Methods and applications of single-molecule fluorescence microscopy
Molecular Horizons PhD Exit Seminar - Stefan Mueller
32.G01 or Zoom
The molecular mechanisms that support life depend on the fate of small populations of molecules. The functions of biomolecules, however, are traditionally characterised through bulk-phase biochemical methods that only report on ensemble averages. Such methods are not sensitive to population heterogeneity or short-lived reaction intermediates. Single-molecule methods that allow for the direct observation of individual molecules have significantly advanced our understanding of biology in the past two decades. The goal of the work described in my PhD thesis was the development and use of single-molecule techniques to answer biological questions about the process of DNA replication. DNA replication, the copying of the genome prior to cell division, is essential to all life forms.
We developed a method to synthesise and visualise individual DNA molecules containing various roadblocks to DNA replication, such as UV damage or protein blocks. We observe replication of these individual DNA molecules by a fully in-vitro reconstituted S. cerevisiae replisome and observe an unexpected plasticity.
However, single-molecule techniques have a large caveat. The high complexity of the workflows and the resulting low throughput often limit the application of single-molecule techniques. To address this issue and make single-molecule methods more widely usable, we developed a high-throughput method, to characterise enzymes involved in DNA metabolism within minutes.