EP1194591A2

EP1194591A2 - Method and kit for determining dna double/single strand breaks and device for carrying out the controlled filtration of, in particular, dna molecules with well filter plates

Info

Publication number: EP1194591A2
Application number: EP00949369A
Authority: EP
Inventors: Werner E. G. MÜLLER; Heinz C. SCHRÖDER; Renato Batel
Original assignee: Schroder Heinz C
Current assignee: Schroder Heinz C
Priority date: 1999-07-19
Filing date: 2000-07-18
Publication date: 2002-04-10
Also published as: AU6275100A; WO2001006003A2; DE19933078B4; DE19933078A1; WO2001006003A3

Abstract

The invention relates to, among other things, a method for determining double strand breaks which comprises the following steps: a) depositing cells and/or tissues to be examined on specific filters; b) lysis of the cells/tissue with at least one detergent, with at least one chaotropic substance, and with at least one fluorescence dye that binds specifically a DNA double strand; c) elution of the DNA fragments, released by the lysis, through the specific filters, and; d) carrying out a fluorescence spectroscopic evaluation, whereby only one eluate fraction is collected per well.

Description

Method and kit for determining DNA double / single strand breaks and device for the controlled filtering of, in particular, DNA molecules with well filter plates

The invention relates generally to Methods and kits for determining DNA double or single strand breaks.

In particular, the invention relates in particular to a method and a kit for determining DNA double-strand breaks, corresponding uses, devices for the controlled filtration of liquid media containing well, in particular DNA molecules and / or other molecules / molecular complexes, with well filter plates and various uses.

A number of assays for determining DNA double-strand breaks are known from the prior art. These are in particular neutral filter elution, pulsed field gel electrophoresis and the neutral comet assay (Elia et al., Pharmacol. Ther. 51: 291-327; 1991; Sarkaria et al, Radiat. Res. 150 : 17-22; 1998; Prize et al .. Int. J. Radiat. Biol. 74: 173-184; 1998).

In filter elution, the cells to be examined for DNA damage are placed on filters and first treated with a neutral lysis solution to remove non-DNA components. The DNA remains on the filter. The filter with the DNA on it is then slowly passed through with a suitable solution for several hours, the washed-out solution being collected in several fractions. The distribution of the DNA between the collected fractions is measured. This procedure is very time-consuming (work usually takes more than 10 hours), complicated to carry out; susceptible to failure (frequent leakage of the filter and filter holder), and it can only be used for the simultaneous determination of double-strand breaks in a relatively small number of samples.

Pulse field gel electrophoresis is an agarose gel electrophoresis which can detect double-strand breaks, which were only induced with a 1 Gy ionizing radiation, but which has inherent problems in measuring the strand break repair. Pulsed field gel electrophoresis is best performed with cell suspensions, but not with tissue samples. Costly equipment is required to implement this method. The procedure is time-consuming (approx. 24 hours) and allows only a relatively small number of samples to be determined at the same time.

In the comet assay ("single-cell gel electrophoresis"), DNA strand breaks are measured in single cells. For this purpose, the cells are suspended in "low melting point" agarose on a slide. The slide is then placed in a lysis buffer and then in the electrophoresis buffer. During electrophoresis, the damaged DNA migrates towards the anode, forming a comet's tail. The greater the extent of the damage (number of strand breaks), the larger the resulting tail. Double strand breaks are measured under neutral conditions and single strand breaks under alkaline conditions. A disadvantage of this method is that it is not possible to determine DNA damage in tissues. In addition, the method for evaluation requires a costly image analyzer and experienced personnel for the interpretation of the results. Finally, the time for sample preparation before lysis is relatively long.

EP 0 359 249 discloses i.a. an apparatus and a method for filtering liquid media with well filter plates in combination with microtiter plates. In this device, a connection for applying a vacuum is provided below the respective filter. The disadvantage here is the relatively complicated design of the device, and in addition, a controlled filtering of liquid media is not always guaranteed during the method. In the commercially available MultiScreen vacuum filtration system (Millipore GmbH), the vacuum required for slowly sucking through the samples, which is necessary for the method described in this application for determining DNA double-strand breaks, cannot be obtained with the aid of the vacuum slide switch on this device build up.

From the above, the problem arises of at least partially eliminating the disadvantages listed above. The problem that arises in particular is to provide a method and a kit for the determination of DNA double-strand breaks, which have a high sensitivity and accuracy and at the same time a rapid determination of the DNA damage (working time less than 3 hours) with a high sample throughput (e.g. parallel Allow determination of a larger number of samples, for example of 96 samples when using 96-well microtiter plates). In addition, the problem is to provide an apparatus and a method that ensure improved controllability of the filtration. There is an urgent need for such a procedure, for example in medicine (determination of the optimal radiation dose for radiation therapy patients) or in environmental monitoring (proof of the effect of genotoxic substances).

This problem is solved according to the invention by a method according to claim 1, a kit according to claim 3, devices according to claims 4 and 5, methods according to claim 6, and uses according to claim 10.

In the method according to the invention, the cells and / or tissues to be examined are first placed on specific filters. These can be commercially available filter types suitable for this purpose, for example MultiScreen filtration plates from Millipore GmbH (Eschborn, Germany) with Durapore membranes (e.g. 0.22 μm pore size). Depending on the length of the DNA, membranes with smaller or larger pore sizes can be advantageous.

The cells / tissues are then lysed in the presence of at least one detergent, for example SDS (sodium dodecyl sulfate), at least one chaotropic substance, for example urea, and at least one substance which complexes metal ions and inhibits DNA repair enzymes, for example EDTA, or at least one protease, for example proteinase K. Already in this step, at least one fluorescent dye that binds a specific DNA double strand can be added. The substance that complexes metal ions and inhibits DNA repair enzymes is necessary because firstly metal ions for cross-linking can lead to wetting of the DNA and thereby impair its elution and can lead to an incorrect assessment of the proportion of intact DNA and secondly because DNA repair enzymes lead to an elimination of the DNA damage to be measured. Alternatively, a protease can be used, since this lyses DNA repair enzymes during lysis, which would otherwise lead to an elimination of the DNA damage to be measured.

The DNA fragments released by the lysis are then eluted through the filters at a controlled, constant elution rate and a fluorescence spectroscopic evaluation is then carried out. The elution is carried out in particular by the devices according to the invention for the controlled filtration of liquid media with filter plates.

In the process according to the invention, use is made of the knowledge that after the cell lysis and the disintegration of the DNA-histone complexes taking place simultaneously in the lysis solution, the released DNA molecules are washed out with the lysis / elution solution. The elution of the DNA molecules is faster the shorter they are ("sieve effect"). The method thus allows the number of double-strand breaks in the DNA to be determined.

