WO2014000872A1

WO2014000872A1 - Purification of nucleic acids

Info

Publication number: WO2014000872A1
Application number: PCT/EP2013/001815
Authority: WO
Inventors: Viorel Rusu; Gerbert Schaap
Original assignee: Magnamedics Gmbh
Priority date: 2012-06-26
Filing date: 2013-06-20
Publication date: 2014-01-03
Also published as: DE102012012523A1; DE102012012523B4

Abstract

Method for the purification of nucleic acids and/or fractions of nucleic acids from biological processes by adsorption onto silica gel particles in the presence of an aqueous solution of kosmotropic and chaotropic salts and subsequent desorption, and means suitable for this method.

Description

Purification of nucleic acids

The invention relates to a method for purifying nucleic acids and / or

Fractions of nucleic acids from biological processes and appropriate

Medium.

Nucleic acids are understood to mean a chemical umbrella term for

Deoxyribonucleic acid (DNA) and ribonucleic acid (RNA); nucleic acids

are chain-like molecules whose single building blocks are the nucleotides.

Nucleic acids in the context of the present invention are preferred

genomic nucleic acids. Nucleic acids are obtained in various biological processes in the

Usually along with other ingredients and / or reagents used for

subsequent procedures are disturbing and must be removed.

Thus one obtains by breaking up biological cells (called lysis) beside the

Deoxyribonucleic acid (DNS) other cell ingredients, for example

prevent biological processes such as polymerase chain reaction (PCR).

Using Polymerase Chain Reaction (PCR), tiny amounts can be obtained

Duplicate DNS in such a short amount of time that it works without problems

be detected, investigated and reused. Different by-products and / or reagents, such as primers,

Primer dimers, protein complexes (DTNB), enzymes, work-up reagents

(dye terminator) additives and / or salts, which again before further

Investigations must be removed.

There are several methods for purifying nucleic acids

biological processes known (Mol.Gen.Genet. 229, 445-452, EP

0389063 B2, J.Clin.Microbiol., 2003, 41 (6) and 2440-3, DE 100 33 991 A1,

BESTÄTlGUHGSKOPie AT454471, EP 1278834 A1, WO 2007 035750, US 2009 191566, EP 2363476 A1, US 2012059160, US 2009 306359, WO 2011 124705 and US 2007

087385). From EP 1 608 752 B1 a method for the separation of genomic nucleic acids from biological cells is known. The cells are treated with a reagent in which one component causes the lysis of the cell and a second component causes the precipitation of the nucleic acid on a carrier as a solid phase. The solid phase may contain a magnetic core.

Thereafter, the carrier is separated with the precipitated nucleic acid and the nucleic acid isolated with a selective eluent.

A disadvantage of the process known from EP 1 608 752 B1 is that the precipitation is less selective and the purification process requires a long period of time.

From US 5,898,071 A a method for purifying genomic DNA is known wherein the nucleic acids are adsorbed to magnetic microparticles consisting of a magnetic metal oxide core coated with a carboxyl group or an aminosilane. The magnetic core can be encased as a functional group with streptavidin.

From ACS Nano (2011), 5 (2), 1259-1266 is known to be mesoporous

Kieselgelmaterialien have large surface areas and pore sizes. It is used as a coating or capping of iron (II) oxide particles

Aminopropyltriethoxysilane used.

The object of the present invention is the provision of a fast and selective purification process for nucleic acids.

It was a method for the purification of genomic nucleic acids or fractions of nucleic acids from biological processes by adsorption on Kieselgel particles in the presence of cosmotropic and chaotropic salts and subsequent desorption found, which is characterized in that the nucleic acids from biological processes in a buffer solution of

Cosmotropic and chaotropic salts and polyethylene glycol with a

Molecular weight in the range of about 2 and 10 kDa, the nucleic acids adsorbed on acid group-free silica gel particles having a magnetic core and with a pore size in the range of 5 to 50 nm and an inner surface in the range of 0.1 to 2000 m ² / g the adsorbate is separated and, if appropriate, washed, and the nucleic acids or the fractions of nucleic acids are eluted with water or conventional additives.

