WO2003095646A1 - Procede d'isolement de l'acide nucleique - Google Patents

Procede d'isolement de l'acide nucleique Download PDF

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Publication number
WO2003095646A1
WO2003095646A1 PCT/IB2003/001822 IB0301822W WO03095646A1 WO 2003095646 A1 WO2003095646 A1 WO 2003095646A1 IB 0301822 W IB0301822 W IB 0301822W WO 03095646 A1 WO03095646 A1 WO 03095646A1
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WO
WIPO (PCT)
Prior art keywords
solid phase
nucleic acid
process according
chaotrope
sample
Prior art date
Application number
PCT/IB2003/001822
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English (en)
Inventor
Arne Deggerdal
Vidar Skagestad
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Qiagen As
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.)
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Publication date
Application filed by Qiagen As filed Critical Qiagen As
Priority to AU2003230070A priority Critical patent/AU2003230070A1/en
Publication of WO2003095646A1 publication Critical patent/WO2003095646A1/fr

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    • 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 process for isolating nucleic acid from a nucleic acid-containing sample, and to a kit therefor.
  • nucleic acid detection and manipulation including hybridisation, amplification, sequencing and other processes generally require nucleic acid to have been isolated from contaminating material.
  • contaminating material may include proteins, • carbohydrates, lipids and polyphenols. Accordingly, a variety of approaches have hitherto been used in the isolation of DNA or RNA.
  • US5234809 describes a procedure to isolate DNA from biological samples which uses a chaotropic agent together with a silica based nucleic acid binding solid phase. Guanidine hydrochloride at pH 3 to 5 or guanidine thiocyanate at higher pH, combined with other salts, is used as the chaotropic agent. After binding of the DNA to the solid surface, the solid phase may be washed with the chaotropic agent to remove any biological contamination followed by treatment with 70% ethanol to remove the chaotrope. The DNA is eluted using water.
  • US 5990302 describes a method for isolating RNA which is also performed in the presence of a chaotrope.
  • a sample is mixed with an acidic solution containing a lithium salt, a chaotropic agent and a nucleic acid-binding carrier to absorb the RNA onto the carrier.
  • the RNA-bound carrier is isolated from the liquid phase and eluted.
  • Magnetic silica particles are used as the nucleic acid-binding carrier, although silica, cellulose, nitrocellulose, latex and hydroxyapatite are all mentioned as possible carriers.
  • W096/18731 also uses magnetic particles to bind nucleic acid.
  • the magnetic particles are polystyrene-based and polyurethane-coated and a detergent is used instead of a chaotrope.
  • US 5705628 discloses a method of separating polynucleotides, especially DNA, by binding the polynucleotides to a magnetic micro particle having a functional group-coated surface.
  • Carboxyl groups are mentioned as the functional group and are coated on the surface of a silica based micro particle. This method requires the presence of salt and a polyalkylene glycol such as polyethylene glycol at a concentration in the range 7 to 13%.
  • magnétique particles are available from the agent suppliers for use not as a binding phase for isolating nucleic acid, but as a starting material for the production of affinity materials.
  • magnetic beads bearing on their surface carboxylic acid moieties and composed of a highly cross-linked polystyrene are known for use in coupling reactions with' proteins, peptides, oligonucleotides or other target specific molecules.
  • a bi- functional cross-linking reagent such as carbodiimide is first coupled to the carboxylic acid moieties and then used as a reactive group to couple the target specific molecules via primary a ino groups.
  • silica-based nucleic acid binding solid phases the use of a silica surface has its drawbacks in that • it is difficult to control the synthesis and preparation of silica-based solid phases.
  • the present invention provides a process for isolating nucleic acid from a nucleic acid-containing sample, which comprises:
