DE19933078B4 - Method and kit for determining DNA double / single strand breaks and device for the controlled filtering of liquid media containing in particular DNA molecules and / or other molecules / molecule complexes with well filter plates - Google Patents

Abstract

Among other things, a method for determining DNA double-strand breaks is provided, with the following steps: DOLLAR A a.) Application of cells and / or tissues to be examined onto specific filters; DOLLAR A b.) Lysis of the cells / tissues with at least one detergent, at least one chaotropic substance and at least one fluorescent dye that binds specifically to the DNA double strand; DOLLAR A c.) Elution of the DNA fragments released by the lysis through the specific filters; DOLLAR A d.) Carrying out a fluorescence spectroscopic evaluation, DOLLAR A or DOLLAR A a.) Placing cells and / or tissues to be examined on specific filters; DOLLAR A b.) Lysis of the cells / tissues with at least one detergent, at least one chaotropic substance; DOLLAR A c.) Elution of the DNA fragments released by the lysis through the specific filters; DOLLAR A d.) Adding at least one fluorescent dye which binds specifically to the DNA double strand to the eluates obtained; DOLLAR A e.) Carrying out a fluorescence spectroscopic evaluation.

Description

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

The invention relates in particular on the one hand, a method and a kit for determining DNA double-strand breaks, corresponding Uses, devices for controlled filtering in particular DNA Mokeküle and / or other molecules / molecular complexes containing liquid media with well filter plates and various uses.

A number of assays for determining DNA double-strand breaks are known from the prior art. In particular, neutral filter elution, pulsed field gel electrophoresis and neutral comet assay (Elia, MC, DeLuca, JG, Bradley, MO: Significance and measurement of DNA double strand breaks in mammalian cells. Pharmacol. Ther. 51: 291 - 327; 1991; Sarkaria, JN, Bush, C., Eady, JJ, Peacock, JH, Steel, GG, Yarnold, JR: Comparison between pulsed-field gel electrophoresis and the comet assay as predictive assays for radiosensitivity in fibroplasts Radiat. Res. 150: 17-22; 1998; Prize, KM, Ahnstrom, G., Belli, M., Carlsson, J., Frankenberg, D., Kiefer, J., Lobrich, M., Michael, BD, Nygren, J., Simone, G., Stenerlow, B .: A review of dsb induction data for varying quality radiations, 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 over a period of 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 (working time usually more than 10 hours), complicated to carry out (it can only be handled by experienced personnel); 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. The threshold dose for the detection of DNA double strand breaks using the filter elution method is 5-10 Gy (Bradley, MO, Kohn, KW: X-ray induced DNA double strand break production and repair in mammalian cells as measured by neutral filter elution. Nucleic Acids Res. 7: 793-804; 1979).

In pulse field gel electrophoresis is an agarose gel electrophoresis with which DNA double strand breaks can be detected which were only induced with a 1 Gy ionizing radiation, the inherent however Has problems with the measurement of strand break repair. The Pulsed field gel electrophoresis can be performed on best with cell suspensions but not with tissue samples. to execution this method is costly equipment and the existence required by experienced personnel. The procedure is time consuming (Duration about 24 hours) and enables only a simultaneous determination of relatively few samples.

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. While the damaged DNA migrates towards the anode during electrophoresis forming a comet's tail. The greater the extent of the damage (Number of strand breaks), the bigger the resulting tail. The assay can be neutral or alkaline Conditions (> pH 13) carried out become. Double strand breaks are under neutral conditions and single strand breaks under measured alkaline conditions. A disadvantage of this method is that provisions of DNA damage are not possible in tissues. Furthermore needed the process of evaluating a costly image analyzer as well experienced staff for the implementation and especially for the interpretation of the results. Finally, the time for sample preparation relatively high before lysis.

In AN 1992, 390836 BIOSIS a Method for determining DNA damage disclosed, in which a alkaline filter elution with specific filters is used, on the hemolymph marine invertebrate is given.

In AN 1994, 505825 BIOSIS with the help of an alkaline filter elution single strand breaks and Cross-links detected in the mussel's DNA.

In AN 1993, 455306 BIOSIS with the help of an alkaline filter elution using bleomycin-Fe (II) complex Cross-linking of DNA detected.

In AN 1999, 241493 BIOSIS with the help of an alkaline filter elution using PicoGreen, a specific double strand binding fluorescent dye single strand breaks in the DNA from cancer patients detected.

EP 0 359 249 discloses, inter alia, 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, wherein moreover, controlled filtration of liquid media is not always guaranteed in the process. In the commercially available MultiScreen vacuum filtration system (vacuum manifold from 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, can also be used with the vacuum slide switch do not build on this device.

It follows from the above the problem listed above To eliminate disadvantages at least partially. The resulting The problem is in particular a method and a kit for Determination of DNA double strand breaks to provide a have high sensitivity and accuracy and at the same time a quick determination of the DNA damage (working time less than 3 hours) with a high sample throughput (e.g. parallel determination a larger number of Samples, for example of 96 samples when using 96-well microtiter plates) enable.

There is also the problem in providing an apparatus and a method that a Ensure improved controllability of the filtration.

For such a process is an urgent need, for example in medicine (determination of the optimal radiation dose for radiation therapy patients) or with environmental monitoring (proof the effect of genotoxic substances).

This problem is solved according to the invention a method according to claim 1, a kit according to claim 19, uses according to claims 41, 42 and 43, devices according to claims 44 and 51, method according to the claims 56 and 57, and uses according to claims 58, 59 and 60 solved.

In the method according to the invention be first the cells and / or tissues to be examined on specific filters given. These can be commercially available filter types suitable for these purposes act, for example MultiScreen filtration plates from the company Millipore GmbH (Eschborn, Germany) with Durapore membranes (e.g. 0.22 μm pore size; material: Polyvinylidene difluoride, PVDF). Depending on the DNA length, membranes can advantageous with smaller or larger pore sizes his.

Then the cells / tissues lyses in the presence of at least one detergent, for example SDS (sodium dodecyl sulfate), at least one chaotropic substance, for example urea, and complexing at least one metal ion and substance which inhibits DNA repair enzymes, for example EDTA, or at least one protease, for example Proteinase K. Schon in this step at least one specific DNA duplex can be used binding fluorescent dye can be added; However, it is also possible this Dye only after the subsequent elution add. Metal ion complexing and DNA repair enzymes inhibitory substance is necessary because firstly metal ions for cross-linking of DNA can and thereby impair their elution and become a faulty one assessment share of intact DNA can and secondly, DNA repair enzymes to eliminate the measuring DNA damage to lead. Alternatively, a protease can be used because it does in the lysis of DNA repair enzymes that would otherwise become a Elimination of the DNA damage to be measured would result.

After that, through the lysing released DNA fragments eluted through the filters at a controlled, constant elution rate and then carried out a fluorescence spectroscopic evaluation. The Elution is particularly by the devices according to the invention for controlled filtration of liquid Media carried out with well filter plates.

In the method according to the invention one takes advantage of the knowledge that after the cell lysis and the taking place simultaneously in the lysis solution Disintegration of the DNA-histone complexes the released DNA molecules with the lysis / elution solution be washed out. The elution of the DNA molecules is faster the faster shorter they are; i.e. at DNA molecules with an increased Number of double-strand breaks many pieces very early be eluted while at little damaged DNA the strands washed out later become. The method thus allows the number of Double strand breaks in the DNA.

It is beneficial if the lysis in the presence of at least one metal ion complexing and DNA repair enzyme inhibiting substance and in the presence of at least one protease, because the for the respective substances interact with the advantages mentioned above.

In the lysis step, this is advantageously Concentration of the chaotropic substance 0.5 to 9 mol / l, in particular 2 to 4 mol / l, as there is sufficient cell lysis in this area and denaturation is effected without the quench effect becoming too strong.

In addition, the concentration is the substance complexing metal ions and inhibiting DNA repair enzymes advantageously 0.005 to 0.05 mol / l, since in this range sufficient effectiveness guaranteed is.

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 pH values).

In the lysis step, this is advantageously Concentration of the protease 0.01 to 5 mg / ml, since outside sufficient protein digestion is no longer guaranteed in this area is (at lower concentrations) or a destruction of the DNA through possible foreign activities in the protease preparation (DNases) possible is (at higher Concentrations).

Advantageously, per well only one eluate fraction can be collected in this way to ensure efficient work, because it with the inventive method possible is statements about the number of breaks only with a total fraction per well.

It is particularly advantageous if the concentration of the chaotropic substance in the lysis step 2.25 mol / l is, since at this concentration an optimum between denaturation on the one hand and dyeability (with the fluorescent dye only little quench effect) on the other hand is present.

It is also an advantage if the concentration of metal ion complexing and DNA repair enzymes inhibiting substance of the lysis step is 0.02 mol / l, as a result adequate removal of traces of heavy metals (which too Cross-link DNA can) while at the same time greatly reducing the impairment of the measurements Quench effect can be ensured.

The following embodiments are advantageous because they have proven themselves in practice:
The concentration of the detergent (especially SDS) is 0.00175 mol / l.
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 an EDTA complexing metal ion and DNA repair enzyme inhibiting substance.
Proteinase K is used as protease.
SDS is used as a detergent.
The pH during lysing is 10.0.

It is also an advantage if step a.) 1. Alternative at a temperature of 15 ° C to 35 ° C and / or Step b.) 1. Alternative at a temperature of 15 ° C to 35 ° C, and / or Step c.) 1. Alternative at a temperature of 15 ° C to 35 ° C and / or Step d.) 1. Alternative at a temperature of 15 ° C to 35 ° C and / or Step a.) 2. Alternative at a temperature of 4 ° C to 15 ° C and / or Step b.) 2. Alternative at a temperature of 4 ° C to 15 ° C and / or Step c.) 2. Alternative at a temperature of 4 ° C to 15 ° C and / or step d.) 2. alternative at a temperature of 4 ° C to 15 ° C and / or step e.) 2. alternative at a temperature of 4 ° C up to 15 ° C carried out (it is especially important that the Temperature within a series of measurements is at least almost constant) because these temperature ranges proven to have.

Finally, the fluorescent dye PicoGreen from extremely reliable and thus successfully proven.

This also applies to the use of TOTO, YOYO, 7-aminoactinomycin D, DAPI, Hoechst 33258 and Hoechst 33342 as DNA double strand selective (under selective is in the context of the invention also approximately to be understood selectively) binding fluorescent dye ([TOTO = (1,1 '- (4,4,7,7-tetramethyl-4,7-diazaundecamethylene) bis-4- (3-methyl-2,3-dihydro (benzo 1,3-thiazole) -2-methylidene) quinolinium tetraiodides), YOYO = (1,1 '- (4,4,7,7-tetramethyl-4,7-diazaundecamethylene) bis-4- (3-methyl-2,3-dihydro (benzo-1,3-oxazole ) -2-methylidene) quinolinium tetraiodide)], bisbenzimide dyes [Hoechst 33258 = (bisbenzimide, 2 '- (4-hydroxyphenyl) -5- (4-methyl-1-piperazinyl) -2,5'-bi-1H-benzimidazole) and Hoechst 33342 = (bisbenzimide, 2 '- (4-ethoxyphenyl) -5- (4-methyl-1-piperazinyl) -2,5'-bi-1H-benzimidazole)], DAPI = (4,6-diamidino-2-phenylindole) and 7-AAD = (7-aminoactinomycin D)).