In comparison to known cleaning methods, cleaning can be carried out very quickly. With the aid of the purification method according to the invention, individual fractions of the nucleic acids (nucleotides) can be selectively separated stepwise.

Compared to the method for the separation of nucleic acids according to EP 1 608 752 B1, no aggregation and precipitation of the nucleic acids occurs, but the separated nucleic acids are adsorbed on the acid group-free silica gel particles with a magnetic core.

For the method according to the invention, nucleic acids are precipitated

cleanse various biological processes.

In a preferred embodiment of the present invention, the purification of the nucleic acids takes place from the cell contents obtained by lysis of biological cells. In another preferred embodiment of the present invention, the purification of the nucleic acids from PCR processes, for example by separation of dNTPs, primers, enzymes, dye terminators, additives or salts. In a further preferred embodiment of the present invention, the purification of the nucleic acids takes place for the separation of fractions of nucleic acids.

The inventive method is based on an adsorbent

acid group-free silica gel particles having a magnetic core and having a pore size in the range of 5 to 50 nm, preferably 5 to 30 nm and an inner surface in the range of 0.1 to 2000 m ² / g, preferably 10 to 1000 m ² / g, carried out.

The particles generally have a diameter in the range from 20 nm to 500 μm, preferably from 200 nm to 10 μm, in particular from 500 nm to 1.3 μm.

The adsorbent from acid group-free silica particles with a

Magnetic core can be prepared by coating particles containing iron oxide with silica gel. The coating of iron oxide-containing particles with silica gel is known per se (J. Colloid Interface Sci. 1968, 26, 62 to 69, Langmuir 2005, 21, 10763 to 10769, J. Colloid Interface Sei 2005, 283, 392 to 396).

The coating of iron oxide-containing particles with silica gel for the process according to the invention can be carried out, for example, as follows:

, A suspension of iron oxide-containing particles in an alcohol (e.g., isopropanol) is added to tetraethyl orthosilicate (TEOS) with vigorous stirring in the presence of ammonia. The thickness of the

Coating can be controlled by the amount of tetraethyl orthosilicate added. The coated iron oxide-containing particles are washed with an alcohol (eg methanol) and stored in water.

For the process according to the invention, particularly acid group-free silica gel particles which consist of a mesoporous layer which is applied to the magnetic core and has a layer thickness in the range from 10 to 100 nm are preferred.

Magnetic cores for the acid group-free silica gel particles according to the invention may be per se known particles of iron oxide (eg Fe ₂ O 3 and / or Fe 3+ 4) and silicon dioxide, polystyrene and / or polyvinyl alcohol.

Iron oxide particles with a mesoporous silica gel coating, as produced in the presence of polyethylene glycol as a porogen, are preferably used for the process according to the invention.

More preferably, acid group-free silica gel particles consisting of a mesoporous layer coated on the magnetic core and having a layer thickness in the range of 10 to 100 nm, the magnetic cores maghemite and / or magnetite in the range of 30 to 95 wt % and have a mean diameter in the range of 10 nm to 500 pm, wherein the silica gel particles have a mean

Diameter in the range of 20 nm to 500 pm, preferably 200 nm to 10 pm, more preferably 300 nm to 1, 5 pm have.

Buffer solutions for the process according to the invention generally contain aqueous solutions of cosmotropic and chaotropic salts and

Polyethylene glycol. Cosmotropic ions in aqueous solutions of salts act as precipitants for proteins and nucleic acids. They enhance hydrophobic effects and thereby promote and aggregate and precipitate the proteins and nucleic acids via hydrophobic interactions. Chaotropic ions in aqueous solutions of salts reduce hydrophobic effects and thereby increase the solubility of proteins and nucleic acids in water.

Cosmotropic and chaotropic ions can be represented in the so-called Hofmeister series.