  • the present invention provides a kit for isolating nucleic acid from a nucleic acid-containing sample, which kit comprises: (a) a chaotrope; and (b) a nucleic acid binding solid phase capable of binding nucleic acid in the presence of the chaotrope, wherein the solid phase bears acid groups on its surface.
  • a solid phase which bears acid groups on its surface is capable of non-specific or non-covalent direct binding of nucleic acids.
  • Such solid phases are found to be as efficient as silica surfaces for the isolation of nucleic acids.
  • the present invention provides an advantage in that acid groups on the surface of the solid phase represent a more controlled chemistry for the synthesis and preparation of the solid phase.
  • An acid surface for example an organic acid surface such as a carboxylic acid surface is also more useful than the corresponding silica surface.
  • covalent coupling of the nucleic acid to the acid may be effected in si tu .
  • the concentration of acid groups on the surface should be above l ⁇ mol/g solid phase, preferably above lO ⁇ mol/g and more preferably above lOO ⁇ mol/g solid phase.
  • the isoelectric point of the solid phase surace is below pH 8, preferably below pH 6 and more preferably below pH 4.
  • carboxy, sulpho and aryloxy groups may be mentioned carboxy, sulpho and aryloxy groups.
  • the carboxy or sulpho groups may be linked to the solid phase by alkylene or arylene groups so as to form carboxylic or sulphonic acids.
  • Aryloxy groups such as phenoxy groups may also be so linked and may incorporate further aromatic or aliphatic moieties.
  • Carbon atoms in each type of organic acid may be substituted with heteroatoms.
  • the presence of such heteroatoms and the optional presence of further functional groups on the surface may each contribute to the properties of the solid phase, especially to the hydrophilicity of the solid phase.
  • the preferred solid phase is hydrophilic because too hydrophobic a solid phase (for instance where there is too a high a concentration of polystyrene) will tend to give problems with nucleic acid binding.
  • the solid phase bearing acid groups on its surface may be prepared by various different methods.
  • a solid phase comprising a silica or non-silica surface may be modified by a reactant to introduce the acid groups.
  • Such surfaces may be modified by carboxylic acids, for example by using an epoxy coating of silica followed by carboxylic introduction.
  • carboxylic acids for example by using an epoxy coating of silica followed by carboxylic introduction.
  • these may be transformed into a carboxylic acid coated surface by a variety of different means.
  • acid anhydrides are introduced onto the hydroxyl-based surface.
  • an epoxy coating of the ⁇ hydroxyl moieties is added to the solid surface, followed by carboxylic acid introduction by various means.
  • the solid phase is an organic solid phase rather than one based on silica.
  • Silicia-based solid phases tend to give poorer yields and are more difficult to synthesize. It is preferred, if starting from a silica- based surface, to apply an organic coating which includes acid groups or on which is subsequently introduced acid groups. It is therefore preferred that the solid phase is substantially free of silica or at least free of surface- exposed silica.
  • the nucleic acid-containing sample typically comprises a biological sample such as a cellular sample.
  • the biological sample may or may not need to be pretreated, depending on its structure. For example, in the case of plant or fungal cells or solid animal tissue, pretreatment would be required as is known in the art. Samples stored in the form of a solid phase such as a paraffin section may also need pretreatment. Samples may be from foodstuffs, environmental samples or clinical samples and may contain prokaryotic or eukaryotic cells or other moieties such as mycoplasmas, protoplasts or viruses. Blood products are an important area for nucleic acid isolation and the present invention is particularly applicable to whole blood and other blood products such as plasma, serum and buffycoat .
  • the nucleic acid to be isolated may be DNA, RNA or a modified form thereof. Where the nucleic acid is DNA, this may be ds or ss DNA. Where the nucleic acid is RNA, this may be rRNA, mRNA or total RNA.