In the lysis step, the concentration of the chaotropic substance is advantageously 2 to 4 mol / 1. since sufficient cell lysis and denaturation is effected in this area without the quench effect becoming too strong. In addition, the concentration of the substance complexing metal ions and inhibiting DNA repair enzymes is advantageously 0.005 to 0.05 mol / 1, since sufficient activity is guaranteed in this range. It is advantageous if the pH value is set to 7 to 11 during the lysis step, since optimal cell lysis is observed in this area without DNA unwinding (separation of the two DNA strands at higher ones) pH values).

It is particularly advantageous if the concentration of the chaotropic substance in the lysis step is 2.25 mol / 1, since at this concentration there is an optimum between denaturation on the one hand and colorability (with the fluorescent dye; only a slight quench effect) on the other hand. Furthermore, it is advantageous if the concentration of the metal ion complexing and DNA repair enzyme inhibiting substance of the lysis step is 0.02 mol / 1, since this means that sufficient traces of heavy metals (which can lead to crosslinking of the DNA) are removed with a strong reduction of the quench effect impairing the measurements can be ensured.

The following embodiments are advantageous because they have proven themselves in practice: the concentration of the detergent (in particular SDS) is 0.00175 mol / 1; the concentration of the protease in the lysis step is 0.5 mg / ml; it is used as a chaotropic substance urea; it is used as a metal ion complexing substance and DNA repair enzyme inhibiting substance EDTA; Proteinase K is used as protease; SDS is used as detergent; the pH during lysing is 10.0. After all, the fluorescent dye PicoGreen has proven to be extremely reliable and therefore advantageous.

The method has the following advantages: 1. The method requires only a small amount of time (approximately one hour). 2. The process is highly sensitive; only small amounts of sample DNA are required (about 30 ng DNA per well; this corresponds to about 3000 cells or 25 μg tissue).

3. The method allows a high sample throughput (96 determinations per hour when using a filtration device with a 96-well microfilter plate).

4. The procedure can be carried out with non-radioactive DNA.

5. The method can be used not only for cells but also for tissue samples.

6. The procedure is simple and does not require any special expertise. The following kit for determining DNA double-strand breaks has the advantages mentioned above: The kit contains at least one detergent, at least one chaotropic substance, at least one fluorescent dye which binds a specific DNA double-strand and at least one specific filter type.

The following embodiments of the kit are advantageous for the above reasons: The kit contains at least one substance which complexes metal ions and inhibits DNA repair enzymes. The kit contains at least one protease. The concentration of the chaotropic substance during lysis is 2 to 4 mol / 1. The concentration of the metal ion complexing and DNA repair enzyme inhibiting substance during lysis is 0.005 to 0.05 mol / l. The protease is Proteinase K. The concentration of the protease in the lysis step is 0.01 to 5 mg / ml.

The subsequent uses of the method according to the invention and the kits according to the invention also have the surprising and advantageous properties listed above. In particular, it concerns uses for:

• Detection of DNA double-strand breaks and cross-links in human, animal or vegetable tissues and cells, in bacteria and unicellular organisms, in cell lines of human, animal or vegetable origin and subcellular fractions, as well as in isolated DNA;

• Detection of DNA repair in human, animal or plant tissues and cells, in bacteria and unicellular organisms, in cell lines of human, animal or plant origin, subcellular fractions, cell lysates and cell extracts (isolated molecules);

• To measure the occurrence of DNA double-strand breaks and cross-links and their repair after radiation, chemotherapy and / or treatment with genotoxins in human and animal sample material, tissue sections and cell suspensions;

• Measurement of the occurrence of DNA double-strand breaks and cross-links and their repair in the tissues and cells of patients during radiation therapy;

• Detection of DNA double-strand breaks in frozen examination materials (for example biopsy samples) after their gentle homogenization in liquid nitrogen or liquid air in the presence of an anti-freeze agent (for example dimethyl sulfoxide);

• Evidence of the occurrence of DNA double strand breaks and cross-links and their repair in the event of occupational exposure of persons who come into contact with DNA-damaging substances or radiation either during their work (e.g. flight attendants, X-ray doctors) or as a result of accidents (e.g. reactor accidents); """.-,",

5 PCT / EP00 / 06842

• Measurement of the occurrence of DNA double strand breaks and cross-links and their repair in test organisms, tissues, cells or cell lines during water monitoring.

The devices according to the invention for the controlled filtering of liquid media containing in particular DNA molecules and / or other molecules / molecular complexes with well filter plates, with at least one well filter plate and at least one corresponding microtiter plate are the following embodiments:

The first embodiment (the first device) is characterized by two separate gas spaces arranged above and below the filter plate, at least one sealing device which interacts with a container and the well filter plate and thus creates a separation of the two gas spaces, a device for connecting the two Gas spaces and a device for pressure equalization between the environment and a gas space.

One gas space is located above the filter plate and the micro titer plate arranged below it. in the wells of which the medium to be filtered is applied beforehand (before inserting the filter plate into the device). A second gas space, separated from the first gas space, is located below the filter plate. First, both gas spaces are connected to one another by means of a tap arranged in a bore (this embodiment has proven itself in an advantageous manner), so that the pressure in both gas spaces is the same. Both gas spaces are via the suction device, for example a suction pipe connected to a corresponding vacuum pump. evacuated or a negative pressure was established with respect to the environment and then the two gas spaces were separated from one another (for example by closing the tap). The previously closed device for pressure equalization between the surroundings and the upper gas space is now carefully opened to the surroundings (i.e. to the outside atmosphere), so that the pressure in the upper gas space can be increased very slowly and in a metered manner compared to the gas pressure in the lower gas space. In this way, a controlled filtering of the liquid medium is possible, in particular for the solutions for measuring DNA double-strand breaks.

The "suction pressure" required in conventional devices and methods (contact pressure of the sealing device arranged between the filtration plate and the corresponding microtiter plate, in particular in the form of a rubber ring, for sealing between the two gas spaces) is generally so high that controlled filtration is not possible. The advantage of the principle used in the device according to the invention is that the filtration process can be carried out at a constant speed without the occurrence of the initial pressure jump which occurs in suction devices which are operated by applying a negative pressure as a result of the suction process (pressing process on the sealing ring).

