WO2001006003A2 - Procede et necessaire pour la determination de cassures bicatenaires ou monocatenaires d'adn, et dispositif pour la filtration controlee en particulier de molecules d'adn avec des plaques de filtration a puits - Google Patents

Procede et necessaire pour la determination de cassures bicatenaires ou monocatenaires d'adn, et dispositif pour la filtration controlee en particulier de molecules d'adn avec des plaques de filtration a puits Download PDF

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Publication number
WO2001006003A2
WO2001006003A2 PCT/EP2000/006842 EP0006842W WO0106003A2 WO 2001006003 A2 WO2001006003 A2 WO 2001006003A2 EP 0006842 W EP0006842 W EP 0006842W WO 0106003 A2 WO0106003 A2 WO 0106003A2
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Prior art keywords
dna
substance
lysis
strand breaks
cells
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PCT/EP2000/006842
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German (de)
English (en)
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WO2001006003A3 (fr
Inventor
Werner E. G. MÜLLER
Heinz C. SCHRÖDER
Renato Batel
Original Assignee
Mueller Werner E G
Schroeder Heinz C
Renato Batel
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Application filed by Mueller Werner E G, Schroeder Heinz C, Renato Batel filed Critical Mueller Werner E G
Priority to AU62751/00A priority Critical patent/AU6275100A/en
Priority to EP00949369A priority patent/EP1194591A2/fr
Publication of WO2001006003A2 publication Critical patent/WO2001006003A2/fr
Publication of WO2001006003A3 publication Critical patent/WO2001006003A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5025—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures for parallel transport of multiple samples
    • B01L3/50255—Multi-well filtration
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/06—Fluid handling related problems
    • B01L2200/0689—Sealing
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0809—Geometry, shape and general structure rectangular shaped
    • B01L2300/0829—Multi-well plates; Microtitration plates
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/04—Moving fluids with specific forces or mechanical means
    • B01L2400/0475—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure
    • B01L2400/0487—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure fluid pressure, pneumatics
    • B01L2400/049—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure fluid pressure, pneumatics vacuum

