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Single molecule fluorescence spectroscopy (SMFS) has become an established biophysical technique since its first demonstration 35 years ago, along the way stimulating a revolution in the study of DNA sequence, structure and function. In this chapter we summarise the physical and chemical techniques underlying these advances, with emphasis on the selection of fluorophores for single molecule work and their conjugation to DNA. We then highlight a few key applications of SMFS including next generation sequencing technologies, the determination of DNA structures at nanometre resolution and the dissection of protein-DNA interactions one complex at a time.

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