SINGLE CELL GEL ELECTROPHORESIS

Abstract

A carrier plate and a liquid sample are provided for the use of a single cell gel electrophoresis device for screening a subject for at least a DNA-damage, in particular a specific DNA-damage, in relation to a predisposition for and/or the presence of a disease. The liquid sample has cells of the subject. A single cell gel electrophoresis on the sample of the subject is performed by positioning the sample accommodated on the carrier plate in the generated homogenous electrical field and controlling strength and direction of the electrical field while the sample is positioned in the gel electrophoresis device. The DNA-damage is determined with a high reproducibility, with a coefficient of variation (CV) of 15% or less, in particular 10% or less, in particular 5% or less.

Claims

1. A method for screening a subject for at least a DNA-damage in relation to a predisposition for and/or a presence of a disease, wherein the method comprises the steps of: providing carrier plate and a liquid sample; wherein the liquid sample comprises cells of the subject; providing a gel electrophoresis device, wherein the gel electrophoresis device is capable of: positioning at least one carrier plate in a homogenous electrical field generated by at least one pair of electrodes; and controlling strength and direction of the electrical field generated at the position of the samples accommodated on the carrier plate; performing a single cell gel electrophoresis on the sample of the subject by: positioning the sample accommodated on the carrier plate in the generated homogenous electrical field and controlling strength and direction of the electrical field while the sample is positioned in the gel electrophoresis device; determining the DNA-damage with coefficient of variation CV of 15% or less.

2. The method according to the previous claim 1, wherein a lab-to-lab, a plate-to-plate and a spot-to-spot coefficient of variation is 15% or less.

3. The method according to claim 1, wherein the screening is performed on at least two aliquots of the subject liquid sample.

4. The method according to claim 1, wherein a biological variance between different screened subjects is determined.

5. The method according to claim 1, wherein a biological variance and/or determined DNA-damage is capable to indicate a predisposition for and/or the presence of the disease, in particular for a specific type of disease, a specific type of cancer, a aggressiveness of cancer, environmental variation of biological response of a cell line, effectiveness of a therapy.

6. The method according to claim 1, wherein before performing the single cell gel electrophoresis the cells of the liquid sample are separated, in particular wherein the single cell gel electrophoresis is performed on lymphocytes and/or biomarker carrier cells.

7. The method according to claim 1, wherein the liquid sample accommodated on the carrier plate is not flattened on the carrier plate.

8. The method according to claim 1, wherein the determination of the DNA-damage is performed in a one spotone image manner, wherein one image of one sample is required for the determination of the DNA-damage present in the sample.

9. The method according to claim 1, wherein the screening method is utilised in personalised medical therapy and/or a bio-monitoring.

10. The method according to claim 1, wherein per aliquot 400 to 75 cells are evaluated to determine the DNA-damage, in particular 400 to 100, or 400 to 200, or 200 to 75, or 200 to 100 cells per aliquot are evaluated to determine the DNA-damage.

11. The method according to claim 1, wherein the volume of the liquid sample is 15 L or less per aliquot.

12. A method of using a single cell gel electrophoresis device for screening a subject for at least a DNA-damage in relation to a predisposition for and/or a presence of a disease, wherein the gel electrophoresis device is capable of: generating a homogenous electrical field generated by at least one pair of electrodes; the method comprising: providing a carrier plate and a liquid sample, wherein the liquid sample comprises cells of the subject; positioning the sample on the carrier plate; positioning the sample in the generated homogenous electrical field; and controlling a strength and a direction of the electrical field while the sample is positioned in the gel electrophoresis device; wherein the DNA-damage is determined with coefficient of variation CV of 15% or less.

13. The method according to the claim 12, wherein a lab-to-lab, a plate-to-plate and a spot-to-spot coefficient of variation is 15% or less.

14. The method according to claim 12, wherein the screening is performed on at least two aliquots of the subject liquid sample.

15. The method according to claim 12, wherein a biological variance between different screened subjects is determined.

16. The method according to claim 12, wherein a biological variance and/or the determined DNA-damage is capable to indicate a predisposition for and/or the presence of the disease, in particular for a specific type of disease, a specific type of cancer, a aggressiveness of cancer, environmental variation of biological response of a cell line, effectiveness of a therapy.

17. The method according to claim 12, wherein before performing the single cell gel electrophoresis the cells of the liquid sample are separated, in particular wherein the single cell gel electrophoresis is performed on lymphocytes and/or biomarker carrier cells.

18. The method according to claim 12, wherein the liquid sample accommodated on the carrier plate is not flattened on the carrier plate.