Definitions

  • the invention relates generally to Methods and kits for determining DNA double or single strand breaks.
  • the invention relates in particular to a method and a kit for determining DNA double-strand breaks, corresponding uses, devices for the controlled filtration of liquid media containing well, in particular DNA molecules and / or other molecules / molecular complexes, with well filter plates and various uses.
  • a number of assays for determining DNA double-strand breaks are known from the prior art. These are in particular neutral filter elution, pulsed field gel electrophoresis and the neutral comet assay (Elia et al., Pharmacol. Ther. 51: 291-327; 1991; Sarkaria et al, Radiat. Res. 150 : 17-22; 1998; Prize et al .. Int. J. Radiat. Biol. 74: 173-184; 1998).
  • the cells to be examined for DNA damage are placed on filters and first treated with a neutral lysis solution to remove non-DNA components.
  • the DNA remains on the filter.
  • the filter with the DNA on it is then slowly passed through with a suitable solution for several hours, the washed-out solution being collected in several fractions. The distribution of the DNA between the collected fractions is measured.
  • This procedure is very time-consuming (work usually takes more than 10 hours), complicated to carry out; susceptible to failure (frequent leakage of the filter and filter holder), and it can only be used for the simultaneous determination of double-strand breaks in a relatively small number of samples.
  • Pulse field gel electrophoresis is an agarose gel electrophoresis which can detect double-strand breaks, which were only induced with a 1 Gy ionizing radiation, but which has inherent problems in measuring the strand break repair. Pulsed field gel electrophoresis is best performed with cell suspensions, but not with tissue samples. Costly equipment is required to implement this method. The procedure is time-consuming (approx. 24 hours) and allows only a relatively small number of samples to be determined at the same time.
  • DNA strand breaks are measured in single cells.
  • 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.
  • the damaged DNA migrates towards the anode, forming a comet's tail.
  • the greater the extent of the damage (number of strand breaks) the larger the resulting tail.
  • Double strand breaks are measured under neutral conditions and single strand breaks under alkaline conditions.
  • a disadvantage of this method is that it is not possible to determine DNA damage in tissues.
  • the method for evaluation requires a costly image analyzer and experienced personnel for the interpretation of the results.
  • the time for sample preparation before lysis is relatively long.
  • EP 0 359 249 discloses i.a. an apparatus and a method for filtering liquid media with well filter plates in combination with microtiter plates.
  • a connection for applying a vacuum is provided below the respective filter.
  • the disadvantage here is the relatively complicated design of the device, and in addition, a controlled filtering of liquid media is not always guaranteed during the method.
  • MultiScreen vacuum filtration system Millipore GmbH
  • the vacuum required for slowly sucking through the samples which is necessary for the method described in this application for determining DNA double-strand breaks, cannot be obtained with the aid of the vacuum slide switch on this device build up.
  • the problem arises of at least partially eliminating the disadvantages listed above.
  • the problem that arises in particular is to provide a method and a kit for the determination of DNA double-strand breaks, which have a high sensitivity and accuracy and at the same time a rapid determination of the DNA damage (working time less than 3 hours) with a high sample throughput (e.g. parallel Allow determination of a larger number of samples, for example of 96 samples when using 96-well microtiter plates).
  • the problem is to provide an apparatus and a method that ensure improved controllability of the filtration. There is an urgent need for such a procedure, for example in medicine (determination of the optimal radiation dose for radiation therapy patients) or in environmental monitoring (proof of the effect of genotoxic substances).
  • the cells and / or tissues to be examined are first placed on specific filters.
  • filters can be commercially available filter types suitable for this purpose, for example MultiScreen filtration plates from Millipore GmbH (Eschborn, Germany) with Durapore membranes (e.g. 0.22 ⁇ m pore size).
  • MultiScreen filtration plates from Millipore GmbH (Eschborn, Germany) with Durapore membranes (e.g. 0.22 ⁇ m pore size).
  • membranes with smaller or larger pore sizes can be advantageous.
  • the cells / tissues are then lysed in the presence of at least one detergent, for example SDS (sodium dodecyl sulfate), at least one chaotropic substance, for example urea, and at least one substance which complexes metal ions and inhibits DNA repair enzymes, for example EDTA, or at least one protease, for example proteinase K.
  • at least one fluorescent dye that binds a specific DNA double strand can be added.
  • the substance that complexes metal ions and inhibits DNA repair enzymes is necessary because firstly metal ions for cross-linking can lead to wetting of the DNA and thereby impair its elution and can lead to an incorrect assessment of the proportion of intact DNA and secondly because DNA repair enzymes lead to an elimination of the DNA damage to be measured.
  • a protease can be used, since this lyses DNA repair enzymes during lysis, which would otherwise lead to an elimination of the DNA damage to be measured.
  • the DNA fragments released by the lysis are then eluted through the filters at a controlled, constant elution rate and a fluorescence spectroscopic evaluation is then carried out.
  • the elution is carried out in particular by the devices according to the invention for the controlled filtration of liquid media with filter plates.
  • the concentration of the chaotropic substance is advantageously 2 to 4 mol / 1. since sufficient cell lysis and denaturation is effected in this area without the quench effect becoming too strong.
  • the concentration of the substance complexing metal ions and inhibiting DNA repair enzymes is advantageously 0.005 to 0.05 mol / 1, since sufficient activity is guaranteed in this range. It is advantageous if the pH value is set to 7 to 11 during the lysis step, since optimal cell lysis is observed in this area without DNA unwinding (separation of the two DNA strands at higher ones) pH values).
