[go: up one dir, main page]

WO2017203225A1 - Isolement d'acides nucléiques - Google Patents

Isolement d'acides nucléiques Download PDF

Info

Publication number
WO2017203225A1
WO2017203225A1 PCT/GB2017/051428 GB2017051428W WO2017203225A1 WO 2017203225 A1 WO2017203225 A1 WO 2017203225A1 GB 2017051428 W GB2017051428 W GB 2017051428W WO 2017203225 A1 WO2017203225 A1 WO 2017203225A1
Authority
WO
WIPO (PCT)
Prior art keywords
alkanol
volume
solution
miscible
lithium
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.)
Ceased
Application number
PCT/GB2017/051428
Other languages
English (en)
Inventor
Stephen John Minter
Georgios Patsos
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.)
Revolugen Ltd
Revolugen Ltd
Original Assignee
Revolugen Ltd
Revolugen Ltd
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Revolugen Ltd, Revolugen Ltd filed Critical Revolugen Ltd
Publication of WO2017203225A1 publication Critical patent/WO2017203225A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • 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
    • C12N15/1017—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by filtration, e.g. using filters, frits, membranes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
    • C12M47/06—Hydrolysis; Cell lysis; Extraction of intracellular or cell wall material
    • 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
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/06—Lysis of microorganisms

