response part can be around the layer of non-magnetic response core or can be the particle that is placed among the non-magnetic response core.
Nucleic acid: polynucleotide can be for example PNA of DNA, RNA or synthetic DNA analogue.The single chain compound and the double-stranded heterocomplex of any kind are also contained in the scope of this term in these three kinds of chain types.
Release, wash-out: by contacting to remove the material that the overwhelming majority is combined in solid phase material surface or hole with solution or composition.
RNA: include but not limited to messenger RNA(mRNA) (mRNA), transfer RNA (tRNA) and ribosome-RNA(rRNA) (rRNA).
Sample: contain or suspect the liquid that contains nucleic acid.The typical sample that can be used for the inventive method comprises body fluid, for example blood (it can be the anticoagulation of finding in collecting blood sample as usually), blood plasma, serum, urine, seminal fluid, saliva, cell culture, tissue extract etc.The sample of other type comprises solvent, seawater, industrial water sample, foodstuff samples and environmental sample (for example soil or water), vegetable material, eukaryote, bacterium, plasmid and virus, fungi and from procaryotic cell.
Solid phase material: material with the surface that can attract nucleic acid molecule.The form of material can be particle, microparticle, nano particle, fiber, pearl, film, filter (filter) and other upholder (for example test tube and micropore).
Replace: refer to that at least one hydrogen atom on the group is substituted by non-hydrogen group.It should be noted that about substituting group unless spell out in addition, it means and can exist multiple spot to replace.
The present invention relates to be used for obtaining quickly and easily the method for RNA from biological sample.Described method has been used solid phase bond material and acidic solution, and RNA contained nucleic acid source from sample discharges and enters in the described acidic solution.Selection has is not at first implementing the direct described solid phase bond material that discharges the RNA ability from biological sample under the situation of any pre-cracking with destruction cell or virus.Directly discharging RNA by the solid phase effect enters sour environment and the RNA that discharges is captured to described solid phase subsequently under acidic conditions and make minimum degradation.In addition, the applicant finds to reclaim Yeast Nucleic Acid from the sample with RNA enzymic activity and does not need to rely on adding RNA enzyme deactivation compound or protein, for example guanidinesalt, high density chaotropic agent or RNA enzyme arrestin and antibody.
In fact described method can be used for catching and extract RNA from protein-RNA mixture, intact cell and virus.Can the method according to this invention from any biological sample (particularly intact cell and virus) that contains nucleic acid, extract RNA.The common source of these materials includes but not limited to bacterial cultures or precipitation, blood, urine, cell, body fluid (for example urine, phlegm, seminal fluid, CSF, blood, blood plasma and serum) or from the tissue homogenate thing.Method of the present invention can not need to make them to stand to be applied under the situation of other preliminary step and comprise following sample: live, intact cell dead or apoptosis and tissue, the perhaps bacterium of Pei Yanging, plant or animal cell line.Especially, do not need to use any pre-destruction or cracking fully.Extract RNA in the cell from suspension (promptly from biofluid or cell culture) and can and discard substratum from for example low-speed centrifugal sedimentation cell.Use disorganization well known in the art method to extract RNA from complete tissue or organ, described method is for example by using manual homogenizer or automatic homogenizer (for example Waring agitator) or other tissue homogenizer to carry out homogenate.Described homogenate can by strainer (for example cheese cloth) with remove bulk matter or can be under low speed centrifugal prepared product with the separating particles material.
The inventive method is fast, only needs just can finish usually in several minutes.Importantly, the RNA of present method gained has enough purity makes it can be used for clinical or other downstream application, for example is used for reversed transcriptive enzyme self or then carries out polymerase chain reaction (PCR) amplification (RT-PCR), rna blot analysis and external translation.Advantageously, before using present method, do not need isolated cell, only need simple equipment to implement present method.Needs cracking or ethanol sedimentation step in advance not before handling sample according to the inventive method.Do not need not use yet be used for lysing cell or virus stain remover or from the liquid material.
In one embodiment of the invention, the selected biological sample (liquid that for example contains cell and/or virus) that contains RNA simply mixes to form mixture with acidic solution.Described sample and mixture only need contact in mixture the short several seconds.Do not need to carry out other processing.When described mixture forms or afterwards, described mixture and solid phase bond material merge, select such solid phase bond material, it has is not at first implementing the direct ability that discharges RNA from biological sample under the situation of any pre-cracking with destruction cell or virus.Directly discharging RNA by described particulate effect enters sour environment and the RNA that discharges is captured to fast these particles subsequently under acidic conditions and make minimum degradation.Remove supernatant, and randomly clean the solid phase that contains nucleic acid with one or more cleaning buffer solutions.If desired, can follow the described solid phase of wash-out with the RNA that dissociates from solid phase.In one embodiment, use basic solution from described solid phase or particle eluted rna.Usually, the ideal concentration that is used for the alkali of this purpose is at least 10
-4M, preferably about 1mM is to about 1M.
In another embodiment, if desired, method of the present invention can be implemented by randomly using RNA enzyme inhibitors (for example aurin tricarboxylic acid (aurin tricarboxylic acid), DTT or DEPC).Those skilled in the art can select other RNA enzyme inhibitors to be used for this purpose.
All described steps can be carried out in single container or on the single upholder fast continuously, and do not need special equipment (for example whizzer).Described method can be transformed the automatic platform that is used for handling in the serial or parallel mode a large amount of samples.All combinations and cleaning step are preferably finished at short notice, preferably are no more than 1 minute.Cleaning step can preferably carry out in 10 seconds.Wash-out preferably carries out being no more than in 1 minute.In an exemplary arrangement, the 100 microlitre samples that contain the RNA source mix with 100 microlitre acidic solutions in 1.5 milliliters of Eppendorf tubes, by the of short duration mixing of vortex.Be added in magnetic in the acidic solution then in conjunction with microparticle, with mixture vortex mixing 30 seconds.On the magnetic force platform with supernatant and described particle separation.Clean particle twice with 200 microlitre acidic solutions, clean twice with 200 microliters of water.With the particle after cleaning alkaline eluant mesoscale eddies mixing 1 minute, with eluted rna.
Solid phase material
In one embodiment, RNA extracting method use solid phase bond material of the present invention in conjunction with RNA, allows RNA and other sample composition to be separated with fast thus.Selection has is not at first implementing the direct solid phase bond material that discharges the ability of nucleic acid from biological sample under the situation of any pre-cracking with destruction cell or virus.The material that is used for bind nucleic acid in the methods of the invention comprises the matrix of determining its size, shape, porosity and mechanical property.The form of described matrix can be particle, microparticle, fiber, pearl, film and other upholder (for example test tube and micropore).Multiple concrete material and its preparation are described in applicant's common unsettled U.S. Patent Application Publication 2005/0106576,2005/0106577,2005/0106589,2005/0106602,2005/0136477 and 2006/0234251.
In one embodiment, described material also comprises near the covalently bound nucleic acid binding moiety that is in surface or its, and it allows to catch and in conjunction with the nucleic acid molecule of different lengths.The surface not only refers to the periphery of solid phase material, also refer to any in the solid phase material can be near the surface in hole district.
In another embodiment, described material also comprises the nucleic acid binding moiety that is near the non-covalent connection surface or its, and it allows to catch and in conjunction with the nucleic acid molecule of different lengths.The nucleic acid binding moiety of described non-covalent connection by with the surface on the electrostatic attraction of oppositely charged residue link to each other with described solid substrate, perhaps link to each other with described surface by hydrophobic gravitation.