  • the concentration of the chaotropic substance in the lysis step is 2.25 mol / 1, since at this concentration there is an optimum between denaturation on the one hand and colorability (with the fluorescent dye; only a slight quench effect) on the other hand. Furthermore, it is advantageous if the concentration of the metal ion complexing and DNA repair enzyme inhibiting substance of the lysis step is 0.02 mol / 1, since this means that sufficient traces of heavy metals (which can lead to crosslinking of the DNA) are removed with a strong reduction of the quench effect impairing the measurements can be ensured.
  • the concentration of the detergent in particular SDS
  • the concentration of the protease in the lysis step is 0.5 mg / ml
  • Proteinase K is used as protease
  • SDS is used as detergent
  • the pH during lysing is 10.0.
  • the fluorescent dye PicoGreen has proven to be extremely reliable and therefore advantageous.
  • the method has the following advantages: 1. The method requires only a small amount of time (approximately one hour). 2. The process is highly sensitive; only small amounts of sample DNA are required (about 30 ng DNA per well; this corresponds to about 3000 cells or 25 ⁇ g tissue).
  • the method allows a high sample throughput (96 determinations per hour when using a filtration device with a 96-well microfilter plate).
  • the procedure can be carried out with non-radioactive DNA.
  • the method can be used not only for cells but also for tissue samples.
  • the procedure is simple and does not require any special expertise.
  • the following kit for determining DNA double-strand breaks has the advantages mentioned above:
  • the kit contains at least one detergent, at least one chaotropic substance, at least one fluorescent dye which binds a specific DNA double-strand and at least one specific filter type.
  • the kit contains at least one substance which complexes metal ions and inhibits DNA repair enzymes.
  • the kit contains at least one protease.
  • the concentration of the chaotropic substance during lysis is 2 to 4 mol / 1.
  • the concentration of the metal ion complexing and DNA repair enzyme inhibiting substance during lysis is 0.005 to 0.05 mol / l.
  • the protease is Proteinase K.
  • the concentration of the protease in the lysis step is 0.01 to 5 mg / ml.
  • the devices according to the invention for the controlled filtering of liquid media containing in particular DNA molecules and / or other molecules / molecular complexes with well filter plates, with at least one well filter plate and at least one corresponding microtiter plate are the following embodiments:
  • the first embodiment (the first device) is characterized by two separate gas spaces arranged above and below the filter plate, at least one sealing device which interacts with a container and the well filter plate and thus creates a separation of the two gas spaces, a device for connecting the two Gas spaces and a device for pressure equalization between the environment and a gas space.
  • One gas space is located above the filter plate and the micro titer plate arranged below it. in the wells of which the medium to be filtered is applied beforehand (before inserting the filter plate into the device).
  • both gas spaces are connected to one another by means of a tap arranged in a bore (this embodiment has proven itself in an advantageous manner), so that the pressure in both gas spaces is the same.
  • Both gas spaces are via the suction device, for example a suction pipe connected to a corresponding vacuum pump. evacuated or a negative pressure was established with respect to the environment and then the two gas spaces were separated from one another (for example by closing the tap).
  • the previously closed device for pressure equalization between the surroundings and the upper gas space is now carefully opened to the surroundings (i.e. to the outside atmosphere), so that the pressure in the upper gas space can be increased very slowly and in a metered manner compared to the gas pressure in the lower gas space.
  • a controlled filtering of the liquid medium is possible, in particular for the solutions for measuring DNA double-strand breaks.
  • the "suction pressure" required in conventional devices and methods contact pressure of the sealing device arranged between the filtration plate and the corresponding microtiter plate, in particular in the form of a rubber ring, for sealing between the two gas spaces
  • the advantage of the principle used in the device according to the invention is that the filtration process can be carried out at a constant speed without the occurrence of the initial pressure jump which occurs in suction devices which are operated by applying a negative pressure as a result of the suction process (pressing process on the sealing ring).
  • the second embodiment (second device) is characterized by two separate gas spaces arranged above and below the filter plate, at least one sealing device which interacts with a container and the well filter plate and thus creates a separation of the two gas spaces, and a pressurizing device which functions for a gas space ,
  • One gas space is located above the filter plate and the microtiter plate arranged below, in the wells of which the medium to be filtered is applied beforehand (before inserting the filter plates into the device).
  • a second gas space, separated from the first gas space, is located below the feed plate.
  • a pressure which is higher than the lower gas space, in particular 0.01 bar is applied to the medium to be filtered in one of the two gas spaces, preferably in the upper one, by means of the pressurizing device. In this way, a controlled filtering of the liquid medium is possible, in particular for the solutions for measuring DNA double-strand breaks.
  • a method according to the invention which uses one of the filtration devices according to the invention, has the surprising and advantageous properties mentioned above. This also applies to the corresponding uses.
  • the invention further relates to a method for determining DNA single-strand breaks, the use of several fluorescent dyes, a kit for determining DNA single-strand breaks, use of the kit and uses of the method and the kit.
  • DE 197 24 781 AI discloses inter alia a micro method for the rapid determination of DNA damage and its repair using the fluorescent dye Picogreen.