In particular, the low filtration / elution rates in the range of a few microliters per minute required in the method according to the invention for measuring DNA double-strand breaks cannot be achieved by means of conventional devices and methods, but are not a problem for the devices and methods according to the invention. The second embodiment (second device) is characterized by two separate gas spaces arranged above and below the filter plate, at least one sealing device which interacts with a container and the well filter plate and thus creates a separation of the two gas spaces, and a pressurizing device which functions for a gas space , One gas space is located above the filter plate and the microtiter plate arranged below, in the wells of which the medium to be filtered is applied beforehand (before inserting the filter plates into the device). A second gas space, separated from the first gas space, is located below the feed plate. After the medium has been applied to the respective wells of the feed plate, a pressure which is higher than the lower gas space, in particular 0.01 bar, is applied to the medium to be filtered in one of the two gas spaces, preferably in the upper one, by means of the pressurizing device. In this way, a controlled filtering of the liquid medium is possible, in particular for the solutions for measuring DNA double-strand breaks.

A method according to the invention, which uses one of the filtration devices according to the invention, has the surprising and advantageous properties mentioned above. This also applies to the corresponding uses.

These are uses: for the detection and quantification of DNA strand breaks, of cross-linking of DNA molecules with one another or with other molecules, and of further DNA damage; for binding studies, to study DNA-protein or receptor-ligand interactions and to determine the dissociation / association constants of these reactions; for the filtration of radioactive materials with optional autoradiographic detection of the radioactivity remaining on the filters; for the filtration of infectious samples; for the filtration of oxygen sensitive samples; for filtration under sterile conditions.

In particular, the invention further relates to a method for determining DNA single-strand breaks, the use of several fluorescent dyes, a kit for determining DNA single-strand breaks, use of the kit and uses of the method and the kit.

DE 197 24 781 AI discloses inter alia a micro method for the rapid determination of DNA damage and its repair using the fluorescent dye Picogreen. In the method described there, the cells to be examined are first lysed with the aid of SDS with a 9 molar urea solution. About 200 mmol / l EDTA and a corresponding fluorescent dye, in particular Picogreen, which binds specifically DNA single-strand or DNA double-strand are added. A sodium hydroxide solution with a pH of approx. 12.5 is added and then evaluated by fluorescence spectroscopy. With this method, the high concentration of urea is necessary to ensure the desired denaturation. In addition, approx. 200 mmol / 1 EDTA must be used to ensure complete complexation of the metal ions that crosslink the DNA. The disadvantage of this method is the use of large amounts of substance, which lead to a high quench effect in the fluorescence evaluation and can, under certain circumstances, falsify the results. In addition, the sensitivity of the above method is sometimes unsatisfactory, especially for the determination of DNA strand breaks in (shorter) DNA molecules of lower organisms (important for the investigation of the effects of environmental pollutions) and for the determination of DNA strand breaks in radiation therapy patients in the dose range used for radiation therapy (0.5 - 3.0 Gy single dose).

From the above, the problem arises to at least partially eliminate the disadvantages listed above. The problem that arises in particular is to provide a method and a kit for the determination of single-strand DNA breaks which have a low quench effect, a high sensitivity and accuracy and a good reproducibility and at the same time a rapid determination of the DNA damage (working time less than 3 hours ) with a high sample throughput (eg parallel determination of 96 samples in 96-well microtiter plates or determination of even larger numbers of samples by using microtiter plates with an even larger number of wells).

This problem is solved according to the invention by a method according to claim 7 and a kit according to claim 9. In addition, the use of the method and uses of the kit are claimed according to claim 10.

In the method according to the invention, cells and / or tissues to be examined are first lysed with at least one detergent, for example SDS (sodium dodecyl sulfate) and at least one chaotropic substance solution, for example urea. The lysis takes place in the presence of at least one substance which complexes metal ions and inhibits DNA repair enzymes. At least one fluorescent dye that specifically binds single or double-stranded DNA is added in the lysis step or the subsequent step.

The step following the lysis is to add at least one solution to the suspensions containing the lysed cells and / or tissue, which allows the cell / tissue suspension to be adjusted to an alkaline pH and the at least one metal ion complexing and DNA repair enzyme inhibitory substance.

Due to the additional addition of at least one substance complexing metal ions and inhibiting DNA repair enzymes in the step following the lysis, the sensitivity and accuracy is completely surprising than with the method disclosed in DE 197 24 781 A1.

It is important in the process according to the invention that a fluorescent dye is used which is either specifically single-stranded or specifically double-stranded. The background to this requirement is the fact that the dye must adhere specifically to either single or double strands of the DNA. to determine the proportion of the respective strand types. For example, cyanine dyes such as PicoGreen can be used as selective or as approximately selective double-strand binding and OliGreen as selective single-strand binding. The method according to the invention is based on the following principle: In the double-stranded DNA, two polynucleotide chains (DNA single strands) are linked to one another via hydrogen bonds between two complementary bases. Both polynucleotide chains are wound around each other to form a right-handed double spiral (helix). Due to this structure, the two DNA single strands of DNA remain attached to each other even if breaks occur in them. Such single strand DNA breaks are among the most common types of DNA damage. A prerequisite for the two single strands of DNA to remain together is that the breaks - which is usually the case - are offset against one another on the two strands. If one exposes the DNA or the entire cell to a strongly alkaline solution, the hydrogen bonds between the single DNA strands are released. However, since the molecules are wrapped around each other, they do not fall apart immediately. Both single strands first have to be wrenched from each other. DNA molecules, the single strands of which have breaks, emerge faster than intact DNAs.

The proportion of double-stranded DNA can then be determined with the aid of fluorescent dyes which bind specifically DNA double-strand or which bind specific DNA single-strand by means of fluorescence spectroscopic analysis. The fluorescence of these dyes is dependent on the amount of double-stranded or single-stranded DNA, so that by measuring the intensity of the fluorescence emission in the region of the maximum of the fluorescence emission spectrum, either the increase in the single strands or the decrease in the double strands is determined is to be determined. As a rule, the lysis, which is cooled in the dark and with ice, takes place directly in the corresponding wells of a microtiter plate and takes about 30 to 60 minutes. Subsequently, the lysed cells and / or tissues are mixed with at least one solution (particularly pH 11 to 12) which is used to adjust the DNA unwinding (step b) and which has a concentration, in particular from 0.005 to 0.01 mol / 1, a substance complexing metal ions and inhibiting DNA repair enzymes, for example EDTA (see above).

At this pH, the double strands of the DNAs evolve - as explained above - so that the DNA single-strand breaks can be determined at regular, preferably regular intervals by conventional fluorescence spectroscopic analysis. The individual steps should take place under temperature-controlled conditions to ensure good reproducibility. Typical temperature values in the range from 0 ° C to + 30 ° C can be used depending on the individual case.