These matrix of material that have the nucleic acid conjugated group of covalently or non-covalently connection can be any suitable materials.Preferred substrate material is selected from: silicon-dioxide, glass, insoluble synthetic polymer, insoluble polysaccharide and metallic substance and with the magnetic response material of silicon-dioxide, glass, synthetic polymer or insoluble polysaccharide bag quilt, described metallic substance is selected from metal, metal oxide and metallic sulfide.Exemplary materials comprises surface functional group bag quilt or the functionalized material based on silicon-dioxide that is linked to each other by covalency, and described functional group is used to destroy cell and attracts nucleic acid.Can also comprise suitable functionalisation of surfaces based on the material of carbohydrate and polymer materials with this surface functionality.Comprise as the surface functional group of nucleic acid conjugated group and can destroy the cellularstructure integrity and to make nucleic acid be attracted to any group of solid support.Such group includes but not limited to the material of hydroxyl described below, silanol group, carboxyl, amino, ammonium, quaternary ammonium He quaternary alkylphosphonium salt and uncle's sulfonium salt type.Wherein, the material with quaternary ammonium, quaternary phosphine or uncle's sulfonium group is preferred.
For many application, preferred described solid material is the particulate form.Preferably, described particle size is more preferably less than about 10 microns less than about 50 microns.Small-particle is easier to be dispersed in the solution, has higher surface area/volume ratio.Bigger particle and pearl also can be used for using in gravitational settling or the centrifugation method.Two or more different size particulate mixtures can be preferred in some applications.
Preferably described solid phase also can comprise the magnetic response part, and it is the form of paramagnetic or super paramagnetic microparticle normally.Described magnetic response partly allows by magnetic field suction and manipulation.Such magnetic corpuscular comprises magnetic metal oxide or metallic sulfide core usually, described core be adsorbed usually or covalently bound layer center on to protect magnetic components.The nucleic acid conjugated group is layer covalent attachment therewith, wraps thus by described surface.Described magnetic metal oxide core is ferriferous oxide or iron sulfide preferably, and wherein iron is Fe
2+Or Fe
3+Or the two all has.The magnetic-particle that is wrapped in the organic polymer layers is open, for example sees United States Patent (USP) 4,654, in 267,5,411,730 and 5,091,206 and publication (Tetrahedron Lett., 40 (1999), 8137-8140) in.Bag with number of different types shell is had commercial offers by magnetic-particle.As instructing in U.S. Patent Application Publication 2005/0106576,2005/0106577,2005/0106589,2005/0106602,2005/0136477 and 2006/0234251, described shell can functionalised.
Commodity magnetic silica or magnetic polymer particles can be used as the parent material that preparation can be used for magnetic solid phase bond material of the present invention.The polymer beads with surperficial carboxyl of adequate types is known to have commodity to be called SeraMag
TM(Seradyn) and BioMag
TM(Polysciences andBangs Laboratories).The silica magnetic particle of adequate types is known to have commodity to be called MagneSil
TM(Promega).Chemicell GmbH (Berlin) provides the surface to have carboxyl or amino silica magnetic particle.
The joint group that contains trialkoxysilane groups at an end can be connected to the surface of the metallic substance of metallic substance or bag quilt, described bag for example be that bag is by the magnetic-particle of silicon-dioxide or glass by metallic substance.Preferred trialkoxy silane compound has following formula R
1-Si (OR)
3, wherein R is a low alkyl group, R
1Be to be selected from the organic group of straight chain, side chain and ring and to comprise 1-100 atom.Described atom preferably is selected from C, H, B, N, O, S, Si, P, halogen and basic metal.Representative R
1But group is 3-aminopropyl, 2-cyanoethyl and 2-propyloic and as the group that contains cutting part of more abundant description hereinafter.In a preferred embodiment, three alcoxyl silane-based compounds comprise can cut middle body and active group terminal portions, wherein said reactive group can by with the reaction of tertiary amine, tertiary phosphine or organosulfur one step change quaternary salt or uncle's salt into.
Found and can in the process of using fluorion, such joint group be placed in metallic particles and bag by the surface of the metallic particles of glass or silicon-dioxide.Described reaction can be carried out in organic solvent, and described solvent comprises lower alcohol and aromatic solvent (comprising toluene).Suitable fluorine source has solvability preferably in these organic solvents, described fluorine source comprises cesium fluoride and fluoridizes tetraalkylammonium salt.
Nucleic acid contained in can be used for some solid phase bond materials of the inventive method is in conjunction with (nucleic acid binding, NAB) group can be used as dual purpose.The NAB group attracts and combination has all lengths and based composition or sequence nucleic acid, polynucleotide and oligonucleotide.They also can have certain ability that discharges nucleic acid from cell envelope.The nucleic acid conjugated group comprises: for example, and carboxyl, amine and season or uncle's group or more than a kind of mixture of these groups.Amido can be NH
2, alkylamine and dialkylamine.Preferred nucleic acid conjugated group is season or uncle's group (QR
2 +Or-QR
3 +), it comprises trialkyl quaternary ammonium group (NR
3 +), quaternary phosphine base (PR
3 +), it comprises San Wan Ji Phosphonium or San Fang Ji Phosphonium or mixed alkyl Fang Ji Phosphonium group, and uncle's sulfonium base (SR
2 +).Described solid phase can contain more than a kind of nucleic acid conjugated group described herein.Can use particulate mixture more than a kind of size.Can also use above-mentioned solid phase bond material and multiple other to have or do not have the mixture of the solid phase bond material of NAB group.Also can use and contain season or uncle's group (QR
2 +Or-QR
3 +) solid phase material, wherein the R base is the alkyl of at least 4 carbon atoms, it is bind nucleic acid especially effectively, but also can use as few as the alkyl and the aryl of a carbon atom.These solid phase materials keep bonded nucleic acid with high strength very, opposing removing or wash-out described nucleic acid under the known most of elution requirements of prior art.Bonded nucleic acid is invalid to the most of known elution requirement of low ionic strength and high ionic strength for removing.Different with the conventional anionite-exchange resin that contains DEAE and PEI group, no matter the pH of reaction medium how, season or uncle's solid phase material be retainer belt positive electricity all.
Preferred embodiment is used such solid phase bond material, and wherein said nucleic acid conjugated group links to each other with described matrix by the connecting key of alternative cutting.Cut off described connecting key effectively with any bind nucleic acid and solid phase " disconnection ".Can cut off the key that can cut in the joint but the method for demolition purpose nucleic acid is not cut off described connecting key by any chemistry, enzyme, photochemistry or other specificity.These can cut solid phase material and comprise the solid support part, and it comprises aforesaid matrix.Be used to attract link to each other with the surface of described solid support by cutting shank in conjunction with (NAB) part with the nucleic acid of bind nucleic acid.U.S. Patent Application Publication 2005/0106576,2005/0106577,2005/0106602,2005/0136477 and 2006/0234251 has been described has the suitable material that can cut connecting key, and wherein disclosure is incorporated this paper into by reference.
The shank that can cut preferably is selected from the organic group of straight chain, side chain and ring, and comprises 1-100 atom.Described atom is preferably selected from C, H, B, N, O, S, Si, P, halogen and basic metal.The exemplary adapter group is the group of hydrolyzable cutting.Example comprises carboxylicesters and acid anhydrides, thioesters, carbonic ether, thiocarbonic ester, carbamate, imide, sulphonamide, sulfimide and sulphonate.In a preferred embodiment, handle the described connecting key that cuts with alkaline aqueous solution.Another kind of exemplary adapter types of radicals is to stand the group of reductibility cutting, is for example comprised the disulfide linkage (S-S) of the plurality of reagents cutting of phosphine and mercaptan (for example sulfur alcohol, mercaptoethanol and DTT).Another kind of representative groups is the organic group that contains peroxide bridge (O-O).Peroxide bridge can be by mercaptan, amine and phosphine cutting.But the another kind of representational joint group that group is the enzymatic cutting that cuts.Exemplary group comprises ester (it can be cut by esterase and lytic enzyme), acid amides and peptide (it can be cut by proteolytic enzyme and peptase), glycosyl (it can be cut by Glycosylase).Another kind of representativeness can cut group be can cut 1,2-dioxy tetramethylene (dioxetane) part.These materials contain dioxy tetramethylene part, and it can be thermal decomposited or be caused and fragmentation by chemistry or enzyme reagent.Remove blocking group and produce the decomposition that oxygen anion has promoted dioxy tetramethylene ring.By cutting peroxide O-O key and C-C key generation fragmentation according to known method.The dioxy cyclobutane that can cut has been described in a plurality of patents and publication.Representative example comprises United States Patent (USP) 4,952,707,5,707,559,5,578,253,6,036,892,6,228,653 and 6,461,876.
