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

Definitions

• the present invention relates to a method and a kit for the purification of
• nucleic acids with a nucleic acid-binding carrier material are described.
• a known process for purifying nucleic acids is the so-called "charge-switch process.”
• a nucleic acid-binding phase which contains predominantly weakly basic polymers such as poly bis-tris, poly tris, polyhistidine, polyhydroxylated amines, chitosan or triethanolamine is used in a first
• the pH value is brought into contact with a nucleic acid-containing sample in which the nucleic acid-binding phase has a positive charge, which favors the binding of the negatively charged nucleic acids to the phase
• Charge-switch principle set a second pH, which is higher than the pKs value of the nucleic acid-binding phase, to invert the positive charge, or to neutralize.
• the pH adjustment above the pKs value of the nucleic acid binding groups of the solid phase promotes the detachment of the bound nucleic acids from the nucleic acid binding phase.
• the purified nucleic acids are usually processed further. These include, for example, amplifications, such as the polymerase chain reaction (PCR), enzymatic reactions, such as restriction, ligation, phosphorylation, dephosphorylation or RNA transcription, hybrid capture assays and electrophoresis. These "downstream" reactions are often less tolerant of higher salt concentrations, so that often a desalting step must be performed prior to processing the nucleic acids. For direct reuse of the purified nucleic acids without additional rebuffering or desalting steps, it is therefore desirable that the elution buffer be not too rich in salt and furthermore not too basic.
• amplifications such as the polymerase chain reaction (PCR)
• enzymatic reactions such as restriction, ligation, phosphorylation, dephosphorylation or RNA transcription
• hybrid capture assays hybrid capture assays
• electrophoresis electrophoresis
• the elution capacity increases with the pH and salt concentration of the elution buffer.
• the chromatographic requirements are therefore in direct contradiction to the requirements regarding DNA processing.
• the prior art therefore attempts in particular to promote the elution of the nucleic acids by selecting suitable nucleic acid binding groups and pH conditions.
• the present invention is therefore based on the object to provide a method for the purification of nucleic acids, which allows the direct further processing of the sample.
• a method is to be provided which allows elution at low salt concentrations.
• the present invention solves this problem by a method for purification of nucleic acids, in which a specific nucleic acid binding phase is used.
• the nucleic acid-binding phase has nucleic acid-binding groups A which have a pKa value of 8 to 13, and also bond-weakening groups N which are neutrally charged at the binding pH value used.
• the process for purification with this nucleic acid binding phase comprises the following steps:
• the present invention relates to the purification of nucleic acids by means of a nucleic acid-binding phase which, in addition to nucleic acid-binding groups A, is also tion-weakening groups N has.
• the nucleic acid binding groups A according to the invention have a pKa of 8 to 13. The binding of the nucleic acids takes place at a pH below the pKa value of at least one of these groups A. The groups A therefore take up a proton and are thereby positively charged, which leads to the fact that the nucleic acid-binding phase negatively charged
• Nucleic acids can bind. Elution occurs at a pH above the binding pH, thereby decreasing the positive charge of the nucleic acid binding phase.
• the additionally present groups N are charge-neutral at the binding pH and preferably also at the elution pH.
• the groups N therefore influence the binding strength of the groups A in several respects: (1) They interrupt the arrangement of the groups A and thus influence the binding strength of the nucleic acids to the nucleic acid binding phase by the reduced density of A groups. (2) They can be described as functional groups that are weak (for example by van der Waals).
• the groups N can cause a steric shielding of the nucleic acid binding groups A with increasing size or number and thus a reduction of the binding strength. The groups N therefore cause a uniform, pH-independent reduction of
• Nucleic acid binding strength The nucleic acids bound thereby to a reduced extent can be more easily detached from the nucleic acid binding phase.
• the groups N have the function of reducing the binding strength or number of groups A in a targeted and controlled manner and thus to set a desired frequency in the nucleic acid-binding phase according to the invention. The higher the proportion of
• Groups N is, the lower the proportion of groups A and the lower the charge density and the weaker nucleic acids are bound to the nucleic acid binding phase.
• the ratio of groups A to groups N can be adjusted in a targeted manner by the production process, so that it is possible to obtain the desired nucleic acid binding strength in advance or by the process for preparing the corresponding nucleic acid-binding phase (for example by polymerization, (poly -) condensation and in particular by the coating of a support material) set.
• the purification process according to the invention advantageously allows the elution at low salt concentrations, or low ionic strength, so that the direct further processing in a subsequent biotechnological method, in particular a PCR reaction is possible.
• the concept of weakening the binding of the nucleic acids by the use of the groups N leads to surprisingly good results.
• the prior art requires optimum binding of the nucleic acid for the process optimization and then dedicates itself to the optimization of the binding and in particular elution conditions.
• a weakening of the bond strength was considered to be disadvantageous in relation to the expected yield, for example because the nucleic acids in the binding step could only insufficiently bind to the nucleic acid-binding phase or be eluted during washing. From these points of view, the concept according to the invention of the successful and gentle purification of nucleic acids, in spite of the reduction of the binding strength by the presence of the groups N, is to be regarded as extremely surprising.
• the proportion of A groups based on the N groups may be 1% to 99%, 1 to 50%, preferably 1% to 25%.