Definitions

  • the present invention relates to the isolation of RNA from biological material (e.g. cells) by use of a lysis procedure and processing of the lysate to isolate RNA. More particularly, the invention involves the immobilisation of nucleic acids comprising RNA from the lysed biological material on a solid support and purification of the RNA on the support prior to elution of the RNA from the support for collection.
  • a particular aim of the present invention is to provide a process which is relatively quick and straightforward to practise and which produces RNA of high purity in good yield.
  • the invention also relates to compositions for use in the lysis procedure.
  • RNA isolated from biological material (e.g. cells) is of importance in a number of fields, e.g. research and clinical diagnosis.
  • biological material e.g. cells
  • clinical cell-containing samples from a patient may be subjected to a procedure (including the step of cell lysis) to isolate RNA which is then analysed by procedures well known in the art.
  • Such analysis may be for the purpose of identifying RNA from a particular bacterium (to determine whether or not the patient has been infected by that bacterium).
  • a method of extracting RNA substantially free from DNA from biological susceptible to lysis comprising the steps of:
  • RNA RNA
  • the isolated RNA is highly pure, there being little risk of contamination by lithium salts in view of the relatively low amounts thereof employed in the process of the invention.
  • the method of the invention results in the isolation of substantially pure and substantially intact RNA.
  • the method of the invention may be used for isolating RNA from a wide range of RNA- containing samples of biological origin.
  • the sample from which the RNA is to be isolated may be, or may comprise, cells, for example animal (including mammalian) cells, plant cells, bacterial cells, or yeast or other fungal cells.
  • the sample from which the RNA is to be isolated may be, or may comprise, one or more viruses.
  • the cells, virus(es) or other RNA-containing sample from which the RNA is to be isolated may originate from or have been present in a tissue sample or body fluids such as blood, sputum, urine or CSF In certain embodiments (e.g.
  • the sample may additionally be treated with lysozyme to "weaken" the cell walls prior to lysis.
  • the biological material e.g. cells
  • a lysis solution which comprises water, dissolved lithium (present as ions) at a concentration of 0.5M to 1 .OM and 20-60% by volume in the solution of miscible alkanol, so as to release RNA from the material.
  • the lysis solution is "pre-formulated” so that the lysis solution of the desired composition may be added to the biological material.
  • the lysis solution may comprise the stated components, although should be free of agents that prevent nucleases breaking down DNA. It is particularly preferred that the lysis solution does not include any anionic surfactant (so the lysis solution preferably does not include any SDS).
  • the lysis solution may consist essentially of the stated components, or consist of these components. Most preferably, the lysis solution consists essentially of water, at least one lithium salt such that the total lithium concentration is 0.5M to 1 .0M, and 20-60% by volume of miscible C ⁇ 3 alkanol.
  • the lithium salt preferably comprises a lithium halide, which is preferably the sole lithium salt in the lysis solution.
  • the lithium salt is preferably lithium chloride.
  • the concentration of lithium in the lysis solution is 0.7M to 1 .0M, and even more preferably 0.85M to 0.95M. A concentration of about 0.9M is particularly preferred.
  • the lysis solution preferably comprises 35% to 45% by volume of miscible alkanol, which is preferably provided by ethanol and/or isopropanol (which if present together make up the stated concentration range). More preferably, the lysis solution comprises about 40% by volume of miscible alkanol. It is particularly preferred that the miscible alkanol is iso-propanol (present as the sole miscible alkanol).
  • the lysis procedure will generally involve thoroughly admixing the biological material to be lysed with the lysis solution (e.g. by pipetting). Lysis may be effected at ambient temperature (e.g. 15°C to 25°C) without the need for additional heating. A time period of 15-25 minutes, e.g. about 20 minutes, is generally suitable to produce a lysed composition.
  • the lysed composition is treated with a solid support capable of immobilising RNA.
  • the solid support is preferably a porous material.
  • the solid support as used for the method of the invention may be a silica or silica-based material.
  • the silica or silica- based material may be a glass, preferably a borosilicate glass.
  • the support may comprise a fibrous material, and preferably comprises fibres of a silica or silica-based material.
  • the filter preferably comprises borosilicate glass fibres.
  • the support is in the form of a fibrous sheet or membrane in which the fibres are of a borosilicate glass. Filters of the type available under the designation POREX F are suitable for use in the invention.
  • the solid support is provided as a filter (in the form of a sheet or membrane) in a spin tube of the type well known in the art.
  • the filter in the spin tube is preferably a sheet or membrane comprising borosilicate fibres.
  • more than one such filter or sheet is provided in the spin tube.
  • the lysed composition resulting from step (i) of the method may be introduced into the spin tube which is then centrifuged (although we do not preclude the possibility that the lysis treatment of step (i) may be effected in the spin tube itself). During centrifugation, the lysed composition passes through the porous support and is thereby treated whereby the nucleic acids comprising RNA (that is ultimately to be isolated) become bound to the solid support and the majority of impurities (proteins etc.) pass through the solid support (filter) and are collected in the supernatant, which may then be discarded.
  • the solid support is washed to remove any remaining impurities and leave substantially pure RNA immobilised on the support. Washing is effected in two stages.
  • the solid support is treated with a first wash solution containing water, 20-90% (preferably 20- 80%) by volume in the first wash solution of miscible alkanol, and optionally dissolved lithium in a concentration up to 3M.
  • the first wash solution may comprise the stated components, may consist essentially of the stated components, or consist of these components.
  • the wash solution may consist essentially of (or consist of) water and 60- 80%, preferably 65-75%, most preferably about 70% by volume of miscible alkanol.
  • the first wash solution consists essentially of water, at least one lithium salt such that the total lithium concentration is up to 3M, and 20-60% by volume of miscible alkanol.
  • the lithium salt (if used) preferably comprises a lithium halide which is preferably the sole lithium salt in the first wash solution.
  • the lithium salt is preferably lithium chloride.
  • the concentration of lithium (if used) in the first wash solution is 0.1 M to 2.5M.
  • the first wash solution which contains lithium preferably comprises 45% to 55% by volume of miscible C!-3 alkanol, preferably about 50% by volume. It is particularly preferred that the miscible alkanol in the first wash solution comprises ethanol, most preferably as the sole miscible C 3 alkanol in the first wash solution.
  • the isolation procedure is being carried out in a spin tube, then the first wash solution is added to the spin tube which is then centrifuged to cause the first wash solution to pass through the filter and remove at least some of the remaining impurities for collection in the supernatant, which is then discarded.
  • a second, and usually final, wash step is then effected with a second wash solution which comprises at least 80% by volume of alkanol, the balance if any being water.
  • the second wash solution preferably consists essentially of alkanol and water.
  • the concentration of alkanol in the second wash solution is preferably 85%-95% by volume, an amount of about 90% by volume being particularly preferred.
  • the alkanol in the second wash solution preferably comprises ethanol and/or isopropanol, and is most preferably ethanol present as the sole alkanol.
  • the second wash liquid is added to the spin tube which is then centrifuged to force the wash liquid through the filter and remove remaining impurities for collection in the supernatant, which is then discarded.
  • the elution solution may comprise (consists essentially of or consists of) a buffered solution of EDTA or a salt thereof.
  • a particular suitable elution solution comprises Tris-HCI in a concentration of about 10mM, and EDTA disodium salt dihydrate at a concentration of about 0.5mM, the solution having a pH of 9.
  • RNase-free water can be used for elution.
  • the elution solution may be added to the spin column which is then centrifuged to cause the elution solution to elute the nucleic acids comprising RNA from the solid support for collection.
  • the collected nucleic acids comprising RNA may then be further analysed as required.
  • RNA obtained by the method of the invention is sufficiently pure to be used for the purpose of further analysis by techniques well known in the art.
  • the RNA may be used, for example, for the purposes of research or for diagnosis of a medical condition, as required.
  • the method of the invention may conveniently be effected on a pellet of the biological material (e.g. cells) to be lysed.
  • the pellet may be produced by centrifugation of a liquid sample containing the biological material, using techniques well known in the art.
  • Fig. 1 shows the results of gel electrophoresis on the product obtained in Example 1 below; and Fig. 2 shows the results of Example 2.
  • Example 1 shows the results of gel electrophoresis on the product obtained in Example 1 below; and Fig. 2 shows the results of Example 2.
  • This Example demonstrates extraction of RNA from a suspension containing ca 10 6 HeLa cells.
  • the suspension was centrifuged to yield a pellet which was then re-suspended in 600 ⁇ of a lysis solution (0.9M lithium chloride in 40% iso-propanol). The admixture was then incubated at room temperature for 20 minutes before the full volume was transferred to a spin column having 4 POREX F filters. The column was spun at 8000 rpm for 1 minute (although any spin-speed of 8000 to 1 1 ,000 rpm would be suitable). The "flow- through" from the column was then discarded.
  • a lysis solution 0.1% lithium chloride in 40% iso-propanol
  • a further spin at 14,000 rpm for 1 minute was then performed to remove all residual alcohol.
  • 150 ⁇ of elution buffer (10mM Tris-HCI, 0.5mM EDTA, pH 9.0) were then added to the column (a volume in range of 100 ⁇ -200 ⁇ would have been equally suitable) prior to the column being incubated at room temperature for 1 minute and then spun at 8000 rpm for 1 minute.
  • the "flow-through”, which is an RNA extract obtained from the HeLa cells was collected in 1 .5ml Eppendorf tubes.
  • an A260/A280 value of 2 to 2.2 is considered pure for RNA.
  • NEB microRNA ladder (microRNA Marker #N2102)
  • lysis solution 0.M LiCI, 40% isopropanol
  • Each admixture was then incubated at room temperature for 20 minutes before the full volumes were transferred to individual spin columns each having 4 POREX F filters. The columns were spun at 8000 rpm for 1 minute (although any spin-speed of 8000 to 1 1 ,000 rpm would be suitable). The "flow-through" from the column was then discarded. Two of the replicates were washed with 70% ethanol (not containing lithium chloride) as a first wash solution whereas the other two replicates were washed with 0.4M LiCI in 50% ethanol as their first wash solution.
  • Example 2 A modification of the procedure described in Example 1 can be used to obtain RNA from gram positive or gram negative bacteria.
  • the bacterial cells are pelleted, then treated in 100 ⁇ of 3mg/ml lysozyme solution (20mM Tris, 2mM EDTA, 1 .2% Triton x-100), and then incubated at ambient temperature for 10 mins prior to addition of the lysis solution. The subsequent steps are as described in Example 1.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
  • Organic Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Analytical Chemistry (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Plant Pathology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Mycology (AREA)
  • Medicinal Chemistry (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Virology (AREA)
  • Cell Biology (AREA)
  • Sustainable Development (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

