Following the DNA ligation of a single duplex using atomic force microscopy

Nick-sealing of a single DNA duplex was studied with the use of atomic force microscopy (AFM). To form a nick between a 47mer DNA and a 24mer DNA, the complementary 71mer template DNA immobilized on an AFM tip was hybridized with the 47mer DNA and brought into contact with the 24mer DNA on a substrate surface. The AFM tip and substrate surface were modified with dendron molecules to ensure the formation of a single DNA duplex. When a single nick in the DNA duplex was sealed by DNA ligase during a pause, an increase in the unbinding force was observed after the pause. The change from 24.0 ± 4.4 piconewtons (pN) to 62.8 ± 14.6 pN matched well with the resulting DNA length (71 bp). Additionally, a 30 s pause showed a 3-fold higher nick-sealing probability (60%) than a 10 s pause, while the probability did not increase with a 120 s pause. In the presence of free 47mer DNAs in solution, the single nick-sealing event could be repeated at other positions.