EP1075546A1

EP1075546A1 - Method and device for isolating nucleic acids

Info

Publication number: EP1075546A1
Application number: EP99922172A
Authority: EP
Inventors: Jürgen Bernhagen; Herwig Brunner; Hubertus Eyb; Stefan Kiesewetter; Brigitte Koch-Pelster; Günter Tovar; Frank Vitzthum
Original assignee: Fraunhofer Gesellschaft zur Forderung der Angewandten Forschung eV
Current assignee: Fraunhofer Gesellschaft zur Forderung der Angewandten Forschung eV
Priority date: 1998-05-04
Filing date: 1999-05-04
Publication date: 2001-02-14
Also published as: IL139393A0; US6511831B1; IL139394A0; IL139394A; HUP0101828A2; AU4036699A; WO1999057313A1; HUP0101828A3; JP2002513590A; JP2002513591A; CA2330819A1; WO1999057314A1; CA2330790A1; AU3931699A; EP1075548A1; HUP0102123A2; AU746005B2

Abstract

The invention relates to a method for the quantitative isolation of nucleic acids from a sample. According to the method, the sample is decomposed and a DNA mixture consisting only of random sequences is simultaneously or subsequently brought into contact with said sample in such a way that substantially all nucleic acids present in the sample are able to bond with the DNA mixture.

Description

Method and device for isolating nucleic acids

description

The present invention relates to a method and a device for isolating nucleic acids from a sample, in particular a biotic or abiotic material.

The specific isolation of certain DNA sequences from mixtures of different DNA sequences using more or less specified nucleic acids is known. No. 5,650,489 describes the use of random DNA sequences for isolating certain DNA sequences of interest. A sample digestion and the quantitative separation of nucleic acids present in the sample are not described.

However, the quantitative isolation of nucleic acids from certain raw materials still to be digested plays an important role in a large number of scientific, industrial or other areas. Such areas are, for example, environmental analysis, food diagnostics, veterinary diagnostics, epidemiology, soil tests, agricultural engineering, forensic technology, medical diagnostics such as tumor diagnostics, genetic analysis, early detection of epidemic multiple drug-resistant germs, development of more efficient therapies or disease monitoring, basic research the like. As diverse as that - 2 -

Areas of application can be the starting materials for the preparative isolation of the nucleic acids, for example eukaryotic or prokaryotic cells or their homogenates, soil samples, blood samples, body fluids or tissue homogenates. Depending on this starting material or sample material, different digestion methods have to be used in order to make the nucleic acids present in cells and / or cell nuclei accessible for isolation. Digestion processes that are frequently used are, for example, ultrasound and / or enzyme treatment. After the digestion treatment has been carried out, the nucleic acids are isolated, for example by means of gel electrophoresis, ultracentrifugation or affinity chromatography.

Affinity chromatography is essentially based on the ability of nucleic acids to bind reversibly to positively charged and / or positively polar matrices. As a rule, an anionic or polar binding of the nucleic acids to the matrix is first achieved and then the nucleic acid is freed of impurities by using suitable solvents. In a second step, the nucleic acid bound to the matrix is detached from the matrix using a further solvent, for example with a higher ionic strength. Subsequently, the nucleic acid isolated in this way generally has to be deionized again in order to be able to be used for further investigations.

It proves to be disadvantageous that, depending on the sample material used and the processing performed different isolation strategies must be developed and used.

Both DNA and RNA are conventionally also isolated by means of ultracentrifugation, with protease and phenol treatments frequently having to be carried out. This procedure has the disadvantage that high molecular weight DNA in particular is often still contaminated with proteins even after the isolation has been carried out and the molecules are exposed to the risk of breakage due to the acting shear forces. The phenol treatment is also harmful to health and the environment.

Gel electrophoresis, which is often used for nucleic acid isolation, also has disadvantages, inter alia because of the comparatively complicated pre- and post-treatment of the samples or nucleic acids that are necessary.

The technical problem on which the present invention is based is therefore to provide a cost-effective and easy-to-carry out quantitative isolation method for nucleic acids, which, from any biotic and / or abiotic sample material, is highly specific to all or essentially all of the nucleic acids in a particularly simple step during sample digestion in a single step Form insulated and provided.

The invention solves this problem by providing a method for the quantitative isolation of nucleic acids from a sample, the - 4 -

Sample is disrupted and a DNA mixture consisting only of random sequences is brought into contact with the disrupted sample in such a way that all or essentially all of the nucleic acids present in the sample can bind to the DNA mixture. In an advantageous embodiment of the invention, after the remaining sample has been separated off, the nucleic acids bound to the DNA mixture can then be detached from the DNA mixture after an optional washing step and, for example, amplified, detected or used in some other way.

In the context of the present invention, sample digestion is understood to mean the most complete possible release of nucleic acids, optionally the sequence and / or compartment-selective release of nucleic acids from a biological material, ie a sample, which involves destruction of the sample, for example Lysis, or reversible damage to the sample, for example pore formation, can go hand in hand.

The invention thus provides an affinity-chromatographic method for the quantitative, that is to say quantitative, isolation of all or essentially all of the nucleic acids present in a sample, in which the nucleic acids contained in a sample after digestion of the sample with a DNA mixture consisting only of random sequences be brought into contact and this DNA mixture specifically contains the nucleic acids contained in the sample, for example DNA and / or - 5 -

RNA, binds and thus isolated from the other sample components such as carbohydrates, fats etc. The hybridization conditions to be observed, such as temperature and buffer composition, depend on the respective specific insulation task.

