EP3596215A1

EP3596215A1 - Process for concentrating cells from a sample and then isolating nucleic acids from said cells

Info

Publication number: EP3596215A1
Application number: EP18716517.0A
Authority: EP
Inventors: Timo Hillebrand; Monique BRENDEL; Kristin WESSEL
Original assignee: AJ Innuscreen GmbH
Current assignee: AJ Innuscreen GmbH
Priority date: 2017-03-14
Filing date: 2018-03-14
Publication date: 2020-01-22
Also published as: WO2018167138A1; DE102017204267A1; US11702648B2; DE102017204267B4; CN110945124A; US20200255820A1

Abstract

The invention relates to the concentration and isolation of biological cells from a sample and/or the concentration of biological cells from a sample, followed by the isolation of nucleic acids from said cells, wherein the sample is brought into contact with a solid phase which has a rough or structured surface.

Description

METHOD FOR ENRICHING CELLS FROM A SAMPLE AND THE SUBSEQUENT NUCLEIC ACID ISOLATION FROM SUCH CELLS

description

The invention relates to a novel and highly simplified method which allows to enrich biological cells from a sample and to remove from the sample, then release the nucleic acids contained in the cells and isolate the way that the same means, which was used for the accumulation of cells, is also used for the isolation of nucleic acids.

The method can be carried out manually or fully automated. In particular, it simplifies the isolation of nucleic acids from larger sample volumes and in this context also reduces the use of necessary ones

Extraction Reagents.

Under classical conditions, the isolation of DNA from cells and tissues takes place in that the nucleic acid-containing starting materials under strongly denaturing and reducing conditions, partially using protein-degrading enzymes, digested and purified the exiting nucleic acid fractions via phenol / chloroform extraction steps and the nucleic acids by means of

Dialysis or ethanol precipitation from the aqueous phase (Sambrook, J., Fritsch, E.F. and Maniatis, T., 1989, CSH, "Molecular Cloning").

These "classical method" for the isolation of nucleic acids from cells and especially from tissues are very time consuming (sometimes longer than 48 h), require a considerable amount of equipment and are also not feasible under field conditions beyond. In addition, such methods due to the chemicals used such as phenol and chloroform in a not insignificant health hazard.

The classical methodology of isolation of nucleic acids has been described in the

significantly improved in recent years. The "new" methods are based on a method developed and first described by Vogelstein and Gillespie (Proc Natl Acad., USA, 1979, 76, 615-619) for the preparative and analytical purification of DNA fragments from agarose gels combines the dissolution of an agarose containing the DNA band to be isolated in a saturated solution of a chaotropic Salt (NaJ) with a binding of the DNA to glass particles. The DNA fixed to the glass particles is then washed with a washing solution (20 mM Tris HCl [pH 7.2], 200 mM NaCl, 2 mM EDTA, 50% v / v ethanol) and then removed from the carrier particles. This method has undergone a number of modifications to date and is currently used for different methods of extracting and purifying nucleic acids of various origins (Marko, MA, Chipperfield, R. and Birnboim, HG, 1982, Anal. Biochem. 121, 382-387).

[0007] In addition, a large number of reagent systems are present worldwide for the purification of DNA fragments from agarose gels and for the isolation of plasmid DNA from bacterial lysates, but also for the isolation of longer-chain nucleic acids (genomic DNA, total cellular RNA). from blood, tissues or even cell cultures.

All of these commercially available kits are based on the well-known principle of binding nucleic acids to mineral carriers in the presence of solutions of different chaotropic salts and use as carrier materials

Suspensions of finely ground glass powder (e.g., Glasmilk, BIO 101, La Jolla, CA),

Diatomaceous earth (Sigma) or silica gel. (Diagen, DE 41 39 664 AI).