  • the cells to be examined are first lysed with the aid of SDS with a 9 molar urea solution.
  • About 200 mmol / l EDTA and a corresponding fluorescent dye, in particular Picogreen, which binds specifically DNA single-strand or DNA double-strand are added.
  • a sodium hydroxide solution with a pH of approx. 12.5 is added and then evaluated by fluorescence spectroscopy. With this method, the high concentration of urea is necessary to ensure the desired denaturation. In addition, approx.
  • the problem arises to at least partially eliminate the disadvantages listed above.
  • the problem that arises in particular is to provide a method and a kit for the determination of single-strand DNA breaks which have a low quench effect, a high sensitivity and accuracy and a good reproducibility and at the same time a rapid determination of the DNA damage (working time less than 3 hours ) with a high sample throughput (eg parallel determination of 96 samples in 96-well microtiter plates or determination of even larger numbers of samples by using microtiter plates with an even larger number of wells).
  • cells and / or tissues to be examined are first lysed with at least one detergent, for example SDS (sodium dodecyl sulfate) and at least one chaotropic substance solution, for example urea.
  • the lysis takes place in the presence of at least one substance which complexes metal ions and inhibits DNA repair enzymes.
  • At least one fluorescent dye that specifically binds single or double-stranded DNA is added in the lysis step or the subsequent step.
  • the step following the lysis is to add at least one solution to the suspensions containing the lysed cells and / or tissue, which allows the cell / tissue suspension to be adjusted to an alkaline pH and the at least one metal ion complexing and DNA repair enzyme inhibitory substance.
  • a fluorescent dye which is either specifically single-stranded or specifically double-stranded.
  • the background to this requirement is the fact that the dye must adhere specifically to either single or double strands of the DNA. to determine the proportion of the respective strand types.
  • cyanine dyes such as PicoGreen can be used as selective or as approximately selective double-strand binding and OliGreen as selective single-strand binding.
  • the method according to the invention is based on the following principle: In the double-stranded DNA, two polynucleotide chains (DNA single strands) are linked to one another via hydrogen bonds between two complementary bases. Both polynucleotide chains are wound around each other to form a right-handed double spiral (helix).
  • the proportion of double-stranded DNA can then be determined with the aid of fluorescent dyes which bind specifically DNA double-strand or which bind specific DNA single-strand by means of fluorescence spectroscopic analysis.
  • the fluorescence of these dyes is dependent on the amount of double-stranded or single-stranded DNA, so that by measuring the intensity of the fluorescence emission in the region of the maximum of the fluorescence emission spectrum, either the increase in the single strands or the decrease in the double strands is determined is to be determined.
  • the lysis which is cooled in the dark and with ice, takes place directly in the corresponding wells of a microtiter plate and takes about 30 to 60 minutes.
  • the lysed cells and / or tissues are mixed with at least one solution (particularly pH 11 to 12) which is used to adjust the DNA unwinding (step b) and which has a concentration, in particular from 0.005 to 0.01 mol / 1, a substance complexing metal ions and inhibiting DNA repair enzymes, for example EDTA (see above).
  • the double strands of the DNAs evolve - as explained above - so that the DNA single-strand breaks can be determined at regular, preferably regular intervals by conventional fluorescence spectroscopic analysis.
  • the individual steps should take place under temperature-controlled conditions to ensure good reproducibility. Typical temperature values in the range from 0 ° C to + 30 ° C can be used depending on the individual case.
  • the concentration of the chaotropic substance in the lysis step is 2 to 4 mol / 1, since sufficient lysis and denaturation is achieved only in this concentration range, without the fluorescence being excessively reduced as a result of the quench effect chaotropic substance is coming. Furthermore, the concentration of the substance complexing metal ions and inhibiting DNA repair enzymes in the lysis step is advantageously from 0.005 to 0.05 mol / l. This is also advantageous in step b.).
  • the pH value is advantageously (since it has been tried and tested) set to 11 to 12, since lower pH values do not lead to sufficient detoxification of the DNA and higher pH values impair the ability to be colored with the fluorescent dye.
  • the concentration of a substance solution complexing metal ions and inhibiting DNA repair enzymes can surprisingly be less than 200 mmol / 1, whereby this concentration range also apparently suffices for a sufficient removal of heavy metal traces and, moreover, the disruptive quenching effect could be unexpectedly reduced.
  • the entire process (lysis, unwinding and fluorescence spectroscopic evaluation) can also be carried out on 96-well microtiter plates (or microtiter plates with another Well number) can be made.
  • the method according to the invention it is also possible for the first time to carry out a continuous measurement of the DNA unwinding kinetics in one and the same approach without stopping the reaction.
  • the concentration of the chaotropic substance is 2.25 mol / 1, since at this concentration there is an optimum between denaturation on the one hand and colorability on the other hand. It is also advantageous if the concentration of the metal ion complexing and DNA repair enzyme inhibiting substance solution of the first step is 0.02 mol / 1, since a sufficient removal of traces of heavy metals can be ensured while at the same time greatly reducing the quenching effect.
  • the concentration of the metal solution complexing and DNA repair enzyme inhibiting substance solution of the second step is advantageously 0.02 mol / 1, since higher concentrations of some of these substances such as EDTA themselves can induce strand breaks and lower concentrations do not have an efficient metal ion complexing effect or DNA -Repairing enzyme inhibitory effect and no longer sufficient buffer effect.