In the context of the present invention are cosmotropic ions

for example Na ⁺ , Mg ²⁺ and / or S0 ₄ ^{2 "} and / or Ca ²⁺ , Li ⁺ HPO ₄ ^2" called. Preferred cosmotropic ions are Na ⁺ , Mg ²⁺ and S0 ₄ ^{2 "} .

In the context of the present invention are chaotropic ions such as

for example, K ⁺ , CI ^" and NH ₄ ⁺ , Cs ⁺ and N0 ₃ ^" . Preferred chaotropic ions are K ⁺ and Cl ^" .

For example, polyethylene glycol for the process of the invention may have an average molecular weight in the range of about 2 and 10 kDa, preferably in the range of about 3 to 5 kDa, and more preferably 4 to 4.5 kDa.

Water for the process according to the invention is generally deionized water which does not contain the enzyme deoxyribonuclease (DNase).

Buffer solution for the process according to the invention generally contain cosmotropic and chaotropic salts and polyethylene glycol with a

Molecular weight in the range of about 2 and 10 kDa in aqueous solution.

Particularly preferred is an average molecular weight of the polyethylene glycol in the range of 3 to 5 kDa and in the buffer solution in a concentration of 10 to 20 wt.% Is present.

In a particular embodiment of the present invention, nucleic acids can be purified from PCR processes in the range of 80bp and 30kb and separating, the fractionation taking place in the presence of an aqueous additional buffer solution comprising 0.1 to 25% by weight of a cosmotropic salt, 0.1 to 20% by weight of a chaotropic salt, 10 to 20% by weight.

Polyethylene glycol having an average molecular weight in the range of about 2 and 10 kDa, wherein the sum of the individual components does not exceed 100%.

It is particularly preferred here that the concentration of the cosmotropic and chaotropic salts in the aqueous additional buffer solution does not exceed a concentration of 2 M and the fractionation is achieved by a step gradient in the range from 0.1 to 2 M.

After adsorption of the nucleic acids is generally the adsorbate of genomic deoxyribonucleic acids (DNA) or fractions of

Deoxyribonucleic acids to acid group-free silica gel particles in a conventional manner, for example by filtration, separated from the aqueous solution.

In a particular embodiment of the present invention, the adsorbate of genomic deoxyribonucleic acids (DNA) or fractions of deoxyribonucleic acids to acid group-free silica gel particles

Applying a magnetic field are separated from the aqueous solution.

For this purpose, the acid group-free silica gel particles with a

magnetic core adsorbed nucleic acids separated on a magnetic separator and isolated.

The isolated adsorbate can be washed. As the washing medium, a short-chain aliphatic alcohol such as ethanol or isopropyl alcohol is generally used.

In the following step, the nucleic acids are separated from the adsorbate by means of an elution buffer. Elution buffers for the process according to the invention are, for example, aqueous solutions of (hydroxymethyl) -aminomethane (TRIS),

Ethylenediaminetetraacetic acid (EDTA) or DNase-free water.

In a particular embodiment of the present invention, the nucleic acid adsorbed on acid group-free silica gel particles with a magnetic core can be separated in the magnetic field and optionally washed and subsequently separated with an elution buffer.

The process according to the invention can be carried out, for example, as follows:

As an example, the following preferred cases are mentioned:

Purification of the nucleic acids from the cell contents obtained by lysis of biological cells.

Purification of nucleic acids from PCR processes, for example by separation of dNTPs, primers, enzymes, dye terminators, additives or salts.

Purification of nucleic acids for separation of fractions from

Nucleic acids.

As a rule, all nucleic acids are separated from the biological processes by the purification process according to the invention.

In a preferred embodiment of the present invention separate individual fractions of nucleic acids (nucleotides).

The purification of nucleic acids for the separation of fractions of

Nucleic acids can be carried out, for example, as follows:

On the basis of a sequence protocol, one recognizes the size distribution of the individual nucleotides. Depending on their size, the nucleotides become individual

Divided fractions. In a first step, the fraction of the largest nucleotides is separated. For this purpose, we suspended the mixture of nucleotides in the additional buffer and the fraction in question on the silica gel particles absorbed. The separation is carried out with the aid of a magnetic separator. The isolation of the relevant fraction is carried out by washing the particles and elution.