• 1. The process has the following advantages:
• 2. The procedure needed only a small amount of time (performing the entire procedure in one hour).
• 3. The process is highly sensitive; only small amounts of sample DNA are required (about 30 ng DNA per well of the microtiter plate; this corresponds to approximately 3000 cells or 25 μg tissue).
• 4. The method allows a high sample throughput (implementation of 96 determinations per hour when using a filtration device with a 96-well microfilter plate; several filtration devices can be operated in parallel and allow an even higher sample throughput).
• 5. The procedure can be carried out with non-radioactive DNA.
• 6. The method can be used not only for cells but also for tissue samples be applied.
• 7. The implementation the procedure is simple and does not require any special expertise (experienced Staff is not necessary).

The following kit for the determination of DNA double-strand breaks has the advantages mentioned above:
The kit contains at least one detergent, at least one chaotrape substance, at least one fluorescent dye that 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 metal ion complexing and DNA repair enzyme inhibiting Substance.
The kit contains at least one protease.
The concentration of the chaotropic substance during lysis is 0.5 to 9 mol / l, in particular 2 to 4 mol / l.
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.

In an advantageous, proven way and way contains the kit, one

• - Solution A: buffer solution to suspend the cells, for example Tris-EDTA (10 mol / l Tris-HCl and 1 mol / l EDTA with a pH of about 7.4) or any other buffer that does not have the effect of solutions B and should affect C;
• - Solution B: with a concentration of 0.001 to 0.2 mol / l of a detergent (Final concentration in the lysis step: 0.001 to 0.01 mol / l), one Concentration of 4 to 8 mol / l of a chaotropic substance (final concentration in the lysis step: 2 to 4 mol / l), (the individual components of the solution B can also as separate solutions with accordingly changed Concentrations are present);
• - Solution C: with a concentration of 0.001 to 1.0 mmol / l one specific DNA double strand binding Fluorescent dye (final concentration in the lysis step: 0.01 to 10 μmol / l) (The fluorescent dye can also be changed accordingly Concentration of the solution B or one or more solutions the components of the solution B are added),
• - in which of a volume mixing ratio of solution A: Solution B: Solution C of 50: 50: 1 is assumed.

When these mixture ratios change the concentration information of the solutions while maintaining the final concentrations obtained after mixing.

The following embodiments of the kit are advantageous for the above reasons and because they have proven themselves:
Solution B contains at least one substance that complexes metal ions and inhibits DNA repair enzymes.
The concentration of the metal B complexing and DNA repair enzyme inhibiting substance of solution B is 0.01 to 0.1 mol / l, in particular 0.04 mol / l.
The metal complexing and DNA repair enzyme inhibiting substance is EDTA.
Solution B contains at least one protease, in particular proteinase K.
The concentration of the protease in solution B is 0.02 to 10 mg / ml, in particular 1 mg / ml.
Solution A is a Tris-HCl-EDTA solution.
The Tris-HCl-EDTA solution has a concentration of 10 mmol / l Tris-HCl and 1 mmol / l EDTA with a pH of about 7.4.
SDS is used as a detergent.
The SDS concentration in solution B is 0.0035 mol / l.
Urea is used as the chaotropic substance.
The urea concentration in solution B is 4.5 mol / l.

The specific DNA double strand binding fluorescent dye is PicoGreen.

The subsequent uses of the inventive method and the kits of the invention have the surprising listed above and beneficial properties as well.

In particular, this relates to uses for:
Detection of DNA double-strand breaks and cross-links in human, animal or plant tissues and cells, in bacteria and unicellular organisms, in cell lines of human, animal or plant 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);
For measuring the occurrence of DNA double-strand breaks and cross-links and their repair after radiation, chemotherapy and / or treatment with genotoxins (mutagens and cancerogens) in human and animal sample material, tissue sections and cell suspensions;
Detection of DNA double strand breaks and cross-links and their repair in human or animal lymphocytes after their rapid isolation by centrifugation;
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 (as a pre-therapeutic test to determine the individual radiation sensitivity, e.g. on lymphocytes or tumor biopsy material of the patient before radiation, to predict the success of the therapy and / or to determine the success optimal dose with radiation therapy;
Detection of DNA double-strand breaks and cross-links in frozen test materials (e.g. biopsy samples) after their gentle homogenization in liquid nitrogen or liquid air in the presence of an anti-freeze agent (e.g. dimethyl sulfoxide);
Evidence of the occurrence of DNA double-strand breaks and cross-links and their repair in the event of occupational exposure of people who are either at work (e.g. flight attendants, X-ray doctors, workers in chemical companies) or as a result of accidents (e.g. Reactor accidents) come into contact with DNA-damaging substances or radiation;
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 (for example municipal and industrial sewage plant inflows and outflows, river water samples);
Detection of DNA double strand breaks after exposure of cells to topoisomerase II inhibitors;
Evidence of food preservation using radiation;
Detection of DNA damage from cosmic radiation in flight personnel;
Screening for radioprotective substances.

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 room is above the filter plate and the microtiter plate arranged underneath, in their wells beforehand (before inserting the filter plate into the device) the medium to be filtered is applied. Below the filter plate there is a second gas space separated from the first gas space. (In the context of the invention, the terms "above" and "below" are interchangeable, with in usually the statements made above and those that follow refer to the conventional variant). First of all both gas spaces by means of a cock arranged in a bore (this embodiment has proven itself in an advantageous manner) so that in two gas rooms the pressure is the same. Both gas spaces are through the suction device, for example, one connected to a corresponding vacuum pump Suction pipe, evacuated or a negative pressure is created with respect to the environment and then both gas rooms separated from each other (for example by turning the tap). The previously closed device for pressure equalization between The surrounding area and the upper gas space now carefully become the surrounding area towards (i.e. towards the outside atmosphere) open, so that the Pressure in the upper gas space is very slow and doses in comparison to the gas pressure of the lower gas space increased can be. In this way is a controlled filtration of the liquid Medium possible, especially for the solutions for measuring DNA double strand breaks.

The one with conventional devices and methods necessary "suction pressure" (contact pressure of the between the filtration plate and the corresponding microtiter plate arranged sealing device, in particular in the form of a rubber ring, for sealing between the two gas spaces) is usually so high the existence controlled filtration is not possible. The advantage of in the device according to the invention principle applied is that the filtration process with constant speed can be performed without occurrence of suction devices that are created by applying a vacuum operated, occurring initial pressure jump as a result of Suction process (pressing process to the sealing device, in particular sealing ring). In particular those in the method according to the invention necessary for measuring DNA double-strand breaks low filtration / elution rates in the range of a few Microliters per minute are by means of conventional devices and methods not to be implemented, but represent the devices according to the invention and procedures are not a problem.

It is advantageous if the well filter plate and the corresponding microtiter plate using spacers from each other are spaced so that when evacuating also between the individual Wells of the microtiter plate and the filter plate arranged gas spaces are evacuated become.

The following embodiments are advantageous because they have proven themselves in practice:
The suction device is a suction pipe.
The device for pressure equalization between the environment and a gas space is a valve.
The device for connecting the two gas spaces is a tap arranged in a bore. (The hole itself connects the two gas spaces directly with each other).
The sealing device is a rubber ring.

Finally, it is beneficial if the device is made of acrylic glass because this material for the most part Alpha and beta rays absorbed and thus also radioactively labeled Substances can be filtered / eluted safely.

The two other embodiments (second and third device) are characterized by two separate, upper and Gas spaces arranged below the filter plate, at least one with one container and the well filter plate cooperating and thus a separation of the two gas rooms generating sealing device and a functioning for a gas space Pressurizing.

One gas space is located above the filter plate and the microtiter plate arranged underneath, in the wells of which the medium to be filtered (before inserting the filter plates into the device) to be applied. A second gas space, separated from the first gas space, is located below the filter plate. After the medium has been applied to the respective wells of the filter 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 is a controlled filtration of the liquid Medium possible, especially for the solutions for measuring DNA double-rod breaks.

The following embodiments are advantageous because they have proven themselves in practice:
The pressurization device is a control valve; however, it can also have such a valve only as a component.
The device consists of acrylic glass (handling of radioactive preparations possible).
A circumferential frame or two guide rails are arranged between the two plates (both plates cannot be pressed together so tightly when the pressure in the upper gas space rises that the pressure between the filter plate and the microtiter plate no longer corresponds to the pressure in the lower gas space).
The sealing device is a rubber ring.

A method according to the invention, which a used the filtration devices according to the invention, has the above surprising and beneficial properties. This also applies to the corresponding ones Uses.

It is about uses:
for the detection and quantification of ANA strand breaks, of cross-linking of DNA molecules with one another or with other molecules, and of further DNA damage;
for binding studies, to investigate 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 / air sensitive samples;
for filtration under sterile conditions.

DE 197 24 781 A1 discloses, among other things, 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 specifically binding single-stranded or double-stranded DNA, in particular Picogreen, are added. A sodium hydroxide solution with a pH of approximately 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, approximately 200 mmol / l EDTA must be used in order to ensure complete complexation of the metal ions which crosslink the DNA.

This method is disadvantageous the use of high amounts of substance, which leads to a high quenching effect lead in the fluorescence evaluation and the results may strongly falsify can.

In addition, the sensitivity the above method is partially unsatisfactory, in particular for the Determination of DNA strand breaks in (shorter) DNA molecules from lower organisms (important for the study of the effects of environmental pollution) and for the determination of DNA strand breaks for radiation therapy patients in the dose range used for radiation therapy (0.5 - 3.0 Gy single dose).

It follows from the above the problem listed above To eliminate disadvantages at least partially. The resulting The problem lies particularly in a method and a kit for determination of single strand DNA breaks to provide the low quench, high sensitivity and have accuracy and good reproducibility and at the same time a quick determination of the DNA damage (working time less than 3 hours) with a high sample throughput (e.g. parallel determination 96 samples in 96-well microtiter plates or determination of even larger numbers of samples by using microtiter plates with an even larger number on wells).

In the method according to the invention be first cells and / or tissues to be examined with at least one detergent for example SDS (sodium dodecyl sulfate) or Triton X-100 (t-octylphenoxypolyethoxyethanol) and at least one chaotropic substance solution, for example urea solution or Guanidine HCl solution, lysing offset. The lysis is carried out in the presence of at least one Substance that complexes metal ions and inhibits DNA repair enzymes instead of. At least one specific DNA single strand or DNA double strand binding Fluorescent dye is used during the lysis step or after the lysis added the following 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. By adding at least one substance that complexes metal ions and inhibits DNA repair enzymes to the Lyse's next step is completely surprising in sensitivity and accuracy higher than that in DE 197 24 781 A1 disclosed method.