  • the following embodiments are advantageous because they have proven themselves in practice: the concentration of the detergent (SDS) is 0.00175 mol / 1; it is used as a chaotropic substance urea; it is used as a metal ion complexing substance and DNA repair enzyme inhibiting substance EDTA; during lysis the pH is 10.0; the pH of the resulting solution after adding the solution of the second step is 11.5.
  • the following kit for the determination of single DNA strands has the advantages mentioned above:
  • the kit contains at least one detergent, at least one chaotropic substance solution, the concentration of which is 2 to 4 mol / l during lysis, at least one metal ion complexing and DNA repair enzyme inhibiting substance solution, at least one solution for adjusting the pH required for DNA unwinding (step b.), which contains a substance complexing metal ions and inhibiting DNA repair enzymes, and at least one fluorescent dye specifically binding single-stranded DNA or double-stranded DNA.
  • the concentration of the substance complexing metal ions and inhibiting DNA repair enzymes during lysis is 0.005 to 0.1 mol / 1;
  • the pH when the suspensions containing the lysed cells / tissue are mixed with the solution for adjusting the pH required for DNA unwinding is 11 to 12;
  • the concentration of the metal ion complexing and DNA repair enzyme inhibiting substance after setting the pH required for DNA unwinding is 0.005 to 0.1 mol / 1;
  • SDS is used as detergent; urea is used as the chaotropic substance;
  • EDTA is used as the substance which complexes metal ions and inhibits DNA repair enzymes; PicoGreen is used as the fluorescent dye.
  • Figure 1 This figure shows the DNA strand breaks (indicated as SSF x (-1)) in the gills of Mytilus galloprovincialis after treatment of the DNA in the TE homogenate (TE: 10 mmol / 1 Tris-HCl, 1 mmol / 1 EDTA; pH 7.4) with bleomycin, determined using the method according to the invention (single-strand method) (black bars) and the method disclosed in DE 197 24 761 AI (white bars). Examples of performing the assay (single-strand method):
  • DNA unwinding is carried out as follows at pH 11.50: 250 ⁇ l of the NaOH-EDTA solution are added to each sample (to a pH of 11) To obtain 50, d.) Measurement of the intensity of the fluorescence emission over a period of up to 1 h, for example every 5 min; when using PicoGreen: excitation: 480 nm; Emission: 520 ran.
  • the fluorescence blank value is measured as the fluorescence of a mixture of 25 ⁇ l TE buffer, 25 ⁇ l lysis solution with PicoGreen and 250 ⁇ l of the NaOH-EDTA working solution, e.) Calculation: Since every sample has to be corrected with regard to the blank value subtracted from the values of the mean of the blank. Multiple determinations are carried out (usually 4 - 6 determinations). The percentage of double-stranded DNA (dsDNA) for the untreated (e.g. unirradiated or untreated cells) at time 0 is set as 100% dsDNA.
  • dsDNA double-stranded DNA
  • SSF Strand Scission Factor
  • the tissue must be kept in liquid nitrogen immediately after removal.
  • tissue per well of a 96-well microtiter plate is approx. 25 ⁇ g.
  • Homogenization buffer TE buffer (see above) or other suitable buffer with 10% cryoprotector (dimethyl sulfoxide).
  • the method described here is able to detect the DNA single-strand breaks that occur during bleomycin treatment even when incubated in the presence of a concentration of 2.5 ⁇ mol / 1 bleomycin. As the bleomycin concentration increases, there is a concentration-dependent increase in DNA single-strand breaks (increase in SSF x (-1)).
  • FIG. 2 shows the dependence of the occurrence of DNA double-strand breaks, expressed in the form of the SSF x (-1) value, in HeLa cells after incubation of the cells for 4 hours in the presence of different concentrations of bleomycin (double-strand method; 3800 cells / well).
  • Figure 3 is a perspective view of an embodiment according to the inventive device for controlled filtration by means of suction;
  • Figure 4 is a perspective view of an embodiment according to the inventive device for controlled filtration by means of a pressurizing device.
  • 96-well filter plates (filtration plates), on the wells of which 96 independent membranes are welded, e.g. MultiScreen filtration plates from Millipore GmbH (Eschborn, Germany) (e.g. GV, 0.22 ⁇ m Durapore).
  • a device for controlled filtration shown in detail below by means of a suction device or a pressurizing device.
  • TE buffer 10mM Tris-HCl, 1mM EDTA, pH 7.4
  • Lysis solution 40 mM EDTA, 4.5 M urea, 0.1% by weight SDS, pH 10
  • a cell suspension is prepared in TE buffer with a pH of 7.4 with a cell density of 150,000 / ml. (approx. 3500 - 4500 cells / 25 ⁇ l, approx. 30 ng DNA).
  • the lysis of the cells takes place in the wells of the 96-well filtration plates for 40 min at room temperature in the dark. 25 ⁇ l of lysis solution with PicoGreen are slowly added to 25 ⁇ l of cell suspension (pipetted on the filter membrane surface).
  • Fluorescence blank values are obtained by pipetting 25 ⁇ l lysis solution with PicoGreen to 25 ⁇ l water (or TE buffer) in portions of the cell suspension in steps 2 and 3.
  • DFR DNA Filter Ratio
  • the tissue must be frozen and stored in liquid nitrogen immediately after removal (storage for shorter periods can also take place at - 80 ° C).
  • the amount of tissue per well of the microtiter plate is usually approx. 25 ⁇ g.
  • Homogenization buffer TE buffer (see above) or other suitable buffer with 10% cryoprotector (dimethyl sulfoxide, glycerin etc.).
  • FIG. 2 shows a concentration-dependent increase in the DNA double-strand breaks [SSF x (-1) values] after incubation of the lines with bleomycin.
  • the suction filtration device shown in FIG. 3 consists of the following parts:
  • 1 acrylic glass attachment consisting of an outer acrylic glass frame and an acrylic glass plate sitting on it.
  • a fitted ventilation pipe (la) with a valve connected to it for regulating the vacuum in the upper vacuum chamber (not shown in FIG. 3).
  • the upper flow valve (lb) can be used to separate or connect the upper vacuum chamber, (lc) hole in the acrylic glass attachment, which can be opened or closed by the flow valve.
  • the filtration device has an acrylic glass attachment (1) which is placed on a base plate (7). Thanks to the silicone seal (6) in the lower edge of the acrylic glass attachment, the space under the attachment can be sealed airtight. An empty microtiter plate (5) and a feed plate (3) already loaded with samples are arranged under this acrylic glass attachment in such a way that the feed plate comes to rest on the microtiter plate and on the bottom plate (see FIG. 3). The fact that there is a white of the feed plate directly above the corresponding white of the microtiter plate is ensured by the fact that the two plates are fitted exactly into the acrylic glass attachment, so that it is not possible to move the plates against each other.