The another kind of joint group that can cut is the two keys of C-C that are rich in electronics, and it can be transformed into unsettled 1,2 dioxy tetramethylene part.At least one substituting group on two keys is connected to described pair of key by O, S or N atom.Two keys of electron rich and singlet oxygen (singlet oxygen) reaction produce unsettled 1,2 dioxy tetramethylene cyclic group, and its quick at normal temperatures fragmentation produces two carbonyl fragments.
Have that the another kind of solid phase material that can cut the joint group has ketenes dithioacetals (ketenedithioacetal) but as cutting part, as United States Patent (USP) 6,858,733 and 6,872,828 is disclosed.By the enzymatic oxidn of peroxidase and hydrogen peroxide, the oxidisability cutting of the two keys of ketenes dithioacetals experience.
Structure shown in but described cutting part can have is included in the analogue that has replacement on acridine (acridan) ring, wherein R
a, R
bAnd R
cAll be organic group, it contains 1 separately to about 50 non-hydrogen atoms that are selected from C, N, O, S, P, Si and halogen, wherein R
aAnd R
bCan be connected and form ring.Multiple other can cut group and it will be apparent to those skilled in the art that.But another group has the joint group that the solid phase material that can cut the joint group has the light cutting, for example the aromatic oxide and the ester of nitro replacement.According to known reactions, adjacent nitrobenzyl ester is cut by UV-light.
Acidic solution
The acidic solution that is used for the inventive method generally comprises has any aqueous solution that is lower than neutral pH, and preferred described solution has the pH in the 1-5 scope, more preferably from about 2-4.Described acid can be organic or inorganic acid.Can use mineral acid for example hydrochloric acid, sulfuric acid and perchloric acid.Can use to comprise monocarboxylic acid, dicarboxylic acid, tricarboxylic acid and amino acid whose organic acid, and the salt of described acid.Representative acid comprises formic acid, acetate, trifluoroacetic acid, propionic acid, oxalic acid, propanedioic acid, succsinic acid, pentanedioic acid and citric acid, glycine and L-Ala.Salt can have any water-soluble gegenion, preferred as alkali or alkaline-earth metal ions.The acidic solution that comprises transition metal salt also can be used for implementing the present invention.Preferred transition metal comprises Fe, Mn, Co, Cu and Zn salt.
Unlike other method of extracting RNA by chemical cracking, used acidic solution does not contain stain remover or chemical cracking agent for example from liquid material (as guanidinesalt) in present method.There are not to be used for the organic solvent of these functions, for example DMF or DMSO arbitrarily.Under the situation that does not have other soluble additive, the combination of described acidic medium and described solid phase bond material is enough to allow extract complete RNA from sample, or even the sample that contains the RNA enzyme also is like this.
Described sample can be mixed mutually with described acidic solution, by in this acidic solution, providing solid phase mixture and described solid phase be merged simultaneously.As an alternative, described sample can at first mix with described acidic solution to form mixture, then described mixture and described solid phase is merged.
Cleaning solution
If you are using, can be used for implementing cleaning solution of the present invention can help from conjunction with removing other composition the RNA.In one embodiment, cleaning solution can comprise and the used same or analogous acidic solution of integrating step.Have been found that it is favourable cleaning with acidic solution, this may be in order to remove remaining RNA enzymic activity.Use the water of neutral pH or damping fluid further to clean to be used in the described acid that neutralizes before the wash-out.Should preparation or treating water and damping fluid do not have the RNA enzymic activity to guarantee them.
Elution reagent
In one embodiment, by with described solid phase material with discharge reagent that institute enters solution in conjunction with RNA contact from described solid phase wash-out in conjunction with RNA.Described solution should dissolve and be enough to preserve the RNA of release.Being eluted in the RNA that discharges in the solution should be compatible with the downstream molecules biological procedures.In another embodiment, being used for from the reagent of solid phase bond material release nucleic acid is to work by the cut joint group that cutting solid phase bond material exists.A kind of preferred reagent is at least 10
-4The strong alkaline aqueous solution of M.Concentration is at least 10
-4The solution of the alkali metal hydroxide of M, ammonium hydroxide, tetra-alkyl ammonium hydroxide, alkaline carbonate and alkalimetal oxide can be effectively on the cutting solid phase fly-cutting and eluted rna.When cutting group and being two sulphur (S-S) group, wash-out/cutting reagent will contain the disulfide bond reduction agent, for example mercaptan (as sulfur alcohol, mercaptoethanol or DTT) when described.When cutting group and being peroxide (O-O) key, wash-out/cutting reagent will contain reductive agent, for example mercaptan, amine or phosphine when described.But cut group enzymatic when cutting when described, wash-out/cutting reagent will contain suitable enzyme.Ester needs esterase or lytic enzyme; Acid amides or peptide bond need proteolytic enzyme or peptase; The glucosides group needs Glycosylase.When the described group that cuts is 1, during 2-dioxy tetramethylene part, can the described dioxy tetramethylene of thermal cutting, elution reagent can be above-mentioned basic solution.When described cut group be can bring out 1, during 2-dioxy tetramethylene part, wash-out/cutting reagent will contain chemistry or enzyme reagent to produce the cutting that unstable oxygen anion is induced described group by removing blocking group.When the described group that cuts is to be transformed into unstablely 1, during the two key of the electron rich C-C of 2-dioxy tetramethylene, wash-out/cutting reagent will contain singlet oxygen source (for example light-sensitive coloring agent).Known in the art and visible light and molecular oxygen reaction produce these dyestuffs of oxygen singlet excited state, comprise for example rose bengal (RoseBengal), Eosin Y (Eosin Y), alizarin red S (Alizarin Red S), Congo red (CongoRed) and orange G (Orange G), fluorescein(e) dye, rhodamine, Erythrosin B, CHLOROPHYLLINE trisodium salt, teichmann's crystals salt, haematoporphyrin, methylene blue, Viola crystallina, malachite green (Malachite Green) and soccerballene.
In another embodiment, be used for being selected from applicant's common unsettled U.S. Patent Application Publication 2005/0106589 disclosed composition from the reagent that comprises season NAB group of solid phase bond material release RNA.
Described release steps can at room temperature be implemented, but can use any suitable temperature.As if eluting temperature be not crucial for the success of isolating nucleic acid method of the present invention.Preferred room temperature, but elevated temperature can increase elution rate in some cases.
Test kit of the present invention
In another embodiment, be provided for implementing the test kit of the inventive method.The test kit of isolated nuclei ribosomal ribonucleic acid comprises at least a solid phase bond material and acidic solution from sample according to the present invention, and selected solid phase bond material is selected to has the ability that directly discharges nucleic acid under at first not carrying out any pre-cracked situation from biological sample.Described solid phase bond material comprises matrix, and it can be the form of particle, microparticle, magnetic-particle, fiber, pearl, film and other upholder (for example test tube and micropore).Described matrix covalently or non-covalently is connected with nucleic acid binding moiety, randomly by cutting joint.
Described nucleic acid binding moiety comprises the group of at least a type, and it is selected from carboxyl, NH
2, alkylamine, dialkyl amino, quaternary ammonium group (comprising the trialkyl ammonium), quaternary phosphine base (comprising three alkane base Phosphonium, three fragrant basic Phosphonium or the basic Phosphonium base of mixed alkyl virtue) and uncle's sulfonium base.
Described acidic solution as a kind of key element in the test kit of the present invention comprises that usually pH is lower than any aqueous solution of neutral.Preferably, described solution has the pH in the 1-5 scope, more preferably from about 2-4.Described acid can be organic or inorganic acid.Mineral acid for example hydrochloric acid, sulfuric acid and perchloric acid is an available.Can use to comprise monocarboxylic acid, dicarboxylic acid, tricarboxylic acid and amino acid whose organic acid, and the salt of described acid.Representative acid comprises formic acid, acetate, trifluoroacetic acid, propionic acid, oxalic acid, propanedioic acid, succsinic acid, pentanedioic acid and citric acid, glycine and L-Ala.Salt can have any water-soluble gegenion, preferred as alkali or alkaline-earth metal ions.The acidic solution that comprises transition metal salt also is available in the embodiment of this invention.Preferred transition metal comprises Fe, Mn, Co, Cu and Zn salt.