The invention therefore provides in an advantageous manner that all or essentially all nucleic acids can be removed quantitatively from a sample by means of the DNA mixture consisting only of random sequences. The method according to the invention is therefore highly specific with regard to the isolated class of substances, namely nucleic acids, but is equally able to isolate all or almost all of the nucleic acids present in the sample. The separation of essentially all nucleic acids means the separation of at least 85%, preferably 90%, particularly preferably 95% and in particular 99% of all nucleic acids present in the sample.

The invention advantageously enables the steps of sample digestion, in particular nucleic acid isolation, nucleic acid purification, and their detection to be combined in a single step, that is to say a one-step method. This integration creates the basis for methods and devices that further accelerate nucleic acid diagnostics and reduce the risk of cross-contamination, so that sensitivity and specificity are increased. Automation of this technology is also made easier. The invention enables - 6 -

thus advantageously a universal, universal, standardized, inexpensive and automatable nucleic acid diagnostics for gene and genome analysis with a multitude of possible uses. The invention comprises a method that can be carried out quickly, requires only a small amount of material and reduces the use of costly and toxic substances.

In the context of the present invention, a DNA mixture consisting only of random sequences is understood to mean a mixture of DNA random sequences, which are also referred to as random primers, which is not specifically put together for a nucleic acid to be specifically isolated, but rather has any desired nucleotide permutation , so that all nucleic acids present in the sample, in particular all nucleic acids with a chain length sufficient for hybridization, are bound indiscriminately. The DNA random sequences have an essentially uniform, but any chain length, for example from 5 to 50, preferably an average chain length from 15 to 30. The DNA mixture consisting only of random sequences thus has a large number of different DNA molecules in single-strand form , the sequence of which is composed at random.

The preparation of this DNA mixture is carried out in such a way that in the course of the DNA synthesis the four nucleosides involved in DNA construction, that is to say deoxyadenosine, deoxyguanosine, deoxycytidine and deoxythymidine and, if appropriate, because synthons, that is, their structurally analogous modifications, are used per DNA chain extension step in a mixing ratio of 1: 1: 1: 1. As a result, all four nucleosides are inserted with the same probability for each position in a DNA molecular chain. A DNA mixture with random DNA sequences each of 20 nucleotides in length, for example, represented by the sequence 5 '(N) ₂₀ 3', where N stands for deoxyadenosine, deoxyguanosine, deoxythymidine, deoxycytidine and / or synthons, thus represents a mixture of all single-stranded DNA molecules with a chain length of 20 nucleotides, that is 4 ²⁰ different DNA molecules. The number of different DNA random sequences per DNA mixture according to the invention is therefore 4 ^X , where x is the chain length or number of nucleotides of the DNA random sequence.

In a particularly advantageous embodiment of the invention, the invention relates to a aforementioned method, the 4 ^X random sequences present in the DNA mixture occurring in the same, preferably in essentially molar, proportions. The DNA mixture used according to the invention is therefore not developed with a view to a specific isolation task, but rather represents a DNA mixture that can be used for any DNA or RNA isolation task without any specific DNA or RNA specificity DNA mixture is only specific to the extent that it can separate nucleic acids, ie DNA or RNA, from other substances such as proteins, sugars or the like. According to the invention, however be provided to distinguish DNA from RNA by suitable selection of the binding or hybridization conditions between nucleic acid to be isolated and DNA mixture or the dissolution conditions (temperature, ionic strength, etc.). The method according to the invention can therefore advantageously be carried out specifically for DNA or RNA.

Of course, the DNA mixture to be used according to the invention can also contain other modified bases or nucleosides such as deoxyinosine, uridine, pseudouridine, N ² -dimethyl-guanosine, N ⁶ -isopentenyl-adenosine, and / or synthons. According to the invention, provision can also be made to use pentoses, that is to say RNA building blocks, instead of the deoxygen pentoses. In the context of the present invention, a DNA mixture may also be understood to mean an RNA mixture.

In connection with the present invention, a sample is understood to mean any organic, inorganic or biotic / abiotic material, provided that it contains a nucleic acid, ie DNA or RNA. Accordingly, a sample can be a prokaryotic or eukaryotic cell or a cell homogenate, viruses, for example in the form of a virus suspension, blood, sperm, lymph or other body fluid, organ or tissue preparation, water or soil samples, liposomes, yeast, cell organelles , Cell nucleus, plant homogenates, amber or other. The sample preferably has water or a physiological salt or buffer solution as a solvent. - 9 -

The invention has the advantage that highly specific nucleic acids can be isolated from a sample already during sample digestion in a single step and obtained in pure form, so that these can be used directly for other analysis or preparation steps, such as a PCR method . The procedure according to the invention advantageously does not require any costly and time-consuming adaptation steps of the method to the respective insulation task. Rather, the method according to the invention can be used directly for any insulation task without further modification. The method according to the invention can thus be used in the course of almost any electrical, chemical, physical or chemical-physical digestion of organic or inorganic material.