  • the suction pipe forms the end of a hole in the base plate, the other end of which is in the middle of the base plate of the lower chamber.
  • Two narrow spacers (4) which are placed between the feed plate and the microtiter plate, prevent the wells of the microtiter plate from being closed when the vacuum is applied by sitting too tightly on the feed plate and thus being separated from the lower chamber space. If the connection between the lower and upper chamber with the hoof of the flow valve is open, both the lower and the upper chamber are evacuated after the vacuum has been applied. When the vacuum is applied, this connection must always be open to ensure a uniform evacuation of the two chambers. Then the connection between the two chambers is closed by turning the flow valve by 180 °. The two chamber rooms are then completely separated from each other.
  • the upper chamber is ventilated by opening the valve on the ventilation pipe integrated in the acrylic glass attachment, the lower chamber remaining evacuated. Due to the resulting higher negative pressure in the lower chamber compared to the upper chamber, the samples pipetted into the weus of the feed plate are sucked through the feeds that bleed the bottom of the weus into the lower chamber. the filtrate being collected in the microtiter plate.
  • the filtration speed can be regulated using the valve on the ventilation pipe. A filtration rate of 300 ⁇ l / 15 min per weü is recommended to carry out the procedure for the determination of DNA double strand breaks.
  • the vacuum applied remains switched on during the entire feeding process. After complete feeding, the acrylic attachment can be removed, whereby the feed in the microtiter plates is available for further examinations.
  • the new feature of the apparatus shown in FIG. 3 in comparison to conventional apparatuses is that first by connecting the opening 7a to the vacuum source both in the The same negative pressure is present in the upper and in the lower chamber, thus sealing the apparatus from the outside.
  • DNA is separated from the upper and lower chamber by reversing the flow valve 1b.
  • the pressure in the upper chamber is then set in such a way that the solution is sucked through the feeds at a continuous speed which is adapted to the experimental conditions.
  • Patent application EP 0359249 describes a feeding device in which a microfilter plate and a microtiter plate are also used.
  • the principle underlying this apparatus differs from that of the first feeding device described in the previous application.
  • a vacuum is first generated in both chambers in the device described here, which is then reduced in one (upper) chamber until the filtration process takes place at the desired speed.
  • a clamp is not used in the device described here.
  • the advantage of the construction described in the present application is that it avoids the contact pressure which is required in other apparatuses for the production of the airtight seal and which would lead to an initial rapid passage of liquid through the feed.
  • a constant liquid flow through the feed is essential for the application of the feeding devices we need for the DNA strand break determinations.
  • the pore size of the feeds of the feed plates we use for DNA strand breakage determination is so small that feeding due to gravity is not possible. Due to the small pore size, feeding according to the principle described in the patent application EP 0359249 for the example cell culture could not take place.
  • the pressure feeding device shown in FIG. 4 consists of the following parts:
  • a control valve (lg ", eg Festo GR-M5B) with a connection hole to the space above the plate, which is also used for connection a compressed air source (e.g. house compressed air, nitrogen bottle or aquarium pump) is used.
  • a compressed air source e.g. house compressed air, nitrogen bottle or aquarium pump
  • the strength of the pressure must be easily adjustable at the pressure queue itself.
  • the liquid that has passed through the feed is removed from the microtiter plate A feeding rate of approximately 300 ⁇ l / 15 min per WeU is suitable for carrying out the method for determining DNA double-strand breaks.
  • the two plates can be removed from the acrylic glass housing and the FUtrate in the microtiter plate can be further examined (The funct The staple in this apparatus is simply to provide an airtight seal above the feeding plate and an exact positioning of the feeding plate and the microtiter plate.)
  • the new feature of the apparatus shown in FIG. 4 compared to conventional apparatuses is that the feeding of the solutions pipetted into the water of the 96-WeU feed plate by applying an overpressure above the feed plate and not by applying a vacuum ( Vacuum) is carried out below the feed plate.
  • An advantage of the principle used in this apparatus is that the feeding process can be carried out at a constant speed without the occurrence of the initial pressure jump which occurs in suction devices which are operated by applying a negative pressure as a result of the suction process (pressing process on the sealing ring).
  • the overpressure in the room above the 96-WeU feed plate can be, in addition to the pressure queues mentioned above, e.g. can also be generated by applying an aquarium air pump.
  • people are protected from radiation by the 10 mm thick radiation-absorbing acrylic plates.
  • the advantages of the pressure feeding device described here compared to conventional apparatus are particularly easy to use. They enable feeding even when there is no compressed air source. This means that this device can also be used for examinations outside of a laboratory equipped with a compressed air source, for example on a research ship (examination of DNA damage in marine organisms). Furthermore, the special construction of the pressure feeding device enables shielding of radiation when using the devices for the filtration of radioactive materials. Pressurized cylinders containing inert gases can be used for the filtration processes of oxygen-sensitive substances. Comparable devices for microfilter plates are not currently available.