La présente invention décrit un procédé d'extraction d'ARN sensiblement exempt d'ADN provenant d'un matériau biologique sensible à la lyse, le procédé comprend les étapes de : (a) exécution de la lyse dudit matériau biologique avec une solution de lyse qui comprend de l'eau, du lithium dissous sous une concentration de 0,5 M à 1,0 M, et de 20 à 60 % en volume en solution d'alcanol miscible pour produire une composition lysée ; (b) le traitement de la composition lysée avec un support capable d'immobiliser l'ARN ; (c) la séparation du support solide avec l'ARN immobilisé du liquide de la composition lysée ; (d) l'exécution du lavage avec une première solution de lavage comprenant de l'eau, de 20 à 90 % en volume dans la première solution de lavage d'alcanol miscible, et éventuellement du lithium dissous sous une concentration allant jusqu'à 3 M, et par la suite avec une seconde solution de lavage qui comprend au moins 80 % en volume d'alcanol, le reste le cas échéant étant de l'eau ; et (e) l'élution de l'ARN du support solide lavé.
PCT/GB2017/051428 2016-05-24 2017-05-22 Isolement d'acides nucléiques Ceased WO2017203225A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB1609115.9 2016-05-24
GBGB1609115.9A GB201609115D0 (en) 2016-05-24 2016-05-24 Isolating nucleic acids