The residue of separation in the magnetic field contains nucleotides smaller than the largest nucleotides. In the same way, fractions of smaller nucleotides with corresponding additional buffers are separated from the residue.

The present invention also provides a means for purifying nucleic acids and / or fractions of nucleic acids from biological

Containing processes

• Buffer solution of cosmotropic and chaotropic salts and

Polyethylene glycol having a molecular weight in the range of about 2 and 10 kDa

Acid group-free silica gel particles having a magnetic core and having a pore size in the range of 5 to 50 nm and an inner surface in the range of 0.1 to 2000 m ² / g,

• additional buffer solution,

0.1 to 25% by weight of a cosmotropic salt,

0.1 to 20% by weight of a chaotropic salt,

10 to 20 wt.% Polyethylene glycol with a middle

Molecular weight in the range of about 2 and 10 kDa, wherein the sum of the individual components does not exceed 100%, and

• Elution buffer. The nucleic acids obtained by the method according to the invention have a purity of greater than 90%, preferably greater than 95%, and particularly preferably greater than 99%.

Examples:

example 1

Iron oxide particles with a mesoporous silica gel coating in the presence of polyethylene glycol 10 ml of iron oxide particles (eg MagSi-S beads from MagnaMedics) with a concentration of 20 mg / ml are separated in a magnetic field and then in 30 ml of polyethylene glycol (average molecular weight 400) 10 ml of isopropanol and 2 ml of water with vigorous stirring. To this mixture are added 2 ml of a 25% by weight ammonia solution and 0.75 ml of tetraethyl orthosilicate (TEOS). The coating takes place within 6 hours

even stirring.

The coated iron oxide-containing particles are washed with 40 ml of water. The final concentration of acid group-free silica gel particles is 20 mg / ml.

Example 2

Purification of a PCR product of salts, enzymes, nucleotides and primers

10 μΙ iron oxide particles with a mesoporous silica gel coating according to Example 1 with an inner surface area of 20 m ² / g are added to 20 μΙ PCR sample. To this is added 30 μl of a buffer solution (20% by weight of PEG 4000 NaCl 0.5M KCl 0.5M MgC 0.1 M) with shaking and the sample is allowed to come to room temperature. The resulting sample is placed in a magnetic separator and the magnetic particles removed. The supernatant is separated.

The magnetic particles with adsorbed DNA are washed twice with 100 μΙ_ 70 v / v% ethanol.

The magnetic particles are removed from the washing solution again with the magnetic separator and the washing process is repeated 4 to 5 times.

The magnetic particles with the adsorbed DNA are dried in air.

The isolation of the DNA fragments is carried out with an elution buffer, 40 μΙ (Dnase free water or DNase free 0.1 M Tris / HCl, pH 6.4) by shaking the sample.

The eluted magnetic particles are separated again in a magnetic separator. The supernatant solution contains the purified DNA.

Example 3

DNA purification

PCR is purified according to Example 2.

A sequence protocol prepared according to DNA Base readout shows a purity of the DNA of 95%.

Example 4 On the basis of a sequence protocol, one recognizes the size distribution of the individual nucleotides. Depending on their size, the nucleotides become individual

Divided fractions. According to the present example, the following 3 fractions are obtained:

300 to 800 bp

300 to 500 bp and

150 to 300 bp

Apart from the isolation of the individual fractions of the nucleotides, by-products of the production process (e.g., enzymes, salts and

Buffer solutions) separated.

Example 5 Isolation Nucleotide Fractions in the range of 150 to 300 bp

In a first step, the fraction of the largest nucleotides (greater than 300 bp) is separated. In a sample tube 100 μΙ DNA fragments, 20 pl iron oxide particles with a mesoporous silica gel coating according to Example 1 with an inner surface of 20 m ² / g and 72 μΙ an additional buffer (20% PEG 4000 NaCl 0.1 M KCl 0.1 M MgCl ₂ 0.1 M ) with shaking and let the sample come to room temperature.

