The quantitative detection of DNA single-strand breaks is of great importance for many areas of biomedical research and diagnostics. The “Fluorescence-detected Alkaline DNA Unwinding” (FADU) assay assists in the investigation of mechanisms of DNA damage and repair following DNA strand breaks upon exposure to chemicals. Prof. Alexander Bürkle and his team at the University of Konstanz have automated and optimised the original FADU assay, which was published in 1981, to make it more efficient and quicker than before. This also benefits the large ongoing EU project MARK-AGE that is testing human ageing-related biomarkers using thousands of blood samples. In addition, the optimised method will potentially help to reduce the number of animal experiments.
Standard methods to measure DNA damage and repair, for example “alkaline elution” or the “comet” assay, are very time- and labour-intensive since some of the steps involved need to be carried out manually. The original, manually performed FADU assay is also very technically demanding in addition to requiring large numbers of cells. “The experiments I did with the manual FADU assay were quite frustrating. And it quickly became clear to me that this sensitive measurement method would only be used by a broader scientific community once it had been automated, the throughput had been increased and the whole method was made more convenient,” said Prof. Bürkle, explaining what motivated him to optimise the FADU assay. The original FADU assay comprises several pipetting steps that need to be carried out relatively slowly and with high precision in order to prevent the alkaline solution mixing with the lysate. In addition, care must be taken to protect the samples from light and to precisely control the ambient temperature. “All this is possible of course when the assay is performed manually; nevertheless it takes a long time, is rather inconvenient and is only reproducible with a small number of cells,” said the biologist explaining the disadvantages of the manual FADU assay.
Manual FADU assays generally function just like automated assays. The assay starts with cell lysis, followed by the addition of an alkaline solution on top of the cell lysate so that a second layer is formed, thus avoiding any mixing with the lysate. The diffusion of the alkaline solution gradually causes the DNA double strands to unwind, starting from the chromosome ends and internal DNA breaks. A neutralising solution is then added to stop the DNA strands unwinding. A fluorescent dye is subsequently added which enables the selective quantification of the remaining double-stranded DNA. “The use of a broad range of different controls enables us to obtain information on the number of DNA strand breaks at the time of cell lysis,” summarised Bürkle.
The researchers’ major motivation for optimising the original FADU assay was to save resources and time while massively increasing throughput. Bürkle and his team were able to achieve their goals by automating the assay. Automation was achieved by using liquid handling and other devices specifically manufactured in the university’s scientific workshops. Miniaturisation of the assay, along with automation, led to a dramatic increase in throughput and the replacement of the manual pipetting steps by pipetting robots. “We were also able to greatly reduce the amount of cell material needed for the assay. In addition, the assay is carried out in microtitre plates,” said Bürkle summarising the advantages of the optimised FADU assay.
The major advantages over standard “comet” assays are the large degree of automation of the assay making it possible to reduce the number of people required to perform the assay, the dramatically increased throughput of samples and the massive reduction of the time required for the measurement cycles. The total assay time required for a typical experiment to assess DNA strand repair with the optimised assay is now four to five hours. “However, the most important thing is that the use of machines now provides us with standardised and reproducible measurement results,” said Professor Bürkle.
Thanks to the improvement of the original FADU assay by Bürkle and his team, the new FADU assay is now more flexible than before and can also be used for a broad range of applications, for example in the field of basic scientific research into DNA damage and DNA repair. In addition, the assay is also suitable for routine measurements involving a large number of samples. “We believe that the optimised assay is particularly suited to epidemiological studies involving large numbers of volunteers in whom DNA damage and repair needs to be assessed,” said Bürkle who believes that this particular advantage would be greatly beneficial for the ongoing MARK-AGE EU project that is seeking to identify the biomarkers of human ageing using thousands of blood samples. In addition, the automated FADU assay might in future also be used for the screening of chemical substances that damage DNA or affect DNA repair. Such tests involve suitable test cells (tumour cell lines or primary cells such as leukocytes or fibroblasts) that are exposed to substances to be tested or to irradiation. When the exposure to chemicals or irradiation leads to DNA single-strand breaks in the cells or temporary DNA single-strand breaks in the course of cellular DNA repair activity, these breaks can be quantitatively assessed with the optimised FADU assay.
The new method also has the potential to reduce the number of animal experiments since it makes it possible to directly investigate human cells. Chemical substances are usually tested for their carcinogenic effect in rodents. Such tests also lead to a large number of false-positive results, with the consequence that the substance under investigation is erroneously grouped as carcinogenic. Thanks to the new, optimised FADU assay, the researchers now expect to be able to reduce the number of false-positives as well as using the in vitro tests to reduce the number of experimental animals in science as a whole. Prof. Bürkle and his colleague Dr. María Moreno-Villanueva were recently awarded the Ursula M. Händel Animal Welfare Prize by the German Research Foundation, a prize which is given to scientists who are considered to have made exemplary and sustained efforts to improve the welfare of animals in research, in particular with regard to the reduction, regulation and replacement of animals. The researchers from Konstanz intend to further optimise the FADU assay: “The FADU machine, which is still in the laboratory prototype stage, will in future be brought to series-production readiness in cooperation with external partners,” said Prof. Alexander Bürkle expressing his hopes for the future.
Prof. Dr. Alexander Bürkle
Dr. María Moreno-Villanueva