A practicable for a variety of different applications process for the isolation of nucleic acids is shown in US 5,234,809 (boom). There is described a process for isolating nucleic acids from nucleic acid-containing starting materials by incubating the starting material with a chaotropic buffer and a DNA-binding solid phase. The chaotropic buffers realize both the lysis of the

Starting material as well as the binding of the nucleic acids to the solid phase. The method is well suited for isolating nucleic acids from small sample quantities and finds its practical application especially in the field of isolation of viral nucleic acids.

Specific modifications of these methods relate to the use of novel support materials, which show applicational advantages for certain questions (WO-A 95/34569). Further improved extraction variants relate to the use of novel lysis / binding buffers.

The patent EP 1135479 discloses that for the adsorption of nucleic acids to the skilled person known and used silicate materials and so-called antichaotropic salts can be used very efficiently and successfully as part of lysis / binding buffer systems. Advantage of this procedure is that through the

Bypassing the use of chaotropic salts significantly reduces health Endangerment of the extraction systems. However, for the efficient isolation of nucleic acids from a complex biological sample, in particular high salt concentrations (> 1.5 M) are required, in particular with regard to the highest possible extraction of nucleic acid in the lysis buffer. Thus, the patent discloses that lysing buffers used contain salt concentrations between 1.5 M - 3 M. In the patent DE 4321904 a method is described that when combining chaotropic high salt buffers with alcoholic components efficient isolation of nucleic acids is possible.

The analysis of the prior art illustrates impressively that there are a variety of possibilities, nucleic acids to solid support materials, especially mineral support materials based on silicon or even to magnetic or paramagnetic silicate support materials or magnetic or paramagnetic materials that carry functional groups, the same function as silicate materials, to bind, to subsequently wash and to detach the nucleic acids from the substrate again. The materials are as membranes (resp.

Glasfaserfiiese) before or in the form of micro or nanomaterials. For the

Binding mediation of the nucleic acids to the carrier materials are both so-called.

chaotropic salts as well as so-called. Antichaotropic salts or mixtures thereof used. All of these methods work independently of the mineral carrier materials used for the binding of the nucleic acids according to the same process flow:

1. Lysis of the sample

2. Optionally adding the binding buffer necessary for the binding of the nucleic acid

3. contacting the reaction mixture with a nucleic acid-binding solid phase (carrier material) and subsequent attachment of the nucleic acid to this material)

4. Washing the nucleic acid bound to the carrier material

5. detachment of the bound nucleic acid from the carrier material

The prior art discloses that methods for the isolation of nucleic acids from small-volume samples work very efficiently. If you want big ones

Sample volumes are processed, then lose these methods significantly in efficiency and are also in their implementation increasingly complicated and expensive. This concerns above all the isolation of nucleic acids from whole blood samples.

A solution variant for the isolation of DNA from larger amounts of blood (> 200 μΐ) based on the fact that the sample is mixed in the usual way with a larger volume of lysis buffer, subsequently adding a binding buffer and the approach, for example by means of a "funnel device" successively via a filter membrane for connecting the

Nucleic acid passes. Such commercially available kits allow the use of more than 1 ml samples for the isolation of nucleic acid. However, these processes require a significantly higher proportion of extraction reagents and are expensive to carry out.

Another widely used solution variant for the isolation of nucleic acids from large-volume blood samples based on the fact that the erythrocytes are selectively destroyed in first steps and then pelleted the nucleated cells by centrifugation. This drastically reduces the sample volume. It continues to work with the obtained nucleated cells. A disadvantage of such a method are the

to be performed process steps of the lysis of the erythrocytes and the pelleting of nucleated cells. Automation of these steps requires a centrifuge. It is known that the technical implementation of a centrifuge in an automated process for the isolation of nucleic acids is complicated and expensive.

Further possibilities for processing larger sample volumes with the aim of isolating nucleic acids, in particular from pathogenic microorganisms, are based on the use of so-called immunomagnetic methods. These methods are based on the use of e.g. magnetic beads coupled to a capture molecule (e.g., with a target specific antibody). These beads are contacted with the biological sample and incubated for several hours. The target is to specifically bind to the antibody. The beads are subsequently separated and washed. Then the detachment of the bound targets takes place. In the case of the planned isolation of nucleic acids from the bound analytes, the bound analyte is lysed and subsequently carries out a known nucleic acid isolation.