• the nucleic acid binding phase is modified to introduce groups A and N. According to the invention there are various embodiments for this purpose, which are explained in more detail below.
• the nucleic acid-binding phase is provided with different ligands (ligand I and ligand II), the ligands I having at least one group A and the ligands II having at least one group N.
• ligand I and ligand II ligands
• the ligands I having at least one group A
• the ligands II having at least one group N.
• FIG. 1A This embodiment is illustrated in FIG. 1A.
• the groups A and N are then in the immediate vicinity, wherein polymer coatings can be formed.
• the presence of the N-group-carrying ligands II decreases the number of groups A, thereby reducing the binding strength of the nucleic acids. This particular when using a substrate. Due to the groups N, therefore, the density of the groups A on the support surface is reduced, which results in a reduction of the binding affinity to the nucleic acids.
• the term "ligand” or “ligand” denotes a surface functionalization by which a carrier material with at least one group A and / or a group N is preferably modified in order to provide the nucleic acid-binding phase according to the invention.
• the ligands I and II are preferably bound to the support material and may, for example, be present as monomers, dimers, oligomers or polymers of reactive single molecules.
• the groups A and the groups N as part of with the
• Support material reacting compounds directly or via a linker or spacer bonded to a carrier material.
• this modification is achieved in that the carrier material is brought into contact with a mixture comprising the different ligands I and II.
• the ligands can also form a polymer coating.
• the support material may further be provided with groups A and initiator molecules T, wherein one or more groups N are bonded to the initiator molecules. This embodiment is shown in Figure 1 B). The groups N are then present with respect to the surface of the support material "above" the groups A. It is also possible to attach the groups A via initiator molecules as well.
• the carrier material is modified with nucleic acid binding ligands, wherein within a ligand one or more groups A and one or more groups N occur.
• nucleic acid binding ligands wherein within a ligand one or more groups A and one or more groups N occur.
• the support material is provided with groups A (either directly or for example bound via an initiator molecule T) which carry one or more compounds which contain a group N (see also FIG. 2A).
• groups N can also be in the form of an unbranched chain or as a branched tree structure or as a corresponding mixture.
• groups N for example, 1 to 100 groups N, 1-20, preferably 1-10 and particularly preferably 1-5 groups N per ligand may occur.
• other groups A and N can also be introduced.
• This linking strategy results in a relatively homogeneous bond plane with nearly pure A-group population and one or more planes joining outward and reducing the bond strength of groups A through N-group population.
• the support material is provided with groups A (either directly or for example bound via an initiator molecule T) which carry a mixture of N groups and A groups (see also FIG. 2B).
• groups A can first be bound to the carrier material.
• a - containing fuselage ligands are then linked compounds which introduce at least one or more groups N.
• the groups N may be randomly distributed (see, for example, Figure 2B, right figure), have an alternating sequence (see, for example. Figure 2B, left figure) or be arranged as a block copolymer. Also combinations are possible. Suitable methods for forming the corresponding A and N-containing functionalizations / ligands are described in detail below.
• the support material is modified with groups A and N, wherein the groups A are sterically shielded by compounds which have at least one, preferably a plurality of groups N. Also for this embodiment, there are different variants.
• the steric shielding preferably takes place by means of oligomers or polymers having groups N. These can be bound, for example, to the ligands having the group (s) A (see also FIG. 3B). However, they can also be arranged adjacent thereto, provided the steric shielding is ensured (see also FIG. 3A).
• the N-group oligomers / polymers shield groups A by forming more or less ordered secondary structures (e.g., "random coils" or helices) as a kind of cap against the ambient environment and thereby attenuate the binding of the nucleic acids
• Oligomers / polymers may also be formed from the N groups and further introduced as block copolymers.
• initiator groups T for example by silanization
• graft groups A containing monomers by means of ATRP (atom transfer radical polymerization).
• ATRP atom transfer radical polymerization
• a second monomer having neutral groups N can be introduced.
• a copolymer is then formed on the support consisting of a first homopolymer carrying anion exchange groups and a second homopolymer linked to the first and bearing neutral groups N. The amount of the second monomer carrying the groups N can be used to control the length of the polymer chain formed and thus the strength of the steric shielding.
• the extent of steric shielding can therefore be determined by the chain length and the
• the monomers described in this application can be used to form the N-bearing oligomers / polymers , which will be described in detail.
• Particularly suitable are polyacrylates.
• Preferred polymers used are polyacrylates according to the following formula:
• a particularly suitable reagent for achieving steric shielding is glycidyl methacrylate, from which neutral diol groups N are formed after hydrolysis.
• an initiator molecule T can be applied.
• a first monomer for example an N, N-dimethylaminopropyl methacrylate, for example, is polymerized on the support by means of ATRP.
• These groups are then sterically screened in a second polymerization step.
• a second monomer can be introduced, for example glycidyl methacrylate from the diol groups N which are neutral after hydrolysis.
• the steric effect prevents the close contact of the nucleic acid with the A groups, so that they can not be so strongly bound and eluted from the nucleic acid binding phase at low ionic strength and thus low salt concentration.
• the steric shielding of the N groups can also be achieved by "bulky” substituents, for example branched alkyl radicals, such as isopropyl, diisopropyl, tertiary-butyl, aliphatic or aromatic rings, either as carbon rings or as heterocycles
