Background
Techniques
Applications
The Comet-assay is a
single cell based technique that allows to detect and quantitate DNA
damages. Therefore the assay uses nuclei embedded in agarose and
exposed to an electric field. This technique was first described in
1984 by Johanson and Oesling and was later modified by Singh and
coworkers in it´s alcaline version to detect single strand DNA
breaks.
The Comet-assay is used in our group to study heat induced DNA damage and to learn about DNA damage and repair patterns. Also the general mechanisms of the Comet-assay and the underlying principles are explored in our group.
For Comet-FISH primary methodical questions are currently under research. But also the question of UV-A induced DNA damages are studied using this new technique. Additionaly the integration of multiple fluorescence labels are tried to enhance the techniques capabilities. This technique is used to study the distribution of DNA damages induced by different genotoxic sources in the complete human genome.
The Comet-assay is used in our group to study heat induced DNA damage and to learn about DNA damage and repair patterns. Also the general mechanisms of the Comet-assay and the underlying principles are explored in our group.
For Comet-FISH primary methodical questions are currently under research. But also the question of UV-A induced DNA damages are studied using this new technique. Additionaly the integration of multiple fluorescence labels are tried to enhance the techniques capabilities. This technique is used to study the distribution of DNA damages induced by different genotoxic sources in the complete human genome.
Techniques
The Comet-assay
is used in the alkaline and neutral version described in the
literature. Additionally we use neutral Comet-assay protocols adopted
to human biomonitoring requests with enhanced reproducability and
sensitivity. Besides primary human blood cells we use cell cultures and
tissues in our Comet-assays. To study the mechanistical processes
underlying the Comet-assay we developed the Video-Comet-assay, which
allows to perform the electrophoresis under visual control. Also
2D-Comet-assay is applied. To analyse the resolution regime of the
Comet-assay we analyse several restriction endonuclease generated
distribution patterns.
Setup of the online electrophoresis
Sample Time series of Video-Comet-assay
The Comet-FISH technique is performed according to the protocol developed by us. We painting probes, single copy gene probes as well as region specific probes, with signal enhancement. Recentrly we established the hybridisation of directly labeled PNA probes for telomere labeling.
Sample Images of Comet-FISH speciment
A second majour intrest in our Comet-assay studies is to make the assay as reproducable as possible. Therefore we incooperate standard cells into our slides, which can be identified by their incooperated halogenated nucleotides.
Setup of the online electrophoresis
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Microelectrophoresis tank: The tank picks up an standard slide. To the connecting pieces at the top the buffer system is conected. |
The microelectrophoresis tank installed under a upright microscope. The images are recorded with a 40x isolated water objective. |
Sample Time series of Video-Comet-assay
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| The image series shows an excample electrophoresis of H2O2 induced DNA strand breaks and alcaline lysis. The images were taken every 30 secounds for a total of 30 minutes. |  |
2D electrophoresis of comets:
The first electrophoresis was performed from left to right, the second
from bottum to top. |
The Comet-FISH technique is performed according to the protocol developed by us. We painting probes, single copy gene probes as well as region specific probes, with signal enhancement. Recentrly we established the hybridisation of directly labeled PNA probes for telomere labeling.
Sample Images of Comet-FISH speciment
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| Undamaged chromosome in COMET-FISH | Damaged chromosome in Comet-FISH | |
A second majour intrest in our Comet-assay studies is to make the assay as reproducable as possible. Therefore we incooperate standard cells into our slides, which can be identified by their incooperated halogenated nucleotides.
Applications
The biological
applications -we apply our Comet-assay techniques to- are found under
the other links of the DNA Damage and Repair research.
| DNA Damage and Repair Topics | |||
| UV-A
Induced DNA Damage |
DNA Double Strand Repair | DNA Repair Home |
Intrinsic Oxidative
DNA Damages |