These methods are expensive and extremely expensive because they are always based on the use of antibodies coupled to magnetic beads. In addition, such methods are also not robust, since the beads with the coupled antibodies must always be cooled. Another related method can be found in the Journal of Virology (Vol. 83, No.10, 2009). This is the immunomagnetic accumulation of

Viral antigen in urine samples. In this case, 1.5 ml of sample can be used. The method uses magnetic beads which are coupled to a protein (ApoH). These beads serve to bind virus antigen from the sample. Incubation of sample with beads is 2h. Thereafter, the beads are separated and washed several times. Subsequently, the lysis of the virus is carried out by means of a lysis buffer. The lysis buffer is then placed on a filter column and the nucleic acid bound in a conventional manner to the filter column, washed and subsequently isolated the viral nucleic acid. This process is again expensive and expensive to work with.

These methods show that it is possible to process larger sample volumes. However, it also turns out that the enrichment of the cells and the isolation of the nucleic acids from these cells are two separate processes and that the means used for the accumulation of the cells can not be used for the isolation of the nucleic acids ,

The patent US 7,964,364 discloses another method. Here is

described that cells from a sample bind to a solid phase. This solid phase is provided with a nonspecific capture ligand. This coating consists of carbohydrates or carbohydrate derivatives. Preferably, microorganisms bind. The solid phase is magnetic particles. After binding, the solid phase is removed from the sample and the cells are lysed. Below are

Conditions set that the released nucleic acid can bind to the magnetic particles. After washing, the DNA is eluted. This is what the

surface-modified magnetic particles on the one hand of the adsorption of cells from a sample and subsequently for the isolation of the nucleic acid from the previously separated cells. Similarly, in the patent US 7,776,580 and in the

Published patent application DE 10 2010 031 401 disclosed methods and means. Cells or cellular components are bound to magnetic particles and separated from the sample, the cells are lysed and the released nucleic acid is bound to the same magnetic particles and subsequently isolated. The for the connection of the

Nucleic acids or extraction chemistry used for the isolation of the nucleic acids corresponds exactly to the state of the art. Thus, these methods are an improvement, since the particles used for cell binding in a further function also allows the isolation of nucleic acids of the bound and separated cells. However, the disadvantage is the use of magnetic particles. Since they are all nano- or microparticles, the separation of these particles is difficult and very time-consuming, especially with viscous samples.

Thus, these methods are complicated and always require a hardware Solution that allows for magnetic separation. In addition, it has been shown that magnetic micro- or nanoparticles have only a very limited binding capacity for nucleic acids. In addition, the surfaces of the particles often still have to be chemically modified, which means extra time and cost. It is also important to mention that nanomaterials may pose significant health risks.

The present invention solves the problem of the combined accumulation of cells and subsequent isolation of the nucleic acids in a surprisingly simple manner.

The invention was based on the following surprising observation. When a sample containing cells (e.g., a blood sample) is contacted with a plastic material having a rough surface (e.g., roughened polypropylene), the cells bind to the plastic material. There are no capture ligands on the material as known in the art.

The term "rough surface" should be understood to mean that it is not smooth by touching or by viewing the surface, but it may also be a surface having a structure (eg grooves). This structure eliminates the smoothness of the surface, even though the structure, ie, the grooves, itself may be smooth, and according to the invention such surfaces are referred to as "structured surfaces". If, by viewing or touching the surface, it is not possible to detect whether a surface is smooth or rough, a test may be performed by directing a laser beam at that surface. With a smooth surface, the laser is only in

Main direction reflected on the surface. On rough surfaces, scattering takes place in all spatial directions. Such a test has been described on the web page of the University of Kiel (http: //www.tf.uni- kield (^ matwis amat semitech_cn ka ^

For a person skilled in the art it is easy to find out whether a surface has the necessary roughness for a binding of cells or nucleic acids. He just has to make an appropriate experiment with the surface to be tested by bringing an aqueous solution of biological cells into contact with this surface and checking that the cells are tethered.