19. The method according to claim 12, wherein the determination of the DNA-damage is performed in a one spotone image manner, wherein one image of one sample is required for the determination of the DNA-damage present in the sample.

20. The method according to claim 12, wherein the screening is utilised in personalised medical therapy and/or a bio-monitoring.

21. The method according to claim 12, wherein the cell concentration in the liquid sample is 200-400 cell per sample.

22. The method according to claim 12, wherein per aliquot 75 to 400 cells are evaluated to determine the DNA-damage, in particular 100 to 400, or 200 to 400, or 75 to 200, or 100 to 200, or 75 to 100 cells per aliquot are evaluated to determine the DNA-damage.

23. The method according to claim 12, wherein the volume of the liquid sample is 15 L or less per aliquot.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0094] The subject matter of the invention will be explained in more detail in the following text with reference to exemplary embodiments which are illustrated in the attached drawings, in which:

[0095] FIG. 1 single spot;

[0096] FIG. 2 section of the single spot of FIG. 1;

[0097] FIG. 3 prior art image of sectional spot;

[0098] FIG. 4 close view of a single spot as depicted in FIG. 1;

[0099] FIG. 5 bar diagram with DNA-damage in % for three labs;

[0100] FIG. 6 prior art image of DNA-damage (T) of 30 samples (S); and

[0101] FIG. 7 diagram showing DNA-damage (T) several samples (S).

DETAILED DESCRIPTION OF THE INVENTION

[0102] The reference symbols used in the drawings, and their meanings, are listed in summary form in the list of reference symbols. In principle, identical parts are provided with the same reference symbols in the figures.

[0103] FIG. 1 shows a whole single spot 1 of a liquid sample on a carrier plate. The liquid sample includes cell of a subject to be screened. The screening is conducted to for screening a subject for at least a (specific) DNA-damage in relation to a predisposition for and/or a presence of a disease.

[0104] The term single shot is represented as a single image respectively single photo taken from the sample.

[0105] The liquid sample la is applied on the carrier plate. The carrier plate is positioned in a homogenous electrical field generated by at least one pair of electrodes of a gel electrophoresis device. By controlling strength and direction of the electrical field generated at the position of the samples accommodated on the carrier plate a single cell gel electrophoresis (SCGE) is performed. The single cells are depicted as whitish dots. The dots are also called comets, as the single cell gel electrophoresis (SCGE) is also called comet assay.

[0106] The positioning of the carrier plate, respectively the liquid sample, and the controlling of the electrical field is performed by a single cell gel electrophoresis device (not shown).

[0107] FIG. 2 shows a section of the single spot of FIG. 1. The single comets, each representing a single cell, show a head on the left side and a tail at the right side. The orientation of the comet depends on the orientation of the single cells in the electrical field. From the ration between the tail and the head of the comet/single cell the DNA-damage can be determined.

[0108] The concentration in the spot is approximately 200-400 cell per sample. Accordingly, an overlap of individual comets is minimized compared to the prior art (Gyori et. al, Redox Biology 2 (2014)), where at least a ten-fold of cells is recommended. A corresponding output image according to the prior art (FIG. 1B/Gyori et. al, Redox Biology 2 (2014)) shown in FIG. 3.

[0109] FIG. 3 shows a section of a spot in an image with valid comets (A), invalid comets (B) and outlier comets (C). The overlap of several comets for example in different depth of the sample results in outlier comets and invalid comets. Such comet (B and C) cannot be evaluated and a determination of the DNA-damage is not possible for a significant number of cells/comets.

[0110] In contrast to that according to the claimed invention the comets/cell are separated from each other and an overlap also in the depth of the sample can be minimized (see FIG. 4). This can lead to a more reliable and/or easy to automatize evaluation of the comets/single cells in the liquid sample. Accordingly, the DNA-damage of the screened liquid sample can by highly reproducible.

[0111] Prior art documents as Azqueta et al.: A comparative performance test of standard, medium-and high-throughput comet assays; Toxicology in Vitro, volume 27, pp. 768-773, 2013 (short: Azqueta et al.) and Collins et al.: Controlling variation in the comet assay; frontiers in Genetics, volume 5, article 359, October 2014 (short: Collins et al.) describe the current common knowledge of standardisation and potential for optimisation regarding the precision of the comet assay.