The invention advantageously provides a aforementioned method, the DNA mixture consisting only of random sequences being immobilized on a carrier. If, according to this preferred embodiment, the DNA mixture is bound to a sample carrier, in a further preferred embodiment the DNA mixture, which consists only of random sequences, can be brought into contact with the sample under the influence of an electric field, for example a pulsed electric field or a field constant voltage are carried out in such a way that electrohybridization takes place, as is described, for example, in DE 196 281 717 or US Pat. No. 5,632,957, which are included in this regard in the present disclosure. The negatively charged - 10 -

Accordingly, nucleic acids initially accumulate in the anode region of a device suitable for carrying out the method according to the invention, in which the immobilized random sequences are advantageously also located, so that locally high nucleic acid concentrations arise in the region of these random sequences, which favor hybridization kinetically and thermodynamically. In a further preferred embodiment of the invention, the polarity can be washed rigorously in a further preferred embodiment of the invention due to the changed kinetic and thermodynamic conditions in the cathode region, as is described, for example, in DE 196 281 717 or US Pat. No. 5,632,957, which is so far described in the present Disclosure content are included. In this way, a further specificity of the method according to the invention, for example with regard to an enrichment of certain nucleic acid species, can be achieved following the separation of all nucleic acid species.

In a further preferred embodiment, the invention relates to a aforementioned method, the DNA mixture consisting only of random sequences being in free, that is to say unbound, form. If, according to this particularly preferred embodiment of the invention, the DNA mixture consisting only of random sequences is freely available, in a preferred embodiment it must have a negative net charge in order to support the nucleic acid purification by means of electrophoretic and dielectrophoretic effects. According to the invention, an electric field is preferably applied, preferably an inhomogeneous electric field. In this field - 1 1 -

the DNA mixture and the released nucleic acids of the sample move in the same direction towards the anode, so that their concentration increases there locally and hybridization is kinetically and thermodynamically favored. In addition, there is the advantage that substances of other electrical properties are separated by this method.

The invention therefore provides in a particularly preferred embodiment that at least one electric field, for example a pulsed electric field and / or a field of constant electric voltage, is applied when the nucleic acids are brought into contact with the either immobilized or free DNA mixture consisting only of random sequences becomes. The number and parameters of the electrical fields to be used depend on the specific insulation and cleaning task. Suitable parameters for pulsed electric fields are, for example, in Kinosita and Tsong, Nature 268 (1977), 438 to 441, Proc. Natl. Acad. Be. USA 74 (1977), 1923 to 1927 or Benz and Zimmermann, Bioelectrochem. Bioenergy. 7 (1980), 723 to 739 and for constant fields in Pollard-Knight et. al., WO 97/41219 (1997), which are included in the present disclosure. Since a significant advantage of the method according to the invention is achieved when the method according to the invention is carried out in one stage, that is to say the sample digestion takes place simultaneously with the nucleic acid purification, the invention provides in a preferred embodiment that an electric field is also present during the sample digestion, for example a - 12 -

pulsed electrical field and / or a field of constant electrical voltage is used. The use of pulsed electric fields leads to the electroporation and osmotic lysis of biological materials such as cells. This releases the nucleic acids present in the biological materials and makes them accessible for purification. Here, too, the number of fields and their technical parameters depend on the respective task, i.e. the material to be digested and the solvent. Of course, it is provided according to the invention to vary the number and parameters of the electrical field (s), such as the number of pulses, field strength or pulse frequency, in the course of a process cycle according to the invention, for example to unlock higher field strengths and larger number of pulses and to electro-hybridize at lower field strengths and or to perform pulse counts. When using pulsed electrical fields or fields of constant electrical voltage for nucleic acid isolation, it is possible according to the invention to lyse only certain cell types in a sample of different cell types via the choice of treatment parameters. This can be used to separate certain nucleic acids of a cell type from other cell types and thus also their nucleic acids. During the digestion of the sample, substances can be introduced into the cells or micelles using the electric fields, for example cell fusion, which later support or specify the lysis and / or nucleic acid release or serve as a marker for later detection. - 13 -

According to the invention, a pulsed electric field with field strengths of 2 to 100 kV / cm, in particular 5 to 50 kV / cm, and a pulse length or time constant of approximately 0.5 μs to 50 ms is particularly preferred both for sample digestion and optionally for electro-hybridization used. Of course, however, different electrical parameters can also be used for the electro-hybridization. The action of the electric field and the electrical breakthrough create pores in the lipid layers that make up the cell membrane. According to the invention, 1 to 1,000,000, preferably 100 to 20,000, pulses can be applied to the cells. The pulse shape can be, for example, rectangular, exponentially decreasing or sinusoidal, preferably in radio frequency. The electrical field can be generated by both AC and DC voltage. The field strength is preferably above the critical voltage V _c across the membrane of the cell to be disrupted. Usual conditions for cell disruption or nucleic acid release from biological materials by means of electrical fields are, for example, in Vitzthum et. al., FEBS Abstract (1998), 155, US Pat. No. 5,235,905, US Pat. No. 5,447,733 or US Pat. No. 5,393,541, which in this respect are included in the disclosure content of the invention.

It is also preferred according to the invention to support cell disruption by using high temperatures, for example from 30 ° C. to 95 ° C. - 14 -

Of course, it is also possible to carry out the sample digestion and / or the nucleic acid release with other parameters of the electric field and / or to provide chemical agents, for example chaotropic substances or detergents, which support cell digestion, for example SDS, lipases, proteases such as that Proteinase K or DMSO (dimethyl sulfoxide), urea, guanidinium thiocyanate or guanidinium hydrochloride.

However, it is preferred to carry out the method according to the invention of sample digestion, nucleic acid purification and, if appropriate, nucleic acid detection without the addition of such substances, for example exclusively in water, physiological saline or buffer solution without the addition of chemicals.