Publications (1)

Publication Number Publication Date
WO2017203225A1 true WO2017203225A1 (fr) 2017-11-30

Family

ID=56369877

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/GB2017/051428 Ceased WO2017203225A1 (fr) 2016-05-24 2017-05-22 Isolement d'acides nucléiques

Country Status (2)

Country Link
GB (1) GB201609115D0 (fr)
WO (1) WO2017203225A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020165602A1 (fr) * 2019-02-15 2020-08-20 RevoluGen Limited Procédé de purification

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004094635A2 (fr) * 2003-04-16 2004-11-04 Gentra Systems, Inc. Compositions et procedes d'utilisation d'un support solide pour purifier de l'arn
WO2006052680A1 (fr) * 2004-11-05 2006-05-18 Qiagen North American Holdings, Inc. Compositions et procedes pour purifier des acides nucleiques provenant de reactifs de stabilisation
WO2007008722A2 (fr) * 2005-07-13 2007-01-18 Sigma-Aldrich Co. Procede permettant d'isoler l'arn de sources biologiques
CN102071186A (zh) * 2009-11-19 2011-05-25 北京金麦格生物技术有限公司 一种从生物样品中快速分离病毒核酸的试剂盒及方法
WO2013074927A1 (fr) * 2011-11-16 2013-05-23 University Of Georgia Research Foundation, Inc. Procédé pour isoler l'arn total dans des cellules
WO2016077294A1 (fr) * 2014-11-14 2016-05-19 Corning Incorporated Procédés et kits pour la purification d'arn post-tiv