In a proven and thus advantageous manner, the concentration of the chaotropic substance in the lysis step is 2 to 4 mol / 1, since sufficient lysis and denaturation is achieved only in this concentration range, without the fluorescence being excessively reduced as a result of the quench effect chaotropic substance is coming. Furthermore, the concentration of the substance complexing metal ions and inhibiting DNA repair enzymes in the lysis step is advantageously from 0.005 to 0.05 mol / l. This is also advantageous in step b.). In this concentration range, sufficient removal of metal ions is achieved, which leads to cross-linking of the DNA and thus to impairment can lead to the unwinding of DNA and secondly a sufficient inhibition of the DNA repair enzymes, which eliminate the existing DNA damage again, without this leading to an excessive quenching effect. The pH value is advantageously (since it has been tried and tested) set to 11 to 12, since lower pH values do not lead to sufficient detoxification of the DNA and higher pH values impair the ability to be colored with the fluorescent dye.

In the process according to the invention, in addition to those listed above, several facts are surprising and are decisive for the success of the process: the sensitivity to the substance following complex addition and inhibition of at least one metal ion and DNA repair enzyme inhibiting in the step following the lysis is completely surprising Accuracy higher than that in the method disclosed in DE 197 24 781 AI. In addition, the use of a less than 4.5 molar concentrated solution of a chaotropic substance, for example urea, in the lysis step is surprising, since DE 197 24 781 AI considered this high concentration to be mandatory (see also general textbooks such as, for example : "Biochemie", Stryer, 4th edition, Spektrum der Wissenschaft Verlag, Heidelberg, 1990, pp: 32 - 33; use of an 8 molar urea solution in lysis for denaturation). The colorability with the fluorescent dye (binding of the dye to the DNA) improves unexpectedly. Because of this fact, the accuracy increases dramatically compared to the method disclosed in DE 197 24 781 AI. Furthermore, the concentration of a substance solution complexing metal ions and inhibiting DNA repair enzymes can surprisingly be less than 200 mmol / 1, whereby this concentration range also apparently suffices for a sufficient removal of heavy metal traces and, moreover, the disruptive quenching effect could be unexpectedly reduced.

In contrast to the FADU method (Birnboim and Jevcak, Cancer Res. 1981. 41: 1889-1892), the entire process (lysis, unwinding and fluorescence spectroscopic evaluation) can also be carried out on 96-well microtiter plates (or microtiter plates with another Well number) can be made. With the method according to the invention it is also possible for the first time to carry out a continuous measurement of the DNA unwinding kinetics in one and the same approach without stopping the reaction.

It is particularly advantageous if the concentration of the chaotropic substance is 2.25 mol / 1, since at this concentration there is an optimum between denaturation on the one hand and colorability on the other hand. It is also advantageous if the concentration of the metal ion complexing and DNA repair enzyme inhibiting substance solution of the first step is 0.02 mol / 1, since a sufficient removal of traces of heavy metals can be ensured while at the same time greatly reducing the quenching effect. The concentration of the metal solution complexing and DNA repair enzyme inhibiting substance solution of the second step (unwinding) is advantageously 0.02 mol / 1, since higher concentrations of some of these substances such as EDTA themselves can induce strand breaks and lower concentrations do not have an efficient metal ion complexing effect or DNA -Repairing enzyme inhibitory effect and no longer sufficient buffer effect. The following embodiments are advantageous because they have proven themselves in practice: the concentration of the detergent (SDS) is 0.00175 mol / 1; it is used as a chaotropic substance urea; it is used as a metal ion complexing substance and DNA repair enzyme inhibiting substance EDTA; during lysis the pH is 10.0; the pH of the resulting solution after adding the solution of the second step is 11.5.

In principle, such a pH value (11.5) should be unsuitable for drafting (denaturing), since, for example, the method of alkaline filter elution (see also Kohn, Pharmacol. Ther., 1991, 49: 55-77; Kohn et al ., Bioche istry 1976, 15: 4629-4637) a pH between 12.3 and 12.7 must be used. In spite of the relatively low pH value, there is still a detangling which also allows kinetic determinations of the detangling to be carried out.

Completely surprisingly, it was also found that the fluorescence values are more stable at this pH. A significant advantage over the known method (DE 197 24 761 AI) is the possibility not only of the so-called SSF value (Strand scission factor, Meyn and Jenkins, Cancer Res. 43, 5668; 1983) from the fluorescence values after approx. 20 Minutes after adding the appropriate solutions to the cells and / or tissues, but also to determine the tendency of the DNA denaturation curve (plotting the intensity of the fluorescence emission, which is proportional to the proportion of double-stranded DNA, over time). There is a linear relationship between the slope x (-1) and the SSF x (-l) value.

Finally, the fluorescent dye PicoGreen has proven to be extremely reliable and therefore advantageous.

The advantages of the method described here are that

1. it enables the determination of the DNA integrity even at low taxa, the DNA of which is comparatively low in complexity;

2. it allows the determination of DNA integrity with a sensitivity that is more than twice as high as the known method (DE 197 24 761 AI);

3. it can be performed on microtiter plates (high sample throughput) in three hours;

4. the results can be given both as SSF and as the rate of time-dependent DNA denaturation (= slope of the DNA denaturation curve); and

5. the pH at which the DNA is unwinding during the unwinding step and is therefore not subject to any shift over time which leads to a change in the fluorescence of the fluorescent dye and thus to a falsification of the measurement result.

The following kit for the determination of single DNA strands has the advantages mentioned above: The kit contains at least one detergent, at least one chaotropic substance solution, the concentration of which is 2 to 4 mol / l during lysis, at least one metal ion complexing and DNA repair enzyme inhibiting substance solution, at least one solution for adjusting the pH required for DNA unwinding (step b.), which contains a substance complexing metal ions and inhibiting DNA repair enzymes, and at least one fluorescent dye specifically binding single-stranded DNA or double-stranded DNA. The following embodiments of the kit are to be regarded as advantageous in practice for the above reasons and because of their proven effectiveness: the concentration of the substance complexing metal ions and inhibiting DNA repair enzymes during lysis is 0.005 to 0.1 mol / 1; the pH when the suspensions containing the lysed cells / tissue are mixed with the solution for adjusting the pH required for DNA unwinding is 11 to 12; the concentration of the metal ion complexing and DNA repair enzyme inhibiting substance after setting the pH required for DNA unwinding is 0.005 to 0.1 mol / 1; SDS is used as detergent; urea is used as the chaotropic substance; EDTA is used as the substance which complexes metal ions and inhibits DNA repair enzymes; PicoGreen is used as the fluorescent dye.