All DNA fragments larger than 300 bp are absorbed on the particles. The rest of the fragments (less than 300 bp) remain in solution.

The obtained sample is placed in a magnetic separator and the magnetic particles are removed. The supernatant is separated. To obtain a greater purification, the first step is repeated. In the next step, 150 μΙ of the supernatant with 82.8 μΙ of

Additional buffer (20% PEG 4000 NaCI 0.1 M KCl 0.1 M MgCl ₂ 0.1 M) and 20 μΙ of the magnetic particles mixed with shaking; then the sample comes to room temperature.

All DNA fragments larger than 150 bp are adsorbed on magnetic particles. The rest of the fragments (less than 150 bp) remain in solution and are discarded. The obtained samples from both separation steps (including repetition) are placed in a magnetic separator and the magnetic particles are removed. The supernatant is separated.

The magnetic particles are removed from the washing solution again with the magnetic separator and the washing process is repeated 4 to 5 times. The magnetic particles with the adsorbed DNA are dried in air.

The eluted magnetic particles from the purification steps are separated again in a magnetic separator. The supernatant solution contains the purified DNA separated by the individual fractions.

Example 6

Isolation nucleotide fractions ranging from 300 to 500 bp In a first step, the fraction of the largest nucleotides (greater than 500 bp) is separated. In a sample tube 100 μΙ DNA fragments, 20 μΙ iron oxide particles with a mesoporous silica gel coating according to Example 1 with an inner surface of 20 m ² / g and 72 μΙ an additional buffer (20% PEG 4000 NaCl 0.1 M KCl 0.1 M MgCl ₂ 0.1 M ) with shaking and let the sample come to room temperature.

All DNA fragments larger than 500 bp are absorbed on the particles. The remainder of the fragments (less than 500 bp) remain in solution.

The obtained sample is placed in a magnetic separator and the magnetic particles are removed. The supernatant is separated.

In order to obtain a greater cleaning, the first step is repeated.

In the next step, 150 μΙ of the supernatant with 82.8 μΙ of

All DNA fragments larger than 300 bp are adsorbed on magnetic particles. The remainder of the fragments (smaller than 300 bp) remain in solution and are discarded.

The obtained samples from both separation steps (including repetition) are placed in a magnetic separator and the magnetic particles are removed. The supernatant is separated.

The adsorbed DNA magnetic particles are washed twice with 100 pL of 70v / v% ethanol. The magnetic particles are removed from the washing solution again with the magnetic separator and the washing process is repeated 4 to 5 times.

The magnetic particles with the adsorbed DNA are dried in air.

Example 7

Isolation nucleotide fractions in the range of 300 to 800 bp

In a first step, the fraction of the largest nucleotides (greater than 800 bp) is separated.

In a sample tube 100 μΙ DNA fragments, 20 μΙ iron oxide particles with a mesoporous silica gel coating according to Example 1 with an inner surface of 20 m ² / g and 72 μΙ an additional buffer (20% PEG 4000 NaCl 0.1 M KCl 0.1 M MgCl ₂ 0.1 M ) with shaking and let the sample come to room temperature.

All DNA fragments larger than 800 bp are absorbed on the particles. The remainder of the fragments (less than 800 bp) remain in solution.

The obtained sample is placed in a magnetic separator and the magnetic particles are removed. The supernatant is separated. In order to obtain a greater cleaning, the first step is repeated.

In the next step, 150 μΙ of the supernatant with 82.8 μΙ of

All DNA fragments larger than 800 bp are adsorbed on magnetic particles. The remainder of the fragments (less than 800 bp) remain in solution and are discarded.

The magnetic particles with adsorbed DNA are washed twice with 100 μΐ_ 70 v / v% ethanol. The magnetic particles are removed from the washing solution again with the magnetic separator and the washing process is repeated 4 to 5 times.

