Peptide Nucleic Acids as Tools for Single-Molecule Sequence Detection and Manipulation
Hagar Zohar, Craig L. Hetherington, Carlos J. Bustamante, and Susan J. Muller
05/18/2011
In an early example of physical mapping of single DNA molecules, individual biotinylated PNA-DNA complexes conjugated to streptavidin were directly visualized via electron microscopy.1 The streptavidins served as the electron microscopy markers and appeared as ‘beads’ located at the target sites. In a later example, high-resolution mapping of YOYO-1-stained DNA using fluorophore-conjugated PNAs was achieved by concurrent scanning near-field optical and atomic force microscopy (SNOM/AFM).2 In an application-oriented example, high-throughput mapping of TOTO-3-stained DNA using fluorophore-conjugated bisPNAs was achieved using a continuous flow device and direct linear analysis (DLA), a scheme that provides the spatial locations of the PNAs along the extended DNA molecule.3,4 The fluorescently labeled DNA was analyzed as it flowed past multicolor confocal fluorescence detectors with single-fluorophore sensitivity. An alternative approach to high-throughput mapping of DNA using bisPNAs was also achieved via purely electrical detection of individual DNA molecules moving through synthetic nanopores 4-5 nm in diameter.5 The region of the DNA with a PNA bound was detected as it threaded through the pore. Here the presence of the bound PNA generated a signal due to the displacement of electrolytes from the nanopore by the PNA-DNA complex and no additional modifications to the PNA were required.