• "bulky” substituents for example branched alkyl radicals, such as isopropyl, diisopropyl, tertiary-butyl, aliphatic or aromatic rings, either as carbon rings or as heterocycles
• These sterically hindering groups N can also be used in the form of oligomers / polymers.
• the steric shielding can therefore be controlled in particular by (i) the proportion of additional introduced groups N; (ii) the structure of the compounds carrying the group N; (iii) the selection or structure of group N and (iv) the chain length of the N-group oligomers / polymers.
• a particularly good steric shielding of the group (s) A can thus take place by groups N which occur in the same ligand or are arranged adjacent thereto. Also, a bond to the group A is possible.
• the steric shielding increases with the size of the group (n) N-containing compound and also with the size of the group N, with both an increase in the chain length, as well as a stronger branching of the carbon chains or an increase in the ring size in cyclic groups increases the steric shielding.
• the carrier material is modified with a combination of all the linking strategies described above. Examples are in
• Figure 4 shown.
• the support material in direct attachment of the groups A, the sterically shielding groups N and initiator molecules T, the initiator molecules are still equipped with other sterically shielding groups N have (see, for example. Figure 4 A).
• This type of linking can be carried out, for example, with the usual groups N (see, for example, FIG. 4B).
• the support material can, for example, be equipped with groups A (bonded directly or via an initiator molecule T) and attached thereto carry a mixture of N groups and A groups, where the groups N are completely or partially present as a sterically inhibiting group (see FIG 4D and 4E).
• the method is carried out with a nucleic acid-binding phase having a support material, which is modified with groups A, wherein only a part of the support material is occupied by groups A.
• Figure 5 shows an embodiment of this concept. This embodiment also makes it possible to elute the nucleic acids under conditions which permit the direct further processing of the nucleic acids, because in particular low salt concentrations can be used in order to elute the nucleic acid from the nucleic acid-binding phase.
• the deficit of A groups on the support material according to the invention can be achieved by using the compound which introduces the group A in excess relative to the support material.
• the carrier material has a smaller amount of potential binding sites to which the groups A can be attached.
• Another embodiment involves the chemical or physical pretreatment of the support material, which causes a reduction in the binding sites or a reduced reactivity of the binding sites.
• the group A-bearing compound may be applied in admixture with another compound that also binds to the support material. Due to the competing binding, the proportion of A groups on the surface of the support material decreases.
• the A-group deficient embodiment may be further combined with one or more of the above-described embodiments for modifying the support material with groups A and N.
• the equipment of the carrier with groups A in the deficit is particularly advantageous when using a silica support material, since it has silanol groups.
• the groups A are introduced by means of silanes or a silane mixture.
• a minimum amount of silane is needed for the complete coating of the carrier. This minimum amount is defined by the specific surface area of the carrier material. If the amount of silane (s) is insufficient to cover the surface of the support, the charge density can be reduced. By the amount of silane to be applied, therefore, the amount of groups A and thereby the binding strength of the nucleic acid-binding phase can be adjusted.
• the support material has a silica surface which is coated with a silane containing groups A, the amount of silane being 0.1 to 50 ⁇ mol (micromole, also referred to herein as umol), preferably 0.1 to 10 ⁇ mol. It is crucial to reduce the amount of silane until the elution at lower ionic strengths or lower (desired) salt concentrations is possible. This can, for example, be experimentally tested and checked by means of a chromatogram. The amount of silane can therefore preferably be optimized depending on the carrier used in order to achieve the desired binding / elution properties.
• the nucleic acid binding phase is a solid phase.
• the groups A, the weakening groups N and the initiator molecules T may for example be bound to a solid support material. Details are described below in detail.
• the use of a solid phase facilitates the separation of the bound nucleic acids from the sample. According to one embodiment, therefore, a separation of the solid phase or of the unbound supernatant takes place after the binding of the nucleic acids.
• Suitable carriers for the nucleic acid binding groups are, for example, oxidic materials.
• oxides such as Al 2 O 3 , TiO 2 , ZrO 2 , Ta 2 O 5 , SiO 2 and polysilicic acid are particularly suitable, with SiO 2 or polysilicic acids as carrier material being preferred.
• the carriers also include organic polymers such as polystyrene and its derivatives,
• nucleic acid-binding groups may also be linked to polysaccharides, in particular hydrogels such as agarose, cellulose, dextran, Sephadex, Sephacryl, chitosan.
• nucleic acid-binding groups may also be attached to inorganic carriers such as, for example, glass or metal surfaces, such as, for example, glass or metal surfaces. Gold, to be bound. Magnetic particles are particularly advantageous in handling.
• nucleic acid-binding groups A and / or the groups N can be bound to these carriers directly or else via other chemical molecules, for example initiator molecules or linkers. They can also be part of a larger molecule. If the carrier material does not have suitable functional groups as binding sites, these can be introduced in a manner known per se.
• carrier materials include non-magnetic and magnetic particles, column materials, membranes, and surface coatings.
• functionalized carriers such as tubes, membranes, nonwovens, paper,
• Reaction vessels such as PCR tubes, "Eppendorf tubes”, multiplates, chips and microarrays called.
• the nucleic acid binding phase is positively charged in both binding and elution.
• Soluble polymers may, for example, have alternating and random groups A and N (see, for example, FIG. 2B).