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Abstract

L'invention concerne, entre autres, un procédé de détermination de cassures bicaténaires d'ADN, qui comprend les étapes suivantes: a) dépôt des cellules et/ou des tissus à analyser sur des filtres spécifiques; b) lyse des cellules/tissus avec au moins un détergent, au moins une substance chaotrope et au moins un colorant fluorescent fixant spécifiquement un double brin d'ADN; c) élution des morceaux résultant de la cassure d'ADN, libérés par la lyse, à travers les filtres spécifiques; d) évaluation par spectroscopie à fluorescence. Pour un puits, seulement une fraction d'éluat est collectée.
PCT/EP2000/006842 1999-07-19 2000-07-18 Procede et necessaire pour la determination de cassures bicatenaires ou monocatenaires d'adn, et dispositif pour la filtration controlee en particulier de molecules d'adn avec des plaques de filtration a puits WO2001006003A2 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
AU62751/00A AU6275100A (en) 1999-07-19 2000-07-18 Method and kit for determining dna double/single strand breaks and device for carrying out the controlled filtration of, in particular, dna molecules with well filter plates
EP00949369A EP1194591A2 (fr) 1999-07-19 2000-07-18 Procede et necessaire pour la determination de cassures bicatenaires ou monocatenaires d'adn, et dispositif pour la filtration controlee en particulier de molecules d'adn avec des plaques de filtration a puits