Test kit can also comprise elution reagent, and one or more optional cleaning buffer solutions and other test kit conventional component, for example service manual, experimental program, damping fluid and thinner.Elution reagent can be selected from strong alkaline aqueous solution, and for example concentration is at least 10
-4Alkali metal hydroxide or the solution of ammonium hydroxide of M (preferably about 1mM is to about 1M); Disulfide bond reduction agent, for example mercaptan (comprising sulfur alcohol, mercaptoethanol or DTT); Superoxide reductive agent (for example mercaptan, amine or phosphine) and enzyme (for example esterase, lytic enzyme, proteolytic enzyme, peptase, Glycosylase or peroxidase).In one embodiment, wherein the solid phase bond material contains and can cut joint, for example can be by the electron rich thiazolinyl that is cut with the singlet oxygen source reactant, and described test kit can comprise aforesaid light-sensitive coloring agent.
Embodiment
Embodiment 1. is used for the solid phase material of isolation of RNA.Synthetic by San Ding Ji Phosphonium NAB group with can cut the functionalized magnetic-particle of aryl thioesters connecting key.
A) preparation magnet.In 1 hour, the gas argon is blown in 3 liters of I type water in 5 litre flasks.Under argon gas, add dense NH
4OH (28%, 180 milliliter).In about 1 hour, add 50 milliliters of 2M FeCl in 1M HCl by dropping funnel
2With 200 milliliters of 1MFeCl in 1M HCl
3Mixture.By following step with solid collection in two flasks, pour in the flask that has outside disc-shaped magnet with 500-600 milliliter part, and pour out supernatant at every turn.By in 500-600 milliliter I type water, being attracted to magnet with ultra-sonic dispersion then and pouring out supernatant and clean described solid.Repeat described process and be approximately 8.5 until the pH of supernatant.The content that merges in two bottles makes described magnet preserve with about 500 milliliters cumulative volume.
B) 3-methyl aminopropyl trimethoxysilane (149.8 gram) is joined in 500 ml flasks, feed argon gas.Described flask is placed after the ice bath, slowly add acryloxy trimethyl silane (119.6 gram) with syringe.Stir described reaction 5 minutes, remove ice bath, continue to stir 2 hours.Promptly use product without being further purified.
C) magnet bag quilt.To be diluted to 140 milliliters from a certain amount of magnet slurry that contains 5.0 gram magnet of step a) with I type water, ultrasonic to mixture.Add ethanol (1.25 liters) after 15 minutes.Add dense NH after 30-45 minute
4OH (28%, 170 milliliter).To restrain the solution and the 13.5 gram Si (OEt) of silicomethane esters from 1.5 of step b)
4Ethanolic soln in 90 fens clock times, divide three parts and join in the reaction.Add the solution of 3.75 gram silicomethane ester cpds in the 20-30 milliliter ethanol then, stir the mixture and ultrasonic again 90 minutes.Stirring is spent the night.Mixture is advanced in two 1 liter of flasks with every part of 500 milliliters of transfers, rely on the described particle of magnetic resolution.Use the HCl (before putting back to mixture on the magnet, keeping 10 minutes) in the I type that the is diluted in water of 4 * 250 ml methanol, 2 * 250 milliliters of I type water, 1 * 250 milliliter of pH1,4 * 250 milliliters of I type water, 4 * 250 ml methanol and 2 * 250 milliliters of acetone to clean described solid successively.Air-dry solid spends the night.In this step process, silicomethane ester generation hydrolysis causes producing hydroxy-acid group.
D) will place 30 milliliters of thionyl chloride from magnetic carboxylic acid functionalized particle's (1.0 gram) of previous step, reflux 4 hours.Excessive thionyl chloride is poured out from magnetic retention.Use CH
2Cl
2Clean described particle for several times, continue next step.
E) with 0.22 gram 1, dithio-hydroquinone and 0.52 milliliter of diisopropyl ethyl amine are handled and are suspended from 50 milliliters of CH
2Cl
2In the acyl chlorides functionalized particle.To mixture ultrasonic 5 minutes, stir with the track shaking table and to spend the night.Utilize magnetic resolution to use CH successively
2Cl
2, 1: 1CH
2Cl
2/ CH
3OH, CH
3OH, 1: 1CH
2Cl
2/ CH
3OH and CH
2Cl
2Clean described solid.With the solid air dried overnight.
F) handle previous step particle (about 0.9 gram) and 25 milliliters of CH with 0.81 gram tributylphosphine
2Cl
2Mixture.To described mixture ultrasonic 5 minutes, stir with the track shaking table and to spend the night.Utilize magnetic resolution to use CH successively
2Cl
2, 1: 1CH
2Cl
2/ CH
3OH, CH
3OH, 1: 1CH
2Cl
2/ CH
3OH and CH
2Cl
2Clean described solid.With the solid air dried overnight.
G) handle previous step particle (about 0.8 gram) and 25 milliliters of CH with 0.25 gram 4-chloromethyl benzoic acid chlorides and 0.52 milliliter of diisopropylethylamine
2Cl
2Mixture.To described mixture ultrasonic 5 minutes, stir with the track shaking table and to spend the night.Utilize magnetic resolution to use CH successively
2Cl
2, 1: 1CH
2Cl
2/ CH
3OH, CH
3OH, 1: 1CH
2Cl
2/ CH
3OH and CH
2Cl
2Clean described solid.Collect solid and dried overnight.
H) handle previous step particle (about 0.7 gram) and 25 milliliters of CH with 0.41 gram tributylphosphine
2Cl
2Mixture.To described mixture ultrasonic 5 minutes, stirred totally 7 days with the track shaking table.Utilize magnetic resolution to use 1 successively: 1CH
2Cl
2/ CH
3OH and CH
3OH cleans described solid.Collect and drying solid.
The solid phase material of embodiment 2. larger particle size.Synthesize San Ding Ji Phosphonium NAB group and can cut the functionalized magnetic-particle of aryl thioesters connecting key.
A) 3-methyl aminopropyl trimethoxysilane (149.8 gram) is joined 500 ml flasks, charge into argon gas.Described flask is placed after the ice bath, slowly add acryloxy trimethyl silane (119.6 gram) with syringe.Stir described reaction 5 minutes, remove ice bath, continue to stir 2 hours.Promptly use product without being further purified.
B) with 140 milliliters of I type water and 1.25 liters of alcohol dilution 5.0 gram commodity magnet (Strem catalog number (Cat.No.) 93-2616,1-5 micron).Add dense NH after 30-45 minute
4OH (28%, 170 milliliter).Will be from the 1.5 gram silicomethane esters and the 13.5 gram Si (OEt) of step b)
4Ethanolic soln in 90 fens clock times, divide three parts to join in the reaction.Add the gram of 3.75 in 20-30 milliliter ethanol silicomethane ester cpds solution then, stir the mixture and ultrasonic again 90 minutes.Stirred overnight.Mixture advanced with every part of 500 milliliters of transfers in two 1 liter the flask, rely on the described particle of magnetic resolution.Use the HCl (before putting back to mixture on the magnet, keeping 10 minutes) in the I type that the is diluted in water of 4 * 250 ml methanol, 2 * 250 milliliters of I type water, 1 * 250 milliliter of pH 1,4 * 250 milliliters of I type water, 4 * 250 ml methanol and 2 * 250 milliliters of acetone to clean described solid successively.Air-dry solid spends the night.In this step process, silicomethane ester generation hydrolysis and cause producing hydroxy-acid group.
D) will place 30 milliliters of thionyl chloride from magnetic carboxylic acid functionalized particle's (1.0 gram) of previous step, reflux 4 hours.Excessive thionyl chloride is poured out from magnetic retention.Use CH
2Cl
2Clean described particle for several times, continue next step.