• the side chains of the A groups may also be screened off by groups N, in particular alkyl radicals, for example diisopropyl radicals on the nitrogen.
• the nucleic acid binding groups A are ion exchangers according to one embodiment; preferably anion exchangers.
• Preferred groups A which have proven useful for the binding of nucleic acids are amino groups, preference being given to primary, secondary and tertiary amino groups. These may be substituted or unsubstituted.
• the amines may bear substituents, for example alkyl, alkenyl, alkynyl or aromatic substituents, moreover, the hydrocarbon chains may also be closed in a ring.
• the hydrocarbon chains can also have heteroatoms, such as oxygen, nitrogen, sulfur or silicon, or branches.
• the amino groups as groups A pKs values of 8 to 13, preferably 9 to 13, particularly preferably from 10 to 12 on.
• the group A may be part of a compound which forms an oligomer or polymer by polymerization or condensation and is therefore particularly suitable for the formation of the ligands.
• chain-forming compounds are amino-containing acrylates such as N- (3-aminomethyl) methacrylamide, N- (3-aminoethyl) methacrylamide, N- (3-aminopropyl) methacrylamide, N- (3-aminoisopropyl) methacrylamide N, N-dimethylacrylamide, N, N-diethylacrylamide, N, N Diisopropylacrylamide N, N- (dimethylamino) ethylacrylamide, N, N- (dimethylamino) ethyl acrylate, N, N- (dimethylamino) ethylmethacrylamide, N, N- (dimethylamino) ethyl methacrylate,
• N, N- (dimethylamino) propylacrylamides N, N- (dimethylamino) propyl acrylate, N, N- (dimethylamino) propylmethacrylamides, N, N- (dimethylamino) propylmethacrylate, N, N- (diethylamino) ethylacrylamide, N, N- (Diethylamino) ethylacrylate, N, N- (diethylamino) ethylmethacrylamide, N, N- (diethylamino) ethylmethacrylate, N, N- (diethylamino) propylacrylamide, N, N- (diethylamino) propylacrylate,
• DMAEMA 2- (dimethylamino) ethyl methacrylate
• 2- (diisopropylamino) ethyl methacrylate 2- (dimethylamino) ethyl methacrylate (DMAEMA) and 2- (diisopropylamino) ethyl methacrylate.
• N N- (dimethylamino) propyl methacrylate is particularly preferred.
• the group A may be present in a silane, preferably in a reactive silane.
• Reactive silanes are compounds which have hydrolytically unstable Si bonds, for example Si-N or Si-O bonds.
• Examples of reactive silanes containing at least one group A are:
• n 1 to 5
• R is a C1 to C6, preferably a C1 to C3 alkyl group; and * amino, aminomethyl, aminoethyl, aminopropyl, dimethylamino, diethylamino,
• Corresponding reactive silanes can be used to introduce groups A. Particular preference is given to diethylaminopropyltrimethoxysilane (DEAPS), dimethylaminopropyltrimethoxysilane and N, N-diisopropylaminopropyltrimethoxysilane.
• DEAPS diethylaminopropyltrimethoxysilane
• N N-diisopropylaminopropyltrimethoxysilane.
• the binding of the nucleic acids takes place at a pH of 2 to 8, preferably 4 to 7.5.
• This information refers to the pH during binding and thus in the sample.
• the method according to the invention can therefore be carried out even under very mild conditions, and can be carried out almost in the neutral range. Due to the fact that the protonatable groups of the nucleic acid-binding phase have a pKa value of 8 to 13, preferably 9 to 13 and particularly preferably 10 to 12, these are sufficiently positively charged even at relatively neutral pH values, to allow the effective binding of the nucleic acids. Therefore, the bond can - if desired - be carried out under very mild conditions.
• the binding may be preceded by at least one customary lysis step in order to liberate the nucleic acids.
• nucleic acid release occurs at a pH above the binding pH.
• groups A are less positively charged in the elution, whereby the release of the nucleic acids is favored.
• the reduction in the proportion of groups A and / or the presence of the bond-weakening groups N causes a reduced binding of the nucleic acids to the support material. As stated above, this has the consequence that the elution can be carried out even at low salt concentrations.
• the elution pH is preferably below the pK value of groups A.
• the elution preferably takes place at a pH of 8 to 11, preferably at a pH of 8 to 11, depending on the nucleic acid-binding group A or nucleic acid-binding phase used. Value of 8.0 to 9, more preferably 8.5 to 9. In principle, however, higher pH values could also be used. At the preferred low pH values, however, particularly advantageous results are achieved, since the nucleic acids can still be released at low salt concentrations and the conditions are also mild. To enable the direct further processing of the isolated nucleic acids in the elution buffer, this preferably has a low salt concentration as stated. This is made possible by the inventive design of the nucleic acid-binding phase.
• the salt concentration is therefore, according to one embodiment, from 1 mM to 1000 mM, more preferably from 1 mM to 200 mM, 1 mM to 250 mM, or 1 mM to 100 mM.
• Suitable salts may include chlorides of the alkali and alkaline earth metals or ammonium, other salts of mineral acids, acetates, borates and compounds such as tris, bis-tris and organic buffers such as MIS, CHAPS, HEPES and the like. The same applies to the binding buffer.
• Suitable substances for elution are also known in the art.
• the salt concentration is unchanged in the binding step and in the elution step or is slightly raised during the elution. Preferably, however, the concentration is not increased such that the subsequent reactions are hindered.
• the temperature during binding and elution may be the same or raised during elution.
• the wash buffers preferably in a concentration of 1 mM to 1000 mM, more preferably from 1 mM to 200 mM, 1 mM to 250 mM, or 1 mM to 100 mM.
• the buffer may be organic compounds such as carbohydrates and preferably organic solvents such as, for example, alcohols, polyols, polyethylene glycols, ethers, polyethers, dimethyl sulfoxide, acetone or acetonitrile.
• the wash buffers should not have interfering amounts of the corresponding organic ingredients so as not to interfere with the downstream applications.
• Charge-neutralizing groups N such as hydroxyl groups, diol groups, triol groups, saccharides, epoxide groups, C 1 -C 6 alkyl, alkene, or alkyne groups, polyol groups, ethers, polyethers, halides or imides can serve as bond-weakening groups N.
• hydrophilic groups N the ion exchanger remains readily wettable with aqueous buffers.
