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EP1171584A1 - Kit d'extraction d'adn magnetique pour plantes - Google Patents

Kit d'extraction d'adn magnetique pour plantes

Info

Publication number
EP1171584A1
EP1171584A1 EP00923575A EP00923575A EP1171584A1 EP 1171584 A1 EP1171584 A1 EP 1171584A1 EP 00923575 A EP00923575 A EP 00923575A EP 00923575 A EP00923575 A EP 00923575A EP 1171584 A1 EP1171584 A1 EP 1171584A1
Authority
EP
European Patent Office
Prior art keywords
dna
kit
positively charged
charged groups
cells
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Withdrawn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
EP00923575A
Other languages
German (de)
English (en)
Inventor
Maria-Luisa Maccecchini
Mitchell T. Gore
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.)
Annovis Inc
Original Assignee
Annovis Inc
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 Annovis Inc filed Critical Annovis Inc
Publication of EP1171584A1 publication Critical patent/EP1171584A1/fr
Withdrawn legal-status Critical Current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
    • C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
    • C—CHEMISTRY; METALLURGY
    • 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
    • C12N15/1013—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 by using magnetic beads

Definitions

  • the present invention generally relates to the field of methods and kits for the extraction of DNA and specifically to methods and kits for the extraction of DNA from plants.
  • Methods for extracting DNA from plants include:
  • the Dellaporta method Dellaporta. S.L., et al. 1983. A plant DNA minipreparation: Version II. Plan Mol Biol Rep 1 : 19-21 , where DNA is precipitated to produce a very crude preparation, with a lot of the ethanol insoluble contaminants present in the solution.
  • a variant of the Dellaporta method introduces an additional step of purifying the DNA via ultracentrifugation through a cesium chloride (CsCl) gradient for several hours or overnight.
  • CsCl cesium chloride
  • CTAB cetyltrimethylammonium bromide
  • CTAB is a cationic detergent, which will form an insoluble complex with the DNA in the presence of low concentrations of salt, such as a 0.5 M sodium chloride (NaCl) solution.
  • salt such as a 0.5 M sodium chloride (NaCl) solution.
  • NaCl sodium chloride
  • the original lysis solution contains about 1.4 M NaCl.
  • the DNA binds with the CTAB when the NaCl concentration is decreased to 0.5 M.
  • This method is also time consuming and. because the procedure does not use organic solvents, an additional step needs to be included to clean up the DNA: a final extraction with organic solvents to rid the preparation of polysaccharide and phenolic compounds.
  • U.S. Patent No. 5,705.628 to Hawkins discloses the use of magnetic microparticles with a coating including functional groups, specifically carboxyl or negatively charged groups, for the purification and isolation of DNA by binding and elution.
  • International Application WO 96/18731 by Deggerdal. et al. discloses a method for the isolation of nucleic acids by the binding to and elution from a solid support, preferably magnetic beads, in the presence of a detergent, preferably an anionic detergent such as SDS or SARCOSYLTM.
  • U.S. Patent No. 5,650,506 to Woodard, et al. discloses the use of glass fiber membranes bearing positive surface charges for DNA purification by binding to and elution from the glass fibers.
  • a commercially available method for the extraction of DNA from bacterial cells is marketed as ISOLATETM by Annovis, Inc. (catalog number 2- 0300-85).
  • the protocol includes the following steps: suspending bacterial cells in a buffer (pH 8.0), lysing the cells by mixing the suspension with an alkaline - detergent solution (0.2 M NaOH, 1% SDS).
  • a method and kit for the extraction of DNA from plants which quickly yields plant DNA with a high level of purity.
  • the method isolates DNA (genomic, chloroplast. and/or mitochondrial DNA) using immobilized anionic groups, preferably on a chromatographic substrate or more preferably magnetic beads derivatized with anionic groups such as diethylaminoethyl (DEAE) via an anion-exchange interaction.
  • the purified DNA is then eluted with ions (typically a salt solution).
  • RNA can be removed by digestion with RNAse.
  • the method described herein can be used with any plant material and has been demonstrated to be efficacious with the following representative types of plants: arabdopsis seedlings, barley embryos, tobacco leaves, tomato leaves, soybean hypocotylis and cultured cells, white beans hypocotylis and roots, young and old pine needles.
  • the process generally includes the steps of: grinding plant material to make a tissue extract, lysing the plant cells, removing cell debris, and binding the plant DNA to an immobilized or insoluble material such as magnetic beads, where it is separated into pure form.
  • plant material is first ground to a powder in liquid nitrogen and then incubated in lysis buffer (0.1 M Tris-HCl, pH 8.0, 0.1 M EDTA. pH 8.0, 0.25 M NaCl and 100 microgram/ml proteinase K) in the presence of a surfactant or nonionic detergent such as N-laurylsarcosine (SARKOSYLTM), TRITONTM or NONIDETTM P-40, which acts to solubilize cell components and lyse the cells. Representative detergents are listed in Table I.
  • Cell debris is then removed by centrifugation and the supernatant, which contains the DNA and other soluble cell components, is collected.
  • the DNA is then bound to an immobilized or insoluble material having anionic groups bound thereto, such as magnetic beads derivatized with DEAE. This material is mixed with the supernatant, bound to the DNA, and then removed from the supernatant using a magnetic separator.
  • the beads are subsequently washed and then the DNA is preferably eluted from the beads by adding NaCl to a final concentration of 1.0 M.
  • the DNA could be removed by binding to DEAE chromatographic material or filler material, which is separated by washing, centrifugation, or other methods known to those skilled in the art.
  • the method and kit produces DNA of equal purity to CsCl gradient methods at yields that are equal to, or better than, the prior art, Dellaporta and CTAB methods.
  • the specific problems of the Dellaporta method (use of a CsCl column), and CTAB method (use of cationic detergents), are avoided by this method.
  • the method and kit also extracts and purifies the DNA more quickly than the CsCl method.
  • the method including the precipitation step takes a total of approximately 2.5 hours; without the precipitation step it takes less than 2 hours (1 hour and 50 minutes) to extract and purify the plant DNA: approximately 1 hour to lyse the cells, 5 minutes to bind the DNA to the magnetic beads. 5 minutes to wash the beads, 5 minutes to separate the DNA from the beads.
  • the preferred method described herein produces a very pure DNA preparation via an anion-exchange interaction where DNA is replaced as the binding species on the anion-exchange matrix by chloride ions or other negatively charged ions derived from any of a variety of salts. If a traditional anion-exchange column were used at the point where the beads are introduced, the columns would likely clog, preventing collection of bound DNA, or if the elution was effected with strong acid or base, significant levels of contaminates would co-elute with the DNA.
  • the DNA bound to the beads can be thoroughly mixed with the wash solutions, contaminates are more easily removed than they are in traditional column formats, resulting in a more pure preparation. Moreover, because the DNA bound to the beads is not sheared or compressed, when pelleted, the attached DNA consists of longer and more intact strands.
  • the lysis methods for plant genomic DNA and bacterial plasmid DNA are different.
  • the lysis method for bacterial plasmid DNA uses alkaline conditions in the presence of anionic detergent in the form of SDS, while the lysis method for plant genomic DNA uses a nonionic detergent.
  • the type of detergent used affects the remaining steps in each method. Since SDS is anionic (negatively charged, like DNA), it must be removed from the solution in the plasmid procedure before the beads are introduced, otherwise the SDS would compete for binding with the plasmid DNA on the positively charged beads. In the ISOLATETM plasmid extraction system, the SDS is removed from the solution by adding potassium acetate to form an insoluble precipitate with the chromosomal DNA.
  • the aggregate of SDS with bacterial genomic DNA can be easily separated from the soluble plasmid DNA.
  • no precipitation is needed prior to the binding step, because the nonionic non-charged detergent used in the methods and kits described herein does not compete with the DNA for binding sites, since the genomic DNA is the desired binding species for the DEAE groups on the beads. Therefore, the nonionic detergent does not need to be removed, whereas SDS and other anionic detergents are negatively charged and do compete with DNA for anionic binding sites and therefore would need to be removed.
  • N-lauroylsarcosine at a final concentration of between 0.1 % and 10%. If desired, 25 - 200 ⁇ g of RNase A can be added at this point. Alternatively, the final resuspended pellet can be treated with RNase. Incubate 30 minutes to 1 hour at 50-60°C.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Health & Medical Sciences (AREA)
  • Biomedical Technology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Plant Pathology (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Saccharide Compounds (AREA)