In a further preferred embodiment, it can be provided to detect the nucleic acids following the binding of the nucleic acids or the detachment of the nucleic acids to or from the free or immobilized DNA mixture, an amplification optionally being carried out by means of PCR before detection. The nucleic acids purified from the sample can be detected directly in the sample carrier, for example optically, for example spectrophotometrically, luminometrically, radioactive or else electrically. In a particularly preferred embodiment, it is possible to carry out the sample digestion, the sample purification and the detection in a one-step process in one and the same sample carrier, preferably one during the entire process - 15 -

electric field is applied, which ensures sample digestion, nucleic acid purification and detection. The amplification of the nucleic acids to be detected which may be carried out can, according to the invention, be an electrical isothermal amplification as described in WO 98/02573, WO 93/15224 or WO 95/25177 and which are included in the present disclosure.

In a particularly advantageous manner, provision can be made to expose the sample, which has been brought into contact with the preferably immobilized DNA mixture consisting only of random sequences, to ultrasonic influence, for example at 16 to 40 kHz, in order to achieve sample digestion, in particular cell digestion. This simultaneously leads to heating of the reaction mixture of sample and, preferably immobilized, DNA mixture, so that the DNA contained in the sample is denatured and can bind to the, preferably immobilized, DNA mixture on cooling. Furthermore, the DNA is fragmented, which improves binding to the immobilized DNA mixture. The influence of ultrasound can therefore also be used or only to support the binding of the nucleic acids to the DNA mixture.

In a particularly preferred embodiment of the invention it is provided that after the affinity binding of the nucleic acids present in the sample to the DNA mixture, impurities are removed by means of suitable buffer solutions or water. After the washing step has taken place, the affinity - 16 -

bound nucleic acid can be detached from the DNA mixture by increasing the ambient temperature, for example to at least 70 ° C., for example boiling temperature, in the presence of a suitable solvent, for example a buffer solution or water. Detachment can also take place by increasing the ionic strength of the dissolving buffer or by changing the pH. According to the invention, it is also possible to carry out the washing step and / or an elution using the electrical field described, that is to say to carry out an electro-stringent washing, as described, for example, in Hintsche, Medical Genetics 11 (1999), 12-13, Cheng et al. , Anal. Chem. 70 (1998), 2321-2326, Cheng et al. , Nat Biotechnol 16 (1998), 541-546, DE 196 28 171.7 or US Pat. No. 5,632,957, which are included in the disclosure content of the present invention. The nucleic acid obtained is free from impurities and can be amplified in particular by PCR methods, even in the presence of the immobilized DNA mixture.

The present invention also relates to a device, in particular an affinity matrix, for isolating nucleic acids from a sample, in particular for carrying out a method mentioned above, comprising a DNA mixture consisting only of random sequences and described above, which is immobilized on a matrix. The random sequences of the DNA mixture are preferably coupled via their 3 'ends, so that when a subsequent amplification is preferably carried out, for example by means of PCR, no by-products - 17 -

arise. The invention therefore provides a device, in particular an affinity matrix, with which the aforementioned method can be carried out, in particular with the aid of which nucleic acids can be isolated from any sample in a simple and inexpensive manner.

The device according to the invention comprises a matrix which can be designed, for example, as an electrode, for example made of stainless steel, platinum, silver, gold, aluminum and / or graphite, as a membrane, as beads or columnar gel and as a support or basic structure for the only DNA mixture consisting of random sequences acts. It can also be provided to use magnetic particles, for example beads, as a matrix.

The invention advantageously provides for the material for the matrix to be a chemically and physically largely inert material, such as glass or plastic, for example polystyrene or polypropylene, or a correspondingly inert electrode material.

In particular, the material must be suitable, temperature differences in an interval between 10 ° C and 95 ° C, pH differences in an interval between 0 to 14 and sodium or potassium chloride ion concentrations in an interval of 10 itiM to 2 M without significant change in Tolerate material properties. In addition, the material used must be insoluble in water, detergents and surfactant mixtures as well as chemically - 18 -

inert to chaotropic reagents such as isoguanidine thiocyanate.

The surface of the matrix is modified in an advantageous manner, for example by the application of biomolecules which bind with high affinity to the surface of the matrix material. Such a biomolecule immobilized on the matrix can be, for example, streptavidin. However, the matrix surface can also be modified in such a way that a covalent bond between the random sequences of the DNA mixture and the matrix is made possible. Accordingly, the matrix can have amino groups which can bond via a dialdehyde spacer or a dialdehyde compound molecule, for example glutaraldehyde, to form a Schiff base with an amino function introduced, for example, at the 5 'end of the DNA random sequences. According to the invention, it can be provided that the matrix is cleaned before modification of its surface, for example with nitric acid. In addition, the invention provides, in an advantageous embodiment, for silanizing the surface of the matrix before the modification.

The random sequences of the DNA mixture accordingly also have modifications for the purpose of immobilization on the matrix, preferably at the 3 'or 5' end. Such modifications can be, for example, biomolecules, such as biotin, which are linked to the 5 'end of the DNA random sequence and which bind with high affinity to other biomolecules immobilized on the matrix, such as, for example, streptavidin. It can also be provided that amino functions in the - 19 -

Introduce random DNA sequences so that they can covalently bind with the formation of a Schiff base with aldehyde groups immobilized on the matrix. The 5 'or 3' end of each random sequence can thus have, for example, an amino group or other customary functional groups such as epoxy or succinimide esters.

In any case, the modified DNA random sequences in single strand form, that is to say the DNA mixture, and the modified matrix are brought into contact with one another, so that the DNA mixture is immobilized on the matrix. The matrix loaded with the DNA mixture to be used according to the invention is also referred to in the context of the present invention as the affinity matrix.

