EP2176410A1 - Procédé de séparation de biomolécules ne contenant pas de protéines, en particulier d'acides nucléiques, à partir d'échantillons contenant des protéines - Google Patents

Procédé de séparation de biomolécules ne contenant pas de protéines, en particulier d'acides nucléiques, à partir d'échantillons contenant des protéines

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Publication number
EP2176410A1
EP2176410A1 EP08786435A EP08786435A EP2176410A1 EP 2176410 A1 EP2176410 A1 EP 2176410A1 EP 08786435 A EP08786435 A EP 08786435A EP 08786435 A EP08786435 A EP 08786435A EP 2176410 A1 EP2176410 A1 EP 2176410A1
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EP
European Patent Office
Prior art keywords
protein
samples
biomolecules
nucleic acids
solid phase
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP08786435A
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German (de)
English (en)
Inventor
Vera HOLLÄNDER
Stefanie SCHRÖER
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Qiagen GmbH
Original Assignee
Qiagen GmbH
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Filing date
Publication date
Application filed by Qiagen GmbH filed Critical Qiagen GmbH
Publication of EP2176410A1 publication Critical patent/EP2176410A1/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers

Definitions

  • the present invention relates to a method for separating non-protein-containing biomolecules, in particular nucleic acids from protein-containing samples, in particular from biological samples such as blood, stool, saliva, sputum or plasma.
  • the prior art discloses a large number of methods for separating non-protein-containing biomolecules, in particular nucleic acids, from biological samples.
  • the proteins contained in the respective samples are usually degraded in the isolation of DNA by lysis with a proteinase or else a mixture of proteinases.
  • the DNA is usually protected from enzymatic degradation during protein degradation by binding the cofactors required for DNase activity by chelators in the lysis buffer.
  • the isolation of RNA presents a particular challenge because RNases are ubiquitous, abundant, active in a wide temperature range, and do not require cofactors. Specific inactivation of all RNases during lysis is therefore not possible. Nevertheless, in order to rapidly inactivate endogenous and exogenous RNases, lysis of the sample with very strongly denaturing lysing reagents, such as e.g. As phenol and / or chaotropic substances.
  • the purification can z. B. for the achievement of high purity, on the binding of non-proteinous biomolecules, in particular nucleic acids, to a solid phase, for. B. silica membranes, done. Such methods for z. B. Nucleic acid purification are known in the art.
  • sample materials that may be of interest for isolation of non-protein biomolecules, particularly nucleic acid isolation, are significantly different in structure and composition.
  • sample materials with an unfavorable nucleic acid / protein ratio very much protein or very few non-protein-containing biomolecules, in particular nucleic acids
  • samples which contain substances which may interfere in later applications place special demands on the method for purifying nucleic acids
  • Such "difficult" sample materials are z.
  • Another particular difficulty arises when it is intended to isolate both DNA and a separate fraction of RNA from a sample.
  • protocols known to those skilled in the art for "lighter" sample materials are known.
  • RNA is first bound to a solid phase.
  • the flow which contains, inter alia, the RNA, is adjusted by adding further reagents so that subsequently the RNA can be bound to a solid phase.
  • Difficult sample materials such as saliva, sputum, blood or stool, which imperatively require treatment with proteinases, so far can not be used in such a method.
  • the proteinase step necessary for complete lysis can not be carried out under the chaotrope concentration necessary for the protection of the RNA.
  • dilution to proteinase-compatible chaotrope concentrations leads to an increased risk of RNA degradation, since RNases are also active under these conditions.
  • a dilute lysate does not permit selective binding of the DNA to a solid phase and thus no separation of the various nucleic acid species. Without a proteinase treatment, however, the yield and quality of the isolated nucleic acid fractions are usually insufficient.
  • the object is achieved by a method according to claim 1 of the present invention. Accordingly, a method is proposed for the isolation of non-protein-containing biomolecules, in particular nucleic acids from protein-containing biological samples, comprising the steps:
  • Protein degradation, the non-protein-containing biomolecules, in particular nucleic acid ⁇ ) are bound to the solid phase.
  • biological samples includes, in particular, body fluids such as blood, semen, cerebrospinal fluid, saliva, sputum or urine, fluids which are obtained when reprocessing blood, such as serum or plasma, Leucocyte fractions or "buffy coat", leech saliva, feces, smears, punctates, dandruff, hair, cutaneous fragments, forensic samples, food or environmental samples containing free or bound biomolecules, in particular free or bound non-proteinaceous biomolecules , in particular nucleic acids, whole organisms, preferably whole non-living organisms, tissues of multicellulars, preferably of insects and mammals, in particular of humans, for example in the form of tissue sections (eg FFPE samples), tissue fragments, or organs, isolated cells, for example in Form of adherent or suspended cell cultures, organelle len, for example, chloroplasts or mitochondria, vesicles, cell nuclei or chromosomes, plants, plant parts, plant
