Manipulation and Isolation of Individual DNA Molecules with Atomic Force Microscope - DNA Nanotechnology: From Structure to Function - page 79

Fig. 5.3 A series of AFM images indicating positioning dissection and isolation of single DNA
fragments. ( a ) AFM image of a pBR322 DNA molecule deposited on a solid substrate. ( b )
The DNA molecule was cut at the selected positions. ( c ) AFM image showing that the small
DNA fragment has been isolated (Reprinted with the permission from Ref. [ 19 ]. Copyright 2004
American Chemical Society)
DNA fragment by the AFM tip, the cantilever was transferred to a PCR system
for single-molecule amplification. Subsequent electrophoresis and DNA sequencing
confirmed that single DNA molecules were isolated and amplified [ 19 ].
The success in cutting, isolating, amplifying, and analyzing single DNA frag-
ments suggests that it is possible to develop a sequencing method based on AFM
manipulation of single DNA molecules. We have proposed a strategy termed
“ordered single-molecule sequencing based on nanomanipulation (OsmSN)” [ 20 ]
that provides a new method to analyze individual DNA molecules. The general
concept of the OsmSN strategy is that DNA fragments are isolated from a long DNA
molecule in a sequential order and subsequently amplified and sequenced in the
same order (Fig. 5.4 ). Therefore, no sequence assembly is needed. To achieve this
goal, a series of techniques including precise nano-dissection, positional isolation,
and biochemical analysis of single DNA molecules have to be developed.
As proof of concept of the OsmSN, two overlapping DNA fragments from
a genome DNA were isolated, amplified, and sequenced successfully [ 21 ]. The
technical difficulties of OsmSN strategy rely on isolating two overlapping fragments
from two different genomes of otherwise identical DNA molecules and sequentially
sequencing the two overlapping fragments in order. To this end, a DNA-labeling
technique was developed so that the target DNA can be recognized and accurately
positioned by AFM imaging (Fig. 5.5 ). In this way, the sequence of a long DNA
molecule can be determined through analyzing the fragments one by one.
At the moment, there are still several challenges before turning OsmSN strategy
into a practical technology. For example, when we try to amplify an unknown DNA
fragment that was isolated by AFM, it seems difficult to design the primers. Extra
techniques that use a linker DNA duplex ligated to the DNA ends may solve this
problem. In addition, currently the throughput to cut and isolate DNA with single-
probe AFM is not sufficient. Therefore, parallel and automatic nanomanipulation
systems with multiple-probe AFM should be developed.