After fixation of the cells to the surface, the cells can be removed with the surface of the sample. The object of the invention, the isolation of the nucleic acids contained in the fixed cells using the same agent, is achieved by lysing the cells with a lysis buffer and thus releases the nucleic acids. To

Release of the nucleic acids, these can also be isolated with the same surface, which was used for the fixation of the cells.

The inventive method combines for the first time the adsorption of cells of a sample to a material with a "structured surface" with the

subsequent isolation and purification of nucleic acids using the same surface. The process can be carried out as a "single-tube process" or in the form of an automated "walk-away process". The used

Carrier material with a "structured surface" is inexpensive and, in contrast to used nanoparticles, harmless.

There is no real limitation of the method with respect to the volume of the sample and also not with respect to the nature of the sample. Especially the last point has a decisive advantage, because it also allows samples to be processed efficiently, which can only be processed with the known methods of nucleic acid isolation consuming, especially samples with high inhibitory potential. The advantage is based on the method according to the invention. In the first step, the cells from a sample are adsorbed to the "textured" or rough surface, which can discard the sample and, in this context, the inhibitory matrix components become. For subsequent nucleic acid isolation then you already have clean cells. This is i.a. also essential in the isolation of DNA from blood samples.

The process sequence is simple, extremely fast and extremely efficient and combines according to the invention an accumulation of cells with the subsequent extraction of the nucleic acid. The invention relates to the disclosed methods and means of the publications DE 10 2015 216 558, DE 10 2015 211 394 and DE 10 2015 211 393. The invention is based on the properties described therein, that nucleic acids on rough or

Surprisingly, the present invention shows that such a material is not only suitable for isolating nucleic acids, but also suitable for binding cells from a sample, thus resulting in the method according to the invention of such a material in one Dual function combined to use.

The essence of the invention is thus that cells of a sample to a "Structured surface" adsorb and subsequently the liberated nucleic acids are in an aqueous environment whose polarity is adjusted by organic substances so that the solubility of the nucleic acid is reduced and subsequently precipitated on the "structured surface" and subsequently the precipitated DNA of the "structured" gray surface is replaced again and is available. Optionally, the precipitated on the rough surface nucleic acid can also be washed and replaced after washing steps.

The process is extremely simple and quick to carry out. It can generally be carried out in the following steps:

1. contacting a sample containing cells with a material having a

"Structured surface" and subsequently adsorption of the cells contained in the sample to this surface

Contains calcium chloride added. This can increase the efficiency of cell adsorption. Removing the surface from the sample and optionally washing the adsorbed cells on the surface.

2. Lysing the cells with a lysis buffer. This lysis buffer may contain chaotropic salts or non-chaotropic salts or mixtures of these two groups. In addition, this buffer may contain other components such as chelating agents, Tris buffer, wetting and dispersing agents, etc. However, the method also works with buffers which contain no salts and e.g. consist only of a detergent, Tris and EDTA. In addition, proteolytic enzymes can also be used.

3. Addition of a binding buffer containing an alcoholic component and others

Adding or adding an alcohol alone or even adding acetone or gasoline. Thus, the nucleic acid of the sample precipitates to the same structured one

Surface to which the cells were previously adsorbed.

4. If necessary Washing and subsequently drying the "structured surface"

5. Final detachment of the nucleic acid from the material with an elution buffer

(Low salt buffer or water).

All these process steps of the cell adsorption, the lysis of the cells, the binding of the nucleic acids, the washing of the bound nucleic acids and the final detachment of the nucleic acids take place in that the "structured surface" by shaking or by pipetting with the respective components in Contact is brought. The method is universally applicable and can be carried out both automatically and manually.

