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
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
  • Y10T436/00—Chemistry: analytical and immunological testing
  • Y10T436/14—Heterocyclic carbon compound [i.e., O, S, N, Se, Te, as only ring hetero atom]
  • Y10T436/142222—Hetero-O [e.g., ascorbic acid, etc.]
  • Y10T436/143333—Saccharide [e.g., DNA, etc.]

Definitions

• the invention relates to a novel buffer formulation for fast separation, purification and highly efficient recovery of long- and/or short-chain nucleic acids.
• the physico-chemical principle of the systems for isolating nucleic acids on the basis of the binding of nucleic acids to the surfaces of mineral supports should thereby consist in the disruption of superordinate structures of the aqueous medium, through which the nucleic acids on the surface of mineral materials, in particular of glass or silica particles, adsorb.
• the disruption of the superordinate structures of the aqueous medium is thereby always carried out in the presence of chaotropic ions and is almost quantitative at high concentrations of these.
• all commercially available systems for the isolation of nucleic acids contain buffer compositions with higher ion strengths of chaotropic salts, for the binding of nucleic acids to a nucleic acid-binding solid phase.
• WO 01/62976 A1 a description is disclosed which comprises the purification of nucleic acids from various reaction assays upon addition of different alcohols, their subsequent precipitation on special solid phases (membranes with specific physical characteristics), wash steps with alcoholic buffers and the final elution of the nucleic acids by means of water.
• U.S. Pat. No. 5,405,951 A and EP 0512767 A1 likewise describe the isolation of nucleic acids by incubation of the sample containing nucleic acids with an alcohol, and the subsequent incubation of the sample with a mineral material. The elution of the nucleic acids is carried out upon the addition of water heated to 60° C.
• nucleic acids is carried out by adjusting the solution containing nucleic acid with additives in such a way that it contains monovalent and multivalent cations as well as an alcohol, brings them afterwards into contact with the solid phase, subsequently washes the support and releases the nucleic acid from the solid phase.
• Ammonium chloride, sodium chloride and/or calcium chloride are used as monovalent salt components
• magnesium chloride, calcium chloride, zinc chloride and/or manganese chloride are used as multivalent salt components.
• WO/34463 A1 describes a method for isolating nucleic acids, in which the nucleic acids are bound to a solid phase, wherein the conveyance of the binding is carried out by binding buffers on the basis of so-called antichaotropic salts and an alcoholic component.
• the bound nucleic acids are washed with wash buffers known in themselves and finally eluted by the addition of a low-salt buffer.
• the ion strengths of the so-called antichaotropic salts come to at least 0.1 M-10M.
• the so-called antichaotropic salts for the binding of nucleic acids to a solid phase are, without exception, chlorides.
• WO 89/08257 A1 indicates that citrates belong to the group of antichaotropic salts.
• the property characterized in WO 89/08257 A1 is to be understood in the context of the immobilization of proteins and has no relationship at all to nucleic acids or methods for the isolation and purification of nucleic acids.
• a method and a test kit were produced which allow a highly efficient recovery of both long- and short-chain nucleic acids, and removing of undesired nucleic acids.
• the method is simple and fast to carry out.
• the invention aims particularly at such uses in which specific nucleic acid fragments from complex reaction assays (PCR, restriction assays, sequencing assays, marking assays) are purified and recovered highly efficiently and quickly, in order to convey them to a subsequent reaction.
• complex reaction assays PCR, restriction assays, sequencing assays, marking assays
• chaotropic salts are salts that destroy regular structures of liquid water based on the formation of hydrogen bonds, in that they inhibit the formation of H 2 O cage structures necessary for salvation.
• chaotropic salts are thiocyanates, iodides or perchlorates. They bring about denaturation of proteins, the increase in the solubility of nonpolar substances in water as well as the destruction of the hydrophobic interaction.
• antichaotropic components are defined as substances that enhance regular structures of liquid water based on the formation of hydrogen bonds.
• antichaotropic components are ammonium, sodium or potassium salts. They do not bring about denaturation, but enhance hydrophobic forces and the increase in hydrophobic interactions.
• non-chaotropic components are, for example salts, that are between chaotropic and anti-chaotropic salts, and include for example, magnesium chloride or aluminium chloride.