It is important in the method according to the invention that he uses a fluorescent dye that is either specifically single-stranded or is specifically double-stranded. Background for this Requirement is the fact that the dye is specific either on single strands or specifically on double strands who has to stick DNA to determine the proportion of the respective strand types. For example can as selective or as approximate selectively double-stranded cyanine dyes such as PicoGreen and Dyes from the TOTO range [TOTO (1,1 '- (4,4,7,7-tetramethyl-4,7-diazaundecamethylene) bis-4- (3-methyl-2,3-dihydro (benzo-1,3-thiazole) -2 -methylidene) quinoliniumtetrajodide) YOYO (1,1 '- (4,4,7,7-tetramethyl-4,7-diazaundecamethylene) bis-4- (3-methyl-2,3-dihydro (benzo-1,3-oxazole) -2- methylidene) quinoliniumtetrajodide)], Bisbenzimide dyes [Hoechst 33258 (bisbenzimide, 2 '- (4-hydroxyphenyl) -5- (4-methyl-1-piperazinyl) -2,5'-bi-1H-benzimidazole) and Hoechst 33342 (bisbenzimide, 2 '- (4-ethoxyphenyl) -5- (4-methyl-1-piperazinyl) -2,5'-bi-1H-benzimidazole)], DAPI (4,6-diamidino-2-phenylindole) and 7-AAD (7-aminoactinomycin D) and as selective single-strand binding OliGreen can be used.

The process according to the invention is based on the following principle:
In 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 DNA single-strand breaks are among the most common DNA changes (DNA damage). A prerequisite for the two single strands of DNA to remain together is that the breaks - which is usually the case - on the two single strands of DNA are offset from one another. If one exposes the DNA or the entire cell to a strongly alkaline solution, the hydrogen bonds between the DNA single strands are released. However, since the molecules are wrapped around one another, they do not fall apart immediately. Both single strands first have to be wrenched from each other. DNA molecules, whose single strands have breaks, emerge faster than intact DNAs.

The proportion of double-stranded DNA can then with the help of specifically DNA double strand binding or specific DNA single-stranded fluorescent Dyes about determine a fluorescence spectroscopic analysis. The fluorescence of this Dyes depend on the amount of double or single strand DNA, see above that about the Measurement of the intensity the fluorescence emission in the region of the maximum of the fluorescence emission spectrum to determine either the increase in the single strands or the decrease in the double strands is.

Usually it takes place in the dark and chilled with ice Lysis takes place directly in the corresponding wells of a microtiter plate and takes about 30 to 60 minutes. The approaches should not be mixed and / or shaken because the individual DNAs are very sensitive. Then be the lysed cells and / or tissues with at least one for adjustment of the pH value required for DNA unwinding (step b) (in particular pH 11 to 12) serving solution which has a concentration, in particular from 0.005 to 0.01 mol / l, a metal ion complexing and DNA repair enzyme inhibiting Has substance, for example EDTA (see above).

At this pH value - as above explained - the double strands of DNAs, so that in preferably at regular intervals over a conventional fluorescence spectroscopic analysis to determine the DNA single-strand breaks can. The individual steps should be under temperature controlled Conditions expire to ensure good reproducibility can. Usual temperature values in the 0 ° C range up to + 30 ° C depending on the individual case.

In proven and therefore more advantageous Way is the concentration of the chaotropic substance in the lysis step 0.5 up to 4 mol / l, in particular 2 to 4 mol / l, since only in this concentration range sufficient lysis and denaturation is achieved without it an excessive reduction in fluorescence due to the quench effect the chaotropic substance comes.

Furthermore, in an advantageous manner, it is proven the concentration of metal ion complexing and DNA repair enzymes inhibiting substance in the lysis step 0.005 to 0.05 mol / l. This is also advantageous in step b.). In this concentration range firstly, sufficient removal of metal ions is achieved, the cross-linking of DNA and thus impairment lead to the unwinding of DNA can and secondly, sufficient inhibition of DNA repair enzymes, which restores the existing DNA damage eliminate without doing this there is too strong a quench effect.

The pH value is advantageous (there proven) set to 11 to 12 since lower pH values do not lead to sufficient ones Lead to unwinding of DNA and higher pH values the dyeability interfere 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:
Due to the additional complexing of at least one metal ion and DNA repair enzyme inhibiting substance in the step following the lysis, the sensitivity and accuracy is completely surprising than that in the DE 197 24 781 A1 disclosed method.

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 from the DE 197 24 781 A1 this high concentration has been regarded as mandatory (see also general textbooks such as: "Biochemie", Stryer, 4th edition, Spektrum der Wissenschaft Verlag, Heidelberg, 1990, pp: 32-33; use of an 8 molar urea solution in the lysis for denaturation).

Unexpectedly, the dyeability with the fluorescent dye improves (binding ability of the fluorescent dye to the double-stranded DNA): due to this fact, the accuracy compared to that in DE 197 24 781 A1 disclosed method dramatically.

Furthermore, the concentration a substance solution complexing metal ions and inhibiting DNA repair enzymes surprisingly be less than 200 mmol / l, with this concentration range also apparently for a sufficient removal of heavy metal traces is sufficient and Furthermore unexpectedly the annoying Quench effect could be reduced.

The implementation of the entire procedure (lysis, Untangling and fluorescence spectroscopic evaluation) can be in contrast on the FADU method (see also FADU (Fluorometric analysis of the DNA unwinding: Birnboim, H.C. and Jevcak, J.J. Fluorimetric method for rapid detection of DNA strand breaks in human white blood cells produced by low doses of radiation, Cancer Res. 1981, 41: 1889 - 1892) also on 96-well microtiter plates (or microtiter plates with a other well number).

With the method according to the invention is it possible for the first time a continuous measurement of the kinetics of DNA unwinding in one and take the same approach without stopping the reaction.

It is particularly advantageous if the concentration of the chaotropic substance is 2.25 mol / l, because at this concentration is an optimum between denaturation on the one hand and dyeability on the other hand is present.

It is also an advantage if the concentration of metal ion complexing and DNA repair enzymes inhibitory substance solution of the first step is 0.02 mol / l, since it is still sufficient Removal of traces of heavy metals at the same time strong Reduction of the quench effect can be ensured.

The concentration of the metal ions Complexing and DNA repair enzyme inhibiting substance solution of the second step (unwinding) is advantageously 0.02 mol / l, because higher Concentrations of some of these substances, e.g. EDTA itself induce strand breaks can and lower concentrations do not complex metal ions efficiently Effect or DNA repair enzyme inhibiting effect and insufficient buffering effect exercise more.

The following embodiments are advantageous because they have proven themselves in practice:
The concentration of the detergent (especially SDS) is 0.00175 mol / l.
It is used as a chaotropic substance, urea.
It is used as an EDTA complexing metal ion and DNA repair enzyme inhibiting substance.
SDS is used as a detergent.
The pH of the lysis 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) for the Unwrapping (denaturation) may be unsuitable, for example in the method of alkaline filter elution (see also Kohn, K.W .: Principles and practice of DNA filter elution: Pharmacol. Ther., 1991, 49: 55-77; Kohn, K.W., Erickson, L.C., Ewig, R.A. and Friedman, C.A., Fractionation of DNA from mammalian cells by alkaline elution, Biochemistry 1976, 15: 4629 - 4637) a pH between 12.3 and 12.7 must be used. Despite of the relatively low pH there is nevertheless a unwinding that also allows kinetic To make provisions of the unwinding.

It was also completely surprisingly found that the fluorescence values are more stable at this pH. A major advantage over the known method ( DE 197 24 761 A1 ) is the possibility, not only the so-called SSF value (strand scission factor, see Meyn, RE and Jenkins, WT (1983), Cancer Res. 43, 5668-5673) from the fluorescence values after approx. 20 minutes after adding the corresponding To determine solutions to the cells and / or tissues, but also the inclination of the DNA denaturation curve (plotting the intensity of the fluorescence emission, the proportion of double-stranded DNA in the appropriate concentration range of the DNA depending on the choice of the fluorescent dye is to be determined against time t). There is a linear relationship between the slope x (-1) value and the SSF x (-1) value; please refer 4 ,

Furthermore, it is advantageous if the first step (step a of the method defined in claim 63) of the method according to the invention at a temperature of 0 ° C to + 5 ° C and the two further steps (steps b and c of claim 63 defined procedure) at a (constantly selected) temperature of + 15 ° C to + 30 ° C, as these temperature ranges have proven themselves: at higher temperatures, the DNA is untwisted too quickly to ensure exact kineti to be able to carry out cal measurements, while at increasingly lower temperatures the unwinding is progressively slower, which unnecessarily increases the time required to carry out the method. In the case of shorter DNA molecules, such as, for example, of lower organisms, the use of lower temperatures can be advantageous due to the faster unwinding of shorter DNA molecules.

Finally, the fluorescent dye PicoGreen as extremely reliable and thus successfully proven.

• 1. es die Bestimmung der DNA-Integrität auch bei niedrigen Taxa ermöglicht, deren DNA eine vergleichsweise geringe Komplexität besitzt;
• 2. es die Bestimmung von DNA-Integrität mit einer mehr als zweifach höheren Empfindlichkeit als das bekannte Verfahren ( DE 197 24 761 A1 ) erlaubt;
• 3. es auf Mikrotiterplaten (Vorteil: hoher Probendurchsatz) in drei Stunden durchgeführt werden kann;
• 4. die Ergebnisse sowohl als "Strand Scission Factor" (SSF) als auch als Geschwindigkeit der zeitabhängigen DNA-Denaturierung (= Neigung der DNA-Denaturierungskurve) angegeben werden können; und
• 5. der pH-Wert bei der bei dem Entwindungsschritt der DNA stabil ist und somit mit der Zeit keiner Verschiebung unterliegt, die zu einer Veränderung der Fluoreszenz des zur Färbung

der DNA benutzten Fluoreszenzfarbstoffs und damit zu einer Verfälschung des Meßergebnisses führt.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 is the determination of DNA integrity with a sensitivity that is more than twice as high as the known method ( DE 197 24 761 A1 ) allowed;
• 3. it can be carried out on microtiter plates (advantage: high sample throughput) in three hours;
• 4. The results can be given both as "Strand Scission Factor" (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 stable in the unwinding step and is therefore not subject to a shift with time, which leads to a change in the fluorescence of the dye

the fluorescent dye used in DNA and thus leads 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 detex gene, at least one chaotropic substance solution whose concentration during lysis is 0.5 to 4 mol / l, in particular 2 to 4 mol / l, at least one substance solution complexing metal ions and inhibiting DNA repair enzymes, at least one solution to adjust the pH value required for DNA unwinding (step b.), which contains a substance that complexes metal ions and inhibits DNA repair enzymes, and at least one fluorescent dye that specifically binds single-stranded or double-stranded DNA.

The following embodiments of the kit are to be regarded as advantageous for the above reasons and because of their proven practice:
The concentration of the metal ion complexing and DNA repair enzyme inhibiting substance during lysis is 0.005 to 0.1 mol / l.
The pH when the suspensions containing the lysed cells / tissues are mixed with the solution for adjusting the pH required for DNA unwinding is 11 to 12.
The concentration of the metal ion complexing substance and DNA repair enzyme inhibiting substance after the pH value required for DNA unwinding is 0.005 to 0.1 mol / l.
SDS is used as a detergent.
Urea is used as the chaotropic substance.
EDTA is used as a substance that complexes metal ions and inhibits DNA repair enzymes.
PicoGreen is used as the fluorescent dye.

