Visualization of an oriC region on an isolated single whole-genome DNA with triplex forming PNA probe using fluorescence microscopy

This paper presents a methodology for analyzing intact Mb-size DNA which enables to obtain spatial information on the position of the specific sequence in single molecular level. We utilized the complex of Qdot-streptavidin conjugate and biotinylated PNA as a triplex forming fluorescent probe and tried to visualize a region of the origin site of chromosomal replication (
ori
C) on an intact genomic DNA of a hyperthermophilic archaeon,
Thermococcus

kodakaraensis
KOD1. To improve the spatial resolution along the DNA, the DNA was dragged and elongated in the solution by the optical tweezers. PNA binding to homopurine sequence in
ori
C region of KOD1 was confirmed by the gel electrophoresis mobility shift assay and fluorescence optical mapping in single molecular level was demonstrated. However, we have not yet detected the
ori
C region specifically due to the difficulty of the complete elimination of the non-specific binding between Qdot-streptavidin conjugate and DNA.