[0112] Collins et al. describes a method for standardising the treatment of the tested cells. Two partner laboratories (short: labs) carried out experiments with TK-6 lymphoblastoid cells treated with 0.25 mM methylmethanesulphonate (MMS) for 3 h. Furthermore the sample is biological influenced and therefore not applicable to an arbitrary experiment, but is manipulating the biological properties of the cells. This limits the application of Collins method for usage in real screening a subject for a DNA-damage in relation to a predisposition and/or presence of a disease. Such a manipulation is depends to a high degree on the activity and specificity of the enzyme used for the treatmant of the cells, as for example formamidopyrimidine DNA glycosylase (FPG) converts 8-oxoguanine and some other oxidised purines to breaks. Depending on the enzyme more or less breaks are produced which are building the comet in the evaluated comet assays.

[0113] As stated by Collins et al. page 3, right column: one laboratory had more variable results than the other. According to data shown by Collins the determined DNA-damage between two labs differs roughly by a factor of 2 to 3 meaning by roughly 30%. Such a CV is also called lab-to-lab coefficient of variation. The lab-to-lab CV can be view as inter-laboratory comparison giving an inside on the actual reproducibility of the determined DNA-damage.

[0114] In contrast to that according to the claimed invention the a lab-to-lab coefficient of variation (CV) can be 15% or less, in particular 10% or less, in particular 8% or less, in particular 7.5% or less, in particular 5% or less, in particular 3% or less, in particular 2% or less, in particular 1% or less.

[0115] FIG. 5 shows the determined DNA-damage in % as % DNA in tail (T) for three labs, namely LabA, LabB and LabC in a bar chart, wherein data according to prior art is depicted as horizontally striped and data according to the present invention is depicted as diagonally striped bars. It can be seen, that the variation in the data according to prior art (Azqueta et al.) varies significantly more, than the data obtained according to the invention. TK-6 cells (human-derived lymphoblastoid cell line) were used for the experiments. TK-6 is a popular cell line.

[0116] Due to the high reproducibility a biological variance in the screened liquid sample of the subject can be evaluated. In contrast to that, according to the prior art (horizontally striped bars in FIG. 5) the biological variance is covered by the variation of the single cell gel electrophoresis and the subsequent determination of the DNA-damage. The high reproducibility makes the use of the screening according to the invention reliable and applicable to other systems and evaluations. The low lab-to-lab coefficient of variation enables a determination of the DNA-damage and a corresponding screening of a subject for a DNA-damage in relation to a predisposition for and/or a presence of a disease.

[0117] Based on the high variation between the different labs, Collins' data require an internal standard, as already mentioned by Collins. This is not the case for the highly standardised and highly reproducible method and use according to the invention, which offers a timely stable and homogenous field. It can also include a well-defined and strict protocol including ready to use reagents. Any internal standard would only be used to make sure that the assay is working appropriate.

[0118] According to Collins et al. the data of the comet assay vary from spot-to-spot quiet significantly, as shown reproduced in FIG. 6 (corresponding to FIG. 3 of Collins et al.). In FIG. 6 the DNA-damage (T) of 30 samples (S) is depicted in %, wherein the squared represent data without and the circles data with a normalization according to the prior art. In contrast to that FIG. 7 shows a similar set of data for 5 samples (S). The insert depicts an enlarged section of data shown FIG. 7. The data according to the present invention (FIG. 7) show a significant smaller variation than data according to prior art (FIG. 6). Accordingly, the reproducibility on a spot-to-spot basis improved in the present invention. The spot-to-spot coefficient of variation (CV) can be 15% or less, in particular 10% or less, in particular 8% or less, in particular 7.5% or less, in particular 5% or less, in particular 3% or less, in particular 2% or less, in particular 1% or less. In this context spot-to-spot CV can be viewed as variation of the determined DNA-damage on a spot-to-spot basis. A spot being one liquid sample respectively one aliquot of the liquid sample screened.

[0119] Additionally, also an evaluation of the DNA-damage on a plate-to-plate basis gives rise to a coefficient of variation (CV) which can be 15% or less, in particular 10% or less, in particular 8% or less, in particular 7.5% or less, in particular 5% or less, in particular 3% or less, in particular 2% or less, in particular 1% or less. The plate-to-plate CV can be determined form single spots (aliquots) arranged on different carrier plates. The results are similar to the once shown in FIG. 7.

[0120] State of the art determination of a DNA-damage can be performed on none manipulated cell sample with an average damage with a coefficient of variation of approximately 30-40%. According to the claimed invention the coefficient of variation (CV) of 15% or less can also be obtained. Such a CV can also be obtained in disproportionally (over- or under-proportionally) damaged cells, as for example a simulated damage introduced by X-ray.

[0121] While the invention has been described in present preferred embodiments of the invention, it is distinctly understood that the invention is not limited thereto, but may be otherwise variously embodied and practised within the scope of the claims.