A special configuration for Determination of DNA single-strand breaks contains:

• - Solution A: buffer solution to suspend the cells, for example a metal ion Complexing substance containing substance that inhibits DNA repair enzymes, Buffer solution free of metal ions with a pH of about 7.4 or any other buffer that but not the effect of the solutions B, C and D should influence (for example by a salary the effect of EDTA on divalent metal ions);
• - Solution B: with a concentration of 1 to 8 mol / l of a chaotropic agent, 0.01 to 1.0% by weight of a detergent and 10 to 200 mmol / l one Substance that complexes metal ions and inhibits DNA repair enzymes with a pH of 9.0 to 10.5;
• - Solution C: with a concentration of 0.001 to 1 mmol / l one specific DNA single-strand or DNA double strand binding fluorescent dye;
• - Solution D: with a concentration of 0.02 to 0.2 mol / l of a basic substance and / or a buffer effective in the alkaline range,
• - in which of a volume mixing ratio of solution A: Solution B: Solution C: solution D of 50: 50: 1: 500 is assumed.

The components of solution B can also be used as separate solutions with corresponding changes of the concentrations are used. The fluorescent dye the solution C can also be the solution B or the solutions added to one of its components with a corresponding change in concentration become.

The following explanations have proven to be advantageous in practice:
Solution A is a Tris-HCl-EDTA solution.
The Tris-HCl-EDTA solution contains 10 mmol / l Tris-HCl and 1 mmol / l EDTA at a pH of around 7.4.
The chaotropic agent is urea.
The detergent is SDS.
The metal complexing and DNA repair enzyme inhibiting substance is EDTA.
Solution C is a PicoGreen solution in DMSO.
The basic substance of solution D is sodium hydroxide and / or the buffer tris, triethanolamine or Glycine / NaOH.
Solution D contains EDTA (EDTA traps divalent cations that can lead to cross-linking of the DNA and helps to stabilize the pH).
The EDTA concentration of solution D is 5 to 100 mol / l.

The following uses indicate the surprising listed above and beneficial properties as well.

It is about uses for:
Detection of DNA single-strand breaks and / or alkali-labile sites (DNA modifications such as, for example, apurine sites which change into single strands in the presence of an alkaline solution) in human, animal or vegetable tissues and cells, in bacteria and single cells, in human, animal cell lines or of 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);
Measurement of the occurrence of DNA single-strand breaks and / or alkali-labile sites and their repair after radiation, chemotherapy and / or treatment with genotoxins (mutagens and cancerogens) in human and animal sample material, tissue sections and cell suspensions;
Detection of DNA single-strand breaks and / or alkali-labile sites and their repair in human or animal lymphocytes after their rapid isolation by centrifugation;
Measurement of the occurrence of DNA single-strand breaks and / or alkali-labile spots and their repair in the tissues and cells of patients during radiation therapy (as a pre-therapeutic test to determine the individual radiation sensitivity, for example on lymphocytes or tumor biopsy material of the patient before radiation, to predict the success of the therapy and / or to determine the optimal dose for radiation therapy;
Detection of DNA single-strand breaks and / or alkali-labile sites 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 / or alkali-labile spots and their repair in the event of occupational exposure of people who are either at work (e.g. flight attendants, X-ray doctors, workers in chemical companies) or as a result of accidents (e.g. reactor accidents) with DNA - harmful substances or radiation come into contact;
Measurement of the occurrence of DNA single-strand breaks and / or alkali-labile spots and their repair in test organisms, tissues, cells or cell lines during water monitoring (e.g. municipal and industrial sewage plant inflows and outflows, river water samples);
Evidence of food preservation using radiation;
Detection of DNA single-strand breaks and / or alkali-labile spots by cosmic radiation in flight personnel;
Determination of the unwinding and its reversal as well as for the detection of strand breaks in DNA-RNA hybrids and double-stranded RNAs;
Measurement of the unwinding and its reversal in modified DNAs.

The following examples serve In order to explain the invention.

The abbreviations "M" and "mM" stand for the units below mol / l or mmol / l.

Show it:

1 : This figure shows the dependence of the SSF x (-1) value in human lymphocytes on the radiation dose (60-cobalt); determined using the method according to the invention (single strand method) (black bars) and the in DE 197 24 761 A1 disclosed procedure (white bars).

2 : 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 / l Tris-HCl, 1 mmol / l EDTA ; pH 7.4) with bleomycin, determined using the method according to the invention (single-strand method) (black bars) and the in DE 197 24 761 A1 disclosed procedure (white bars).

3 : This figure shows the determination of the DNA strand breaks (indicated as SSF x (-1)) in Mytilus galloprovincialis after treatment of the DNA in the TE homogenate of gills with hydrogen peroxide; determined with the aid of the method according to the invention (single-strand method).

4 : This figure shows the correlation between the SSF x (-1) values and the slope x (-1) of the DNA denaturation curves of Mytilus galloprovincialis gill tissue when treated with hydrogen peroxide; determined with the aid of the method according to the invention (single-strand method).

5 : This figure shows the dependence of the occurrence of single-strand DNA breaks, expressed in terms of the SSF x (−1) value, on Holothuria tubulosa DNA after exposure to activated bleomycin.

1.) Materials:

The chemicals used in the experiments described below were obtained from E. Merck (Darmstadt, Germany) or from Sigma Chemical Co. (St. Louis, USA). The fluorescent dye PicoGreen was developed by Molecular Pro bes, Leiden, The Netherlands.

2.) Implementation of the assays:

The detection was carried out in the examples described using a fluorescence ELISA plate reader (Fluoraskan II, Labsystems, Helsinki, Finland); she can also with other fluorescence sensing systems such as one Two-beam photameter and an HPLC fluorescence detector.

3.) Solutions:

• PicoGreen dsDNA quantification reagent stock in DMSO (Molecular Probes};
• TE buffer: 10 mmol / l Tris-HCl, 1 mmol / l EDTA, pH = 7.4;
• lysis solution: 4.5 mol / l urea, 0.1% SDS, 40 mmol / l EDTA, pH = 10.0;
• lysis solution with PicoGreen: dilution the concentrated original PicoGreen solution: 20 μl / ml lysis solution

NaOH-EDTA solution:

From a 0.4 molar NaOH solution containing 20 mmol / l EDTA, is by dilution with a 20 mmol EDTA solution a 0.2 molar NaOH-EDTA solution manufactured. This solution becomes the NaOH-EDTA working solution by further dilution with the 20 mmolar EDTA solution made just before the experiments. The pH will change every time by adding 2.5 ml of the NaOH-EDTA solution to 0.25 ml of the lysis solution plus 0.25 ml of TE buffer or TE-DMSO buffer (TE buffer with 10% DMSO at the execution of the assay with tissue samples). The pH must be 11.50 his.

4.) 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 × 10 ³ cells / ml. (Note: in experiments with lymphocytes about 3500 - 4500 cells / 25 μl, about 30 ng DNA).
• b.) The lysis of the cells takes place directly in the wells of black ones Microtiter plates for 40 min to 1 h in the dark on ice instead. Become 25 μl of cell suspension per well slowly 25 μl lysis solution added with PicoGreen: It must not be mixed or shaken.
• c.) DNA denaturation (DNA unwinding) is left at pH 11.50 as follows expire: 250 μl of the NaOH-EDTA solution are added to each sample (per well), to get a pH of 11.50.
• d.) Measuring the intensity the fluorescence emission over a period of up to 1 h, e.g. every 5 min (corresponds to 5 min the time for reading an entire microtiter plate using the fluorescence ELISA plate reader mentioned above needed becomes; dependent on from the devices used (Fluorimeters) are also shorter measurement intervals possible); when using PicoGreen: excitation: 480 nm; Emission: 520 nm. The fluorescence blank value is the fluorescence of a mixture of 25 μl TE buffer, 25 μl lysis solution with PicoGreen and 250 μl the NaOH-EDTA working solution measured.
• e.) Calculation: Since every sample has to be corrected with regard to the blank value, the mean value of the blank value is subtracted from the values. Multiple determinations of each individual sample and the blank value are carried out (usually 4 - 6 determinations each). The percentage of double-stranded DNA (dsDNA) for the untreated or reference cells (for example cells that were not irradiated or not treated with a substance that induced DNA breakage) at time 0 is set as 100% dsDNA; all other values are calculated according to this definition. The measured effects are expressed as "Strand Scission Factor" (SSF, see above) and calculated after a denaturation 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. SSF = 0 means that the sample has no increased frequency of DNA strand breaks in comparison to the control. Positive values can indicate DNA-DNA or DNA-protein crosslinks. For graphic plots, the negative SSF values in the case of DNA strand breaks are usually multiplied by (−1) (representation of the SSF x (−1) values). The results can also be given in the form of the rate of time-dependent DNA denaturation (= slope in the region of the linear portion of the DNA denaturation curve; usually in the period from 5 to 10 minutes after the start of denaturation). The denaturation curve is obtained by plotting the values of the intensity of the fluorescence emission for the samples or controls at time t (after deduction of the corresponding blank values) against time t.

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 immediately after removal in liquid Nitrogen.
• 2. The amount of tissue needed for a well (world) of a 96-well microtiter plate is usually required is 25 μg.
• 3. 10 mg of the tissue is homogenized 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 (world); then 25 ul the lysis solution added with PicoGreen (see above).
• 6. The procedure for cell suspensions is then followed.

1.) Application of the method

a.) Determination of DNA strand breaks in human lymphocytes after exposure to low radiation doses (radiation with 60 cobalt)

Execution:

During these examinations human lymphocytes from a healthy donor different radiation doses (60-cobalt) exposed and then determined the state of the DNA. The Isolation of the lymphocytes was carried out by centrifugation in preformed Density gradients (at + 8 ° C; 10 min) by means of special centrifuge tubes with perforated plastic inserts over one Polysucrose / Natriummetrizoat density gradient.

Result:

1 shows that the newly developed method described here is able to detect DNA damage at a dose of 0.2 Gy. In contrast, the in DE 197 24 761 A1 described procedure only from a dose of 0.6 Gy ( 1 ) found a significant change in the Strand Scission Factor (SSF), which is a measure of DNA damage (single strand breaks). With increasing dose, the SSF increased almost linearly in the range of 0.2 to 2.0 Gy.

b.) Determination of DNA strand breaks in the Mussel Mytilus galloprovincialis after treatment of the DNA in the TE homogenate of gills with bleomycin

Execution:

Gills from the mussel Mytilus galloprovincialis were homogenized after the in DE 197 24 761 A1 described method in TF-DMSO buffer (DMSO = dimethyl sulfoxide), diluted to a concentration of 30 ng DNA / 25 μl and then treated with iron-activated bleomycin for 3 min on ice. Thereafter, the DNA was both after the in DE 197 24 761 A1 described method and analyzed according to the inventive method. The DNA strand breaks were determined immediately after the incubation.

Result:

2 shows that no evidence of induction of DNA damage is possible with the conventional method. In contrast, the improved method described here is able to detect the DNA damage occurring during bleomycin treatment even when incubated in the presence of a concentration of 2.5 μmol / l bleomycin. As the bleomycin concentration increases, there is a concentration-dependent increase in DNA strand breaks (increase in SSF x (-1)).

c.) Determination of DNA strand breaks in Mytilus galloprovincialis after treatment of the DNA in the TF homogenate with hydrogen peroxide

Execution:

Gills of Mytilus galloprovincialis, which according to in DE 197 24 761 A1 described processes in TE-DMSO buffer were diluted to a concentration of 30 ng DNA / 25 ul and then treated with hydrogen peroxide on ice (15 min). Immediately afterwards, the DNA was analyzed using the method described here.