  • the chaotrope generally comprises a chaotropic ion provided at a concentration sufficiently high to cause the nucleic acid to lose its secondary structure and, in the case of double-stranded nucleic acids, ' to melt. Chaotropes are thought to disrupt hydrogen-bonding in water so as to make denatured nucleic acid more stable than its undenatured counterpart.
  • the chaotrope typically comprises a guanidinium salt, urea, or an iodide, chlorate, perchlorate or (iso) thiocyanate.
  • Preferred chaotropes include guanidinium thiocyanate, and guanidinium hydrochloride .
  • the concentration of chaotrope typically present when contacted with the sample is in the range 2M to 8M.
  • the form of the solid phase includes sheets, sieves, sinters, webs and fibres. Particles are particularly useful as these may be packed in a column or used in suspension and have high binding capacity. Magnetic particles are particularly preferred because of the ease with ' which they merely separated from an associated liquid phase in a magnetic field. Typical materials for use in magnetic particles include magnetic metal oxides especially the iron oxides . Useful magnetic oxides include iron oxides in which, optionally all or a part of the ferrous iron thereof is substituted with a divalent transition metal such as cadmium, chromium, cobalt, copper, magnesium, manganes_e, nickel, vanadium and/or zinc.
  • a divalent transition metal such as cadmium, chromium, cobalt, copper, magnesium, manganes_e, nickel, vanadium and/or zinc.
  • the step of separating the solid phase with the nucleic acid bound thereto from the liquid phase is generally required in order to remove contaminants in the liquid phase. Further ' washing steps may be applied to the solid phase at this point. Any conventional separation step for separating solid phase from liquid phase is applicable, including centrifugation and decanting of the liquid phase from the pelleted solid phase or using a column in which the solid phase is packed and the liquid phase passed through. Where the magnetic solid phase is used, this facilitates separation, which can be carried out in the presence of a magnetic field. Depending on the form in which the isolated nucleic acid is required, a further elution step can be provided. In some cases it may be satisfactory for the nucleic acid to remain bound to the solid phase.
  • nucleic acid may be eluted from the solid phase by applying an elution solution, which may simply be water or a buffer.
  • Typical coupling reagents include bifunctional cross-linking agents such as carbodiimides e . g. (l-ethyl-3- (3 dimethylamino- propyl) or di (cyclohexyl) carbodiimide .
  • nucleic acid whilst the nucleic acid is still non-specifically attached to the solid phase, this may be washed with 70% ethanol and an alcohol soluble coupling reagent, such as one of those described above, may be added. Following incubation, typically at room temperature and a washing step, the nucleic acids may be coupled covalently to the solid phase.
  • Example 1 The present invention is now described in more detail, by way of example only, with reference to the following Example .
  • Hydrophilic magnetic particles are available from numerous sources (Dynal, Chemagen, MicroMod, ChemiCell etc) . Any hydrophilic magnetic particle with, for instance, OH moieties can be transformed into magnetic particles with a surface carboxylic acid (COOH) functionality.
  • COOH carboxylic acid
  • ca 15 mg of MPVA C22 Carboxylic Acid (Chemagen, Germany) with a COOH content of ca 900 umol/g) and ca 15 mg of Dynabeads M270 Carboxylic Acid (Dynal, Norway) , with a COOH content of at least 150 umol/g were utilized.
  • the chaotropi c lysis and binding solution 1 1 .
  • the wash II solution To 150 ⁇ l, 8M LiCl (Sigma) was added 750 ul 96% EtOH. To 600 ul of this solution was added 100 ul water.
  • the wash III solution 10 mM NaCl .
  • the binding condi tions To cultured cells (4xl0 6 HL 6 o) were added 700 ul of the chaotropic lysis and binding solution and 100 ul binding solution 2. The suspension was "sheared" with a syringe. The solution was allowed to lyse for 10 min. The magnetic beads, as a water suspension, were added and allowed to incubate for 1 min. The beads were resuspended in 900 ul washing solution I and again collected on a magnet. The beads were resuspended and washed in 900 ul washing solution II and collected on a magnet. This was repeated with 900ul washing solution III.