The magnetic particles with the adsorbed DNA are dried in air.

The isolation of the DNA fragments is carried out with an elution buffer, 40 μΙ (Dnase free water or DNase free 0.1 M Tris / HCl, pH 6.4) by shaking the sample. The eluted magnetic particles from the purification steps are separated again in a magnetic separator. The supernatant solution contains the purified DNA separated by the individual fractions.

Claims

Process for the purification of genomic nucleic acids or

Fractions of nucleic acids from biological processes

by adsorption on silica gel particles in the presence of cosmotropic and chaotropic salts and subsequent desorption,

characterized,

that the nucleic acids from biological processes in one

Buffer solution of cosmotropic and chaotropic salts and

Polyethylene glycol having a molecular weight in the range of about 2 and 5 kDa,

the nucleic acids are adsorbed to acid group-free silica gel particles having a magnetic core and having a pore size in the range of 5 to 50 nm and an inner surface in the range of 0.1 to 2000 m ² / g,

the adsorbate is separated and optionally washed and the nucleic acids or the fractions of nucleic acids are eluted with water or conventional additives.

A method according to claim 1, characterized in that the acid group-free silica gel particles consist of a mesoporous layer which is applied to the magnetic core and has a layer thickness in the range of 10 to 100 nm.

A method according to claim 1 and 2, characterized in that the acid group-free silica gel particles consist of a mesoporous layer which is applied to the magnetic core and have a layer thickness in the range of 10 to 100 nm,

wherein the magnetic cores maghemite and / or magnetite in the range of 30 to 95 wt% and have a mean diameter in the range of 10 nm to 500 μιη, wherein the silica gel particles have a mean diameter in the range of 20 nm to 500 μητι.

Process according to claims 1 to 3, characterized in that

Nucleic acids from biological processes in an aqueous

Buffer solution is suspended,

0.1 to 25% by weight of a cosmotropic salt,

0.1 to 20% by weight of a chaotropic salt,

10 to 20 wt.% Polyethylene glycol with a middle

Contains molecular weight in the range of about 2 and 10 kDa,

the sum of the individual constituents does not exceed 100%.

Process according to claims 1 to 4, characterized in that the average molecular weight of the polyethylene glycol is in the range of 3 to 5 kDa and is present in the buffer solution in a concentration of 10 to 20% by weight.

Process according to claims 1 to 5, characterized in that nucleic acids are purified and separated from PCR processes in the range of 80 bp and 30 kb,

wherein the fractionation in the presence of an aqueous

Additional buffer solution takes place,

0.01 to 25% by weight of a cosmotropic salt,

0.01 to 25% by weight of a chaotropic salt,

0 to 20 wt.% Polyethylene glycol having an average molecular weight in the range of about 2 and 10 kDa,

the sum of the individual constituents does not exceed 100%.

Process according to claims 1 to 6, characterized in that the concentration of the cosmotropic and chaotropic salts in the aqueous additional buffer solution does not exceed a concentration of 2 M and the fractionation by a step gradient in the Range of 0.1 to 2 M is achieved.

Method according to one of claims 1 to 8, characterized in that the adsorbate of genomic deoxyribonucleic acids (DNA) or fractions of deoxyribonucleic acids to acid group-free silica gel particles is separated by a magnetic field from the aqueous solution.

Method according to one of claims 1 to 9, characterized in that the acid group on free silica particles with a

magnetic core adsorbed nucleic acids in the magnetic field

be separated and optionally washed with a short-chain alcohol and subsequently separated with an elution buffer.

Means purification of nucleic acids and / or fractions of the

Containing nucleic acids from biological processes

• Buffer solution of cosmotropic and chaotropic salts and

• additional buffer solution,

0.1 to 25% by weight of a cosmotropic salt,

0.1 to 20% by weight of a chaotropic salt,

10 to 20 wt.% Polyethylene glycol with a middle

• Elution buffer.