E) with 0.22 gram 1, dithio-hydroquinone and 0.52 milliliter of diisopropyl ethyl amine are handled and are suspended from 50 milliliters of CH
2Cl
2In the acyl chlorides functionalized particle.To mixture ultrasonic 5 minutes, stir with the track shaking table and to spend the night.Utilize magnetic resolution to use CH successively
2Cl
2, 1: 1 CH
2Cl
2/ CH
3OH, CH
3OH, 1: 1 CH
2Cl
2/ CH
3OH and CH
2Cl
2Clean described solid.With the solid air dried overnight.
F) handle previous step particle (about 0.9 gram) and 25 milliliters of CH with 0.81 gram tributylphosphine
2Cl
2Mixture.To described mixture ultrasonic 5 minutes, stir with the track shaking table and to spend the night.Utilize magnetic resolution to use CH successively
2Cl
2, 1: 1CH
2Cl
2/ CH
3OH, CH
3OH, 1: 1CH
2Cl
2/ CH
3OH and CH
2Cl
2Clean described solid.With the solid air dried overnight.
G) handle previous step particle (about 0.8 gram) and 25 milliliters of CH with the diisopropylethylamine of 0.25 gram 4-chloromethyl benzoic acid chlorides and 0.52 milliliter
2Cl
2Mixture.To described mixture ultrasonic 5 minutes, stir with the track shaking table and to spend the night.Utilize magnetic resolution to use 1 successively: 1CH
2Cl
2/ CH
3OH and CH
3OH cleans described solid.Collect solid and dried overnight.
H) handle previous step particle (about 0.7 gram) and 25 milliliters of CH with 0.41 gram tributylphosphine
2Cl
2Mixture.To described mixture ultrasonic 5 minutes, stirred totally 7 days with the track shaking table.Utilize magnetic resolution to use CH successively
2Cl
2, 1: 1CH
2Cl
2/ CH
3OH, CH
3OH, 1: 1CH
2Cl
2/ CH
3OH and CH
2Cl
2Clean described solid.Collect and drying solid.
Embodiment 3. synthetic functionalized magnetic polymkeric substance
Help by magnetic force will contain 25 milligrams of solid pearls (Dynal magnetic COOH pearl, lot number G36710) aliquots containig pours out.Use 3 * 1 ml waters and 3 * 1 milliliters of CH then
3CN cleans pearl, dry 4 hours afterwards.Described pearl is suspended from the 1mLCH that has added 28.8 milligrams of EDC
2Cl
2In, jolting 30 minutes.With 1, dithio-hydroquinone (30 milligrams) solution joins mixture.To pipe ultrasonic 1 minute, jolting was spent the night.Remove supernatant, rely on magnetic force with 4 * 1 milliliters CH
2Cl
2, 1 milliliter of 1: 1 methyl alcohol: CH
2Cl
2, 4 * 1 ml methanol and 4 * 1 milliliters of CH
2Cl
2Clean described pearl.
Described pearl is suspended from 1 milliliter of CH that adds 140 microlitre tributylphosphines
2Cl
2In.With reaction mixture vortex 1 minute, jolting was 3 days altogether.Pour out solvent on the magnet by remaining on.Rely on magnetic force with 4 * 1 milliliters of CH
2Cl
2, 1 milliliter of 1: 1 methyl alcohol: CH
2Cl
2, 4 * 1 ml methanol, 1 milliliter of 1: 1 methyl alcohol: CH
2Cl
2With 4 * 1 milliliters of CH
2Cl
2Clean described pearl.
Handle 1 milliliter of CH with 2 milligrams of 4-chloromethyl benzoic acid chlorides and 52 microlitre diisopropylethylamine
2Cl
2The mixture of middle previous step particle (about 25 milligrams).To described mixture vortex 10 seconds, ultrasonic 5 minutes, stir with the track shaking table and to spend the night.Utilize magneticseparation to use 4 * 1 milliliters of CH successively
2Cl
2, 1 milliliter of 1: 1 methyl alcohol: CH
2Cl
2, 4 * 1 ml methanol, 1 milliliter of 1: 1 methyl alcohol: CH
2Cl
2With 4 * 1 milliliters of CH
2Cl
2Clean described solid.
Handle 1 milliliter of CH with 30 milligrams of tributylphosphines
2Cl
2Mixture with previous step particle (25 milligrams).To described mixture ultrasonic 2 minutes, stirred totally 6 days with the track shaking table.Utilize magneticseparation to use 4 * 1 milliliters of CH successively
2Cl
2, 3 * 1 ml methanol, 2 * 1 ml waters clean described solid.Make pearl stock solution (25 mg/ml) by adding 1 ml water.
Embodiment 4. synthetic functionalized magnetic polymkeric substance
Collect from 2 * 0.535 milliliters of Sera-Mag with magnetic force
TMThe magnetic carboxylic acid ester is modified the magnetic-particle of microparticle suspension (Seradyn) (it contains totally 50 milligrams of particles), and supernatant is poured out.Use 3 * 1 ml waters, 3 * 1 milliliters of CH then
3CN and 3 * 1 milliliters of CH
2Cl
2Clean pearl.Described pearl is suspended from 3.6 milliliters of CH that added 60 milligrams of EDC
2Cl
2In, jolting 30 minutes.
The preparation joint: with 1, dithio-hydroquinone (11.97 gram) is dissolved in 300 milliliters.Described solution is cooled to-78 ℃.In 1 hour, dropwise add 100 milliliters of CH
2Cl
2In the solution of 8.86 gram 4-chloromethyl benzoic acid chlorides and 3.8 milliliters of pyridines.Make reaction soln be warming up to room temperature, keep and spend the night.Clean the 1 impure solid product that restrains with any scavenging solution after finishing and obtain 200 milligrams of pure products.Can from filtrate, separate more amount by chromatography.
Joint among the 400 microlitre DMF (60 milligrams) solution is added in the described mixture.To described pipe ultrasonic 1 minute, jolting was spent the night.Described pearl is divided into two parts of each parts of 25 milligrams, handles respectively.Remove supernatant, utilize magneticseparation to use 4 * 1 milliliters of CH successively
2Cl
2, 1 milliliter of 1: 1 methyl alcohol: CH
2Cl
2, 4 * 1 ml methanol, 1 milliliter of 1: 1 methyl alcohol: CH
2Cl
2With 4 * 1 milliliters of CH
2Cl
2Clean described pearl.
Adding 10 milliliters of CH of 75 microlitre tributylphosphines
2Cl
2The middle described particle that suspends.With described reaction mixture vortex 1 minute, jolting totally 7 days.Described pearl is divided into two parts of each parts of 25 milligrams, handles respectively.Pour out solvent on the magnet by remaining on.Utilize magneticseparation to use 3 * 1 milliliters of CH successively
2Cl
2, 1 milliliter of 1: 1 methyl alcohol: CH
2Cl
2, 4 * 1 ml methanol and 2 * 1 ml waters clean pearl.Make pearl stock solution (25 mg/ml) by adding 1 ml water.
Embodiment 5. is used to extract the acidic solution of RNA.Use simple test macro and prove that the inventive method is reclaiming effectiveness on the RNA and the relative efficiency of estimating multiple condition and reagent.The mixture for preparing 100 microlitre test solns and 100 microlitre foetal calf serums (FBS).The luciferin ribozyme that adds 2 microlitres, 1 microgram/microlitre was with mixture vortex mixing 1 minute.2 milligrams of embodiment 1 particulate suspensions in mixture and the 100 microlitre test solns are merged vortex mixing 30 seconds.On the magnetic force platform, liquid is removed from particle, used 2 * 200 microlitre test solns and 2 * 200 microlitre 0.1%DEPC treating water to clean described particle successively.Extract RNA successively through the following steps, 50 microlitre 50mM NaOH and described particle are merged, vortex mixing 1 minute also removes elutriant.On second ingot dyeing gel, analyze the supernatant of initial association reaction, determine from solution, to remove and be bonded to the amount of described particulate RNA by fluorescent dye.On second ingot dyeing gel, analyze elutriant, determine amount and the quality of the RNA that extracts by described method by fluorescent dye.Use following solution to cause the quantitative combination of RNA, the wash-out overwhelming majority bonded RNA of institute.