Preferably, in particular for an automated sequence using any liquid handling stations, the agent according to the invention can also be in a device for extracting nucleic acids, comprising a hollow body through which a liquid is passed, wherein in this hollow body a material with rough In a preferred embodiment, a pipette tip acts as a hollow body The rough or "textured surface" material has a size such that it can not escape from the pipette tip at the bottom can and thus differs from the magnetic particles described in the prior art (WO 01/05510 Al). In sum, it is necessary that within the pipette tip by the spent material a two / three-dimensional structure arises, to which cells first and subsequently, after lysis of the cells, the released nucleic acids can adsorb.

The used in the pipette tip materials for cell adsorption and subsequent attachment of nucleic acids can be extremely different. It can be used modified plastic materials whose surface is not smooth, but rough or textured. These include so-called composite materials containing blends of polymers and e.g. organic components as well as inorganic components. Essential is only the provision of a roughened or

"Structured surface" (no smooth surface) or the transfer of material into the pipette tip, which leads to the formation of a two / three-dimensional network, whereby the nucleic acids then precipitate on this structure.The architecture of the material is also not limiting (round, rectangular , etc.), which can also be multiple materials (eg several granules).

It is important that the material, which is spent in a pipette tip, at any time can be lapped by a liquid without the liquid must pass through the introduced material. Also, a pipette tip can be used, in which (made of a molded injection molded part) that binding material is already and this no longer needs to be spent in the top. Also advantageous is the use of rough or structured material, which additionally has magnetic or paramagnetic properties. Such a material is then also suitable for manual sample processing in which it is added separately to the sample and is not located in a hollow body. The sample with the contained cells is "pipetted" by means of pipetting operations on the material according to the invention introduced into the pipette tip, the cells adsorb onto the material In a preferred embodiment, calcium chloride may also be added to the sample containing the cells After cell attachment, the pipette tip is immersed in a cavity containing a lysis buffer and possibly also a proteolytic enzyme, lysis of the cells and release of the nucleic acids. The cells are thus no longer on

material according to the invention. According to the invention, the following serve

Process steps now the connection of the liberated nucleic acids to the same inventive material to which the cells were previously. After lysis of the cells, the nucleic acid to be isolated is in an aqueous form.

Subsequently, the conditions necessary for the precipitation of the nucleic acids are set so that the nucleic acid can precipitate on the material that is being dispensed in the pipette tips. The approach is by means of pipetting vertically in the

Pipette tip spent nucleic acid binding material "pipetted over"

Nucleic acids precipitate on the material. Subsequently, wash buffer may optionally be "pipetted" past the nucleic acid binding material, followed by a drying step (e.g., frequent pipetting up and down) Finally, the eluent is again "pipetted" past the vertically arranged nucleic acid binding material, thereby peeling off the bound nucleic acid. The nucleic acid is now available for necessary downstream application. The process is extremely fast and easy to carry out and allows isolation of nucleic acids in extremely high yield and purity.

The present invention, in addition to the simplicity of the implementation of another enormous advantage. Contains a biological sample large amounts of cells containing nucleic acids, so by means of the method according to the invention and the

Materials of the invention are extracted an extremely large amount of nucleic acids. Thus, the method is also ideally suited for the processing of large sample volumes.

The invention will be explained in more detail with reference to embodiments. The embodiments do not represent a limitation of the invention. embodiments

Example 1: Enrichment of cells from an aqueous solution combined with the subsequent extraction of the nucleic acid contained in the cells using a modified pipette tip and using a commercially available extraction machine

The automated extraction was carried out with the InnuPure C16 extraction machine (Analytik Jena AG).

For carrying out a nucleic acid extraction according to the inventive method, the pipette tips were changed so that they correspond to the agent according to the invention. A roughened plastic granulate (4 granules with a diameter of about 2 mm - 4 mm, polypropylene) was loosely placed in the lower third of the pipette tips. The granules do not close the lumen of the pipette tip. This preserves the pipetting function of the pipette tips.