  • protein-containing is understood to mean, in particular, that the sample contains peptides and peptide fragments, but also entire proteins or protein complexes, which may also be substituted, in particular glycosylated, phosphorylated or acetylated ,
  • non-proteinaceous biomolecules is understood to mean, but is not limited to, all biomolecules (other than proteins), such as, for example, lipids, carbohydrates, metabolites, metabolites and, in particular, all types of nucleic acids.
  • biomolecules are understood as meaning, but not limited to, all naturally occurring or artificially introduced molecules in biological samples.
  • nucleic acid in the context of the present invention particularly, but not limited to natural, preferably isolated linear, branched or circular nucleic acids such as RNA, in particular mRNA, siRNA, miRNA, snRNA, tRNA, hnRNA or ribozymes, DNA and the like, synthetic or modified nucleic acids, for example oligonucleotides, in particular primers, probes or standards used for the PCR, nucleic acids labeled with digoxigenin, biotin or fluorescent dyes or so-called PNAs ("peptide nucleic acids").
  • immobilization in the sense of the present invention is understood to mean, in particular but not limited to, a reversible immobilization to a suitable solid phase.
  • Samples like blood, plasma, sputum, stool or saliva.
  • step a) Preference is given in step a) to the non-proteinaceous biomolecules contained in the biological sample, in particular nucleic acids which are essentially completely immobilized on the solid phase.
  • the method according to the invention can also be used is when only a part of the non-protein-containing biomolecules, in particular nucleic acids, is immobilized.
  • step b) the non-protein-containing biomolecules bound to the solid phase, in particular nucleic acid (s), are completely immobilized on the solid phase; however, the present invention is not limited thereto. It has been found that in many applications of the present invention, the method according to the invention can also be used if, during step b), part of the non-protein-containing biomolecules, in particular nucleic acid (s), dissolves or does not dissolve from the solid phase immobilized present.
  • the solid phase is a phase with a high nucleic acid affinity, preferably selected from the group of silica membranes, silica beads, magnetic particles, hydrophilic membranes, hydrophobic membranes, ion exchange matrices, or mixtures thereof.
  • a high nucleic acid affinity preferably selected from the group of silica membranes, silica beads, magnetic particles, hydrophilic membranes, hydrophobic membranes, ion exchange matrices, or mixtures thereof.
  • hybrid-mediated binding of nucleic acids to solid phases such.
  • the ratio of protein to non-protein-containing biomolecules, in particular nucleic acids, in g / g before carrying out the method is> 10: 1, according to a further embodiment> 100: 1, according to a further embodiment> 1000: 1, and according to another embodiment> 10000: l.
  • step a) is carried out with the addition of a chaotropic buffer.
  • a chaotropic buffer This has the advantage that any degradation by RNases or DNases can be largely prevented in advance and the binding of nucleic acids is mediated especially on Silikaoberflä- chen.
  • other suitable for the respective sample material and the selected solid phase lysis reagents are possible, such as. B. the known in the art alkaline lysis of bacteria with subsequent binding z. B. on Anionenhenhenerober horr.
  • the buffer to be used in step a) is preferably determined on the basis of the respective sample material, the biomolecule to be immobilized, and the selected solid phase.
  • the method additionally comprises at least one step a1), which is carried out before step b):
  • the method additionally comprises at least one step c), which is carried out after step b):
  • step b) This has turned out to be favorable for many applications since in this way the proteinases present in step b) can be largely inactivated. Carrying out the enzyme into the eluate, which could prevent or disturb further enzyme- or protein-based applications (eg PCR) there, can also be prevented or largely avoided in many applications.
  • enzyme- or protein-based applications eg PCR
  • Step b) can be carried out with various protein-degrading enzymes (proteases) or a mixture of several proteases.
  • proteases proteinase K and the QIAGEN protease are preferred.
  • step b) can take place during the incubation at a desired temperature.
  • the temperature is preferably adapted to the respective temperature optimum of the selected enzyme or of the enzyme mixture.
  • the incubation is preferably carried out at a temperature of> 0 ° C to ⁇ 100 ° C, preferably between> 4 ° C and ⁇ 80 ° C, more preferably between> 18 ° C and ⁇ 70 ° C, particularly preferably between> 30 ° C. and ⁇ 65 ° C.
  • proteinase K and / or QIAGEN protease are used as protein-degrading enzyme
  • a temperature range of> 40- ⁇ 60 ° C, in particular> 54 ° C to ⁇ 58 0 C is preferred.
  • the protease is preferably applied dissolved in liquid to the solid phase.
  • liquid it is preferred to use solutions which produce optimal conditions for enzymatic degradation (eg, pH, salt compositions and concentrations) of cofactors or other conditions.
  • step b) is carried out in water and / or in unbuffered solutions.
  • the solution of the protease in water surprisingly enough in many applications to allow the protein degradation.
  • the solvent water and the relatively small volume thus allow in many applications in the method according to the invention the access of the proteases to the immobilized protein-containing sample and the expression of the enzymatic activity.
  • step b) is carried out with at least one protease having an activity of> 1 mAU / mg.
  • mAU is meant the activity in which the enzyme liberates folin-positive amino acids and peptides corresponding to 1 ⁇ mol of tyrosine per minute.
