The combined AFM manipulation and fluorescence imaging of single DNA molecules

A combined fluorescence microscope/AFM set-up was constructed,
which enabled the real-time manipulation of single DNA molecules.
Fluorescence images of these TO-PRO-3 intercalated strands could be
taken, while they were stretched with an AFM tip on silanised or
polylysine covered glass surfaces. Characteristic AFM force spectra
of single DNA molecules were achieved and a statistical analysis of
the rupture forces, plateau heights and rupture lengths was
compiled. The wide-field fluorescence images indicated that the DNA
underwent condensation on polylysine to form aggregated rods and
globular structures. Due to the strong unspecific adhesion of the
DNA to the polylysine surface, AFM tip manipulation frequently led
to strand scission. In addition, it was possible to “write”
nm-sized letters of fluorescent DNA by unraveling agglomerated
strands from the tip onto the surface. In contrast, DNA strands on
silane showed far less condensation. Extended single chains were
bound to the surface by the termini or at specific sites along the
double helix. These fixed and straightened strands could be
overstretched laterally to ca. 1.6 times the original contour
length. Chain rupture occurred at the tip position, but
occasionally mid-strand rupture was also observed. An analysis of
the chain fluorescence intensity for different stretching lengths
revealed that the dyes remain intercalated up to the end of the DNA
B-S overstretching transition.