The inventive method was then implemented on the machine InnuPure C16 as follows:

The reagents required for the procedure were in a pre-filled deep well plate. In the first well of the deep well plate, the aqueous cell suspension was spent.

The pipette tip with the material according to the invention was retracted into the cavity. The cell binding is done by pipetting up and down (100 repetitions). In this process, the cells adsorb to the material of the invention contained in the tip. After completion of the pipetting process, the tip was moved out of the cavity. The cells are located on the material inside the tip. The tip was subsequently retracted into a second well of the deep well plate. This cavity contains a commercially available lysis buffer as well as Proteinase K (Lysis Solution CBV, InnuPREP Blood DNA K PC16, Analytik Jena AG). The lysis of the cells located on the material was carried out by pipetting up and down the lysis buffer (200

Repetitions). The cavity was additionally heated. At the end of the process, the released nucleic acid of the cells is in the lysis buffer. The cells are no longer on the material according to the invention. The sample was now drawn into the pipette tip and dispensed into another cavity of the deep well plate. This cavity is prefilled with an alcohol (isopropanol). This solution was then again pipetted up and down by means of the pipette tip. Again, so that the solution flows past the granules (100

Repetitions). Now, the binding of the liberated nucleic acid to the material according to the invention, with which previously the cells were already adsorbed from the sample. After binding of the nucleic acid to the granules were followed by washing steps.

For this purpose, a further 3 wells of the deep well plate were filled with an alcoholic wash buffer. Washed by pipetting up and down the wash buffer (10 reps each).

Following the last washing step, the tip of the invention and the granules contained in it were dried by pipetting air and thus the residual ethanol removed. The elution of the nucleic acids from the granules was carried out in another well of the deep well plate, in which 200 μΐ water were contained as eluent. The nucleic acid was removed from the granules by pipetting up and down the water (120

Repetitions) replaced.

The method is extremely simple and fast and shows that commercially available extraction machines for carrying out the method according to the invention in the combination of cell binding and removal of cells from the initial sample and subsequent extraction of the nucleic acid contained in the cells with the corresponding inventive Means can be used.

The detection of the isolated nucleic acid was carried out by spectrophotometric measurement and gel electrophoresis.

Results of spectrophotometric measurement

A gel electrophoretic analysis of the isolated nucleic acid is shown in FIG. 1.

Shown is the electrophoretically separated in a 0.8% agarose gel nucleic acid isolated by the method according to the invention. The samples were applied from left to right beginning with sample 1. Lane 1 contains a DNA ladder.

Example 2: Enrichment of nucleated cells from different volumes of whole blood samples combined with the subsequent extraction of the nucleic acid contained in the nucleated cells using a modified pipette tip and using a commercially available extraction machine

The automated extraction was again with the extraction machine

InnuPure C16 (Analytik Jena AG).

For carrying out a Nuklemsäureextraktion according to the inventive method, the pipette tips were changed so that they correspond to the agent according to the invention.

A roughened plastic granulate (4

Granules with a diameter of about 2 mm - 4 mm; Polypropylene) introduced loosely. This preserves the pipetting function of the pipette tips.

The process according to the invention was then reacted on the InnuPure C16 machine as follows.

The reagents required for the procedure were in a pre-filled deep well plate. Different amounts of blood (200 μΐ, 300 μΐ, 400 μΐ and 500 μΐ) were spilled into the first well of the deep well plate. In each case, 800 μl of a commercially available erythrocyte lysis buffer (Ery Lysis Solution A, Analytik Jena AG) were used for these blood samples. In addition, 200 μΐ of a 1 M calcium chloride solution was added.