Result:

How 3 shows the method according to the invention is able to detect the DNA damage occurring during hydrogen peroxide treatment even when incubated at a concentration of 13 μmol / l. As the hydrogen peroxide concentration increases, there is a linear increase in the SSF x (-1) value.

d.) Comparison of the SSF x (-1) value with the slope x (–1) the DNA denaturation curves of Mytilus galloprovincialis on treatment with hydrogen peroxide

The experiment was carried out as previously described.

Result:

4 shows that there is a linear relationship between the slope x (-1) values and the SSF x (-1) values. The correlation coefficient be carries 0.971.

Manufacturing the specific DNA preparation with bleomycin for calibration of the method according to the invention (double-strand and single-strand method):

To get reproducible results bleomycin be activated immediately before each experiment.

Activation log:

• 1. To 7-11 mg of Bleomycinum-Mack ^R powder (Heinrich Mack Nachf., Illertissen, Bavaria, Germany) (equivalent to 15 mg of biologically active bleomycin), 5 ml of water and 75 μl of mercaptoethanol are added (the stock solution contains 3 mM bleomycin and is stable in a closed, dark container at + 4 ° C for several months).
• 2. Mix 250 μl of the bleomycin solution (1) with 250 μl of an FeSO ₄ solution (8.35 mg / 5 ml of water). Final concentration of bleomycin: 1.5 mM.
• 3. Mix 500 ul of (2) with 500 μl Dithiothreitol (DTT) solution (8 mg / 5 ml water) and 500 μl Water. Final concentration of bleomycin: 0.5 mM.
• 4. The solution (3) must be fresh daily to the ones you want Concentrations can be diluted with TE buffer. TE buffer: 10mM Tris-HCl, 1mM EDTA; pH = 7.4. Corresponds to 1 mM bleomycin approximately 1 mg / ml.

Treatment of DNA with activated bleomycin:

• 1. The DNA is in TE buffer at a concentration of 1 mg / ml in the dark Solved at night.
• 2. The DNA solution obtained (Step 1) is diluted with TE buffer to a concentration of 5 μg / ml (= working solution).
• 3. 60 ul this working solution is then the with 60 ul desired Bleomycin solution (Solution step 4, see the activation protocol).
• 4. Incubation for 5 minutes at room temperature in the dark.
• 5. Remove four aliquots of 25 μl for further Analysis.

Analysis of single strand DNA breaks

• 1. Pipette 25 μl of the bleomycin-treated DNA solution (Step 5 see Treatment of DNA with activated Bleomycin) per well of a black 96-well microtiter plate (performed as Quadruplicate).
• 2. Add 25 μl of lysis solution as described in the procedure for measuring single strand breaks and their repair.

Analysis of DNA double-strand breaks

• 1. Pipette 25 μl of the bleomycin-treated DNA solution (Step 5 see Treatment of DNA with activated Bleomycin) per well of a 96-well microfilter plate (Execution as a quadruple determination).
• 2. Add 25 μl of lysis solution as described in the procedure for measuring DNA double-strand breaks and their repair.

In 5 shows the dependence of the occurrence of DNA single-strand breaks on Holothuria tubulosa DNA after exposure to various concentrations of activated bleomycin. It can clearly be seen that the SSF x (−1) values increase depending on the concentration.

6 : shows the dependence of the PicoGreen fluorescence on the amount of DNA per well (well native calf thymus DNA (Sigma) was used) (double-strand method);

7 : shows the dependence of the PicoGreen fluorescence on the cell number (HeLa cells) (double-strand method);

8th : represents DNA double-strand breaks in human lymphocytes induced by irradiation with a dose of 0 and 16 Gy (6 MeV linear accelerator) (double-strand method);

9 : represents DNA strand breaks in HeLa cells after incubation of the cells for 4 hours in the presence of different concentrations of bleomycin (the number of cells per well (3800) was 3800) (double-strand method);

10 : represents DNA double-strand breaks (dsb) in different brain areas of spontaneously hypertensive rats (SHR) and normotensive control rats (WKY (double-strand method); (FC: frontal cortex; OC: occipital cortex; H: hippocampus; S: striatum; C : Cerebellum)

11 : represents the dependence of the occurrence of DNA double-strand breaks, expressed in the form of the SSF x (−1) value, on Holothuria tubulosa DNA after exposure to activated bleomycin (double-strand method).

12 : a perspective view of an embodiment according to the device according to the invention for controlled filtration by means of a suction device;

13 : a perspective view of a first embodiment according to the inventive device for controlled filtration by means of a pressurizing device;

14 : A perspective view of a second embodiment according to the inventive device for controlled filtration by means of a pressurizing device.

Examples of the implementation of the Assays (double-strand method):

Materials:

The ones described below Unless otherwise stated, experiments with chemicals were used by E. Merck (Darmstadt, Germany) or Sigma Chemical Co. (St. Louis, USA) acquired. The fluorescent dye PicoGreen was from Molecular Probes, Leiden, The Netherlands.

Carrying out the assay:

The detection takes place in the examples described using a fluorimeter (fluorescence microtiter plate reader; e.g. Fluoroskan II from Labsystems, Helsinki, Finland); she can also with other systems that can detect fluorescence: e.g. Double beam photometer, HPLC fluorescence detector.

Required materials:

• 1. 96-well filter plates (filtration plates), at their wells 96 independent Welded membranes are, e.g. MultiScreen filtration plates from Millipore GmbH (Eschborn, Germany) (e.g. GV, 0.22 μm Durapore, non-sterile, transparent). The Durapore membranes mentioned consist of hydrophilic polyvinylidene difluoride (PVDF); they have only a low protein binding.
• 2. 96-well microtiter plates.
• 3. One detailed below shown device for controlled filtration by means of a suction device or a pressurization 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 the concentrated original PicoGreen solution: 20 μl / ml lysis solution
• 5. Elution solution: prepared by mixing 10 ml of TE buffer, 10 ml of lysis solution, 100 ml bidest. water

Method:

• 1. There is a cell suspension in TE buffer with a pH of 7.4 with a cell density of 150,000 Cells / ml. (Note: in experiments with lymphocytes 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. To 25 μl cell suspension (pipetted on the filter membrane surface) slowly 25 μl lysis solution added with PicoGreen. It must not be mixed or shaken. It recommends the filter before applying the samples by washing with a small Amount of elution solution (about 200 μl per well).
• 3. In a parallel approach (to determine the total DNA per Well; = Total value) is the lysis of the cells in the wells of a black 96-well microtiter plate as described under 2. The total value is then the Fluorescence measured.
• 4. Add 250 ul Aqua bidest. per well of the 96-well filtration plates and the black 96-well microtiter plate.
• 5. After starting the vacuum pump or another vacuum source (House vacuum) become the solutions in the wells of the 96-well filtration plates at low speed (35 μl per well per minute) sucked through the filter (total duration: 7 min). Wells that are not used are sealed with an adhesive tape (parafilm). The filtrates (eluates) are placed in a vacuum holder the black 96-well microtiter plate collected. The fluorescence of the DNA-PicoGreen complexes in the filtrates will then measured.
• 6. It is possible but not absolutely necessary to collect further fractions. In In this case, after 10 min (3 min after the end of the first filtration) 250 μl elution solution in pipetted the wells of the 96-well filtration plate; then will filtered again under vacuum as in step 5 and then again measured the fluorescence.
• 7. If necessary, repeat from step 6 to 20 and after 30 min. The measurement of the fluorescence of the filtrates (eluates) and the Total values can be collected immediately after the last filtration step respectively.
• 8. Measurement of fluorescence (with PicoGreen: excitation: 480 nm; Emission: 520 nm). Fluorescence blank values are obtained by in steps 2 and 3 25 μl lysis solution with PicoGreen at 25 μl Water (or TE buffer) is pipetted in place of the cell suspension. All other steps are carried out as described above.
• 9. Calculation: Multiple determinations are carried out for each sample and for the blank value (4 - 6 determinations each). The samples are corrected for the blank value by subtracting the mean value of the blank value from the measured values. In the case of the collection of several groups, the elution curves are shown in a semilogarithmic graphic, with a linear x-axis (fraction number or elution volume) and a logarithmic y-axis (percentage of double-stranded DNA remaining on the filter after aspiration, based on the total amount of double-stranded DNA ( = Total value, set as 100%)). The results are reported as "DNA Filter Ratio" (DFR). The DFR value is calculated using the following formula: [(tDNA - ΣfDNA n ) / tDNA] × 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 show an increased frequency of DNA double-strand breaks on.

Alternatively, the results can be given in the form of the "Strand Scission Factor" (SSF). In this case, the calculation is based on the following formula:
SSF = log (DFR / DFR)
DFR _p = DFR value for the sample to be determined
DFR _k = DFR value for the control.

Negative values for SSF show an increased proportion on double strand breaks on.

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 immediately after removal in liquid Nitrogen can be frozen and stored (storage for shorter periods can also take place at –80 ° C).
• 2. The amount of tissue needed for one well of the microtiter plate is usually required approx. 25 μg.
• 3. 10 mg of the tissue is homogenized in 1 ml of homogenization buffer in a mortar with a pestle under liquid Nitrogen. Homogenization buffer: TE buffer (see above) or others suitable buffer with 10% cryoprotector (dimethyl sulfoxide, glycerin Etc.).
• 4. Dilute the homogenate 10-fold with TE buffer.
• 5. In each well of the 96-well filtration plate is 25 ul of the homogenate given; DNA become 25 ul the lysis solution added with PicoGreen (see above).
• 6. The procedure for cell suspensions is then followed.

Influence of DNA concentration:

Execution:

To determine the dependency The PicoGreen fluorescence from DNA amount became native calf thymus DNA used.

Result:

6 shows that up to a DNA amount of 80 ng per well there is an almost linear relationship between the fluorescence and the amount of DNA.

Dependence of the PicoGreen fluorescence on the cell count

Execution:

To determine the dependency The PicoGreen fluorescence from the cell count were increasing numbers incubated on cells with PicoGreen containing lysis solution and then the Fluorescence measured.

Result:

7 shows that the PicoGreen fluorescence increases linearly up to a number of 4000 cells per well.

Determination of the sensitivity of the assay (human lymphocytes)

Execution:

During these examinations human lymphocytes after taking blood at different radiation doses exposed and then determines the state of the DNA.

Result:

8th shows that with increasing dose (16 Gy) the amount of double-stranded DNA in the filtrate, ie the amount of DNA double-strand breaks, increases.

The sensitivity of the process can still be considerable be increased by the introduction of a Protease digestion step or by collection and filtration of the Filtrates through a second microfilter plate.

Determination of DNA double-strand breaks in HeLa cells after incubation with bleomycin

Execution:

HeLa cells were known in the presence of various concentrations of bleomycin (a known Inducent of DNA double strand breaks (Byrnes, RW, Petering, DH: DNA double-strand breakage by bleomycin in Ehrlich ascites tumor cells as measured by nondenaturating filter elution. Rad. Res. 137: 162-170; 1994)) incubated for 4 hours , The DNA double-strand breaks were determined immediately after the incubation.