• Non-chaotropic compounds do not enhance or destroy regular structures of liquid water based on the formation of hydrogen bonds.
• Non-chaotropic substances are, for example, those in the middle of the Hofmeister series of salts.
• alcoholic components are all water-soluble alcohols such as methanol, ethanol, propanol, isopropanol, ethylene glycol, polyethylene glycol or glycerine. They can be used alone or in combination.
• the method according to the invention for the separation, purification and recovery of long- and/or short-chain nucleic acids comprises:
• salts can be introduced alone or in combination of at least two salts:
• the alcohol concentrations of the binding buffer are between 20%-90 wt. %, preferably between 40%-70 wt. %.
• the alcohol concentration includes all values and subvalues therebetween, especially including 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80 and 85 wt. %.
• Methanol, ethanol, propanol, isopropanol, ethylene glycol, polyethylene glycol or gycerin can be used as alcohols, alone or in combination.
• the ion strengths for binding to the solid phase in combination with an alcohol are less than 100 mM, preferably less than 50 mM.
• the ion strength includes all values and subvalues therebetween, especially including 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 and 95 mM.
• the solid phase can comprise glass fibre materials, silica gels, suspensions of mineral supports, functionalised magnetic particles; preferably glass fibre materials having a length of from 0.7 ⁇ m to 2 ⁇ m.
• the method according to the invention for the separation, purification and recovery of long- and/or short-chain nucleic acids includes the following steps:
• the binding buffer comprises at least one citric acid salt and at least one alcohol.
• the nucleic acids bound to the solid phase are not washed. Even more preferred is a method where no washing is performed at any stage.
• Subject of the invention is also a test kit comprising at least one alcohol, at least one citric acid salt, a solid phase and an elution buffer.
• citric acid salts in combination with at least one alcohol is the separation, purification and recovery of long- and/or short-chain nucleic acids, in particular of PCR products, restriction assays or sequencing assays.
• citric acid salts in combination with an alcohol can bind long- and short-chain nucleic acids to support materials, in particular glass fiber materials, and to remove them again.
• the inventor of the present invention has also found that the ion strengths necessary for the binding of the nucleic acids must also only be present in millimolar concentrations.
• the use of buffer solutions on the basis of an alcohol and of a citric acid salt allows a highly efficient recovery of nucleic acids from reaction assays with simultaneous separation of undesired by-products. This is particularly the case in the purification of PCR products from PCR reaction mixtures, where primers or primer-dimers in particular have to be separated from the specific amplification products.
• di-ammonium hydrogen citrate, tri-potassium citrate-monohydrate or tri-sodium citrate-dihydrate are introduced as preferred citric acid salts.
• Ammonium dihydrogen citrate, di-sodium hydrogen citrate, sodium dihydrogen citrate, di-potassium hydrogen citrate, potassium dihydrogen citrate can also be introduced.
• the citrates can be used alone or in combination.
• the ion strengths necessary for the binding are smaller than 100 mM, preferably smaller than 50 mM.
• the required alcohol concentrations of the binding buffer are between 20%-90 wt. %, preferably between 40%-70 wt. %. Different alcohols can be introduced; preferably isopropanol is used.
• the method according to the invention for the purification and recovery of DNA fragments from reaction assays is extremely fast and simple to carry out.
• the reaction assays from which the nucleic acid(s) have to be purified are mixed with the binding buffer according to the invention and subsequently conveyed to a centrifuge column (e.g. with a glass fibre material) and centrifuged. Afterwards, the centrifuge column is brought into a new collecting vessel and the DNA fragments eluted by the column surface after addition of water or a low salt buffer (10 mM Tris HCL).
• the method makes possible the purification and recovery of a broad spectrum of sizes of DNA fragments with a very high recovery rate.
• the inventor of the present invention has found that the combination according to the invention of the citric acid salts influences, in terms of ion strength, both the efficiency of the recovery as well as the selectivity regarding the DNA fragments to be purified regarding their fragment length. This observation can be excellently used to develop new areas of application. So the method according to the invention also makes possible an efficient purification of PCR products, restriction assays or sequencing assays. In the case of sequencing assays, the task is to efficiently separate dye-terminators, with simultaneous efficient recovery of the nucleic acid fragments of a wide molecular weight spectrum, in particular also the recovery of very small nucleic acid fragments. The implementation of this task-setting likewise requires only 3 min and is therefore very clearly simpler and faster than all other methods being used until now.