• the group N may be part of a compound which forms an oligomer or polymer by polymerization or condensation.
• Examples of compounds by which groups N can be introduced are acrylates such as butyl acrylate, propyl acrylate, ethyl acrylate, methyl acrylate, glycidyl methacrylate, hydroxyethyl methacrylate (HEMA), glycidoxypropyl methacrylate, glycerol monomethacrylate (mixture of isomers), glycol monomethacrylate and N-acryloxysuccinimide.
• acrylates such as butyl acrylate, propyl acrylate, ethyl acrylate, methyl acrylate, glycidyl methacrylate, hydroxyethyl methacrylate (HEMA), glycidoxypropyl methacrylate, glycerol monomethacrylate (mixture of isomers), glycol monomethacrylate and N-acryloxysuccinimide.
• the group N can also be present in a silane, preferably in a reactive silane.
• Examples of reactive silanes containing the group N are:
• R is a C1 to C6, preferably C1 to C3 alkyl group
• the carrier material comprises initiator groups T, which are at least partially functionalized with compounds which have groups A and / or N. Suitable examples will be explained in detail below.
• the invention further relates to the synthesis or a process for the preparation of a corresponding nucleic acid-binding carrier material. There are different ones for this
• the support material is functionalized with a mixture of at least two different ligands I and II, wherein the ligands I have at least one group A and the ligands II have or represent at least one group N. Details of this embodiment have already been explained in detail above. We refer to the above revelation.
• the compounds carrying a group A are selected or modified so that no further molecules can be bound.
• compounds are then added which carry at least one group N and bind to the initiator molecules T.
• the binding of initiator molecules T to the carrier material takes place in a first step.
• the polymerization can also be controlled so that the groups A and N are incorporated as oligomers, so that block copolymers arise.
• An example of a controlled polymerization reaction which can be used according to the invention is, for example, the so-called "atom transfer radical polymerization" (ATRP) .
• ATRP atom transfer radical polymerization
• the ATRP is characterized in that the concentration of free radicals by addition of a transition metal complex and in combination with a Atom transfer process with an organohalide is lowered so far that chain termination reactions, such as disproportionation or recombination are largely suppressed.
• the functionalization of the carrier material with initiator groups T takes place in a first step. The addition of compounds having at least one group A and attachment of these to the initiator molecules T ensues.
• the groups A are shielded by linking at least one compound which has at least one group N.
• the groups N are preferably present in the form of ON gomers / polymers (see above) which, owing to their three-dimensional structure, cause a steric shielding of the groups A. Details are explained above in connection with the method according to the invention.
• the alternatives AE can also be used in any combination. Details of the nucleic acid binding groups A, the groups N and the initiator molecules T are described in detail above and also apply in the context with the methods of carrier modification listed herein and identify the ingredients used therein. We refer to the above revelation.
• these groups are introduced via monofunctional, bi- or trifunctional reactive silanes or by means of a mixture of at least two differently functional reactive silanes.
• reactive silanes for example, aminosilanes, disilazanes, chlorosilanes or alkoxysilanes can be used.
• the proportion of A groups based on the N groups is 1% to 99%, 1 to 50%, preferably 1% to 25%.
• Trifunctional and bifunctional reactive silanes tend to form thick layers crosslinked by polycondensation on the support.
• monofunctional reactive silanes for example, react with the silanol groups of the support material to form siloxane (Si-O-Si) bonds. This then leads to more monomolecular layers on the support.
• the reaction of the support material with reactive silanes can be carried out in the gas phase or in suspension in a solvent, it being possible for the latter to use organic and preferably aqueous solvents, depending on the chemistry of the reactive silane.
• the support material is first modified with initiator molecules T and in the next step, the groups A and / or N are introduced in the form of monomers.
• the initiator molecules T can serve as a point of attachment for further compounds which are introduced, for example, via (poly) condensation or polymerization, in particular a free-radical polymerization such as in particular ATRP (atom transfer radical polymerization). Examples of initiator molecules T are:
• 2- (chloromethyl) allyltrimethoxysilane or [3- (2-bromoisobutyryl) propyl] ethoxydimethylsilane (BPDS) is used as the initiator molecule.
• R is a C1 to C6, preferably C1 to C3 alkyl group, in particular methyl, ethyl,
• Propyl is i-propyl
• halide-containing silanes by means of ATRP (atom transfer
• ATRP is a special form of Living / Controlled Free Radical Polymerization (LFRP) in which the free radical concentration is lowered by the addition of a transition metal complex and in combination with an atom transfer process with an organohalide, such that chain termination reactions, such as Disproportionation or recombination, be pushed back as far as possible.
• LFRP Living / Controlled Free Radical Polymerization
• this method makes it possible to specifically set the charge density of the carrier surface and thus to influence the binding strength of the nucleic acids.
• the groups A and / or N can be selectively applied.
• nucleic acid-binding carrier materials can be used in particular in the purification process according to the invention.
• the invention further relates to the use of a nucleic acid-binding phase according to the invention for the purification of nucleic acids.
• Nucleic acids within the meaning of the invention include in particular DNA and RNA, in particular genomic DNA, plasmid DNA, as well as PCR fragments, cDNA, miRNA, siRNA, as well as oligonucleotides and modified nucleic acids such as. PMA or LMA. It is also possible to purify viral or bacterial RNA and DNA or nucleic acids from human, animal or plant sources. Furthermore, for a purification according to the invention, DNA / RNA hybrids are also suitable as well as modified nucleic acids.
