Microfluidic Tools for DNA Analysis - DNA Nanotechnology: From Structure to Function - page 134

Fig. 7.19 Types of structural
variations (Reprinted with
permission from Macmillan
Publishers Ltd: Ref. [ 89 ],
copyright 2012)
A straightforward approach to obtain structural information of DNA molecules
is based on DNA stretching (or in other term, elongation) in nanochannels, which
was developed as a method called “optical mapping,” by which the stretched and
stained single DNA molecules can be photographed under a fluorescence micro-
scope directly. Therefore, consistent and uniform linearization of DNA molecules
is the first step, which is critical to the linear DNA mapping. Unfortunately,
long DNA molecules are hard to be introduced into nanoscale fluidic channels
from macroscale world directly because of the steep entropic barrier caused by
stretching (Fig. 7.20 a). Cao and coworkers skillfully took advantage of diffraction,
originally a disadvantageous resolution limiting phenomenon for photolithography,
and developed diffraction gradient lithography (DGL) technique to fabricate the
micro-posts array with gradually reduced gaps in front of the nanochannels to pre-
stretch long DNA molecules before they enter the nanochannels (Fig. 7.20 b) [ 90 ].
Based on this achievement, DNA with different structural variations can be treated
first with enzymes and dyes before introduction into nanochannels. By exquisite
“nick-flap labeling” strategy [ 91 ], the double-stranded DNA molecules were nicked
by nickase to generate single-strand flaps in the presence of polymerase with strand
displacement activity but lacking 5 0 -3 0 exonuclease activity, and the generated
flap sequences were hybridized with sequence-specific fluorophore-labeled probes.
For instances, DNA containing multiple repetitive sequences, inversions, or other
translocations (all contains nicking sites) can be confirmed by the unique flaps along
individual DNA chains [ 91 ]. Xiao, Selvin, and coworkers improved the resolution
of this mapping technique from 2 kb to 100 bp [ 92 ]. They used more fluorescent