• WO/34463 describes, without exception, methods for the isolation and purification of genomic nucleic acids which are isolated from complex biological samples. But it does not describe a method which makes possible the purification and recovery of a nucleic acid already present. For this reason, the isolation of the nucleic acids is also always carried out with buffers which, alongside a salt component, also contain detergents, proteolytic enzymes as well as further additives, the function of which consists in the digestion (lysis) of the biological sample.
• the present invention does not refer to the isolation of genomic nucleic acids from complex biological samples.
• the subject of the present invention is the purification of reaction assays, e.g. PCR reaction assays, wherein subsequently e.g. an amplified DNA fragment with a high recovery rate has to be recovered. No lysis of a biological sample takes place, in the sense in which this is implemented with the buffers of WO/34463.
• WO/34463 describes, without exception, a method for the isolation and purification of genomic nucleic acids, which contains as an obligatory step the washing (several times) of the nucleic acids bound to the solid phase.
• Washing is not only necessary in WO/34463 in order to remove inhibiting substances from the biological samples, it is also necessary in order to wash out the high salt concentrations of the binding buffer used.
• the analysis of the exemplary embodiments shows that the lysis/binding buffers have ion strengths of >1.5 M, as a rule.
• the present invention uses clearly smaller salt concentrations (less than 0.1 M). For these reasons, the advantage according to the invention also consists in getting by without wash steps which have been necessary until now, and thereby clearly simplifying and shortening the operation.
• citric acid salts are adopted for binding nucleic acids to mineral solid phases.
• the invention is, however, based on precisely this observation, all the more so that precisely these salts make it possible to implement a binding of nucleic acids even in the presence of ion strengths of less than 100 mM, in particular less than 50 mM.
• the salts WO/34463 described do not allow for any binding of nucleic acids and their quantitative recovery if they were to be introduced in these low ion strengths.
• WO 89/08257 A1 indicates that citrates belong to the group of antichaotropic salts.
• the property characterized in this application is to be understood in the context of the immobilization of proteins and has no relationship at all to nucleic acids or methods for the isolation and purification of nucleic acids.
• Buffer 1 50 mM di-ammonium hydrogen citrate/62% isopropanol.
• Buffer 2 50 mM tri-sodium citrate-dihydrate/62% isopropanol.
• Buffer 3 50 mM tri-calcium citrate-monohydrate/62% isopropanol.
• Buffer 4 25 mM di-ammonium hydrogen citrate/62% isopropanol.
• Buffer 5 25 mM tri-sodium citrate-dihydrate/62% isopropanol.
• Buffer 6 25 mM tri-calcium citrate-monohydrate/62% isopropanol.
• the mixture was subsequently conveyed to a centrifugation column with a glass fibre filter (AF; Fa. Pall), in order to connect the desired nucleic acid fragment, and centrifuged at 10000 ⁇ g for 1 min.
• the centrifugation column was then inserted into a new 1.5 ml reaction vessel and centrifuged again for 1 min at 8000 ⁇ g after addition of an elution agent (10 mM Tris-HCL).
• the eluted PCR fragments were subsequently analysed on an Agilent Bioanalyzer, and the purity as well as the recovery rates were determined in relation to the non-purified PCR assays.
• the following table contains the recovery rates for each of the different binding buffers P1-P6.

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• 2005
  • 2005-12-07 DE DE102005059217A patent/DE102005059217B4/de not_active Revoked
• 2006
  • 2006-12-07 PL PL06830455T patent/PL1960521T3/pl unknown
  • 2006-12-07 ES ES06830455T patent/ES2373976T3/es active Active
  • 2006-12-07 EP EP06830455A patent/EP1960521B1/fr active Active
  • 2006-12-07 WO PCT/EP2006/069451 patent/WO2007065934A1/fr active Application Filing
  • 2006-12-07 AT AT06830455T patent/ATE525466T1/de active
  • 2006-12-07 DK DK06830455.9T patent/DK1960521T3/da active
• 2008
  • 2008-06-09 US US12/135,465 patent/US20090068662A1/en not_active Abandoned
• 2010
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