The affinity matrix according to the invention can advantageously also be applied to the surface of particles which are exposed or exposed to the digestion in the course of digestion.

The invention also relates to a device for isolating nucleic acids from a sample, in particular for carrying out a method according to the invention, this device comprising a sample chamber with at least two planar electrodes, preferably arranged opposite one another, and at least one non-conductive frame part, the at least two electrodes and the frame part are designed as a wall, cover and / or base part. The invention thus relates to a device mentioned above, also as a sample carrier. - 20 -

records whose dimensions can be adapted to the conditions of the biological material to be examined. This includes orders of magnitude from the area of microsystem technology and chip technology as well as the currently usual sample carrier sizes that are used in laboratories. The dimensions and geometries of the sample carrier are preferably also adapted to the chip format, ie cm or mm format, since this minimizes the amount of consumables and maximizes the sample throughput. In a preferred embodiment, such a chip is constructed according to the principles of microfluidics. In such an embodiment, the frame part can accordingly also be flat, that is to say evenly constructed, with no sample chamber being provided and the electrodes being integrated into the frame part. Furthermore, when designing the device according to the invention as a sample carrier chip, there are advantages with regard to the voltage to be applied, since high field strengths can be achieved even at lower voltages due to the small electrode spacing. A preferred method according to the invention, that is to say sample digestion and nucleic acid purification and, if appropriate, washing and detection can advantageously be carried out in a single sample carrier, an electrical field preferably being applied during the entire method or at certain times, which ensures the said method steps.

The sample carrier according to the invention is accordingly made of conductive elements, the electrodes, and not conductive. - 21 -

tendency elements, the frame part. The electrodes preferably consist or contain aluminum, stainless steel, silver, gold, platinum, graphite or the like, the non-conductive frame parts being made of plastic, glass, silicon or the like or containing these individually or in combination. The geometry of the sample holder is to be designed in such a way that both homogeneous and inhomogeneous electric fields can be generated.

The device according to the invention is thus characterized in that a sample chamber is provided for receiving a sample from a base, cover and wall parts, which has at least two flat electrodes as a wall or base part, the remaining areas of the sample chamber not conductive material, the frame part, is constructed. Preferably, one or more openings can be integrated into the frame parts, for example as input and removal units for introducing and removing sample liquid, which are optionally provided with filters and / or valves. In addition, it is preferred according to the invention to integrate optical or other measuring units between the frame parts, which allow the detection of the isolated nucleic acids.

The dimensions of the sample carrier are preferably from 1 cm ² (chip) to 10 cm x 10 cm x 10 cm (laboratory device). - 22 -

In a particularly preferred embodiment, at least one of the electrodes and / or a region of the frame part has the DNA mixture used according to the invention, which consists only of random sequences, and thus acts equally as a matrix or carrier for the DNA mixture immobilized on the electrode and / or the frame part .

The invention also relates to the use of a DNA mixture consisting only of random sequences, in particular a mixture of the same part, of 4 ^X different DNA random sequences, where x is the chain length of the DNA random sequence, preferably 5 to 50, in particular 15 to 30, for Isolation of nucleic acids from a sample.

Further advantageous embodiments of the invention result from the subclaims.

The invention is explained in more detail by means of exemplary embodiments and associated figures.

The figures show:

FIG. 1 shows a matrix of a device according to the invention,

FIG. 2 shows a device or DNA affinity matrix according to the invention,

FIG. 3 shows an electropherogram, - 23 -

FIG. 4 shows two chromatograms of the binding of standard DNA to untreated and treated glass beads,

5 shows a further embodiment of a device according to the invention in the form of a sample carrier and

Figure 6 shows a further embodiment of a device according to the invention designed as a chip.

Example 1: Device for DNA isolation

FIG. 1 shows a matrix 10 which is designed in the form of a membrane 1. The membrane 1 is coated on its surface with streptavidin molecules 3.

FIG. 2 shows an affinity matrix 100 which was produced by bringing the matrix 10 into contact with 5 'modified chemically or synthetically produced DNA random sequences 7, 7', 7 '' of a DNA mixture in single-strand form, the 5th Modification of the DNA random sequence enables it to bind to the streptavidin molecules 3 with high affinity. FIG. 2 shows that the 5 'ends of the DNA random sequences 7, 7', 7 ¹ 'are each bound to biotin 5. The biotin modification at the 5 'end of the DNA random sequences 7, 7', 7 '' binds with high affinity to the immobilized streptavidin molecules 3, so that a DNA mixture immobilized on a matrix 10 is also included - 24 -

the individual DNA random sequences 7, 7 ', 7' 'is formed.

Example 2: Isolation of DNA from a cell disruption

A) Preparation of the affinity matrix

Bailotini micro glass balls type 3000 from Potters-Ballotini GmbH, Kirchheimbanden, were used for the tests. Their size was specified up to 50 μm. To clean the starting material and prepare the surface for silanization, the glass beads were boiled in nitric acid. 50 g of the glass beads were placed in a 1 liter three-necked flask with a reflux condenser to 500 ml of approximately 7% nitric acid. The mixture was heated to boiling over a heating element and refluxed for 1 hour. In the meantime, a magnetic stirrer was used to stir the fish. After cooling, the supernatant liquid was decanted off. The glass beads were washed three times with MilliQ quality water and filtered through a filter. They were dried in a drying cabinet at 95 ° C. overnight. Since contamination was still visible to the naked eye after drying, the entire cleaning process was repeated again.