Test soln
PH
Test soln
PH
Trisodium Citrate 0.3M 4.0 glycine 0.3M 3.0
Trisodium Citrate 0.3M 3.5 glycine 0.3M 2.5
Trisodium Citrate 0.3M 3.0 glycine 0.05M 2.5
Trisodium Citrate 0.05M 3.0 Sodium glutarate 0.3M 4.0
Potassium ethanoate 0.3M 4.0 Sodium glutarate 0.3M 3.2
Potassium ethanoate 0.05M 4.0 sodium succinate 0.3M 4.0
Potassium ethanoate 0.3M 3.7 sodium succinate 0.3M 3.8
Sodium-acetate 0.3M 4.0
Embodiment 6. extracts RNA from culture of Escherichia coli.Use simple test macro in the intestinal bacteria of from culture, cultivating, to reclaim the effectiveness of RNA to prove the inventive method, and the relative efficiency that is used to estimate multiple condition and reagent.
Precipitate the culture of Escherichia coli of 200 microlitre parts, and remove substratum.The particle of described precipitation and 200 microlitre test solns and 2 milligrams of embodiment 1 is merged vortex mixing 30 seconds.On the magnetic force platform, from particle, remove liquid, use 2 * 200 microlitre cleaning solutions and 2 * 200 microlitre 0.1%DEPC treating water to clean described particle then successively.Isolation of RNA through the following steps merges solution and the described particle of 50 microlitre 50mM NaOH and 20mM tris pH8.8, and vortex mixing 1 minute also removes elutriant.On second ingot dyeing gel, analyze the supernatant of initial association reaction, determine from solution, to have removed and be bonded to the amount of described particulate RNA by fluorescent dye.On second ingot dyeing gel, analyze elutriant, determine amount and the quality of the RNA that extracts by described method by fluorescent dye.Use following solution to cause being recovered to the global RNA of overwhelming majority amount.By contrast, in the 0.1%DEPC treating water, only produce the RNA of degraded in conjunction with precipitation and cleaning particle.
Test soln
Cleaning solution
Acetic acid 0.05M Trisodium Citrate 0.3M pH3
Acetic acid 0.1M Trisodium Citrate 0.3M pH3
Acetic acid 0.2M Trisodium Citrate 0.3M pH3
Trifluoracetic acid 0.05M trifluoracetic acid 0.05M
Trifluoracetic acid 0.1M trifluoracetic acid 0.1M
Trifluoracetic acid 0.2M trifluoracetic acid 0.2M
Embodiment 7. extracts other condition of RNA from culture of Escherichia coli.Use following test acidic solution to carry out colibacillary separation according to the method for embodiment 6, shown in band pattern in the running gel, also produce global RNA.
Test soln
Zinc acetate 0.05M+0.1M ammonium acetate pH4.0
Methyl San Ding Ji Phosphonium methyl sulfate 0.1M-1M
Sodium succinate 0.05M pH3
Embodiment 8. extracts RNA from Armored RNA.Armored
(Ambion Diagnostics, Austin are the ssRNA of protein parcel TX), and it shows as pseudovirion.A kind of Armored RNA of HIV-B sequence comprises fragment and the viral capsid proteins from the gag district, and it is used for testing the present invention and is used for from the method for complex sample isolation of RNA.
Exemplary steps from Armored RNA extraction RNA in blood plasma is as follows.100 microlitre EDTA anticoagulate plasma (Equitech-Bio, Inc., Kerrville, TX) (contain 50 with 5 microlitre ArmoredRNA, 000 copy) (for example 50mM KOAc pH4.0) merges the described mixture of of short duration vortex mixing for 105 microlitre solution of Zu Chenging and 100 microlitre test solns.After 1 minute, 2 milligrams of embodiment 1 particles among described mixture and the 100 microlitre 50mM KOAc (pH4.0) are merged, to gained slurries vortex mixing 30 seconds.On the magnetic force platform, separate described particle, use 2 * 200 microlitre 50mM KOAc (pH4.0) and 2 * 200 microlitre 0.1%DEPC treating water to clean then successively.By with 50 microlitre 50mM NaOH vortex mixed 1 minute and remove and remove solution and come eluted rna.Compare with contrast, 105 microlitres blood plasma/Armored RNA and 2 milligrams of particles merge with the 200 microlitre 0.1%DEPC treating water that replace test soln in contrast.
Use primer to the elutriant that contains RNA being carried out the RT-PCR amplification with amplification gag gene fragment.Use iScript
TMSingle stage method RT-PCR test kit and SYBR
Green (Bio-Rad) utilizes iCycler equipment (Bio-Rad) to carry out amplified reaction to increase and to detect.
Following test soln allows to reclaim the RNA that can increase, as C
TValue significantly is lower than shown in the water contrast.
Test soln
Potassium ethanoate 0.3M pH4.0
Potassium ethanoate 0.05M pH4.0
Acetic acid 0.05M
Acetic acid 0.2M
Trifluoracetic acid 0.05M
Pyridine hydrochloride 0.05M
Hydrochloric acid 0.025M
Hydrochloric acid 4-butyl-phosphonium 0.05M
Potassium ethanoate 0.05M+ acetic acid 0.05M
Zinc acetate 0.05M pH4.0
Potassium ethanoate 0.05M, pH4.0+ trifluoracetic acid 0.05M
50∶50 pH1.8
70∶30 pH2.1
80∶20 pH2.5
Zinc acetate 0.05M, pH4.0+ trifluoracetic acid 0.05M (80: 20)
Magnesium acetate 0.05M pH4.0
Ammonium acetate 0.05M
Tetrabutylammonium acetate ammonium 0.05M
Tetraethyl-ammonium acetate 0.05M
Zinc acetate 0.05M, pH4.0+ trifluoracetic acid (pH2.0,2.5.3.0,3.5)
Zinc acetate 0.05M+ glycine 0.05M pH3.3
Zinc acetate 0.05M+ Trisodium Citrate 0.05M pH3.3
Zinc acetate 0.05M+ Trisodium Citrate 0.05M pH4.2
Zinc chloride 0.05M+ Sodium glycocollate 0.05M pH2.75
Zinc chloride 0.05M+ Trisodium Citrate 0.05M pH2.5
Embodiment 9. extracts RNA from Armored RNA.In an alternative method, (Invitrogen, Carlsbad CA) are used for substituting blood plasma to foetal calf serum (FBS).Compare with contrast, 105 microlitre FBS/Armored RNA and 2 milligrams of particles merge with the 200 microlitre 0.1%DEPC treating water that replace test soln in contrast.As described in embodiment 4, analyze the elutriant that contains RNA by RT-PCR.Except hereinafter listed, most of test soln of embodiment 4 all allows to reclaim the RNA that can increase, as C
TValue significantly is lower than shown in the water contrast.
Test soln
Glycine 0.05M pH2.5
Glycine 0.3M pH2.5
Glycine 0.3M pH3.0
Trisodium Citrate 0.3M pH3.5
Trisodium Citrate 0.1M pH3.5
Trisodium Citrate 0.3M pH3.0
Embodiment 10.Use embodiment 1,2,3 and every kind of solid phase material of 4 and multiple acidic solution all successful implementation be used among the embodiment 8 separating and the method for the ArmoredRNA that is added to blood plasma of increasing.
Solid phase
Acidic solution
Embodiment 1 Cobaltous diacetate 0.05M, pH4.0
Embodiment 1 manganese acetate 0.05M, pH4.0
Embodiment 1 Cobaltous diacetate 0.05M+ Potassium ethanoate 0.05M, pH4.0
Embodiment 2 Potassium ethanoate 0.05M, pH4.0
Embodiment 2 zinc acetate 0.05M, pH4.0
Embodiment 3 zinc acetate 0.05M, pH4.0
Embodiment 4 zinc acetate 0.05M, pH4.0
Embodiment 11. extracts and analyzes HTV RNA from blood plasma.The inventive method is used for extracting RNA from the positive blood plasma of handling from the EDTA anticoagulation of HIV.Use the existence of HIV RNA in COBASAMPLICOR HIV-1 MONITOR TEST ver.1.5 (Roche Diagnostics) test sample.This detection is to be used for the automatization RT-PCR of quantitative HIV-1RNA to detect, the RNA reverse transcription is become the cDNA copy, 155 base-pair sequences in the pcr amplification gag gene high conservative region, with biotin labeled amplicon and the oligonucleotide probe hybridization that combines magnetic-particle, biotin label combines with avidin-horseradish peroxidase conjugate, uses TMB to carry out color developing detection.