Abstract

L'invention concerne un procédé et un kit pour l'extraction d'ADN de plantes, permettant de produire rapidement de l'ADN de plantes présentant un grand degré de pureté. Ledit procédé permet d'isoler de l'ADN (ADN génomique, de chloroplaste et/ou mitochondrien) au moyen de groupes anioniques immobilisés, de préférence sur un substrat chromatographique ou idéalement sur des billes magnétiques dérivées de groupes anioniques, tels que le diéthylaminoéthyle (DEAE), par une interaction à échange d'anions. L'ADN purifié est ensuite élué à l'aide d'ions (généralement une solution salée). L'ARN peut être supprimé par digestion à l'aide de Rnase.
EP00923575A 1999-04-21 2000-04-21 Kit d'extraction d'adn magnetique pour plantes Withdrawn EP1171584A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US13035399P 1999-04-21 1999-04-21
US130353P 1999-04-21
PCT/US2000/010834 WO2000063362A1 (fr) 1999-04-21 2000-04-21 Kit d'extraction d'adn magnetique pour plantes

Publications (1)

Publication Number Publication Date
EP1171584A1 true EP1171584A1 (fr) 2002-01-16

Family

ID=22444292

Family Applications (1)

Application Number Title Priority Date Filing Date
EP00923575A Withdrawn EP1171584A1 (fr) 1999-04-21 2000-04-21 Kit d'extraction d'adn magnetique pour plantes

Country Status (5)

Country Link
EP (1) EP1171584A1 (fr)
JP (1) JP2002541839A (fr)
AU (1) AU4367500A (fr)
CA (1) CA2370656A1 (fr)
WO (1) WO2000063362A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102888397A (zh) * 2012-09-25 2013-01-23 杭州硕航生物科技有限公司 一种利用磁珠提取全血基因组dna的试剂盒及应用

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US7666592B2 (en) 2004-02-18 2010-02-23 Ibis Biosciences, Inc. Methods for concurrent identification and quantification of an unknown bioagent
ES2641832T3 (es) 2004-05-24 2017-11-14 Ibis Biosciences, Inc. Espectrometría de masas con filtración de iones selectiva por establecimiento de umbrales digitales
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WO2006135400A2 (fr) 2004-08-24 2006-12-21 Isis Pharmaceuticals, Inc. Procedes pour l'identification rapide d'organismes recombinants
EP1788859B1 (fr) 2004-09-16 2014-03-19 CropDesign N.V. Evaluation de la croissance d'une racine
KR100601972B1 (ko) 2004-11-03 2006-07-18 삼성전자주식회사 레이저와 비드를 이용한 상 분리에 의한 핵산의 정제 장치및 방법
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Also Published As

Publication number Publication date
JP2002541839A (ja) 2002-12-10
AU4367500A (en) 2000-11-02
WO2000063362A1 (fr) 2000-10-26
CA2370656A1 (fr) 2000-10-26

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