  • step b) is carried out with a (protease) activity of at least> 10 mAU / mg, more preferably> 20 mAU / mg and particularly preferably with a (protease) activity of at least> 30 mAU / mg
  • step b) is carried out for a period of> ⁇ ⁇ ⁇ seconds to ⁇ T seconds, wherein
  • X denotes the numerical value of the (protease) activity of the enzyme used (as described above).
  • X means the average (protease) activity of these enzymes. This has proved useful in many fields of application of the present invention, since on the one hand a protein digestion which is as complete as possible can be ensured, but on the other hand the biomolecules to be isolated are not or only slightly degraded or damaged.
  • step b) is for a period of> ⁇ ⁇ ⁇ seconds to ⁇ T ⁇
  • Step b) is preferred for a period of> ⁇ seconds to ⁇ -y
  • the minimum total volume during the performance of step b) is such that the solid phase, including all the biomolecules immobilized thereon, is completely wetted.
  • the maximum total volume during the performance of step b) is preferably such that the biomolecules to be isolated are not eluted from the solid phase and washed off, z. B. when dripping through a membrane.
  • FIG. 2 shows three absorption spectra of DNA after isolation in three comparative experiments according to Example 1
  • a human saliva sample is collected. Before the collection, the subject had neither eaten nor drunk for Ih to avoid contamination of the saliva sample with food leftovers. During the collection process, the sample is stored on ice. The sample is then subdivided into 200 ⁇ l aliquots and each aliquot is mixed with ImI of the saliva stabilization solution RNAprotect Saliva from QIAGEN and stored for 2 days at 2-8 ° C in the refrigerator. For the subsequent isolation of DNA and RNA from the stabilized saliva samples, the components of the QIAamp Mini Kit and the RNeasy Micro Kit of the manufacturer QIAGEN are used.
  • the samples are centrifuged off according to the manufacturer for 10 min at lOOOOrpm, the supernatant removed by Abpippet Schl and the pellet dissolved by snapping the vessel slightly.
  • the pellet is then dissolved in 350 ⁇ l of the GTC-containing lysis buffer RLT by vortexing.
  • the lysate is applied to the silica membrane, in the QIAamp mini-column, and passed through the membrane by centrifugation for 1 min. At 1000 rpm.
  • RNA was analyzed by means of quantitative real-time RT-PCR using the QuantiTect OneStep RT-PCR kit and primer and sample for the detection of the actin ß transcript. Each sample is analyzed in duplicate. The mean of the duplicate determinations of the three samples is shown in Table 1.
  • the QIAamp column is used to isolate the DNA.
  • the membrane is washed by passing 350 ⁇ l of the guanidine hydrochloride-containing wash buffer AW1.
  • Each 25 ⁇ l of the proteinase K solution contained in the QIAamp Mini Kit is made up to a total volume of 80 ⁇ l with water and applied to the membrane.
  • the washing step is repeated with AWI and then washed with the alcohol-containing wash buffer AW2.
  • the membrane is dried by centrifuging at 14000 rpm for 2 minutes.
  • the DNA is eluted by application of 100 ⁇ l of water and subsequent centrifugation, the elution being repeated with another 100 ⁇ l.
  • the DNA isolation without protease shows very high absorptions in the range less than 250 nm, which are due to impurities.
  • the isolated with the aid of the method according to the invention DNA (Fig.l, samples la-c) is of significantly better quality, since the range of less than 240nm only has a much lower absorption and thus a lower impurity.
  • a curve with a maximum at 260 nm can be recognized, which is due to the absorption by the isolated DNA. (maximum absorption of DNA is 260nm).
  • a saliva sample is collected as described in Example 1, aliquoted, stabilized, refrigerated for 3 days at 2-8 ° C and used for RNA and DNA isolation.
  • the DNA was eluted in 2x 40 ⁇ l of water.
  • the samples 4a-c were used for DNA isolation with proteinase K digestion on the membrane according to the invention, the samples 5a-c were subjected to the same procedure without proteinase K insert.
  • the DNA is obtained from saliva samples by a method involving proteinase K digestion in solution.
  • saliva is collected, stabilized, stored and centrifuged (samples 6a-c). After removal of the supernatant is used for washing of samples of ImI PBS are added to the pellets and mixed by vortexing. The samples are pelleted again by centrifugation at 100rpm for 2 minutes and the supernatant discarded. The pellet is then dissolved in 180 ⁇ l of PBS, mixed with 25 ⁇ l of the proteinase K solution from the QIAamp Mini Kit (QIAGEN) and 200 ⁇ l of the buffer AL and mixed by vortexing. The samples are incubated for 10 min at 56 ° C. Each sample is then mixed with 200 ⁇ l of 100% ethanol and applied to the silica membrane in the QIAamp Mini column. Further DNA isolation is carried out as described for samples 2a-c in Example 1.
  • RNA was analyzed in all cases by quantitative real-time RT-PCR using the QuantiTect OneStep RT-PCR kit and primer and sample to detect the interleukin-8 transcript, all samples showing comparable results, so that comparable NA Content of the samples is to be assumed (data not shown).
  • the DNA yield was determined by measuring the absorbance at 260nm.
  • the mean values of the yields of samples 4, 5 and 6 (a to c) are shown in Table 2.
  • the DNA isolated in this way was used in duplicate for the detection of the gene coding for the 18SrRNA.
  • the samples 1 to 3 were used in each case 2 .mu.l
  • the eluates of samples 4 to 6 were diluted 1:10 with water and used in each case 2 .mu.l.
  • the amplification was carried out in a total volume of 25 .mu.l with a suitable master mix for real-time PCR, such as the QuantiTect SYBRGreen PCR kit from QIAGEN, according to the manufacturer.