The sample preparation methods that test kit provides substitutes with method therefor of the present invention as described below.
HIV RNA extracting method
1. in 100 microlitre 50mM KOAc (pH4), prepare 2 milligrams of embodiment 1 particulate slurries.
2. 100 microlitre 50mM KOAc (pH4) are added to 100 microlitre blood plasma.Point touched the described mixture of vortex, incubated at room 1 minute.
3. plasma solutions is joined the pearl slurries, vortex mixing 30 seconds.
4. remove supernatant, add 200 microlitre 50mM KOAc (pH4).Vortex 5 seconds.
5. repeating step 4.
6. remove supernatant, add the water that 200 microlitre 0.1%DEPC handle.Vortex 5 seconds.
7. repeating step 6.
8. remove all residual buffer liquid.Add 50 microlitre 50mM NaOH, vortex 1 minute.
9. elutriant is transferred to 1.5 milliliters of clean pipes.
10. 150 microlitres, 0.1% DEPC treating water is joined described particle and carry out wash-out once more, vortex 1 minute.
11. first and second elutriants are merged.
12. the elutriant that 50 microlitres are merged joins HIV-1 MONITOR Test, ver.1.5.
After carrying out COBAS AMPLICOR amplification, hybridization and immunity combination, the preparation serial dilutions detects then.When using such scheme, analyze and knownly contain 1.88 * 10
5The plasma sample of HIV particle/milliliter allows to detect the diluent of 1:729 in ELISA.
Embodiment 12. extracts RNA from people's whole blood.Use simple test macro to be used for reclaiming the effectiveness of RNA with proof the inventive method from people's whole blood.As the model of the fresh extraction blood that still contains global RNA, the human T lymphocyte of cultivation (Jurkat) is incorporated in the whole blood (CPD anticoagulation) to estimate the relative efficiency of multiple condition and reagent.
Precipitation collects 7 * 10
5Individual Jurkat cell is removed substratum.Described precipitation and 100 microlitre people whole bloods are merged.Described blood with contain 2 milligrams of embodiment 1 or 2 particulate, 100 microlitre test solns and merge vortex mixing 30 seconds.On the magnetic force platform, remove liquid, use 2 * 500 microlitre cleaning solutions and 2 * 500 microlitre 0.1%DEPC treating water to clean described particle successively from described particle.By following step isolation of RNA, with the solution merging of described particle and 50 microlitre 50mM NaOH and 20mM tris pH8.0, vortex mixing 1 minute removes described solution then.With among the 50 microlitre 100mM zinc acetate pH4 and described elutriant,, elutriant that is neutralized and the particulate 100 microlitre test solns that contain 2 milligrams of embodiment 1 or 2 are merged vortex mixing 30 seconds by the new pearl of following step recombine.On the magnetic force platform, remove liquid, use 2 * 500 microlitre cleaning solutions and 2 * 500 microlitre 0.1%DEPC treating water to clean described particle successively from described particle.By following step isolation of RNA, with the solution merging of described particle and 50 microlitre 50mM NaOH and 20mM tris pH8.0, vortex mixing 1 minute removes described solution then.
The elutriant that contains RNA is carried out RT-PCR and PCR, and it uses primer to RNA and DNA with amplification GAPDH and 18S gene.Use iScript
TMSingle stage method RT-PCR test kit and
Green (Bio-Rad) utilizes iCycler equipment (Bio-Rad) to carry out amplified reaction to increase and to detect.Obtained the positive amplification (C of RT-PCR
TThe C of＜PCR
T).
Solid phase
Acidic solution
Embodiment 1 zinc acetate 0.05M, pH4.0
Embodiment 13,3-dimethylated pentanedioic acid 0.05M, pH3.2
Embodiment 2 zinc acetate 0.05M, pH4.0
Claims (35)
1. extract the method for Yeast Nucleic Acid from the biological sample that contains at least one cell or virus, it comprises:
A) described sample is contacted to form mixture with acidic solution;
B) with described mixture and selected solid phase bond material merging with the ability that under at first not carrying out any pre-cracking situation, from biological sample, directly discharges Yeast Nucleic Acid, wherein do not use chaotropic agent or stain remover to realize cracking, described thus solid phase bond material causes that the cracking of cell and virus is to discharge Yeast Nucleic Acid; With
C) Yeast Nucleic Acid is attached on the described solid phase.
2. the method for claim 1, it also comprises:
D) described sample and the described solid phase that combines Yeast Nucleic Acid thereon are separated;
E) randomly clean described solid phase with at least a cleaning solution; With
F) by with described solid phase material with discharge enter solution in conjunction with RNA reagent contact wash-out institute bonded Yeast Nucleic Acid on the described solid phase.
3. the process of claim 1 wherein the step that forms described sample and the mixture of described acidic solution with the step of described mixture and the merging of described solid phase is carried out simultaneously.
4. the process of claim 1 wherein and carry out the step that described mixture and described solid phase merge again by the mixture that forms described sample and described acidic solution earlier.
5. the process of claim 1 wherein that described solid phase is selected from particle, microparticle, fiber, pearl, film, test tube and micropore.
6. the process of claim 1 wherein that described solid phase comprises matrix part and nucleic acid binding moiety.
7. the method for claim 6, wherein said matrix partly is selected from silicon-dioxide, glass, insoluble synthetic polymer, insoluble polysaccharide, metal, metal oxide and metallic sulfide.
8. the method for claim 6, wherein said matrix partly is selected from bag by the magnetic response material of silicon-dioxide, glass, synthetic polymer or insoluble polysaccharide.
9. the process of claim 1 wherein that described solid phase comprises diameter less than 10 microns microparticle.
10. the method for claim 9, wherein said microparticle is a magnetic responsiveness.
11. the method for claim 9 is wherein used the particulate mixture more than a kind of size.
12. the method for claim 11, wherein the particle of at least a size has nucleic acid binding moiety, and the particle of at least a other size does not have nucleic acid binding moiety.
13. the method for claim 6, wherein said solid phase material also comprises covalently bound nucleic acid binding moiety, and this nucleic acid binding moiety allows to catch and bind rna.
14. the process of claim 1 wherein that described solid phase material also comprises the nucleic acid binding moiety of non-covalent connection, this nucleic acid binding moiety allows to catch and bind rna.
15. the method for claim 1, wherein said solid phase material also comprises the material based on silicon-dioxide, its surface functional group institute that is introduced by covalency is functionalized, described functional group is used to destroy cell and attracts nucleic acid, and described functional group is selected from hydroxyl, silanol group, carboxyl, amino, ammonium, quaternary ammonium He quaternary alkylphosphonium salt and uncle's sulfosalt.
16. the method for claim 1, wherein said solid phase material also comprises the polymer materials of the surface functional group with covalency introducing, described functional group is used to destroy cell and attracts nucleic acid, and described functional group is selected from hydroxyl, silanol group, carboxyl, amino, ammonium, quaternary ammonium He quaternary alkylphosphonium salt and uncle's sulfosalt.
17. the method for claim 13, wherein said nucleic acid binding moiety comprise a plurality of nucleic acid conjugated groups, it is selected from carboxyl, NH
2, alkylamine and dialkyl amino, season or uncle's group or more than a kind of mixture of these groups.
18. the method for claim 17, wherein said nucleic acid binding moiety comprise a plurality of nucleic acid conjugated groups, it is selected from trialkyl quaternary ammonium, San Wan Ji quaternary phosphine, triaryl quaternary phosphine, mixed alkyl aryl quaternary phosphine group and uncle's sulfonium base.