10 g of the dry and cleaned glass beads were added to 200 ml of dry methanol in a 250 ml round neck flask. The flask was purged with argon. 20 ml of 3-aminopropyltrime were - 25 -

thoxysilane (Fluka) added. 0.5 ml of triethylamine was added as catalyst. The mixture was stirred at room temperature for 2 hours. The reaction solution was decanted off and the glass beads were washed with water (MilliQ) and filtered through a suction filter.

First, a potassium phosphate buffer was prepared from 160 ml 0.1 MK ₂ HP0 ₄ and 40 ml 0.1 M KH ₂ P0 ₄ and adjusted to pH 7.5. A 2.5% glutardialdehyde solution was prepared from 10 ml of 25% glutardialdehyde solution and 90 ml of the potassium phosphate buffer. 4 g of the silanized glass beads were added to 40 ml of the 2.5% glutardialdehyde solution and stirred at room temperature for about 1 hour. The glass beads were then washed with water, potassium phosphate buffer and again well with water.

Oligonucleotides (1 μmol per batch) (15-mer, each 15-mer in the same concentration, that is, there is a distribution of all theoretically possible oligomers with the nucleotides A, T, G and C in equal proportions, company Interactiva, Ulm, Germany) were taken up in 1 ml of potassium phosphate buffer. In a 15 ml tube (Greiner GmbH), 1 g of the silanized glass beads activated with glutardialdehyde and 1 ml of the primer solution were added to 4 ml of potassium phosphate buffer. The mixture was briefly cooled in ice. The tube was placed on a roller in the cold room overnight and rolled there for 17 hours. The glass beads were centrifuged in a minifuge (Heraeus) for 5 minutes at 1500 rpm. The supernatant - 26 -

was decanted and initially kept in the refrigerator. The pellet was taken up three times in approximately 6 ml of potassium phosphate buffer and centrifuged to wash out unbound primers. The beads were slurried with water (MilliQ) and half each placed in two Eppendorf cones. One half was frozen in this way, the other half was dried in three 10-minute runs in the Speed vac and also stored in the freezer.

B) Cell disruption and DNA isolation

10 ² to 10 ^{9 of} the cells to be disrupted were placed in 0.05 to 1 ml of a buffer of the composition 137 mM NaCl, 2.7 mM KCl, 1.5 mM KH ₂ P0 ₄ and 8.1 mM Na ₂ HP0 _{4 in a} reaction vessel , pH = 7.0. Then 50 to 500 mg of the oligonucleotide-coated affinity beads were added. To disrupt the cells, ultrasound with a frequency of 16 to 40 kHz was coupled into the resulting mixture via a beaker resonator. The reaction mixture heated up to an average of 80 ° C., which was sufficient to denature the released double-stranded DNA, so that it could bind to the affinity matrix by hybridization when the reaction mixture cooled. A phase separation was brought about by layering the reaction mixture with chloroform, the glass beads accumulating in the chloroform phase due to their high specific weight. The aqueous supernatant, which in addition to cell debris contains all water-soluble components of the cell disruption, was removed and the glass - 27 -

beads, to which the DNA to be isolated adhered, dried in vacuo. In this way, the entire DNA of the sample could be released from the biological sample in a single step and separated from the other components in a preparative manner.

If the DNA was isolated on Dynabeads, 50 μl of cell lysate were mixed with 50 μl of conjugated Dynabeads with a concentration of 5 mg / ml in a buffer of the composition 20 mM Tris / HCl pH = 7.5, 1 M LiCl, 2 mM EDTA. The resulting mixture was incubated here on a roller for 10 minutes and standing for a further 10 minutes. The particles loaded with DNA were magnetically separated and washed twice with 100 μl wash buffer according to the manufacturer's instructions.

C) Elution of DNA from the Affinity Matrix

The DNA-containing affinity matrices were optionally warmed up in water or a buffer of the composition 10 mM Tris, 1 mM EDTA, pH = 8.0. The resulting suspension was heated to boiling for 10 minutes, during which the DNA bound to the affinity matrix dissolved and could be removed with the supernatant in the heat.

Example 3: DNA isolation and PCR

In a first experiment, the feasibility of isolating DNA on immobilized DNA random sequences (15-mers, as in Example 2) was demonstrated. For this purpose, bio- - 28 -

immobilized tinylated DNA random sequences on commercially available streptavidin-coated magnetic particles and compared their DNA binding properties with the binding properties of a commercially available DNA isolation system (DYNAL Direct). A plasmid containing the MIF gene was used as reference DNA, which is released from intact Escherichia coli cells by chemical cell disruption. After plasmid isolation, the MIF gene was amplified with a suitable pair of probes and the PCR products were separated electrophoretically. The results of these comparative investigations are shown in FIG. 3. Lanes 3 and 4 of the electropherogram represent the MIF-PCR product of plasmid DNA, which could be isolated from random DNA sequences. This is contrasted in lanes 5 and 6 with the MIF-PCR products of the plasmid DNA isolated using the commercial system. There is neither a qualitative nor a quantitative difference between the two isolation strategies.

Example 4: Affinity Chromatography

The glass beads prepared as an affinity matrix for DNA isolation according to Example 2 were transferred to an empty HPLC column and examined there for their DNA binding capacity. A major advantage of column chromatography methods over so-called batch methods is their high reproducibility and the possibility of being able to vary the test conditions (flow rate, eluent, temperature) in a simple manner. Figure 4 represents - 29 -

Chromatograms of the binding of standard DNA (25 μg each) at 50 ° C. and a buffer of the composition 10 mM Tris, 1 mM EDTA, 100 mM NaCl, pH = 8.0 to untreated glass beads and to glass beads with immobilized DNA random sequences The difference in the areas under the curves (curve 1: glass beads without a random DNA sequence; curve 2: glass beads with a random DNA sequence) corresponds to the DNA binding capacity of the affinity matrix. The flow rate was 0.1 ml / min in each case. Under the selected conditions, it was found (see FIG. 4) that the DNA binding capacity of the glass beads coated with random sequences exceeded the binding capacity of uncoated glass beads by 7.16 times.