  • the amplification takes place in a suitable real-time amplification device, such. B. the 7700 ABI. From the ct values determined, the mean values are determined using the duplicate determinations of each of three replicates a to c and the standard deviation. The result is shown in Table 3.
  • Table 3 Table 3
  • RNAproduct Saliva from QIAGEN and refrigerate for one day at 2-8 ° C.
  • the components of the RNeasy Micro Kit from the manufacturer QIAGEN are used.
  • the samples are centrifuged off according to the manufacturer for 10 min at lOOOOrpm, the supernatant removed by Abpippet Schl and the pellet dissolved by snapping the vessel slightly.
  • the pellet is then dissolved in 350 ⁇ l of the GTC-containing lysis buffer RLT by vortexing.
  • the lysate is mixed with 350 ⁇ l of 70% ethanol, applied to the silica membrane in the RNeasy Micro column, and passed through the membrane by centrifugation for 1 min at 100 ° C.
  • RNA isolation from each saliva sample, an aliquot (A) for RNA isolation according to the manufacturer's instructions is used.
  • the respective other aliquot (B) is used for RNA isolation by means of the method according to the invention.
  • the membrane is washed by the passage of 350 .mu.l of the guanidine hydrochloride wash buffer RWl.
  • Each 20 ⁇ l proteinase K solution is made up to a total volume of 80 ⁇ l with water and applied to the membrane.
  • a washing step is performed with a mixture of equal portions of Lysis Buffer RLT and 70% ethanol. The wash is then repeated with RWI and then washed with the alcohol-containing wash buffer RPE.
  • RNA is eluted by application of 14 ⁇ l of water and subsequent centrifugation.
  • the RNA thus isolated was analyzed in all cases by means of quantitative real-time PCR using the QuantiTect OneStep RT-PCR kit and primer and sample for the detection of the IL8 transcript in triplicate. In each case 2.5 ⁇ l of the eluates were used in a total volume of 25 ⁇ l. The obtained averages and standard deviations of the triplicate determinations of the samples are shown in Table 4.
  • FFPE samples formalin-fixed and paraffin-embedded tissue samples from rat liver are used. From these samples, cuts of about 20 ⁇ M thickness are made using a microtome and 1 slice per sample is used.
  • RNeasy FFPE Kit components of the RNeasy FFPE Kit, the Allprep DNA / RNA Kit and the QIAamp Mini Kit from the manufacturer QIAGEN are used.
  • the tissues are deparaffinized according to the manufacturer (RNeasy FFPE kit manual) by washing with 1 ml of xylene and, after centrifuging the sample and removing the supernatant, with ImI 100% ethanol. After re-centrifuging the sample and removing the supernatant, the samples are dried for 10 min at 37 ° C. Connecting the samples are added K from the RNeasy FFPE Kit and incubated for 3h min at 56 ° C and 15 min at 80 0 C with 150 ⁇ l of buffer PKD and lO ⁇ l proteinase.
  • the resulting mixture is applied to the silica membrane in the Allprep DNA column (from the Allprep DNA / RNA Mini Kit) and passed through the membrane by centrifugation for 1 min at 14000 rpm.
  • a proteinase K treatment is carried out on the silica membrane by applying 20 ⁇ l proteinase K and 60 ⁇ l water to the membrane and incubating the sample for 10 min at 56 ° C., while the comparison sample ( Sample 2) is not treated with proteinase K.
  • the membranes of both samples are then carried out by carrying out 500 .mu.l of the guanidine hydrochloride-containing wash buffer AWl and then 500 .mu.l of the alcoholic washing buffer AW2.
  • the membrane is dried by centrifuging at 14000 rpm for 2 minutes.
  • the DNA is eluted by applying 30 ⁇ l of water after incubation for one minute by centrifugation.
  • the DNA thus obtained is first checked by means of agarose gel electrophoresis. For this purpose, 5 ⁇ l each of the resulting eluate are diluted with 15 ⁇ l of water and separated on a 0.5% agarose-TAE gel for about 4h at 60V.
  • the ethidium bromide stained gel in both cases shows fragmented DNA, recognizable by a light "smear" spread over a certain size range, the control sample (2) containing substantially smaller fragments than sample 1 after proteinase treatment on the membrane
  • the application of the method according to the invention thus leads to the isolation of larger DNA fragments.
  • the DNA which can be isolated from FFPE samples is always fragmented, since the DNA is already degraded during fixation, embedding and storage.
  • the DNA is covalently cross-linked by formalin fixation with other nucleic acids and especially with proteins, which makes both the isolation of the DNA and the analysis of the DNA by means of amplification techniques extremely difficult.
  • the eluates were used for further analysis by means of quantitative real-time PCR.
  • the isolated DNA was used in duplicate for the detection of a 465 bp amplicon of the prion protein-encoding gene.
  • the eluates were each diluted 1:20 with water and 5 .mu.l of these dilutions used in the real-time PCR.
  • the amplification was carried out in a total volume of 25 .mu.l with a suitable master mix for real-time PCR, such as the QuantiTect SYBRGreen PCR kit from QIAGEN, according to the manufacturer.
  • the amplification was carried out in a suitable real-time amplification device, such as. B. the 7700 ABI. From the determined ct values, the mean values were determined by means of the duplicate determinations of the duplicates and the standard deviation. The result is shown in Table 5.
  • Comparative sample, as well as the treated sample according to the invention, have experienced at the beginning of the DNA isolation process, a three-hour treatment with proteinase K in solution. Nevertheless, the comparatively very short treatment time of 15 minutes causes an improvement in the DNA fragment size and in particular an improvement in the amplifiability of the DNA.

