Figure 2.

Overview of different fluorescent probes developed to detect single DNA molecules using single-molecule fluorescence microscopy. (From left to right) DNA binding dyes such as YOYO-1 and SYTOX Orange (SxO) remain largely non-fluorescent in solution and become highly fluorescent upon interaction with the bases in DNA, enabling direct visualisation during complex biochemical reactions. Fluorescently labelled proteins such as single-stranded binding proteins (SSB) provide a method to visualise long pieces of single-stranded DNA. Modified nucleotides such as 5-bromo-2-deoxyuridine can be incorporated directly into the newly synthesised DNA and labelled by treatment with fluorescently labelled antibodies. Fluorescent dye molecules such as Cy3 and Cy5 can be installed at specific positions in the DNA used in single-molecule fluorescence resonance energy transfer (smFRET) studies to monitor conformational dynamics. DNA methyltransferases (MTases) are able to recognise specific sequences within DNA and covalently link fluorescently labelled cofactors to generate high-resolution optical maps of large DNA fragments.