Manipulation and Isolation of Individual DNA Molecules with Atomic Force Microscope - DNA Nanotechnology: From Structure to Function - page 76

imaging tool that can reveal the topological structures of biomolecules placed on a
solid substrate [ 7 , 8 ]. Therefore, the isolation of biomolecules can be performed
after “seeing.” During AFM operation, the spatial information at the nanometer
scale can be included, which is much difficult for other techniques. Second, AFM
is capable of evaluating the morphology, mechanical property, electrostatic charge,
and even chemistry of the molecular species by various AFM imaging modes, which
is crucial to distinguish the target molecules from a complex sample. Moreover,
isolation of biomolecules with AFM can be conducted on a surface under ambient
conditions. It enables us to avoid the performance in solutions where contaminations
are more likely to be introduced. In addition, the AFM isolation can, if needed, be
realized in a parallel and automatic manner and integrated with other techniques for
conventional bio-analysis.
In this chapter, we present our efforts on developing AFM-based nanomanip-
ulation methods for isolating individual DNA molecules. To achieve this goal,
a series of AFM techniques including precise nano-dissection, positioning iso-
lation, and biochemical analysis of single DNA molecules have to be studied.
AFM operation modes for manipulating single DNA molecules, including cutting,
folding, patterning, and picking up or removing, have been developed. Especially,
the isolated DNA molecules could be amplified successfully by single-molecule
PCR for electrophoresis analysis and subsequent sequencing. Positioning digestion
of DNA strands with nonspecific endonuclease such as DNase I has also been
demonstrated. Several technical problems will be discussed for the purpose of
practical applications in the future.

5.2 AFM Manipulation of Individual DNA Molecules

Although scientists have made considerable progress in arranging and patterning
individual atoms and small molecules on solid substrates since the invention of
scanning tunneling microscopy (STM) [ 9 - 11 ], manipulation of biomolecules has
not been adequately explored with sufficient spatial resolution because of many
practical problems [ 12 ]. We have developed a special method to form complicated
nanopatterns of linear DNA molecules on solid substrates based on the molecular
“cutting” and “pushing” of individual DNA fragments with AFM [ 13 ]. After
stretching and depositing DNA molecules on a mica surface by using a method
termed “molecular combing” [ 14 ], a matrix consisting of a two-dimensional DNA
network was generated (Fig. 5.1 a). Then, elementary units of the network were
cut out and manipulated by the AFM probe to locally form a predesigned pattern
(Fig. 5.1 b-f). Upon cutting a DNA strand, the cutting site can be addressed with
nanometer precision. The gap between the two parts (Fig. 5.1 b) is of the similar
size as the apparent diameter of the DNA strands, indicating that the gap width is
determined by the tip shape.
The loading force applied on the DNA strands can be precisely controlled by
the AFM system. The forces exerted by the tip to break a double-stranded DNA