19. the method for claim 13, wherein said nucleic acid conjugated group is selected from trialkyl quaternary ammonium and San Wan Ji quaternary phosphine group, wherein each alkyl all has at least 4 carbon atoms, and wherein said nucleic acid conjugated group causes that cell and virolysis are to discharge Yeast Nucleic Acid.
20. comprising connecting key by the alternative cutting, the method for claim 6, wherein said solid phase bond material be attached to nucleic acid conjugated group on the matrix.
21. the process of claim 1 wherein that described acidic solution comprises the aqueous solution of pH in the 1-5 scope.
22. the method for claim 21, wherein said acidic solution comprise the aqueous solution of pH in the 2-4 scope.
23. the method for claim 21, wherein said acidic solution comprises organic or inorganic aqueous acid, described organic or inorganic acid is selected from: pyridinium salt, mineral acid, monocarboxylic acid, dicarboxylic acid, tricarboxylic acid and amino acid, and their basic metal, alkaline-earth metal, transition metal, NH
4 +, quaternary ammonium is with quaternary alkylphosphonium salt.
24. the method for claim 2, the reagent that wherein is used for discharging from described solid phase institute's bind rna comprises that alkali concn is the basic solution of 1mM to 1M.
25. the process of claim 1 wherein that described solid phase material comprises the magnetic-particle with three fourth base Phosphonium nucleic acid conjugated groups, described group is connected on the magnetic-particle matrix by the aryl thioesters connecting key that can cut.
26. the method for claim 25, wherein said solid phase material has following formula
Wherein
Representative is by covalently bound joint group functionalization's the magnetic-particle based on silicon-dioxide.
27. the process of claim 1 wherein that described biological sample is selected from bacterial cultures, the sedimentation cell from bacterial cultures, blood, blood plasma, serum, urine, phlegm, seminal fluid, CSF, vegetable cell, zooblast and tissue homogenate thing.
28. from be selected from bacterial cultures, the biological sample of sedimentation cell, blood, blood plasma, serum, urine, phlegm, seminal fluid, CSF, vegetable cell, zooblast and tissue homogenate thing, extract the method for Yeast Nucleic Acid from bacterial cultures, described sample contains at least one cell or virus, and described method comprises:
A) described sample is contacted with acidic solution in the pH1-5 scope to form mixture, wherein said acidic solution comprises organic or inorganic aqueous acid, described organic or inorganic acid is selected from pyridinium salt, mineral acid, monocarboxylic acid, dicarboxylic acid, tricarboxylic acid and amino acid, and their basic metal, alkaline-earth metal, zinc, NH
4 +, quaternary ammonium is with quaternary alkylphosphonium salt;
B) described mixture and the solid phase bond material that comprises matrix part and nucleic acid binding moiety are merged, wherein select described solid phase bond material, make it to have the ability that under at first not carrying out any pre-cracking situation, from biological sample, directly discharges Yeast Nucleic Acid, wherein do not use chaotropic agent or stain remover to realize cracking, described thus nucleic acid conjugated group causes that the cracking of cell and virus is to discharge Yeast Nucleic Acid; With
C) Yeast Nucleic Acid is attached on the described solid phase.
29. the method for claim 28, wherein said solid phase material comprise the magnetic-particle with three fourth base Phosphonium nucleic acid conjugated groups, described group is connected on the magnetic-particle matrix by the aryl thioesters connecting key that can cut.
30. the method for claim 29, wherein said solid phase material has following formula
Wherein
Representative is by covalently bound joint group functionalization's the magnetic-particle based on silicon-dioxide.
31. the method for claim 30, it also comprises:
D) described sample and the described solid phase that combines Yeast Nucleic Acid thereon are separated;
E) randomly clean described solid phase with at least a cleaning solution; With
F) by with described solid phase material with comprise basic solution with discharge enter solution in conjunction with RNA reagent contact wash-out institute bonded Yeast Nucleic Acid on the described solid phase, described basic solution has the alkali concn of 1mM to 1M.
32. the method for isolated nuclei ribosomal ribonucleic acid from be selected from bacterial cultures, the biological sample of sedimentation cell, blood, blood plasma, serum, urine, phlegm, seminal fluid, CSF, vegetable cell, zooblast and tissue homogenate thing from bacterial cultures, described sample contains at least one cell or virus, and described method comprises:
A) described sample is contacted with acidic solution in the pH1-5 scope to form mixture, wherein said acidic solution comprises organic or inorganic aqueous acid, described organic or inorganic acid is selected from pyridinium salt, mineral acid, monocarboxylic acid, dicarboxylic acid, tricarboxylic acid and amino acid, and their basic metal, alkaline-earth metal, transition metal, NH
4 +, quaternary ammonium is with quaternary alkylphosphonium salt;
B) described mixture and the solid phase bond material that comprises magnetic-particle are merged, described magnetic-particle has by cutting aryl thioesters connecting key and is connected to three fourth base Phosphonium nucleic acid conjugated groups on the magnetic-particle matrix, wherein select described solid phase bond material, make it to have the ability that under at first not carrying out any pre-cracking situation, from biological sample, directly discharges Yeast Nucleic Acid, wherein do not use chaotropic agent or stain remover to realize cracking, described thus nucleic acid conjugated group causes that the cracking of cell and virus is to discharge Yeast Nucleic Acid; With
C) Yeast Nucleic Acid is attached on the described solid phase;
D) described sample and the described solid phase that combines Yeast Nucleic Acid thereon are separated;
E) randomly clean described solid phase with at least a cleaning solution; With
F) cut the connecting key that this alternative is cut with cutting reagent, discharge Yeast Nucleic Acid from described solid phase bond material thus.
33. the method for claim 32, the wherein said connecting key that cuts is selected from: the group of hydrolyzable cutting, disulphide group, peroxide bridge, the group that can digestedly cut, can cut 1,2-dioxy tetramethylene part, the wherein said pair of two keys of electron rich C-C, the ketenes dithioacetals compound that key has connected at least one O, S or N atom, but and the light cutting joint group that is selected from nitro substituted aroma ether and ester, wherein said enzyme is selected from esterase, lytic enzyme, proteolytic enzyme, peptase and Glycosylase.
34. the method for claim 33, the group of wherein said hydrolyzable cutting is selected from: carboxylicesters, carboxylic acid anhydride, thioesters, carbonic ether, thiocarbonic ester, carbamate, imide, sulphonamide, sulfimide and sulphonate.
35. the method for claim 34, wherein by cutting the connecting key of described hydrolyzable cutting with the reagent react that comprises basic solution, described basic solution has the alkali concn of 1mM to 1M.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US77151006P | 2006-02-08 | 2006-02-08 | |
| US60/771,510 | 2006-02-08 | ||
| US11/703,459 | 2007-02-07 |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104334726A (en) * | 2012-05-25 | 2015-02-04 | 艾皮斯托姆有限公司 | Nucleic acid extraction |
| CN105008534A (en) * | 2012-09-19 | 2015-10-28 | 贝克曼考尔特公司 | Use of divalent ions, proteases, detergents, and low ph in the extraction of nucleic acids |
| CN106520522A (en) * | 2016-12-06 | 2017-03-22 | 厦门华厦学院 | DNA extraction device and method |
| WO2024017155A1 (en) * | 2022-07-22 | 2024-01-25 | 友康生物科技(北京)股份有限公司 | Lysis composition |
-
2007
- 2007-02-08 CN CNA2007800085506A patent/CN101400689A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104334726A (en) * | 2012-05-25 | 2015-02-04 | 艾皮斯托姆有限公司 | Nucleic acid extraction |
| CN105008534A (en) * | 2012-09-19 | 2015-10-28 | 贝克曼考尔特公司 | Use of divalent ions, proteases, detergents, and low ph in the extraction of nucleic acids |
| CN106520522A (en) * | 2016-12-06 | 2017-03-22 | 厦门华厦学院 | DNA extraction device and method |
| WO2024017155A1 (en) * | 2022-07-22 | 2024-01-25 | 友康生物科技(北京)股份有限公司 | Lysis composition |
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