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Abstract

L'invention concerne un procédé permettant d'isoler des biomolécules ne contenant pas de protéines, en particulier d'acides nucléiques, caractérisé en ce qu'on effectue sur une phase solide, une dégradation des protéines.
EP08786435A 2007-07-27 2008-07-25 Procédé de séparation de biomolécules ne contenant pas de protéines, en particulier d'acides nucléiques, à partir d'échantillons contenant des protéines Withdrawn EP2176410A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE102007035250A DE102007035250A1 (de) 2007-07-27 2007-07-27 Verfahren zum Abtrennen von nicht-proteinhaltigen Biomolekülen, insbesondere Nukleinsäuren aus proteinhaltigen Proben
PCT/EP2008/059774 WO2009016110A1 (fr) 2007-07-27 2008-07-25 Procédé de séparation de biomolécules ne contenant pas de protéines, en particulier d'acides nucléiques, à partir d'échantillons contenant des protéines

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EP2176410A1 true EP2176410A1 (fr) 2010-04-21

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US (1) US20100291658A1 (fr)
EP (1) EP2176410A1 (fr)
JP (1) JP2010534467A (fr)
DE (1) DE102007035250A1 (fr)
WO (1) WO2009016110A1 (fr)

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US10329553B2 (en) 2012-09-03 2019-06-25 Qiagen Gmbh Method for isolating RNA including small RNA with high yield
JP2023503147A (ja) * 2019-11-25 2023-01-26 エーエムペー・バイオテック・ゲーエムベーハー 固体表面に結合させた親和性リガンドを使用する核酸の分離及び単離

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JP2010534467A (ja) 2010-11-11
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