Result:

9 shows a concentration-dependent increase in DNA double-strand breaks after incubation of the cells with bleomycin.

Determination of DNA double-strand breaks in different Brain regions of spontaneously hypertensive rats compared to normotensive ones control rats

Execution:

The studies were on frozen Tissue samples from different brain regions (frontal cortex, occipital Cortex, hippocampus, striatum, cerebellum) of spontaneously hypertensive rats (SHR) and normotensive control rats (WKY). there comparisons were made between areas of the brain that are particularly sensitive to ischemia and areas that are less sensitive. Determination of DNA double-strand breaks in different Brain regions of SHR compared to WKY were identified accordingly Protocol for Tissue samples performed. The age of the animals was 6 months.

Result:

The extent of the double-strand breaks in the DNA was determined. It was found in these investigations that the amount of DNA double-strand breaks was increased very strongly in certain brain regions of the SHR compared to the control rats ( 10 ).

Binding of protamine sulfate to DNA:

In this experiment, 0.6 μg / ml holothuria tubulosa DNA (corresponding to a nucleotide concentration of approx. 1.8 μmol / l) with increasing concentrations of protamine sulfate (0 - 3 mg / ml, 0 - 0.3 mM (Molecular weight of protamine sulfate: 9000) in TE buffer (10 mmol / l Tris-HCl, 0.2 mol / l EDTA; pH = 7.4) directly in the wells of the 96-well filter plates for 30 Incubated for minutes on ice (total volume of the mixture: 300 μl). To the incubation was 0.5 μl PicoGreen solution per Approach added and for Incubated for 10 minutes. After that, at a rate of 20 μl / min filtered through the filters. Then the fluorescence measured in the collected filtrates and that of unfiltered parallel batches.

It turned out that with one Protamine sulfate concentration of 30 ng / ml (3.3 nmol / l) the DNA almost 100% retained on the filters is, whereas at a protamine sulfate concentration of 3 ng / ml (0.3 nmol / l) no retarding effect of the protein was found has been. This means that at a protamine sulfate concentration of about 1 nmol / l about 50% of the DNA is bound. The relationship of protamine sulfate to DNA at this concentration about a protamine sulfate molecule to about 2000 nucleotides is coming.

Binding of histone to DNA:

In this binding assay, 0.6 μg / ml holothuria tubulosa DNA (corresponding to a nucleotide concentration of approx. 1.8 µmol / l with increasing concentrations of histone protein (0 - 30 μg / ml, 0 - 3 μmol / l) (from Calf thymus, type V-S, sigma; Molecular weight of histone: approx. 10,000) in TE buffer directly in the wells of the 96-well filter plates for 30 minutes Incubated ice (total volume of the mixture: 300 μl). After incubation 0.5 μl pico green solution per Approach added and for Incubated for 10 minutes. It was then run through at a rate of 20 μl / min the filters are filtered. The fluorescence in the collected filtrates and that of unfiltered parallel batches was then measured.

It turned out that the DNA from a histone concentration of 3 μg / ml (0.3 μmol / l) almost completely held back by the filters has been. From this one can calculate that at a DNA concentration of 0.6 μg / ml (corresponding to 1.8 μmol / l, related on nucleotide units) a histone molecule approximately 6 nucleotide units the DNA binds. At a histone concentration of 0.3 μg / ml however, no significant retarding effect was observed.

Manufacturing the specific DNA preparation with bleomycin for calibration of the method according to the invention (double-strand and single-strand method):

To get reproducible results bleomycin be activated immediately before each experiment.

Activation log:

• 1. To 7-11 mg of Bleomycinum-Mack ^R powder (Heinrich Mack Nachf., Illertissen, Bavaria, Germany) (equivalent to 15 mg of biologically active bleomycin), 5 ml of water and 75 μl of mercaptoethanol are added (the stock solution contains 3 mM bleomycin and is stable in a closed, dark container at + 4 ° C for several months).
• 2. Mix 250 μl of the bleomycin solution ( 1 ) with 250 μl of a FeSO ₄ solution (8.35 mg / 5 ml water). Final concentration of bleomycin: 1.5 mM.
• 3. Mix 500 ul of (2) with 500 μl Dithiothreitol (DTT) solution (8 mg / 5 ml water) and 500 μl Water. Final concentration of bleomycin: 0.5 mM.
• 4. The solution (3) must be fresh daily to the ones you want Concentrations can be diluted with TE buffer. TE buffer: 10mM Tris-HCl, 1mM EDTA; pH = 7.4. Corresponds to 1 mM bleomycin approximately 1 mg / ml.

Treatment of DNA with activated bleomycin:

• 1. The DNA is in TE buffer at a. concentration of 1 mg / ml in the dark Solved at night.
• 2. The DNA solution obtained (Step 1) is diluted with TE buffer to a concentration of 5 μg / ml (= working solution).
• 3. 60 ul this working solution is then the with 60 ul desired Bleomycin solution (Solution step 4, see the activation protocol).
• 4. Incubation for 5 minutes at room temperature in the dark.
• 5. Remove four aliquots of 25 μl for further Analysis.

Analysis of single strand DNA breaks

• 1. Pipette 25 μl of the bleomycin-treated DNA solution (Step 5 see Treatment of DNA with activated Bleomycin) per well of a black 96-well microtiter plate (performed as Quadruplicate).
• 2. Add 25 μl of lysis solution as described in the procedure for measuring single strand breaks and their repair.

Analysis of DNA double-strand breaks

• 1. Pipette 25 μl of the bleomycin-treated DNA solution (Step 5 see Treatment of DNA with activated Bleomycin) per well of a 96-well microfilter plate (Execution as a quadruple determination).
• 2. Add 25 μl of lysis solution as described in the procedure for measuring DNA double-strand breaks and their repair.

In 11 shows the dependence of the occurrence of DNA double strand breaks on Holothuria tubulosa DNA after exposure to various concentrations of activated bleomycin. It can clearly be seen that the SSF x (−1) values increase depending on the concentration.

Controlled devices Filter liquid Media with well filter plates

Description of the suction filtration device

In the 12 The suction filtration device shown consists of the following parts:

1

Acrylic glass attachment consisting of an outer acrylic glass frame and an acrylic glass plate sitting on it. There is a fitted ventilation tube in the outer acrylic glass frame ( 1a ) with associated valve for regulating the vacuum in the upper vacuum chamber (not shown in 12 ). Through the flow tap ( 1b ) The upper vacuum chamber can be separated or connected depending on the position. ( 1c ) Hole in the acrylic glass attachment, which can be opened or closed using the reversing tap.

2

sealing ring between the acrylic glass attachment and the 96-well filter plate (attached on the inside of the lid of the acrylic glass attachment).

3

96-well filter plate with welded Filters (e.g. Millipore MAGV N22).

4

spacer (Spacer) 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 (from illustration not recognizable) and the acrylic glass attachment against the base plate concludes.

7

Base plate with 4 feet and a hole in the center of the base plate that ends at the top and an inserted suction tube ( 7a ) for connection to a vacuum source (house vacuum or vacuum pump).

working principle

The filtration device has an acrylic glass attachment ( 1 ) on a base plate ( 7 ) is placed. Due to the silicone seal in the lower edge of the acrylic glass attachment ( 6 ) the room under the top can be sealed airtight. An empty microtiter plate ( 5 ) and a filter plate already loaded with samples ( 3 ) arranged so that the filter plate comes to rest on the microtiter plate and on the bottom plate (see 12 ). That there is a well of the filter plate directly above the corresponding well of the microtiter plate is ensured by the fact that the two plates are exactly in the acrylic glass attachment are fitted 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 filter plate, and a lower one, which extends from the bottom of the filter plate to the bottom 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 filter plate after the device has been set up. However, these two chambers (air spaces) are initially through a hole ( 1c ) in the wall of the acrylic glass attachment. Via a flow tap ( 1b ) this connection can be closed or opened. After the correct composition of the individual components of the apparatus, a vacuum source can now be connected to the suction pipe ( 7a ) can be connected. 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 filter 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 filter plate and thus being separated from the lower chamber space. If the connection between the lower and upper chamber is opened with the help of the flow valve, 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 comb spaces are then completely separated. 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 wells of the filter plate are sucked through the filters forming the bottom of the wells into the lower chamber, the filtrate being collected in the wells of 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 well is recommended to carry out the procedure for determining DNA double-strand breaks. The applied vacuum remains switched on during the entire filtration process. After complete filtration, the acrylic attachment can be removed, the filtrate in the microtiter plates being available for further investigations.

difference to other devices

Compared to conventional devices (eg suction filtration device "vacuum manifold" from Millipore GmbH), the new one in 12 Apparatus shown is that first by connecting the opening 7a With the vacuum source, the same negative pressure is present in both the upper and the lower chamber, thus sealing the apparatus from the outside. In the second step, DNA is made by switching the flow tap 1b the connection between the upper and lower chamber separated. By opening the hole 1a and the inflow of outside air controlled by the valve, the pressure in the upper chamber is then adjusted so that the solution is sucked through the filters at a continuous speed and adapted to the experimental conditions. The principle of this design allows slow and continuous suction of solution through the filters 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 Millipore 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 filter. Also, the control valve cannot be reduced so far that the required slow filtration speed (20 μl / min and below when using 96-well microfilter plates with a capacity of 300 μl / well) is achieved, which is used to measure DNA double-strand breaks of the present method are needed.

Our attempts showed that it was too not possible with brackets is to press the filter plates so tightly against the rubber seal so slow Filtration in the presence of a weak vacuum without prior Apply a stronger vacuum (for pressing the filter plates against the rubber seal) comes.

Delimitation of this filtration device from that in the patent application EP 0359249 described filtration device

In the patent application EP 0359249 describes a filtration device in which a microfilter plate and a microtiter plate are also used. However, the principle on which this apparatus is based differs from that of the first filtration device described in the present application. So in the device described here in contrast to that in the patent application EP 0359249 described filtration device first creates a negative pressure in both chambers, which is then in one (above ren) chamber is reduced until the filtration process runs at the desired speed. A bracket is used in the device described here in contrast to that in the patent application EP 0359249 described filtration device not used.

The advantage of the present Application described construction is that this of other equipment for the generation of the airtight seal necessary contact pressure, which leads to a too rapid initial passage of liquid through the filters would, is circumvented.

A constant liquid flow through the filters is essential for the use of the filtration devices we need for DNA strand breakage determinations. It must also be emphasized that the pore size of the filters of the filter plates we use for DNA strand breakage determination is so small that filtration due to gravity is not possible. Due to the small pore size, filtration could be as described in the patent application EP 0359249 the principle described for the example cell culture did not materialize.

This is also in the patent application EP 0359249 The problem solved by the apparatus described there is no longer relevant, since the microfilter plates are now commercially available as relatively inexpensive "disposable articles" (Millipore company). In the filtration devices described in the present application, only the used microfilter plates and the microtiter plates (both "disposable items") come into contact with the examined samples during the filtration process, so that the complicated (or after filtration of radioactive or infectious samples may be difficult to carry out cleaning is not necessary of the devices in contrast to that in the patent application EP 0359249 described apparatus.