The pipette tip with the material according to the invention was retracted into the cavity. The cell binding is done by pipetting up and down (100 repetitions). In this process, the nucleated cells adsorb to the material of the invention contained in the tip. After completion of the pipetting process, the tip was moved out of the cavity. The cells are located on the material inside the tip. Thus, the cells were separated from the actual sample and thus also of inhibitory substances such as hemoglobin. This considerably facilitates the subsequent successful extraction of the nucleic acid. The tip was subsequently inserted into a second well of the deep well. Retracted plate. This cavity contains a commercially available lysis buffer as well as Proteinase K (Lysis Solution CBV, InnuPREP Blood DNA K PC16, Analytik Jena AG). The lysis of the cells located on the material was carried out by pipetting up and down the lysis buffer (200 repeats). The cavity was additionally heated. At the end of the process, the released nucleic acid of the cells is in the lysis buffer. The cells are no longer on the material according to the invention. The sample was now drawn into the tip of the beeper and released into another cavity of the deep well plate. This cavity is prefilled with an alcohol (isopropanol).

This solution was then again pipetted up and down by means of the pipette tip in such a way that the solution flows past the filled material (100 repetitions). Now, the binding of the liberated nucleic acid to the material according to the invention, with which previously the cells were already adsorbed from the sample. After binding the

Nucleic acid to the granules were followed by washes.

These were another 3 wells of the deep well plate with the alcoholic

Wash buffer filled. Was washed by pipetting up and down the wash buffer (jeweisl 10 repetitions.

Following the last washing step, the tip of the invention and the material contained in it were dried by pipetting air, thereby removing the residual ethanol. The elution of the nucleic acids from the granules was carried out in another well of the deep well plate, in which 200 μΐ water were contained as eluent. The nucleic acid was removed from the granules by pipetting up and down the water (120

Repetitions) replaced.

The detection of the isolated nucleic acid was carried out by means of spectrophotometric measurement and gel electrophoresis. It turns out that increasing the amount of blood also increases the yield of nucleic acid. Thus, the method is also excellent for the processing of larger sample volumes.

Results of spectrophotometric measurement

Blood sample 1 (300 μΐ) 51.5 10.3 1.7 2.3

Blood sample 1 (400 μΐ) 72.5 14.5 1.7 2.3

Blood sample 1 (400 μΐ) 58 11.6 1.7 2.2

Blood sample 1 (500 μΐ) 75.5 15.1 1.7 2.1

Blood sample 1 (500 μΐ) 69.5 12.9 1.7 2.1

FIG. 2 shows a gel electrophoresis analysis of the isolated nucleic acid. The nucleic acid which has been electrophoretically separated in a 0.8% agarose gel and isolated by means of the method according to the invention is shown. The samples were applied from left to right beginning with sample 1. Lane 9 contains a DNA ladder. Example 3: Enrichment of the nucleated cells from whole blood samples combined with the subsequent extraction of the nucleic acid contained in the nucleated cells using three differently modified pipette tips and using a commercially available extraction machine. The automated extraction was again carried out with the InnuPure C16 extraction machine (Analytik Jena AG).

For carrying out a Nuklemsäureextraktion according to the inventive method, the pipette tips were changed so that they correspond to the agent according to the invention. For this purpose, pipette tips were modified as follows:

1. Pipette tips in the lower third vertically with roughened plastic granules

Polypropylene (4 granules with a diameter of approx. 2 mm - 4 mm) filled loosely

2. Pipette tips in the lower third vertically with roughened plastic granules

Polypropylene (5 granules with a diameter of approx. 2 mm - 4 mm) loosely filled 3. Pipette tips in the lower third vertically filled with a spiral plastic polyethylene material of a length of 1, 5 cm (as an example of a structured material).

The materials were loosely in the pipette tip so that the pipetting function of the pipette tips remained completely intact. According to the invention, the liquids to be pipetted always moved past the material.

The inventive method was then on the machine InnuPure C16 as follows implemented:

The reagents required for the procedure were in a pre-filled deep well plate. A whole blood sample (500 μΐ) was placed in the first well of the deep well plate. In each case, 800 μΐ of a commercially available erythrocyte lysis buffer (Ery Lysis Solution A, Analytik Jena AG) were transferred to this blood sample. In addition, 200 μΐ of a 1 M calcium chloride solution was added.