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  • 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)
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Abstract

L'invention concerne un procédé servant à isoler l'acide nucléique d'un échantillon contenant un acide nucléique. Ce procédé consiste (a) à utiliser un agent chaotrope ; (b) à utiliser une phase solide servant à la fixation de l'acide nucléique, capable de fixer l'acide nucléique en présence de l'agent chaotrope ; (c) à mettre l'échantillon en contact avec la phase solide, en présence d'une phase liquide contenant l'agent chaotrope ; et (d) à éventuellement séparer la phase solide sur laquelle est fixé l'acide nucléique de la phase liquide, la phase solide comportant des groupes acides sur sa surface.
PCT/IB2003/001822 2002-05-10 2003-05-09 Procede d'isolement de l'acide nucleique WO2003095646A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU2003230070A AU2003230070A1 (en) 2002-05-10 2003-05-09 Isolating nucleic acid

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB0210766.2 2002-05-10
GB0210766A GB0210766D0 (en) 2002-05-10 2002-05-10 Isolating nucleic acid

Publications (1)

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WO2003095646A1 true WO2003095646A1 (fr) 2003-11-20

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Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009020609A2 (fr) * 2007-08-06 2009-02-12 Nanogen, Inc. Isolation de molécules d'acides nucléiques en utilisant des supports solides modifiés
WO2013148346A1 (fr) * 2012-03-28 2013-10-03 Longhorn Vaccines And Diagnostics, Llc Compositions et procédés pour la collecte et l'isolement d'acides nucléiques à partir de spécimens biologiques
US8821885B2 (en) 2007-08-27 2014-09-02 Longhorn Vaccines & Diagnostics, Llc Immunogenic compositions and methods
US9080204B2 (en) 2006-09-12 2015-07-14 Longhorn Vaccines And Diagnostics, Llc Compositions and methods for rapid, real-time detection of influenza a virus (H1N1) Swine 2009
US9212399B2 (en) 2007-10-01 2015-12-15 Longhorn Vaccines And Diagnostics, Llc Biological specimen collection and transport system and method of use
US9416416B2 (en) 2007-10-01 2016-08-16 Longhorn Vaccines And Diagnostics, Llc Biological specimen collection/transport compositions and methods
US9481912B2 (en) 2006-09-12 2016-11-01 Longhorn Vaccines And Diagnostics, Llc Compositions and methods for detecting and identifying nucleic acid sequences in biological samples
US9598462B2 (en) 2012-01-26 2017-03-21 Longhorn Vaccines And Diagnostics, Llc Composite antigenic sequences and vaccines
US9683256B2 (en) 2007-10-01 2017-06-20 Longhorn Vaccines And Diagnostics, Llc Biological specimen collection and transport system
US9976136B2 (en) 2015-05-14 2018-05-22 Longhorn Vaccines And Diagnostics, Llc Rapid methods for the extraction of nucleic acids from biological samples
US10004799B2 (en) 2007-08-27 2018-06-26 Longhorn Vaccines And Diagnostics, Llc Composite antigenic sequences and vaccines
US10816546B2 (en) 2002-07-01 2020-10-27 Sinvent As Binding a target substance
US11041215B2 (en) 2007-08-24 2021-06-22 Longhorn Vaccines And Diagnostics, Llc PCR ready compositions and methods for detecting and identifying nucleic acid sequences
US11041216B2 (en) 2007-10-01 2021-06-22 Longhorn Vaccines And Diagnostics, Llc Compositions and methods for detecting and quantifying nucleic acid sequences in blood samples

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5705628A (en) * 1994-09-20 1998-01-06 Whitehead Institute For Biomedical Research DNA purification and isolation using magnetic particles
US6027945A (en) * 1997-01-21 2000-02-22 Promega Corporation Methods of isolating biological target materials using silica magnetic particles
WO2000069872A2 (fr) * 1999-05-14 2000-11-23 Promega Corporation MATRICE ECHANGEUSE D'IONS DEPENDANT DU pH, UTILISEE POUR ISOLER DES ACIDES NUCLEIQUES
DE19937607A1 (de) * 1999-08-09 2001-02-15 Bilatec Ges Zur Entwicklung Bi Reagenzienkit zur Isolierung von Nukleinsäuren