Technical data of the device and of the device parts:

• Acrylic glass top Dimensions: H × W × D: 4 × 14.5 × 10.5 cm Weight: 0.367 kg
• sealing ring Dimensions: D × W × D: 0.5 × 12.6 × 8.5 cm
• Spacer Dimensions: H × W × D: 0.2 × 12.5 × 0.6 cm Weight: 0.001 kg
• sealing ring Dimensions: D × W × D: 0.6 × 14.3 × 10 cm
• Lower bracket Dimensions: H × W × D: 1.1 × 14.8 × 10.6 cm Weight: 0.278 kg
• 96-well filter plate (e.g. from Millipore) Dimensions: H × W × D: 1.5 × 12.7 × 8.5 cm Weight: 0.055 kg
• Black microtiter plate (e.g. from Nunc) Dimensions: H × W × D: 1.4 × 12.7 × 8.5 cm Weight: 0.05 kg Dimensions of the entire device: H × W × D: 5.4 × 14.5 × 10.5 cm Weight of Total device: 0.755 kg

description the pressure filtration device

In the 13 reproduced pressure filtration device consists of the following parts:

1'

Acrylic glass attachment consisting of an outer acrylic glass frame and an acrylic glass plate sitting on it. There is a fitted tube in the outer acrylic glass frame ( 1a ' ) with inserted adjustable compressed air valve for connection to a compressed air source (e.g. house compressed air or nitrogen bottle). There are two latches on the acrylic glass attachment (not shown in the 13 ) for tightening the lower acrylic glass frame including all device parts in between.

2 '

sealing ring between the acrylic glass attachment and the 96-well filter plate.

3 '

96-well filter plate with welded Filters (e.g. Millipore MAGV N22).

4 '

sealing plate between the 46-well filter plate and the lower acrylic glass frame (only serves to stabilize the filter plate).

5 '

lower Acrylic frame.

6 '

black 96-well microtiter plate (e.g. Maxisorb from Nunc) for collection of the filtrate.

working principle

The filter bar already containing the samples ( 3 ' ) (can be handled without risk of leakage) between the acrylic glass attachment ( 1' ) and lower acrylic glass frame ( 5 ' ) positioned. By closing the two fasteners (not shown in 13 ) the lower acrylic glass frame including all the device parts in between is firmly connected. As a result, the sealing ring (inside the acrylic glass attachment) 2 ' ) pressed onto the outer frame of the top of the filter plate, the air space between the acrylic cover and the filter plate being closed airtight. The apparatus prepared so far is placed on an empty microtitre plate ( 6 ' ) set. By connecting a compressed air source (compressed gas source) to the tube fitted in the acrylic glass attachment ( 1a ' ) With the connected adjustable valve, an overpressure is generated in the chamber above the filter plate, which causes the samples to be filtered. The strength of the overpressure and thus the filtration speed can be regulated via the valve connected to the acrylic glass attachment bar. A filtration rate suitable for carrying out the method for determining DNA double-strand breaks is 300 μl / 15 min per well. The filtrate is collected in the 96-well microtiter plate.

The function of the latches at the apparatus does not consist of an airtight seal between Filtration plate and microtiter plate, but the Space above the filtration plate before applying excess pressure airtight.

difference to other devices

The new in in comparison to conventional apparatus (e.g. vacuum manifold from Millipore GmbH) 13 shown apparatus is that the filtration of the pipetted into the wells of the 96-well filter plate is carried out by applying an overpressure above the filter plate and not by applying a negative pressure (vacuum) below the filter plate. An advantage of the principle used in this apparatus 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). The overpressure in the room above the 96-well filter plate can be generated in addition to the above-mentioned pressure sources, for example by applying an aquarium air pump or by attaching a syringe. In addition, people are protected from radiation by the 10 mm thick radiation-absorbing acrylic plates when using the device for the filtration of radioactive materials during the filtration process.

Technical data of the device and of the device parts:

• Acrylic glass top Dimensions: H × W × D: 2.6 × 15 × 10.6 cm Weight: 0.338 kg
• rubber seal Dimensions: D × W × D: 1 × 13.3 × 9.1 cm
• Acrylic frame Dimensions: H × W × D: 0.7 × 10.2 × 14.8 cm Weight: 0.0725 kg
• rubber seal Dimensions: D × W × D: 0.8 × 8.3 × 12.6 cm
• 96-well filter plate Dimensions: H × W × D: 1.5 × 12.7 × 8.5 cm Weight: 0.055 kg Dimensions of the entire device: H × W × D: 5.3 × 15 × 10.7 cm Weight of Total device: 0.51 kg

Description of another execution the pressure filtration device

In the 14 reproduced pressure filtration device consists of the following parts:

1''

Acrylic glass housing ( 1'' ) consisting of a rectangular base plate ( 1a ' ) and two side plates firmly connected to the long sides ( 1b '' and 1c '' ), as well as a fold-out acrylic glass lid ( 1d '' ). There is a rubber seal on the inside of the fold-out cover ( 1e '' ), which sits on the outer frame of the 96-well filter plate after the lid is closed. There is also a locking clip on the front of the acrylic glass cover ( 1f '' ) for securely pressing the rubber seal onto the 96-well filter plate after closing the device. There is a control valve on the back of the acrylic glass cover ( 1g '' , e.g. Festo GR-M5B) with a connection hole to the room above the filter plate, which also serves to connect to a compressed air source (e.g. house compressed air, nitrogen bottle or aquarium pump).

2 ''

96-well filter plate, inserted through the front opening of the acrylic glass housing along the two guide strips attached to the side plates ( 1h '' ) up to the stop on the acrylic glass block connected to the lower acrylic glass plate ( 1i '' ).

3 ''

black 96-well microtiter plate to collect the filtrate.

working principle

The lid ( 1d '' ) of the acrylic glass housing ( 1'' ), which is connected to it via a hinge, is opened upwards to allow the filter plate to be placed ( 2 '' ) and the microtiter plate ( 3 '' ) inside the housing. The empty microtiter plate is placed on the bottom ( 1a ' ) of the housing. The filter plate, which is already filled with samples, is placed in the two guide strips ( 1h '' ), which is located halfway inside the housing on its side walls ( 1b '' and 1c '' ), inserted (see 14 ). By closing the lid and tightening the locking clip ( 1f '' ) the two plates are pressed against a stop ( 1i '' ) which is located at the end opposite the opening. As a result, the two plates are automatically positioned so that a well of the filter plate is located directly above the corresponding well of the microtiter plate. Closing the lid also opens the bottom of the acrylic glass lid tegrated rubber seal pressed onto the top of the filter plate. This creates a small, airtight space between the acrylic glass cover and the filter plate. Now a pressure source that is connected to the bore and the control valve ( 1g '' ) is connected to the space between the acrylic glass cover and the filter plate. The thus on the liquid column in the wells. pressure exerted on the filter plate enables filtration, the filtration speed being determined by the control valve, which can regulate the amount of gas (air quantity) flowing onto the filter plate down to almost zero. The strength of the pressure should, if possible, be easily adjustable at the pressure source itself. The liquid that has passed through the filter is collected by the microtiter plate. A filtration rate of approximately 300 μl / 15 min per well is suitable for carrying out the method for determining DNA double-strand breaks. After the filtration has been completed and the gas supply has been interrupted, the two plates can be removed from the acrylic glass housing and the filtrate in the microtiter plate can be examined further.

The function of the bracket in this Equipment is just an airtight seal above the filtration plate and an exact positioning of the filter plate and the microtiter plate.

The principle of that described here The apparatus corresponds to that of the second apparatus presented here. The advantage of the third apparatus, however, is that one of easier to lock the 96-well filter plate and the black 96-well microtiter plate possible is.

Technical data of the device:

• Dimensions: H × W × D: 5.3 × 17.4 × 10.5 cm
• Weight: 0.685 kg

use of second and third apparatus in the absence of a compressed air source

Filtration using the second and third apparatus can be even in the absence of a source of compressed air by applying the excess pressure by means of an inflatable (for example with the help of a bicycle pump) Hose or any other compressed air reservoir (gas bottle) will be produced.

Advantages of second and third apparatus

The advantage of those described here two pressure filtration devices compared to conventional, commercially available Equipment is particularly easy to handle. they allow filtration even if there is no compressed air source is. So with these devices also for Examinations outside of a laboratories equipped with a compressed air source, for example a research ship (investigation of DNA damage in marine organisms), possible. Furthermore enables the special design of the two pressure filtration devices shielding against radiation when using the devices for filtration radioactive materials. For filtration processes of oxygen sensitive (air sensitive) substances can instead pressure cylinders containing inert gases are used. Comparable equipment for Microfilter plates are not commercially available.

use of three apparatuses for the filtration of radioactive substances and autoradiography

The three devices are also suitable for the filtration of radioactive materials because of the 10 mm thickness Acrylic glass panels on the outside of the devices a good shielding of the environment from radioactive radiation he follows. Only the microfilter plate and come in the filtration process the microtiter plate, i.e. two commercially available disposable items, is not but the equipment itself is in contact with the radioactive substances. Farther can perform an autoradiographic detection after filtration the one left on the filters radioactivity done by an x-ray film between (micro) filter plate and (micro) titer plate or between (Micro) filter plate and one used instead of the (micro) titer plate about the same size Acrylic glass plate is inserted. The egg laying of the X-ray film can be in a dark room due to the easy handling of the Devices easily carried out become. While of exposure the equipment is wrapped in aluminum foil for light shielding become.

Claims (60)

