Download DNA Methylation studies

DNA Methylation studies
Sadique Syed
DNA methylation is one of the several post-synthetic modifications that normal DNA goes
through after each replication. Methylation does not alter the DNA sequence but alters its
function, and it plays an important role by interfering DNA-protein interactions such as
during transcription.
DNA methylation plays an important role in vertebrates, both in normal
development and in abnormal conditions that lead to diseases like cancer, where high
methylation levels often have been observed. For this reason it is important to estimate the
level of methylation to know how it varies both in diseases and under normal conditions, and
this is helpful for both research and diagnostic purposes. In the present project I studied the
extent of DNA methylation using a method called Luminometric DNA methylation Assay
(LUMA). This is a sensitive and highly reproducible method. It involves DNA digestion by
enzymes that either can or cannot digest methylated DNA; the ratio between their activities
then provides a measure of the extent of methylation. DNA samples from liver cancer were
used to estimate their DNA methylation level. These cells were treated with Trichostatin A
(TSA) a chemical which affects one of the proteins binding to DNA in the cells. Its effect was
observed upon treatment of cells with different concentrations of TSA. The results showed a
gradual decrease in DNA methylation as the concentration of TSA was increased. This dose
dependent decrease in methylation was linear within the tested concentration range from 0 to
800 nM TSA. The methylation level was tested also by another technique known as Nearest
Neighbour Analysis (NNA), which is a sensitive method to detect methylation at minute
levels. It involves digestion of DNA followed by radiolabelling of the newly created DNA
ends and then digestion of this DNA to obtain free nucleotides. If the first cleavage had
occurred next to a methylated base, this base would now be radioactive and could be
identified by its different migration during thin layer chromatography. This method also
showed decreased methylation as a consequence od TSA treatment. With these results it can
be assumed that TSA can be used as future cancer therapy drug.
Another aim of the present project was to further develop the LUMA technique by
using multiple methylation sensitive enzymes. For this purpose I used human peripheral blood
DNA samples. The results show ratios of methylation levels of four blood DNA samples
against different methylation sensitive enzymes.
Degree project in Biology, 2006
Examensarbete i biology, 20p 2006.
Biology Education Centre, Uppsala University, and Centre for Molecular Medicine,
Department of Clinical Neuroscience,, Karolinska Hospital, Solna.
Supervisors, Prof. Tomas Ekström and Mohsen Karimi.