The pipette tip with the material according to the invention was introduced into the cavity. The cell binding is done by pipetting up and down (100 repetitions). In this process, the nucleated cells adsorb to the material of the invention contained in the tip. After completion of the pipetting process, the tip was moved out of the cavity. The cells are located on the material inside the tip. Thus, the cells were separated from the actual sample and thus also of inhibitory substances such as hemoglobin. This considerably facilitates the subsequent successful extraction of the nucleic acid. The tip was subsequently retracted into a second well of the deep well plate. This cavity contains a commercially available lysis buffer as well as Proteinase K (Lysis Solution CBV, InnuPREP Blood DNA K PC16, Analytik Jena AG). The lysis of the cells located on the material was carried out by pipetting up and down the lysis buffer (200 repeats). The cavity was additionally heated. At the end of the process, the released nucleic acid of the cells is in the lysis buffer. The cells are no longer on the material according to the invention. The sample was now in the

The tip of the beeper is drawn up and transferred to another cavity of the deep well plate. This cavity is prefilled with an alcohol (isopropanol).

This solution was then again pipetted up and down by means of the pipette tip. Again, so that the solution flows past the filled material (100 repetitions). Now, the binding of the liberated nucleic acid to the material according to the invention, with which previously the cells were already adsorbed from the sample. After binding of the nucleic acid to the granules were followed by washing steps.

For this purpose, a further 3 wells of the deep well plate were filled with alcoholic washing buffer. Was washed by pipetting up and down the wash buffer (each 10

Repetitions).

Following the last washing step, the tip of the invention and the material contained in it were dried by pipetting air, thereby removing the residual ethanol. The elution of the nucleic acids from the granules was carried out in another Deep well plate well containing 200 μΐ of water as eluent. The nucleic acid was detached from the granules by pipetting up and down the water (120 repetitions).

Results of spectrophotometric measurement

A gel electrophoretic analysis of the isolated nucleic acid is shown in FIG. 3.

Claims

claims

Method for enriching and isolating biological cells from a sample, characterized in that the sample is brought into contact with a solid phase which has a rough or structured surface.

2. The method according to claim 1, characterized in that the sample before the

Contacting a salt of a di- or trivalent cation, preferably

Calcium chloride, is added.

3. The method according to claim 1 or 2, characterized in that it is the rough surface to a non-smooth plastic or rubber surface.

4. The method according to claim 3, characterized in that the non-smooth plastic surface is produced by a 3D printing or by roughening a plastic.

5. The method according to any one of claims 1 to 4, characterized in that rough

Find composite materials as a solid phase use.

6. The method according to any one of claims 1 to 5, characterized in that as solid

Phase roughened pipette tips are used, preferably those which additionally have magnetic or paramagnetic properties.

7. The method according to any one of claims 1 or 2, characterized in that in one or on a smooth hollow body, preferably a pipette tip, a solid phase is introduced or plugged with rough or textured surface, wherein it is preferably in the plugged solid phase is a ring or a sleeve.

8. A method for the enrichment of biological cells from a sample and the subsequent Nuklemsäureisolierung from these cells, characterized by the following

Steps: a) contacting a sample containing biological cells according to any one of Claims 1 to 7

b) Lysing the cells

c) attaching the liberated by the lysis of nucleic acids from the cells to the same material to which according to claims 1 to 7, the cells were attached d) If necessary. Washing and subsequently drying the solid phase

e) Final detachment of the nucleic acid from the material with an elution buffer, preferably with a low salt buffer or water.

9. The method according to claim 8, characterized in that it is manually or fully automated, as a "single-tube process" or in the form of an automated

"Walk-away procedure" is performed.

10. Use of solid phases, preferably pipette tips which have a rough or structured surface, for enrichment and isolation of biological cells from a sample and / or for enrichment of biological cells from a sample and the subsequent nucleic acid isolation from these cells.