The subsequent uses of the method according to the invention and the kits according to the invention also have the surprising and advantageous properties listed above. It is about uses for:

• Detection of DNA single-strand breaks in human, animal or vegetable tissues and cells, in bacteria and single cells, in cell lines of human, animal or vegetable origin and subcellular fractions, as well as in isolated DNA;

• Measuring the occurrence of DNA single-strand breaks and their repair after radiation, chemotherapy and / or treatment with genotoxins in human and animal sample material, tissue sections and cell suspensions;

• Detection of DNA single-strand breaks in frozen test materials (for example biopsy samples) after their gentle homogenization in liquid nitrogen or liquid air in the presence of an anti-freeze agent (for example dimethyl sulfoxide);

• Evidence of the occurrence of DNA single-strand breaks and their repair in the event of occupational exposure of persons who come into contact with DNA-damaging substances or radiation either during their work (e.g. flight attendants, X-ray doctors) or as a result of accidents (e.g. reactor accidents);

• Measurement of the occurrence of DNA single-strand breaks and their repair in test organisms, tissues. Cells or cell lines in water monitoring.

The following examples serve to explain the invention.

Figure 1: This figure shows the DNA strand breaks (indicated as SSF x (-1)) in the gills of Mytilus galloprovincialis after treatment of the DNA in the TE homogenate (TE: 10 mmol / 1 Tris-HCl, 1 mmol / 1 EDTA; pH 7.4) with bleomycin, determined using the method according to the invention (single-strand method) (black bars) and the method disclosed in DE 197 24 761 AI (white bars). Examples of performing the assay (single-strand method):

Materials: The chemicals used in the experiments described below were obtained from E. Merck (Darmstadt, Germany) or from Sigma (St. Louis, USA). The fluorescent dye PicoGreen was purchased from Molecular Probes, Leiden, the Netherlands. Carrying out the assay: The detection in the examples described was carried out using a fluorescence ELISA plate reader (Fluoroskan II, Labsystems, Helsinki, Finland). Solutions:

PicoGreen dsDNA quantification reagent stock in DMSO (Molecular Probes); TE buffer: 10 mmol / 1 Tris-HCl, 1 mmol / 1 EDTA, pH = 7.4; Lysis solution: 4.5 mol / 1 urea, 0.1% SDS, 40 mmol / 1 EDTA, pH = 10.0; Lysis solution with PicoGreen: Dilution of the original PicoGreen solution: 20 μl / ml Lysis solution NaOH-EDTA solution: A 0.4 molar NaOH solution containing 20 mmol / 1 EDTA is diluted with a 20 mmol EDTA solution prepared a 0.2 molar NaOH-EDTA solution. The NaOH-EDTA working solution is prepared from this solution by further dilution with the 20 mmolar EDTA solution immediately before the experiments (pH = 11.50). Procedure: a.) A cell suspension is prepared in TE buffer with a pH of 7.4 (if more convenient, in the centrifugation medium) with a cell density of 150 x 10 ³ cells / ml. (Note: in experiments with lymphocytes about 3500 - 4500 cells / 25μl, about 30 ng DNA), b.) The cells are lysed directly in the wells of black microtiter plates for 40 min to 1 h in the dark on ice. 25 μl of lysis solution with PicoGreen are slowly added to 25 μl of cell suspension per well. It must not be mixed or shaken, c.) DNA unwinding is carried out as follows at pH 11.50: 250 μl of the NaOH-EDTA solution are added to each sample (to a pH of 11) To obtain 50, d.) Measurement of the intensity of the fluorescence emission over a period of up to 1 h, for example every 5 min; when using PicoGreen: excitation: 480 nm; Emission: 520 ran. The fluorescence blank value is measured as the fluorescence of a mixture of 25 μl TE buffer, 25 μl lysis solution with PicoGreen and 250 μl of the NaOH-EDTA working solution, e.) Calculation: Since every sample has to be corrected with regard to the blank value subtracted from the values of the mean of the blank. Multiple determinations are carried out (usually 4 - 6 determinations). The percentage of double-stranded DNA (dsDNA) for the untreated (e.g. unirradiated or untreated cells) at time 0 is set as 100% dsDNA. The measured effects are expressed as "Strand Scission Factor" (SSF) and calculated after a unwinding period of 20 minutes as follows: SSF = log (% dsDNA in the treated sample /% dsDNA in the control sample). Negative values for SSF indicate an increased frequency of DNA strand breaks. For graphic plots, the negative SSF values in the case of DNA strand breaks are multiplied by (-1) (representation of the SSF x (-1) values). Carrying out the assay for tissue samples:

The protocol for tissue corresponds to that for cell suspensions with the following modifications:

1. The tissue must be kept in liquid nitrogen immediately after removal.

2. The required amount of tissue per well of a 96-well microtiter plate is approx. 25 μg.

3. Homogenize 10 mg of the tissue in 1 ml of homogenization buffer in a mortar with a pestle under liquid nitrogen. Homogenization buffer: TE buffer (see above) or other suitable buffer with 10% cryoprotector (dimethyl sulfoxide).

4. Dilute the homogenate 10-fold with TE buffer.

5. 25 μl of the homogenate is added to each well (well); then 25 μl of the lysis solution with PicoGreen (see above) are added.

6. The procedure for cell suspensions is then followed.

Determination of DNA single-strand breaks in the mussel Mvtilus galloprovincialis after treatment of the DNA in the TE homogenate of gills with bleomycin

Procedure: Gills of the mussel Mytilus galloprovincialis were homogenized according to the method described in DE 197 24 761 AI in TE-DMSO buffer (DMSO = dimethyl sulfoxide), diluted to a concentration of 30 ng DNA / 25 μl and then with iron-activated bleomycin for 3 min treated on ice. The DNA was then analyzed both by the method described in DE 197 24 761 A1 and by the method according to the invention. DNA single-strand breaks were determined immediately after the incubation. Result: FIG. 1 shows that the conventional method does not provide evidence of induction of DNA damage (single-strand breaks). In contrast, the method described here is able to detect the DNA single-strand breaks that occur during bleomycin treatment even when incubated in the presence of a concentration of 2.5 μmol / 1 bleomycin. As the bleomycin concentration increases, there is a concentration-dependent increase in DNA single-strand breaks (increase in SSF x (-1)).

FIG. 2: shows the dependence of the occurrence of DNA double-strand breaks, expressed in the form of the SSF x (-1) value, in HeLa cells after incubation of the cells for 4 hours in the presence of different concentrations of bleomycin (double-strand method; 3800 cells / well). Figure 3 is a perspective view of an embodiment according to the inventive device for controlled filtration by means of suction;

Figure 4 is a perspective view of an embodiment according to the inventive device for controlled filtration by means of a pressurizing device.