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE19933078A DE19933078B4 (de) 1999-07-19 1999-07-19 Verfahren und Kit zur Bestimmung von DNA-Doppel-/Einzelstrangbrüchen und Vorrichtung zum kontrollierten Filtrieren von insbesondere DNA-Moleküle und/oder andere Moleküle/Molekülkomplexe enthaltenden flüssigen Medien mit Well-Filterplatten
DE19933078.6 1999-07-19

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WO2001006003A2 true WO2001006003A2 (fr) 2001-01-25
WO2001006003A3 WO2001006003A3 (fr) 2001-12-13

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EP (1) EP1194591A2 (fr)
AU (1) AU6275100A (fr)
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Cited By (3)

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Publication number Priority date Publication date Assignee Title
EP1358936A2 (fr) * 2002-04-29 2003-11-05 Genetix Limited Systeme de vide et ses utilisations
EP1491258A2 (fr) 2003-06-24 2004-12-29 Millipore Corporation Systeme de vide multifonctionnelle
US7588728B2 (en) 2003-06-24 2009-09-15 Millipore Corporation Multifunctional vacuum manifold

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE10111853A1 (de) * 2001-03-05 2002-11-14 Wita Proteomics Ag Verfahren und Vorrichtung zur Aufarbeitung von Proteinen in Gelen

Citations (4)

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Publication number Priority date Publication date Assignee Title
FR2618556A1 (fr) * 1987-07-23 1989-01-27 Adria Procede permettant de detecter si un produit alimentaire a ete irradie
WO1997010055A1 (fr) * 1995-09-15 1997-03-20 Beckman Instruments, Inc. Tubulure a vide pour le traitement en laboratoire d'echantillons liquides multiples
DE19724781A1 (de) * 1997-06-12 1998-12-24 Werner E G Prof Dr Mueller Mikro-Methode zur Schnellbestimmung von DNA-Schäden und deren Reparatur unter Verwendung des Fluoreszenzfarbstoffs Picogreen und ihre Anwendung
DE19725894A1 (de) * 1997-06-19 1998-12-24 Biotechnolog Forschung Gmbh Differentielle Vakuumkammer

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2618556A1 (fr) * 1987-07-23 1989-01-27 Adria Procede permettant de detecter si un produit alimentaire a ete irradie
WO1997010055A1 (fr) * 1995-09-15 1997-03-20 Beckman Instruments, Inc. Tubulure a vide pour le traitement en laboratoire d'echantillons liquides multiples
DE19724781A1 (de) * 1997-06-12 1998-12-24 Werner E G Prof Dr Mueller Mikro-Methode zur Schnellbestimmung von DNA-Schäden und deren Reparatur unter Verwendung des Fluoreszenzfarbstoffs Picogreen und ihre Anwendung
DE19725894A1 (de) * 1997-06-19 1998-12-24 Biotechnolog Forschung Gmbh Differentielle Vakuumkammer

Non-Patent Citations (1)

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Title
DUCORE J.M.: "EDTA alkaline elution characteristics and measurement of DNA damage in unlabeled DNA using Hoechst 33258 fluorescence" ENVIRONMENTAL AND MOLECULAR MUTAGENESIS, Bd. 11, 1988, Seiten 449-460, XP000995522 US *

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1358936A2 (fr) * 2002-04-29 2003-11-05 Genetix Limited Systeme de vide et ses utilisations
EP1358936A3 (fr) * 2002-04-29 2005-01-12 Genetix Limited Systeme de vide et ses utilisations
EP1491258A2 (fr) 2003-06-24 2004-12-29 Millipore Corporation Systeme de vide multifonctionnelle
EP1491258A3 (fr) * 2003-06-24 2005-07-27 Millipore Corporation Systeme de vide multifonctionnelle
JP2008116474A (ja) * 2003-06-24 2008-05-22 Millipore Corp 多機能真空マニフォールド
US7588728B2 (en) 2003-06-24 2009-09-15 Millipore Corporation Multifunctional vacuum manifold
US7824623B2 (en) 2003-06-24 2010-11-02 Millipore Corporation Multifunctional vacuum manifold
EP2027925A3 (fr) * 2003-06-24 2011-01-05 Millipore Corporation Ensemble collecteur
US8007743B2 (en) 2003-06-24 2011-08-30 Millipore Corporation Multifunctional vacuum manifold

Also Published As

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EP1194591A2 (fr) 2002-04-10
AU6275100A (en) 2001-02-05
DE19933078B4 (de) 2004-07-08
DE19933078A1 (de) 2001-02-08

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