Example 5: Nucleic acid isolation using electrical fields using a sample carrier in chip format

FIG. 5 shows a device 200, comprising a sample chamber 210 made up of four electrodes 220, 220 'combined into two opposite electrode pairs and a frame part 230 made of non-conductive materials. The sample carrier is rectangular in cross section and constructed in chip format, that is to say has a size of approximately 1 to 2 cm ² . The top and bottom parts of the sample chamber 210 are not shown. Frame part 230 and the four flat electrodes 220, 220 'form the walls and enclose an internal volume which serves to hold the sample. It can be provided that a distance between the two is arranged opposite one another. - 30 -

Neten and each forming a wall pairs 220, 220 'of 100 nm to 5 mm and an electrode diameter of up to about 1 cm. It is shown that non-conductive regions 240 separate the individual electrodes 220, 220 'of the respective electrode pairs from one another with their connections 225, so that inhomogeneous and approximately homogeneous electric fields can be generated. The formation of inhomogeneous fields can be achieved by applying a voltage to only two or three of four conductive elements 220, 220 '. Preferably, the non-conductive areas 240 between the conductive elements 220, 220 'of a pair of electrodes are chosen to be as small as possible, so that approximately homogeneous electric fields can arise if, for example, the conductive elements, i.e. electrodes 220' as cathode and the conductive elements, i.e. Electrodes 220 act as an anode. The sample chamber 210 has a frame part 230 which has input 260 and removal units 270 provided with filters. The input and removal units 260, 270 are fitted into connection-like non-conductive elements 280 of the frame part 230 in such a way that the sample carrier 200 can be filled and emptied. In order to be able to carry out spectrophotometric or luminometric measurements if necessary, the device 200 has optical units 290 between non-conductive regions 295 and 280 of the frame part 230, so that the optical units can be adapted in such a way that connections are created. The connections 225 of the electrodes 220, 220 'can be used for the electrical detection. It is also shown that random sequences 300 of the invented - 31 -

DNA mixture used according to the invention are immobilized. Provision can also be made to attach the random sequences 300 additionally or exclusively to the non-conductive element 240 in the region of the electrodes 220, 220 '. In order to be able to carry out all process steps using electrical fields, that is to say sample digestion, nucleic acid purification and, if appropriate, nucleic acid washing and detection with the sample carrier 200, one or more corresponding voltage-generating devices must be connected to the sample carrier 200, in order to avoid the various fields required to be able to generate. The device according to the invention is characterized in particular by the fact that parts or areas of the sample chamber 210, that is to say the wall, the base and / or cover part, are constructed from electrodes which can generate at least one electric field in the chamber, in walls, floor or cover openings for sampling, inputs and / or detection may be present.

Using the aforementioned device, the procedure according to the invention initially provides that a sample, such as E. coli cells in water, is introduced into the sample carrier 200. Subsequently, a dielectrophoresis can optionally be provided using an electric field to concentrate or separate certain biological cells or as a prerequisite for an electrofusion, a corresponding voltage being applied to the connections 225 of the electrodes 220, 220 '. An electrofusion can be provided for the electrolysis - 32 -

to support or carry out a marking. An electrical voltage is then applied, for example to generate pulsed electrical fields or an electrical field of constant voltage, in such a way that the biological cells or viruses are electrolyzed, where appropriate the conditions can be set such that only certain biological cells are electrolyzed. In the present case, an electrical field with a field strength of 2 to 100 kV / cm is used. The conditions are then selected such that the nucleic acids released from the disrupted cells use an electrical field with random sequences of the DNA mixture used according to the invention either freely present in the sample chamber or with the conductive and / or non-conductive regions of the sample chamber 210 in the anode region immobilized random sequences 300 of the DNA mixture used according to the invention can be electro-hybridized. A pulse number of 10,000 to 100,000, a time constant of 2 s to 50 μs, a field strength of 1 kV / cm to 10 kV / cm and exponential decrease in pulse during cell disruption and hybridization were used.

Subsequently, if necessary, electrostringent washing can be carried out with reverse polarity and enrichment or de-enrichment of certain nucleic acids and removal of undesired substances, for example amplification inhibitors. Electroelution and, if necessary, amplification of the nucleic acids still present then close in the sample chamber 210 - 33 -

, preferably via an electrical, isothermal PCR, as described in WO 97/47767. Finally, the bound or freely available purified nucleic acids can be detected, preferably electrically (described for example in Hintsche, 1999, op. Cit.)

It is clear to the person skilled in the art that between or after the individual steps described, depending on the isolation strategy, a washing or elution of desired or possibly undesired substances or nucleic acids as well as a change in the electric field with regard to polarity, field strength, pulse number, pulse duration or pulse frequency can take place.

Example 6:

FIG. 6 shows a device 400 according to the invention for carrying out a method according to the invention, the device 400 being constructed from a flat non-conductive frame part 560 and at least two electrodes 500 integrated in this flat frame part 560. The device 400 according to the invention is designed as a chip, that is to say has no sample chamber formed from a wall or cover part. The invention thus also relates to a flat sample carrier, that is to say a chip, made of a non-conductive chip base 560, also referred to as a frame part, and two flat electrodes 500 integrated into this chip base 560. - 34 -

if sequences of existing DNA mixture 550 are immobilized.