Method for determining DNA double-strand breaks with the following steps: a.) Placing cells and / or tissues to be examined on specific filters; b.) Lysis of the cells / tissues with at least one detergent, at least one chaotropic substance, at least one substance that complexes metal ions and inhibits DNA repair enzymes and at least one fluorescent dye that specifically binds the DNA double strand, or lysis of the cells / tissues with at least one detergent, at least a chaotropic substance, at least one protease and at least one specific fluorescent dye binding DNA double-strand, c.) elution of the DNA fragments released by the lysis through the specific filters; d.) carrying out a fluorescence spectroscopic evaluation, whereby only one eluate fraction is collected per well, or a.) placing cells to be examined and / or Fabrics on specific filters; b.) lysis of the cells / tissues with at least one detergent, at least one chaotropic substance and at least one substance that complexes metal ions and inhibits DNA repair enzymes, or lysis of the cells / tissues with at least one detergent, at least one chaotropic substance and at least one protease, c .) Elution of the DNA fragments released by the lysis through the specific filters, d.) Addition of the eluates obtained with at least one specific DNA double strand fluorescent dye; e.) Carrying out a fluorescence spectroscopic evaluation, only one eluate fraction being collected per well. A method according to claim 1, characterized in that the Lysis in the presence of at least one metal ion complexing and DNA repair enzyme inhibiting substance and in the presence at least a protease becomes. Method according to one of claims 1 to 2, characterized in that that at Lysis step the concentration the chaotropic substance is 0.5 to 9 mol / l. Method according to one of claims 1 to 3, characterized in that that at Lysis step the concentration the chaotropic substance is 2 to 4 mol / l. Method according to one of claims 1 to 4, characterized in that that at Lysis step the concentration of metal ion complexing and DNA repair enzymes inhibiting Substance is 0.005 to 0.05 mol / l. Method according to one of claims 1 to 5, characterized in that that at Lysis step the pH is set to 7 to 11. Method according to one of claims 1 to 6, characterized in that that at Lysis step the concentration the protease is 0.01 to 5 mg / ml. Method according to one of claims 1 to 7, characterized in that that the Concentration of the chaotropic substance solution in the lysis step 2.25 mol / l is. Method according to one of claims 1 to 8, characterized in that that the Concentration of metal ion complexing and DNA repair enzymes inhibiting substance in the lysis step is 0.02 mol / l. Method according to one of claims 1 to 9, characterized in that that the Concentration of the detergent in the lysis step is 0.00175 mol / l. Method according to one of claims 1 to 10, characterized in that that the The concentration of the protease in the lysis step is 0.5 mg / ml. Method according to one of claims 1 to 11, characterized in that that as chaotropic substance urea is used. Method according to one of claims 1 to 12, characterized in that that as Substance that complexes metal ions and inhibits DNA repair enzymes EDTA is used. Method according to one of claims 1 to 13, characterized in that that as Protease Proteinase K is used. Method according to one of claims 1 to 14, characterized in that that as Detergent SDS is used. Method according to one of claims 1 to 15, characterized in that that the pH value in the lysis is 10. Method according to one of claims 1 to 16, characterized in that that step a.) 1. Alternative at a temperature of 15 ° C to 35 ° C and / or step b.) 1. Alternative at a temperature of 15 ° C up to 35 ° C and / or step c.) 1. Alternative at a temperature of 15 ° C to 35 ° C and / or Step d.) 1. Alternative at a temperature of 15 ° C to 35 ° C and / or Step a.) 2. Alternative at a temperature of 4 ° C to 15 ° C and / or step b.) 2. Alternative at a temperature of 4 ° C to 15 ° C and / or step c.) 2. Alternative at a temperature of 4 ° C up to 15 ° C and / or step d.) 2. Alternative at a temperature of 4 ° C to 15 ° C and / or Step e.) 2. Alternative is carried out at a temperature of 4 ° C to 15 ° C. Method according to one of claims 1 to 17, characterized in that that as Fluorescent dye PicoGreen is used. Kit for determining DNA double-strand breaks, comprising: at least one detergent, at least one chaotropic substance, at least one substance complexing metal ions and inhibiting DNA repair enzymes, at least one fluorescent dye which binds a specific DNA double strand and at least one specific filter type or at least one detergent, at least one chaotropic substance, at least one protease, at least one specific fluorescent dye binding DNA double strand and at least one specific filter type. Kit according to claim 19, characterized in that the kit at least one metal ion complexing and DNA repair enzyme inhibitory substance and contains at least one protease. Kit according to one of claims 19 to 20, characterized in that that the Concentration of the chaotropic substance during lysis 0.5 to 9 mol / l is. Kit according to one of claims 19 to 21, characterized in that that the Concentration of the chaotropic substance during lysis 2 to 4 mol / l is. Kit according to one of claims 19 to 22, characterized in that that the Concentration of metal ion complexing and DNA repair enzymes inhibiting substance in the lysis is 0.005 to 0.05 mol / l. Kit according to one of claims 19 to 23, characterized in that the Protease Proteinase K is. Kit according to one of claims 19 to 24, characterized in that that the The concentration of the protease in the lysis step is 0.01 to 5 mg / ml. Kit according to one of claims 19 to 25 for determination DNA double strand breaks containing: - Solution A: buffer solution to suspend the cells; - Solution B: with a concentration from 0.001 to 0.2 mol / l of a detergent and with a concentration of 4 to 8 mol / l of a chaotropic substance; - Solution C: with a concentration from 0.001 to 1.0 mol / l of a fluorescent dye that binds DNA specifically, - in which of a volume mix ratio of solution A: Solution B: Solution C of 50: 50: 1 is assumed. Kit according to claim 26, characterized in that solution B at least a substance complexing metal ions and inhibiting DNA repair enzymes contains. Kit according to claim 27, characterized in that the concentration the substance complexing metal ions and inhibiting DNA repair enzymes the solution B is 0.01 to 0.1 mol / l. Kit according to claim 28, characterized in that the concentration the substance complexing metal ions and inhibiting DNA repair enzymes the solution B is 0.04 mol / l. Kit according to one of claims 27 to 29, characterized in that that the Substance that complexes metal ions and inhibits DNA repair enzymes EDTA is. Kit according to one of claims 26 to 30, characterized in that solution B has at least one Contains protease. Kit according to claim 31, characterized in that the protease Proteinase K is. Kit according to one of claims 31 to 32, characterized in that the Concentration of the protease in solution B is 0.02 to 10 mg / ml. Kit according to one of claims 28 to 33, characterized in that that solution A is a Tris-HCl-EDTA solution. Kit according to claim 34, characterized in that the Tris-HCl-EDTA solution is a Concentration of 10 mmol / l Tris-HCl and 1 mmol / l EDTA with a pH of about 7.4. Kit according to one of claims 26 to 35, characterized in that that as Detergent SDS is used Kit according to claim 36, characterized in that the SDS concentration in solution B is 0.0035 mol / l. Kit according to one of claims 26 to 37, characterized in that that as chaotropic substance urea is used. Kit according to claim 38, characterized in that the urea Concentration in solution B is 4.5 mol / l. Kit according to one of claims 26 to 38, characterized in that that the PicoGreen is a DNA double strand binding fluorescent dye. Use of the kit according to one of claims 19 to 40 to carry out of the method according to one of claims 1 to 18. Use of the method according to one of claims 1 to 18 for the detection of DNA double-strand breaks and cross-links in human, animal or plant tissues and cells, in bacteria and unicellular organisms, in cell lines of human, animal or plant origin and subcellular fractions, as well as in isolated DNA; For the detection of DNA repair in human, animal or plant tissues and cells, in bacteria and single cells, in cell lines of human, animal or plant origin, subcellular fractions, cell lysates and cell extracts (isolated molecules); For measuring the occurrence of DNA double-strand breaks and cross-links and their repair after radiation, chemotherapy and / or treatment with genotoxins, mutagens and carcinogens in human and animal sample material, tissue sections and cell suspensions; For the detection of DNA double-strand breaks and cross-links and their repair in human or animal lymphocytes after their rapid isolation by centrifugation; For measuring the occurrence of DNA double-strand breaks and cross-links and their repair in the tissues and cells of patients during radiation therapy; For the detection of DNA double-strand breaks and cross-links in frozen test materials after their gentle homogenization in liquid nitrogen or liquid air in the presence of an anti-freeze agent; To detect 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 or as a result of accidents; For measuring the occurrence of DNA double-strand breaks and cross-links and their repair in test organisms, tissues, cells or cell lines during water monitoring; For the detection of DNA double-strand breaks after exposure of cells to topoisomerase II inhibitors; For the detection of food preservation by means of radiation; For the detection of DNA damage from cosmic radiation in flight personnel; For screening for radioprotective substances. Use of the kit according to one of claims 19 to 40 for the detection of DNA double strand breaks and cross-links in human, animal or vegetable tissues and cells, in bacteria and unicellular organisms, in human, animal or vegetable cell lines Origin and subcellular Fractions, as well as in isolated DNA; For the detection of DNA repair in human, animal or vegetable tissues and cells, in bacteria and unicellular organisms, in human, animal or vegetable cell lines 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, mutagens and carcinogens in human and animal specimens, tissue sections and cell suspensions; To the Detection of DNA double-strand breaks and cross-links and their repair in human or animal Lymphocytes after their rapid isolation by centrifugation; to 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; To the Detection of DNA double-strand breaks and cross-links in frozen test materials their gentle homogenization in liquid nitrogen or liquid air in the presence of an antifreeze; To prove the occurrence of DNA double-strand breaks and cross-links and their repair in case of occupational exposure by people either at work or as a result of of accidents with DNA damaging Substances or rays come into contact; To measure the Occurrence of DNA double-strand breaks and cross-links and their repair in test organisms, tissues, cells or cell lines in water monitoring; To the Detection of DNA double-strand breaks after exposure of cells to Topoisomerase II inhibitors; For proof of food preservation by means of radiation; For the detection of DNA damage by cosmic radiation from flight personnel; For screening for radioprotective Substances. Device for the controlled filtering of liquid media, in particular containing DNA molecules and / or other molecules / molecular complexes, with well filter plates, with at least one well filter plate ( 3 ), at least one corresponding microtiter plate ( 5 ) and a suction device ( 7a ), characterized by: - two separate gas spaces arranged above and below the filter plate; - at least one with a container ( 1 ) and the well filter plate ( 3 ) interacting sealing device and thus a complete separation of the two gas spaces ( 2 ); - An institution ( 1b . 1c ) to connect both gas spaces; - An institution ( 1a ) for pressure equalization between the environment and a gas space. Device according to claim 44, characterized in that the well filter plate and the corresponding microtiter plate by means of spacers ( 4 ) are spaced from each other. Device according to one of claims 44 to 45, characterized in that the suction device is a suction pipe. Device according to one of claims 44 to 46, characterized in that that the Device for pressure equalization between the environment and a gas space is a valve. Device according to one of claims 44 to 47, characterized in that that the Device for connecting both gas spaces one arranged in a bore Rooster is. Device according to one of claims 44 to 48, characterized in that that these consists of acrylic glass. Device according to one of claims 44 to 49, characterized in that that the Sealing device is a rubber ring. Device for the controlled filtering of liquid media, in particular containing DNA molecules and / or other molecules / molecular complexes, with well filter plates, with at least one well filter plate ( 3 ' . 2 '' ), at least one corresponding microtiter plate ( 6 ' . 3 '' ), characterized by: - two separate gas spaces arranged above and below the filter plate; - at least one with a container ( 1' . 1'' ) and the well filter plate ( 3 ' . 2 '' ) interacting sealing device and thus a complete separation of the two gas spaces ( 2 ' . 1e '' ); - a pressurizing device functioning for a gas space ( 1a ' . 1g '' ). Device according to claim 51, characterized in that the Pressurization device is a control valve. Device according to one of claims 51 to 52, characterized in that that these consists of acrylic glass. Device according to one of claims 51 to 53, characterized in that a circumferential frame ( 4 ' ) is arranged. Device according to one of claims 51 to 54, characterized in that that the Sealing device is a rubber ring. Method according to one of claims 1 to 18 for determination DNA double-strand breaks, thereby characterized that a Device according to one of the claims 44 to 55 is used. Process for the controlled filtration of liquid in particular DNA molecules and / or other molecules / molecular complexes containing media, characterized in that a device according to a of claims 44 to 55 is used. Use of a device according to one of claims 44 to 55 to carry out of a method according to one of claims 1 to 18. Use of a device according to one of claims 44 to 55 for the controlled filtration of liquid media. Use of a device according to one of claims 44 to 55 for the detection and quantification of DNA strand breaks, from Cross-linking of DNA molecules with each other or with other molecules as well as other DNA damage; For attachment studies, for the investigation of DNA-protein or receptor-ligand interactions and to determine their dissociation / association constants reactions; For the filtration of radioactive materials with optional autoradiographic Detection of radioactivity remaining on the filters; to Filtration of infectious Rehearse; For filtration of oxygen / air sensitive samples; to Filtration under sterile conditions.