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5705628A (en) * 1994-09-20 1998-01-06 Whitehead Institute For Biomedical Research DNA purification and isolation using magnetic particles
US6027945A (en) * 1997-01-21 2000-02-22 Promega Corporation Methods of isolating biological target materials using silica magnetic particles
WO2000069872A2 (fr) * 1999-05-14 2000-11-23 Promega Corporation MATRICE ECHANGEUSE D'IONS DEPENDANT DU pH, UTILISEE POUR ISOLER DES ACIDES NUCLEIQUES
DE19937607A1 (de) * 1999-08-09 2001-02-15 Bilatec Ges Zur Entwicklung Bi Reagenzienkit zur Isolierung von Nukleinsäuren

Cited By (19)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10816546B2 (en) 2002-07-01 2020-10-27 Sinvent As Binding a target substance
US9481912B2 (en) 2006-09-12 2016-11-01 Longhorn Vaccines And Diagnostics, Llc Compositions and methods for detecting and identifying nucleic acid sequences in biological samples
US9080204B2 (en) 2006-09-12 2015-07-14 Longhorn Vaccines And Diagnostics, Llc Compositions and methods for rapid, real-time detection of influenza a virus (H1N1) Swine 2009
WO2009020609A2 (fr) * 2007-08-06 2009-02-12 Nanogen, Inc. Isolation de molécules d'acides nucléiques en utilisant des supports solides modifiés
WO2009020609A3 (fr) * 2007-08-06 2009-07-16 Nanogen Inc Isolation de molécules d'acides nucléiques en utilisant des supports solides modifiés
US11041215B2 (en) 2007-08-24 2021-06-22 Longhorn Vaccines And Diagnostics, Llc PCR ready compositions and methods for detecting and identifying nucleic acid sequences
US8821885B2 (en) 2007-08-27 2014-09-02 Longhorn Vaccines & Diagnostics, Llc Immunogenic compositions and methods
US9388220B2 (en) 2007-08-27 2016-07-12 Longhorn Vaccines And Diagnostics, Llc Immunogenic compositions and methods
US10004799B2 (en) 2007-08-27 2018-06-26 Longhorn Vaccines And Diagnostics, Llc Composite antigenic sequences and vaccines
US10596250B2 (en) 2007-08-27 2020-03-24 Longhorn Vaccines And Diagnostics, Llc Methods of treating and preventing influenza infections
US9777045B2 (en) 2007-08-27 2017-10-03 Longhorn Vaccines And Diagnostics, Llc Immunogenic compositions and methods
US9416416B2 (en) 2007-10-01 2016-08-16 Longhorn Vaccines And Diagnostics, Llc Biological specimen collection/transport compositions and methods
US11041216B2 (en) 2007-10-01 2021-06-22 Longhorn Vaccines And Diagnostics, Llc Compositions and methods for detecting and quantifying nucleic acid sequences in blood samples
US9212399B2 (en) 2007-10-01 2015-12-15 Longhorn Vaccines And Diagnostics, Llc Biological specimen collection and transport system and method of use
US9683256B2 (en) 2007-10-01 2017-06-20 Longhorn Vaccines And Diagnostics, Llc Biological specimen collection and transport system
US9598462B2 (en) 2012-01-26 2017-03-21 Longhorn Vaccines And Diagnostics, Llc Composite antigenic sequences and vaccines
WO2013148346A1 (fr) * 2012-03-28 2013-10-03 Longhorn Vaccines And Diagnostics, Llc Compositions et procédés pour la collecte et l'isolement d'acides nucléiques à partir de spécimens biologiques
US9976136B2 (en) 2015-05-14 2018-05-22 Longhorn Vaccines And Diagnostics, Llc Rapid methods for the extraction of nucleic acids from biological samples
US10087439B1 (en) 2015-05-14 2018-10-02 Longhorn Vaccines And Diagnostics, Llc Rapid methods for the extraction of nucleic acids from biological samples

Also Published As

Publication number Publication date
GB0210766D0 (en) 2002-06-19
AU2003230070A1 (en) 2003-11-11

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