Examples of performing the Assavs (double-strand method):

Materials: The chemicals used in the experiments described below were purchased from E. Merck (Darmstadt) or Sigma Chemical Co. (St. Louis, USA). The fluorescent dye PicoGreen was obtained from Molecular Probes, Leiden, the Netherlands. Carrying out the assay: In the examples described, detection is carried out using a fluorimeter (fluorescence microtiter plate reader; for example Fluoroskan II from Labsystems). Required materials:

1. 96-well filter plates (filtration plates), on the wells of which 96 independent membranes are welded, e.g. MultiScreen filtration plates from Millipore GmbH (Eschborn, Germany) (e.g. GV, 0.22 μm Durapore).

2. 96-well microtiter plates.

3. A device for controlled filtration shown in detail below by means of a suction device or a pressurizing device.

Solutions:

1. PicoGreen dsDNA quantification reagent stock in DMSO (Molecular Probes)

2. TE buffer: 10mM Tris-HCl, 1mM EDTA, pH 7.4

3. Lysis solution: 40 mM EDTA, 4.5 M urea, 0.1% by weight SDS, pH 10

4. Lysis solution with PicoGreen: Dilution of the original PicoGreen solution: 20 μl / ml lysis solution

5. Elution solution: from 10 ml TE buffer plus 10 ml lysis solution plus 100 ml bidist. Water procedure:

1. A cell suspension is prepared in TE buffer with a pH of 7.4 with a cell density of 150,000 / ml. (approx. 3500 - 4500 cells / 25 μl, approx. 30 ng DNA).

2. The lysis of the cells takes place in the wells of the 96-well filtration plates for 40 min at room temperature in the dark. 25 μl of lysis solution with PicoGreen are slowly added to 25 μl of cell suspension (pipetted on the filter membrane surface).

3. The lysis of the cells in the wells of a black 96-well microtiter plate is carried out as described under 2 in a parallel approach (for determining the total DNA per well; = total value). The fluorescence is then measured from the total value.

4. Add 250 μl Aqua bidest. per well of the 96-well filtration plates and the black 96-well microtiter plate.

5. After switching on the vacuum pump, the solutions in the wells of the 96-well filtration plates are sucked through the filters at a low speed (35 μl per well per minute) (duration: 7 min). The filtrates are collected in a black 96-well microtiter plate located in the vacuum holder under the filtration plate. The fluorescence of the DNA-PicoGreen complexes in the filtrates is then measured.

6. Measurement of fluorescence (with PicoGreen: excitation: 480 ran; emission: 520 nm). Fluorescence blank values are obtained by pipetting 25 μl lysis solution with PicoGreen to 25 μl water (or TE buffer) in portions of the cell suspension in steps 2 and 3.

7. Calculation: Multiple determinations are carried out for each sample and for the blank value (4-6 determinations). The samples are corrected for the blank value by subtracting the mean value of the blank value from the measured values. The results are reported as "DNA Filter Ratio" (DFR). The DFR is calculated according to the formula: [(tDNA - Σ fDNA ") / tDNA] x 100, with n = fraction number; tDNA - Total value of fluorescence; fDNA _n - fluorescence of fraction n. If a fraction is collected, n = 1. Low DFR values indicate an increased number of DNA double-strand breaks. Alternatively, the results can be given in the form of the "Strand Scission Factor" (SSF). In this case, the calculation is based on the formula: SSF = log (DFRp / DFR _k ). DFRp = DFR value for the sample to be determined; DFR _k = DFR value for the control. Negative SSF values indicate an increased number of double strand breaks.

Carrying out the assay for tissue samples:

1. The tissue must be frozen and stored in liquid nitrogen immediately after removal (storage for shorter periods can also take place at - 80 ° C).

2. The amount of tissue per well of the microtiter plate is usually approx. 25 μg.

3. Homogenize 10 mg of the tissue in 1 ml of homogenization buffer in a mortar with a pestle under liquid nitrogen. Homogenization buffer: TE buffer (see above) or other suitable buffer with 10% cryoprotector (dimethyl sulfoxide, glycerin etc.).

4. Dilute the homogenate 10-fold with TE buffer.

5. 25 μl of the homogenate is added to each well of the 96-well filtration plate; 25 μl of the lysis solution with PicoGreen (see above) are added to DNA.

6. The procedure for cell suspensions is then followed.

Determination of DNA double-strand breaks in HeLa cells after incubation with bleomycin. Procedure: HeLa cells were incubated for 4 hours in the presence of various concentrations of bleomycin (inducer of DNA double-strand breaks; Byrnes and Petering, Rad. Res. 137: 162; 1994) , Result: FIG. 2 shows a concentration-dependent increase in the DNA double-strand breaks [SSF x (-1) values] after incubation of the lines with bleomycin.

Devices for the controlled filtration of liquid media with well filter plates

Description of the suction filtration device

The suction filtration device shown in FIG. 3 consists of the following parts:

1 acrylic glass attachment consisting of an outer acrylic glass frame and an acrylic glass plate sitting on it. In the outer acrylic glass frame there is a fitted ventilation pipe (la) with a valve connected to it for regulating the vacuum in the upper vacuum chamber (not shown in FIG. 3). Depending on the position, the upper flow valve (lb) can be used to separate or connect the upper vacuum chamber, (lc) hole in the acrylic glass attachment, which can be opened or closed by the flow valve.

2 sealing ring between the acrylic glass attachment and the 96-well filter plate (attached to the inside of the cover of the acrylic glass attachment).

3 96-well filter plate with welded filters (e.g. Millipore MAGV N22). 4 spacers between the 96-well filter plate and the black 96-well microtiter plate.

5 black 96-well microtiter plate (e.g. Maxisorb from Nunc).

6 Sealing ring, which is attached to the lower edge of the acrylic glass attachment (not recognizable from the illustration) and which seals the acrylic glass attachment against the base plate.

7 base plate with 4 feet and in the middle of the base plate upwards ending bore and inserted suction tube (7a) for connection to a vacuum source.