• the invention further provides a kit for purifying nucleic acids, which comprises a nucleic acid-binding carrier material according to the invention which has nucleic acid-binding groups A with at least one protonatable group having a pKa value of 8 to 13 , preferably 9 to 13, particularly preferably 10 to 12.
• a nucleic acid-binding carrier material according to the invention which has nucleic acid-binding groups A with at least one protonatable group having a pKa value of 8 to 13 , preferably 9 to 13, particularly preferably 10 to 12.
• the kit may further comprise binding, washing and / or elution buffers such as those described above in connection with the purification process.
• it may have lysis and neutralization buffers.
• the kit has a binding buffer, which preferably has at least one of the following features:
• the kit further comprises a washing buffer, which preferably has at least one of the following features:
• (c) it is selected from the group consisting of water, biological buffers, organic buffers, especially Tris, Tris-bis, MIS, MOPS, CHAPS and HEPES.
• the kit further comprises an elution buffer, which preferably has at least one of the following features:
• (c) it is selected from the group consisting of water, biological buffers, organic buffers, especially Tris, Tris-bis, MIS, MOPS, CHAPS and
• nucleic acid binding phase Details of the nucleic acid binding phase as well as the elution conditions are described in detail above and also apply in the context of the kit according to the invention and characterize the components / buffers used therein. We refer to the above revelation.
• Kits according to the invention can be used in particular in the context of the method according to the invention.
• the present methods, kits and nucleic acid-binding solid phases can be used in particular in the field of molecular biology, molecular diagnostics, in forensics, in food analysis and in applied testing.
• the application of the kit according to the invention allows the immediate further processing of the purified nucleic acids in "downstream" applications, in particular in a PCR reaction.
• the pH of the nucleic acid-binding phase can be optimally adjusted with respect to the elution conditions. Accordingly, the elution profile of the nucleic acid-binding phase, in particular the salt concentration and the elution pH, can be controlled or adjusted.
• Nucleic acids that can be purified with the systems of the invention can be present in body fluids such as blood, urine, stool, saliva, in biological sources such as tissue, cells, especially animal cells, human cells, plant cells, bacterial cells and the like, organs such as liver, kidney or lung , In addition, the nucleic acid from support materials such as Swabs, PapSmears, as well as stabilizing
• nucleic acids can be recovered from plant material, bacterial lysates, paraffin-embedded tissue, aqueous solutions or gels.
• the eluted nucleic acids can preferably be further processed directly, so for example in the context of a PCR, RT-PCR, a restriction digestion or a transcription are used. Further purification is not required as long as the elution buffers are designed as described above and preferably have a low salt concentration.
• Figure 1 Schematic overview of the equipment of the support material with the nucleic acid binding groups A and the weakening groups N, which occur on separate ligands (see also claim 2 (i)).
• FIG. 2 Schematic overview of the equipment of the carrier material with the nucleic acid binding groups A and the binding-inhibiting groups N, which occur mixed within a ligand (see also claim 2 (N)).
• A represents a carrier material which has been first provided with groups A and then with groups N;
• B represents an alternating sequence of A and N within a ligand.
• FIG. 3 Schematic overview of the equipment of the carrier material with the nucleic acid-binding groups A and the sterically shielding groups N, which can be combined with the nucleic acid-binding groups A according to the schemes shown in FIGS. 1 and 2. See claim 2 (iii).
• FIG. 4 Schematic overview of the exemplary configuration of the carrier material with the nucleic acid-binding groups A and the binding-inhibiting and / or sterically-shielding groups N, these being formed from a combination of the schemes illustrated in FIGS. 1 to 3. See claim 2 (iv).
• FIG. 5 Schematic overview of the equipment of the carrier material with a deficit of nucleic acid-binding groups A. See claim 3 (iv).
• FIG. 6 Elution profile according to Example A 3 a) at different pH values.
• plasmid DNA was used as model systems for nucleic acids.
• Carrier material Silica / silica gel with a pore size of approx. 150nm. The specific surface area is approx. 25m 2 / g.
• Coating Reagents 2- (chloromethyl) allyltrimethoxysilane, DEAPS; QSP1 buffer (acid acetate buffer).
• the silica gel was separated on a P3 frit and washed successively with 32.3 g Tris / NaCl buffer, twice with 30 ml deionized water, twice with 35 ml methanol and finally with 30 ml methanol.
• the final support AAK01 -10 was dried overnight at 125 ° C.
• the identical support material was modified exclusively with the compound A bearing DEAPS group.
• 70 ml of water, 2.5 ml of QSP1 buffer and 825 ⁇ l of DEAPS with a resulting pH of 5.5 were placed in a three-necked flask and 18 g of silica gel were added.
• the mixture was heated with stirring within 20 min at 95 ° C, stirred for 4 hours at this temperature and then cooled for 1 h with stirring.
• the silica gel was separated on a P3 frit and washed successively with 32.3 g Tris / NaCl buffer, twice with 30 ml deionized water, twice with 35 ml methanol and finally with 30 ml methanol.
• the finished support material AAK01 -30 was dried overnight at 125 ° C. Purification of plasmid DNA with the modified support material AAK01 -10 with determination of the elution point
• the binding was carried out in a buffer 50 mM Tris-HCl pH 7.0, 15% ethanol. For elution, a step gradient of 0% to 100% buffer B was achieved within 23 min
• the support material coated exclusively with the protonatable group A shows the elution point at 160 mM NaCl at a pH of 7.0.
• DEAPS protonatable group A
• the elution point is -700 mM NaCl. The ionic strength necessary for the elution was thus reduced by more than 50%.
• HEMA Hydroxyethyl Methacrylate
• HEMA oligomers are built up on the already bound chlorosilane.
• the mixture is stirred for 4 hours at room temperature and then aspirated directly via a P3 glass filter frit.