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004094635A2 (fr) * 2003-04-16 2004-11-04 Gentra Systems, Inc. Compositions et procedes d'utilisation d'un support solide pour purifier de l'arn
WO2006052680A1 (fr) * 2004-11-05 2006-05-18 Qiagen North American Holdings, Inc. Compositions et procedes pour purifier des acides nucleiques provenant de reactifs de stabilisation
WO2007008722A2 (fr) * 2005-07-13 2007-01-18 Sigma-Aldrich Co. Procede permettant d'isoler l'arn de sources biologiques
CN102071186A (zh) * 2009-11-19 2011-05-25 北京金麦格生物技术有限公司 一种从生物样品中快速分离病毒核酸的试剂盒及方法
WO2013074927A1 (fr) * 2011-11-16 2013-05-23 University Of Georgia Research Foundation, Inc. Procédé pour isoler l'arn total dans des cellules
WO2016077294A1 (fr) * 2014-11-14 2016-05-19 Corning Incorporated Procédés et kits pour la purification d'arn post-tiv

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
"Technical Bulletin: #160", 1 January 2011 (2011-01-01), XP055058775, Retrieved from the Internet <URL:http://dfpcorec-p.internal.epo.org/wf/web/citenpl/citenpl.html> [retrieved on 20130408] *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020165602A1 (fr) * 2019-02-15 2020-08-20 RevoluGen Limited Procédé de purification
CN113423832A (zh) * 2019-02-15 2021-09-21 雷沃卢金有限公司 纯化方法
EP3985112A1 (fr) * 2019-02-15 2022-04-20 RevoluGen Limited Procédé de purification
CN114540343A (zh) * 2019-02-15 2022-05-27 雷沃卢金有限公司 纯化方法

Also Published As

Publication number Publication date
GB201609115D0 (en) 2016-07-06

Similar Documents

Publication Publication Date Title
CN101878304B (zh) 用于从单个样品中分离基因组dna、rna和蛋白质的方法
JP4277115B2 (ja) 核酸の単離および精製
CN106574265B (zh) 高产量分离rna的方法
CA2012777C (fr) Procede d&#39;isolement des acides nucleiques
CN103097532B (zh) 高产量分离包括小靶核酸在内的靶核酸的方法
US20110172405A1 (en) Method for small rna isolation
EP1851313B1 (fr) Méthode pour l&#39;isolation d&#39;acides nucléiques dans laquelle les acides nucléiques sont immobilisés à haute température sur une matrice
EP2942394A1 (fr) Nouveau procédé permettant d&#39;isoler un ADN microbien
JPH0515373A (ja) ヒトゲノムｄｎａの抽出および精製方法
JP2011152142A (ja) 精製ｒｎａの単離試薬及び方法
US9163230B2 (en) Chromatography column and method for isolating nucleic acid
Sun et al. Efficient purification and concentration of viruses from a large body of high turbidity seawater
US12297425B2 (en) Nucleic acid extraction composition, reagent and kit containing the same and use thereof
US10323241B2 (en) Method for recovering short-chain nucleic acids
JP2007509620A (ja) 迅速かつ低コストな核酸の単離方法
EP2010672B1 (fr) Formulations et procédé d&#39;isolement d&#39;acides nucléiques contenus dans des matières premières complexes quelconques et analyse génétique complexe consécutive
JP6713007B2 (ja) 核酸の単離
JPWO2018061877A1 (ja) 核酸を抽出する方法及びそれに用いるキット
WO2017203225A1 (fr) Isolement d&#39;acides nucléiques
JP7479079B2 (ja) カラム方式の単一チューブｐｃｒ用核酸分離のためのバッファ組成物及びこの用途
KR101380909B1 (ko) 핵산 정제용 흡착제 및 그 흡착제를 이용한 정제방법
CN118995383B (zh) 一种磁珠法微生物基因组提取试剂盒及微生物基因组提取方法
CN118599826A (zh) 酚-氯仿法结合柱离心法经DNase I消化的RNA提取纯化方法及提取纯化试剂盒
CN119162283A (zh) 一种噬菌体组富集方法及其试剂盒和应用

Legal Events

Date Code Title Description
NENP Non-entry into the national phase

Ref country code: DE

121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 17728248

Country of ref document: EP

Kind code of ref document: A1

122 Ep: pct application non-entry in european phase

Ref document number: 17728248

Country of ref document: EP

Kind code of ref document: A1