Working principle: The filtration device has an acrylic glass attachment (1) which is placed on a base plate (7). Thanks to the silicone seal (6) in the lower edge of the acrylic glass attachment, the space under the attachment can be sealed airtight. An empty microtiter plate (5) and a feed plate (3) already loaded with samples are arranged under this acrylic glass attachment in such a way that the feed plate comes to rest on the microtiter plate and on the bottom plate (see FIG. 3). The fact that there is a white of the feed plate directly above the corresponding white of the microtiter plate is ensured by the fact that the two plates are fitted exactly into the acrylic glass attachment, so that it is not possible to move the plates against each other. Under the acrylic glass top are two separate air spaces, an upper air space, which is located between the acrylic glass top cover and the top of the feed plate, and a lower one, which extends from the bottom of the feed plate to the base plate. The separation of the two air spaces is achieved by a silicone seal, which is located between the ceiling plate of the acrylic glass attachment and the top (marginal parts) of the lining plate after the device has been set up. However, these two chambers (air spaces) are initially connected to each other by a hole (lc) in the wall of the acrylic glass attachment. This connection can be closed or opened via a flow tap (lb). After the correct composition of the individual components of the apparatus, a vacuum queue can now be connected to the suction pipe (7a). The suction pipe forms the end of a hole in the base plate, the other end of which is in the middle of the base plate of the lower chamber. Two narrow spacers (4), which are placed between the feed plate and the microtiter plate, prevent the wells of the microtiter plate from being closed when the vacuum is applied by sitting too tightly on the feed plate and thus being separated from the lower chamber space. If the connection between the lower and upper chamber with the hoof of the flow valve is open, both the lower and the upper chamber are evacuated after the vacuum has been applied. When the vacuum is applied, this connection must always be open to ensure a uniform evacuation of the two chambers. Then the connection between the two chambers is closed by turning the flow valve by 180 °. The two chamber rooms are then completely separated from each other. Now the upper chamber is ventilated by opening the valve on the ventilation pipe integrated in the acrylic glass attachment, the lower chamber remaining evacuated. Due to the resulting higher negative pressure in the lower chamber compared to the upper chamber, the samples pipetted into the weus of the feed plate are sucked through the feeds that bleed the bottom of the weus into the lower chamber. the filtrate being collected in the microtiter plate. The filtration speed can be regulated using the valve on the ventilation pipe. A filtration rate of 300 μl / 15 min per weü is recommended to carry out the procedure for the determination of DNA double strand breaks. The vacuum applied remains switched on during the entire feeding process. After complete feeding, the acrylic attachment can be removed, whereby the feed in the microtiter plates is available for further examinations.

Difference to other apparatuses: The new feature of the apparatus shown in FIG. 3 in comparison to conventional apparatuses (for example suction-extraction device "vacuum manifold" from Millipore GmbH) is that first by connecting the opening 7a to the vacuum source both in the The same negative pressure is present in the upper and in the lower chamber, thus sealing the apparatus from the outside. In the second step, DNA is separated from the upper and lower chamber by reversing the flow valve 1b. By opening the bore la and the inflow of outside air controlled via the vent, the pressure in the upper chamber is then set in such a way that the solution is sucked through the feeds at a continuous speed which is adapted to the experimental conditions. The principle of this construction allows a slow and continuous suction of solution through the feed with only slight pressure differences between the upper and the lower chamber. This is not possible with conventional suction filtration devices (e.g. vacuum manifold from Mülipore GmbH), since the suction pressure required to start the suction process (contact pressure on the sealing ring) requires a larger pressure difference between the rooms above and below the feed , Also, the control valve cannot be reduced to the point that the required slow filtration speed (20 μl / min and below when using 96-micron filter plates with a volume of 300 μl / weü) is achieved, which is used to measure DNA Double strand breaks are required when using the present method.

Differentiation of this feeding device from the feeding device described in patent application EP 0359249: Patent application EP 0359249 describes a feeding device in which a microfilter plate and a microtiter plate are also used. However, the principle underlying this apparatus differs from that of the first feeding device described in the previous application. In contrast to the feeding device described in patent application EP 0359249, a vacuum is first generated in both chambers in the device described here, which is then reduced in one (upper) chamber until the filtration process takes place at the desired speed. In contrast to the filtration device described in EP 0359249, a clamp is not used in the device described here.

The advantage of the construction described in the present application is that it avoids the contact pressure which is required in other apparatuses for the production of the airtight seal and which would lead to an initial rapid passage of liquid through the feed. A constant liquid flow through the feed is essential for the application of the feeding devices we need for the DNA strand break determinations. It must also be emphasized that the pore size of the feeds of the feed plates we use for DNA strand breakage determination is so small that feeding due to gravity is not possible. Due to the small pore size, feeding according to the principle described in the patent application EP 0359249 for the example cell culture could not take place.

Furthermore, the problem solved in the patent application EP 0359249 by the apparatus described there is no longer current in that the microfilter plates are now commercially available as relatively inexpensive "disposable articles" (Müüpore company). With the feeding devices described in the previous application, only the used microfilter plates and the microtiter plates (both "disposable items") come into contact with the examined samples during the feeding process, so the complicated (or after feeding radioactive or infectious samples may be difficult to carry out cleaning) of the devices.

Description of the pressure filtration device

The pressure feeding device shown in FIG. 4 consists of the following parts:

1 "acrylic glass housing (1") consisting of a rectangular base plate (la ") and two side plates (lb" and lc ") firmly connected to the long sides, as well as a hinged acrylic glass cover (ld"). On the inside of the hinged lid there is a rubber seal (le ") which, after the lid has been closed, sits on the outer frame of the 96-Wü feed plate. There is also a locking clamp (lf ') on the front side of the acrylic glass lid Secure pressing of the rubber seal on the 96-WeU plate after closing the device. On the back of the acrylic glass cover there is a control valve (lg ", eg Festo GR-M5B) with a connection hole to the space above the plate, which is also used for connection a compressed air source (e.g. house compressed air, nitrogen bottle or aquarium pump) is used.

2 "96-WeU-feed plate, introduced via the front opening of the acrylic glass housing along the two guide strips attached to the side plates (lh") up to the stop on the acrylic glass block connected to the lower acrylic glass plate (left ").

3 "black 96 WeU microtiter plate to collect the FUtrate.

Working principle: The lid (ld ") of the acrylic glass housing (1"), which is connected to it via a hinge, is opened upwards in order to place the feed plate (2 ") and the microtiter plate (3") inside the housing ermögüchen. The empty microtiter plate is placed on the bottom (la ") of the housing. The feed plate, which has already been filled with samples, is placed in the two guide strips (lh"), which are located halfway inside the housing on its side walls (lb "and lc") are inserted (see Figure 4). By closing the cover and tightening the locking clip (lf '), the two plates are pushed against a stop (li "), which is located at the end opposite the opening. This makes the two Plates are automatically positioned so that a weU of the feed plate is directly above the corresponding weU of the Mücrotiter plate. By closing the cover, the rubber seal integrated in the underside of the acrylic glass cover is pressed onto the top of the lining plate. This creates a small, airtight space between the acrylic glass cover and the feed plate. Now a pressure queue, which is connected to the space between the acrylic glass cover and the feed plate via the bore and the control valve (lg "), can be activated. The pressure thus exerted on the liquid column in the wells of the feed plate enables feeding, whereby the feeding speed depends on The control valve, which can regulate the amount of gas (air) flowing onto the feed plate down to almost zero, is determined. The strength of the pressure must be easily adjustable at the pressure queue itself. The liquid that has passed through the feed is removed from the microtiter plate A feeding rate of approximately 300 μl / 15 min per WeU is suitable for carrying out the method for determining DNA double-strand breaks.After complete feeding and interruption of the gas supply, the two plates can be removed from the acrylic glass housing and the FUtrate in the microtiter plate can be further examined (The funct The staple in this apparatus is simply to provide an airtight seal above the feeding plate and an exact positioning of the feeding plate and the microtiter plate.)