• the material is first washed several times with 100 mM NaCl / 100 mM EDTA buffer, then washed with deionised water, once with 8 ml of methanol, and twice with 7 ml of methanol.
• the finished carrier material is first washed several times with 100 mM NaCl / 100 mM EDTA buffer, then washed with deionised water, once with 8 ml of methanol, and twice with 7 ml of methanol.
• AAK01 -11 was dried for 14 hours at 60 0 C.
• the support material AAK01-30 modified with DEAPS was reacted and worked up in the same way with HEMA.
• the final product of this reaction was designated AAK01-31. Since the DEAPS as functional group carries only one tertiary amine, but no polymerizable allyl group, no HEMA coupling should take place.
• the support material used was FF silica beads.
• composition elution buffer 2OmM potassium chloride; 50 mM Tris adjusted to pH X in water;
• composition QN buffer 160 mM sodium chloride; 50 mM Tris 15% ethanol in water adjusted to pH 7
• the carrier material used were the magnetic silica beads MagAttract Beads G from QIAGEN:
• composition QN buffer 160 mM sodium chloride; 50 mM Tris 15% ethanol in water adjusted to pH 7
• the mixture reacted with stirring (100 1 / min) at 85 ° C (oil bath temperature) overnight (16h). Thereafter, the oil bath was removed and the suspension was cooled under stirring within 1/4 h.
• the modified support material / the silica gel initiator (KI04) was separated off via a P3 frit and washed there with 7 ⁇ 20 ml of hexane. Thereafter the modified support material at 40 0 C overnight (12h) was dried in a vacuum drying cabinet.
• the starting material for this series of experiments were the silica gel initiators (KI04d) with BPDS as initiator.
• the apparatus was evacuated by means of a membrane pump for 1 minute and then filled with argon. This procedure was performed 3 times. Subsequently, a slight stream of argon was continuously fed into the apparatus to prevent atmospheric oxygen from entering.
• the mixture of ligand and monomer (s) was first treated for 5 minutes in an ultrasonic bath and then the gas space was evacuated for 5 minutes by means of a high vacuum.
• the ligand-monomer mixture was pipetted to the submitted bdy / copper-1-bromide.
• the apparatus was evacuated by means of a membrane pump for 1 minute and then filled with argon. This procedure was performed 3 times. Subsequently, a slight stream of argon was continuously fed into the apparatus. With stirring (200 1 / min), the copper-ligand complex was dissolved within 15 min. Thereafter, 1.2 g of the silica gel initiator Cl 04d were added.
• the apparatus was evacuated by means of a membrane pump for 1 minute and then filled with argon. This procedure was performed 3 times. Subsequently, a slight stream of argon was continuously fed into the apparatus.
• silica gel The support material with the grown-up groups (referred to below as silica gel) was separated directly via a P3 frit and subsequently with one after the other
• silica gel was dried at 40 0 C in a vacuum oven to constant weight.
• KI04d-05 1.29g
• KI04d-07 1.21g
• the resulting anion exchangers (the modified support material or silica gel) and the starting material (the silica gel initiator) were investigated with regard to their pDNA binding capacity, as explained in more detail below, and corresponding elution profiles were recorded.
• silica gel 50.00 mg (+/- 5 mg) of silica gel were weighed into a 2 ml Safe Lock reaction tube from Eppendorf (referred to below as Eppendorf tube), and for a DNA solution, 100 ⁇ g pcmvb per silica gel weighing 100 ⁇ g each with the binding buffer made up to 1 ml.
• the silica gel DNA suspension with a truncated tip was pipetted into a prepared lower frit Tip 20 (hereinafter referred to as Tips) under which a 2 ml Eppendorf tube was provided for collecting the supernatant.
• the carrier material / silica gel (KI04d-01) coated exclusively with the groups A (DMAEMA) showed the elution point at 1032 mM NaCl at a pH of 8.5. Thus, quite high salt concentrations were needed to elute the nucleic acids.
• the elution point was ⁇ 580 mM NaCl. The ionic strength required for the elution was thus reduced by almost 50%.
• the starting material for this series of experiments were the silica gel initiators (KI04e) with BPDS as initiator.
• the apparatus was evacuated by means of a membrane pump for 1 minute and then filled with argon. This procedure was performed 3 times. Subsequently, a slight stream of argon was continuously fed into the apparatus to prevent airborne material from entering.
• the DMF-DMAEMA mixture was pipetted to the bdy / copper-1-bromide.
• the apparatus was evacuated by means of a membrane pump for 1 minute and then filled with argon. This procedure was performed 3 times. Subsequently, a slight stream of argon was continuously fed into the apparatus.
• the copper-ligand complex was dissolved within 15 min. Thereafter, 9 g of the silica gel initiator KI04e were added.
• the apparatus was evacuated by means of a membrane pump for 1 minute and then filled with argon. This procedure was performed 3 times. Subsequently, a slight stream of argon was continuously fed into the apparatus.
• the silica gel KI04e-01 with the grown groups A was separated directly via a P3 frit and subsequently successively with 5 ⁇ 60 ml DMF 5 ⁇ 60 ml THF
• Washed 3 x 60 ml of water Washed 3 x 60 ml of water.
• the apparatus was evacuated by means of a membrane pump for 1 minute and then filled with argon. This procedure was performed 3 times. Subsequently, a slight stream of argon was continuously fed into the apparatus to prevent atmospheric oxygen from entering.
• the DMF-HEMA mixture was pipetted to the bdy / copper-1-bromide.
• the apparatus was evacuated by means of a membrane pump for 1 minute and then filled with argon. This procedure was performed 3 times. Subsequently, a slight stream of argon was continuously fed into the apparatus.
• the copper-ligand complex was dissolved within 15 min. Thereafter, 1.2 g of the silica gel initiator KI04e-01 were added.
• the apparatus was evacuated by means of a membrane pump for 1 minute and then filled with argon. This procedure was performed 3 times. Subsequently, a slight stream of argon was continuously fed into the apparatus. The mixture was stirred 6h at 40 0 C (oil bath temperature) (200 1 / min).
• silica gel KI04e-01 -01 with the grown groups A was separated directly via a P3 frit and subsequently with one after the other
• silica gel KI04e-01 -01 was dried at 40 0 C in a vacuum oven to constant weight.