The method according to the invention can be carried out by a targeted control of the electrodes 500 in chronological order, for example an electrode control 1 for electrical digestion of the biotic or abiotic material (nucleic acid isolation), an electrode control 2 for electrical nucleic acid cleaning, an electrode control 3 for electrical isothermal nucleic acid amplification and an electrode control 4 for electrical detection.

Claims

- 35 claims

1. A method for the quantitative isolation of nucleic acids from a sample, wherein the sample is disrupted and simultaneously or subsequently a DNA mixture consisting only of random sequences is brought into contact with the sample in such a way that essentially all nucleic acids present in the sample bind to the DNA mixing can take place.

2. The method according to claim 1, wherein the nucleic acids bound to the DNA mixture consisting only of random sequences are separated from the DNA mixture after separation of the remaining sample and an optional washing step.

3. The method according to claim 1 or 2, wherein the DNA mixture consisting only of random sequences is immobilized on a support.

4. The method according to claim 1 or 2, wherein the DNA mixture consisting only of random sequences is freely available in solution.

5. The method according to any one of the preceding claims, wherein at least one electric field is applied during the sample digestion and / or the contacting of the nucleic acids with the DNA mixture consisting only of random sequences.

6. The method of claim 5, wherein the electric field is a pulsed electric field or a constant voltage electric field. - 36 -

7. The method according to any one of the preceding claims, wherein the sample is disrupted under the influence of ultrasound.

8. The method according to any one of the preceding claims, wherein the immobilized DNA mixture consisting only of random sequences is brought into contact with the sample under the influence of an ultrasound treatment.

9. The method according to any one of the preceding claims, wherein after the binding of the nucleic acids to the DNA mixture is washed, preferably using an electric field.

10. The method according to any one of the preceding claims, wherein following the binding of the nucleic acids to the DNA mixture consisting only of random sequences or the detachment of the nucleic acids from the free or immobilized DNA mixture consisting only of random sequences, the nucleic acids are detected.

11. The method according to claim 10, wherein the detection is carried out after amplification of the nucleic acids.

12. The method according to claim 10 or 11, wherein the detection is in the form of an optical, spectrophotometric, luminometric, radioactive or electrical detection. - 37 -

13. The method according to any one of the preceding claims, wherein the bound nucleic acids are detached from the DNA mixture by increasing the temperature, in particular to 70 ° C, in the presence of a solvent.

14. The method according to any one of the preceding claims, wherein the sample is present in water, physiological saline or a buffer solution.

15. The method according to any one of the preceding claims, wherein the solvent, the detergent or both water, a physiological saline or a buffer solution.

16. Device for isolating nucleic acids from a sample, in particular for carrying out a method according to one of claims 1 to 15, comprising at least one sample chamber (210) with at least two flat electrodes

(220, 220 ') and at least one non-conductive frame part (230), the at least two flat electrodes (220, 220') and the frame part (230) being designed as a wall, cover or bottom part of the sample chamber.

17. The apparatus of claim 16, wherein the DNA mixture consisting only of random sequences (7, 7 ', 7' ', 300) on at least one of the electrodes

(220,220 ') and / or the frame part (230) is immobilized.

18. The apparatus of claim 16 or 17, wherein the at least two electrodes (220, 220 ') made of gold, - 38 -

Silver, precious metal, silver, aluminum, platinum or graphite exist or include this individually or in combination.

19. Device according to one of claims 16 to 18, wherein the frame part (230) consists of silicon, plastic or glass or comprise these individually or in combination.

20. Device for isolating nucleic acids from a sample, in particular for carrying out a method according to one of claims 1 to 14, comprising a DNA mixture consisting only of random sequences (7,7 ', 7' ', 300), which on a matrix (10) is immobilized.

21. Device according to one of claims 15 to 20, wherein the DNA mixture has 4 ^X different DNA random sequences (7, 7 ', 7'', 300), with x equal to the number of nucleotides per DNA random sequence, preferably with x is 5 to 50, in particular 15 to 30.

22. Device according to one of claims 15 to 21, wherein in the DNA mixture the random sequences

(7, 7 ', 7' ', 300) are present, preferably essentially the same proportions.

23. Device according to one of claims 15 to 22, wherein each random sequence (7, 7 ', 7'', 300) at the 5' or 3 'end is modified, in particular biotinylated. - 39 -

24. The device according to any one of claims 15 to 23, wherein each individual DNA random sequence (7,7 ', 7' ', 300) at the 5' or 3 'end an amino group or other common functional groups such as epoxy, suc - has cinimidester etc.

25. Device according to one of claims 15 to 24, wherein the surface of the matrix (10) has immobilized biomolecules (3) for binding the DNA mixture, in particular streptavidin.

26. Device according to one of claims 15 to 25, wherein the surface of the matrix (10), in particular the at least one electrode, has amino groups to which dialdehyde groups are bonded.

27. The device according to one of claims 15 to 26, wherein the matrix (10) as a membrane (1), electrode

(220,220 ') or columnar angel.

28. Device according to one of claims 15 to 27, wherein the device is applied to the surface of particles.

29. Device for carrying out the aforementioned method, comprising a flat non-conductive frame part (560), at least two flat electrodes (500) integrated in the frame part (560) and one on the frame part (560) and / or the electrodes (500) immobilized DNA mixture consisting only of random sequences

(550).