Difference to other apparatuses: The new feature of the apparatus shown in FIG. 4 compared to conventional apparatuses is that the feeding of the solutions pipetted into the water of the 96-WeU feed plate by applying an overpressure above the feed plate and not by applying a vacuum ( Vacuum) is carried out below the feed plate. An advantage of the principle used in this apparatus is that the feeding process can be carried out at a constant speed without the occurrence of the initial pressure jump which occurs in suction devices which are operated by applying a negative pressure as a result of the suction process (pressing process on the sealing ring). The overpressure in the room above the 96-WeU feed plate can be, in addition to the pressure queues mentioned above, e.g. can also be generated by applying an aquarium air pump. In addition, when using the device for the feeding of radioactive materials during the feeding process, people are protected from radiation by the 10 mm thick radiation-absorbing acrylic plates.

Advantages of the second apparatus: The advantages of the pressure feeding device described here compared to conventional apparatus are particularly easy to use. They enable feeding even when there is no compressed air source. This means that this device can also be used for examinations outside of a laboratory equipped with a compressed air source, for example on a research ship (examination of DNA damage in marine organisms). Furthermore, the special construction of the pressure feeding device enables shielding of radiation when using the devices for the filtration of radioactive materials. Pressurized cylinders containing inert gases can be used for the filtration processes of oxygen-sensitive substances. Comparable devices for microfilter plates are not currently available.

Claims

claims

1.) Method for determining DNA double-strand breaks with the following steps: (a) application of the cells and / or tissues to be examined to specific feeds; (b) lysis of the tissue / tissue with at least one detergent, at least one chaotropic substance, at least one substance complexing meta-ions and inhibiting DNA repair enzymes and at least one fluorescent dye which binds specifically to the DNA double strand, or lysis of the tissue / tissue with at least one detergent, at least one chaotropic substance, at least one protease and at least one specific DNA double strand binding fluorescent dye; (c) Elution of the DNA fragments released by the lysis through the specific feeds, only one eluate fraction being collected per WeU; (d) performing a fluorescence spectroscopic evaluation.

2.) Method according to claim 1, characterized in that in the lysis step as a chaotropic substance urea with a concentration of 2.25 mol 1 and as MetaUionen complexing and DNA repair enzyme inhibiting substance EDTA with a concentration of 0.02 mol / 1 is used, the concentration of the detergent is 0.00175 mol / 1 and the pH during lysis is 10.

3.) Kit for determining DNA double-strand breaks, comprising: at least one detergent, at least one chaotropic substance, at least one substance complexing meta-ions and inhibiting DNA repair enzymes, at least one fluorescent dye which binds a specific DNA double strand and at least one specific feed type or at least one detergent , at least one chaotropic substance, at least one protease, at least one specific DNA double-strand binding fluorescent dye and at least one specific feed type

4.) Device for the controlled feeding of liquid media, in particular containing DNA molecules, with WeU feed plates, with at least one Weü feed plate (3), at least one corresponding microtiter plate (5) and a suction device (7a), characterized by: two separate ones , gas spaces arranged above and below the feed plate; at least one sealing device (2) which interacts with a container (1) and the WeU feed plate (3) and thus creates a complete separation of the two gas spaces; a device (lb, lc) for connecting both gas spaces; a device (la) for pressure equalization between the environment and a gas space.

5.) Device for the controlled feeding of liquid media, in particular containing DNA molecules, with WeU feed plates, with at least one WeU feed plate (2 "), at least one corresponding microtiter plate (3"), characterized by: two separate, top and gas spaces arranged below the feed plate; at least one sealing device (le ") which interacts with a container (1") and the WeU-feed plate (2 ") and thus creates a complete separation of the two gas spaces; a pressurizing device (lg") functioning for a gas space.

6.) Method according to one of claims 1 to 3 for determining DNA double-strand breaks and for controlled feeding of liquid, in particular DNA molecules, characterized in that a device according to one of claims 4 and 5 is used.

7.) Method for the determination of DNA single strand breaks with the following steps: (a) lysis of cells and / or tissues to be examined with at least one detergent, at least one chaotropic substance in the presence of at least one metal complexing and DNA repair enzyme inhibiting substance and at least one DNA single strand or DNA double strand binding fluorescent dye; (b) adding to the suspension containing the lysed cells and / or tissues at least one solution which allows this suspension to be adjusted to an alkaline pH and which has at least one substance which complexes metal ions and inhibits DNA repair enzymes; (c) performing a fluorescence spectroscopic evaluation,

8.) Method according to claim 7, characterized in that in the lysis step as a chaotropic substance urea with a concentration of 2.25 mol / 1, as metal ions complexing and DNA repair enzyme inhibiting substance EDTA with a concentration of 0.02 mol / 1 and as a detergent SDS with a concentration of 0.00175 mol / 1, in step b.) as a metal complexing and DNA repair enzyme inhibiting substance EDTA with a concentration of 0.02 mol / 1 and the pH in the lysis step 10 and in step b.) Is 11.5.

9.) Kit for the determination of DNA single-strand breaks, characterized in that the detergent SDS, the chaotropic substance urea, the metal complexing and DNA repair enzyme inhibiting substance EDTA and the fluorescent dye PicoGreen is used.

10.) Use of the method according to one of claims 1, 2, 6, 7 and 8 and the kit according to one of claims 3 and 9 (a) for the detection of DNA single and double strand breaks in human, animal or vegetable tissues and cells , in bacteria and single cells, in cells of human, animal or vegetable origin and sub-cellular fractions, as well as in isolated DNA; (b) for the detection of DNA repair in human, animal or vegetable tissues and cells, in bacteria and individuals, in cells of human, animal or vegetable origin, subcellular fractions, cell lysates and ZeU extracts (isolated molecules); (c) to measure the occurrence of DNA single and double strand breaks and their repair after radiation, chemotherapy and / or treatment with genotoxins, mutagens and carcinogens in human and animal sample material, tissue sections and ZeU suspensions; (d) for the detection of DNA single and double strand breaks in frozen examination materials after their gentle homogenization in liquid nitrogen or liquid air in the presence of an anti-freeze agent; (e) To detect the occurrence and repair of single and double-stranded DNA breaks in the event of occupational exposure to persons who come into contact with DNA-damaging substances or radiation either at work or as a result of accidents.