Landscapes

• Health & Medical Sciences (AREA)
• Chemical & Material Sciences (AREA)
• Engineering & Computer Science (AREA)
• Biomedical Technology (AREA)
• Life Sciences & Earth Sciences (AREA)
• Genetics & Genomics (AREA)
• Organic Chemistry (AREA)
• Zoology (AREA)
• Wood Science & Technology (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Biotechnology (AREA)
• General Engineering & Computer Science (AREA)
• Molecular Biology (AREA)
• General Health & Medical Sciences (AREA)
• Biophysics (AREA)
• Microbiology (AREA)
• Physics & Mathematics (AREA)
• Crystallography & Structural Chemistry (AREA)
• Biochemistry (AREA)
• Plant Pathology (AREA)
• Analytical Chemistry (AREA)
• Saccharide Compounds (AREA)
• Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
• Solid-Sorbent Or Filter-Aiding Compositions (AREA)
• Sampling And Sample Adjustment (AREA)
• Treatment Of Liquids With Adsorbents In General (AREA)

Priority Applications (5)

Applications Claiming Priority (2)

Publications (1)

Family

ID=42078045

Family Applications (1)

Country Status (7)

Cited By (9)

Families Citing this family (17)

Citations (6)

Family Cites Families (8)

• 2008
  • 2008-12-23 DE DE102008063001A patent/DE102008063001A1/de not_active Withdrawn
• 2009
  • 2009-12-23 US US13/141,878 patent/US9102935B2/en active Active
  • 2009-12-23 JP JP2011541520A patent/JP2012513386A/ja not_active Withdrawn
  • 2009-12-23 CN CN200980152958.XA patent/CN102264902B/zh active Active
  • 2009-12-23 EP EP09804132.0A patent/EP2382315B1/de active Active
  • 2009-12-23 WO PCT/EP2009/067909 patent/WO2010072834A1/de not_active Ceased
  • 2009-12-23 AU AU2009332900A patent/AU2009332900B2/en active Active
• 2016
  • 2016-01-26 JP JP2016012103A patent/JP6072945B2/ja active Active

Patent Citations (6)

Also Published As

Similar Documents

Legal Events