Fig. 1: Preparation of lssDNAc.
From: Preparation of long single-strand DNA concatemers for high-level fluorescence in situ hybridization
a Schematic representation of the extension reaction for lssDNAc. Both nucleotides of 5ʹ and 3ʹ termini of the padlock can complementarily bind to the 3ʹ termini of the adaptor. Next, the padlock is ligated at 5ʹ and 3ʹ termini by DNA ligase to form a closed circle. The adaptor ssDNA can be extended using the closed padlock as a template using RCA. b The amplification products were assayed via agarose gel electrophoresis. The extended products of adaptor ssDNA were amplified by phi29 DNA polymerase (lane 1 for 5 min, lane 2 for 10 min) and Bst 2.0 DNA polymerase (lane 3 for 1 h, lane 4 for 2 h). c The ssDNA adaptors were amplified to form products of different lengths by Bst 2.0 DNA polymerase (lanes 2–14) under three reaction temperatures (45 °C for lanes 2–5, 50 °C for lanes 6–9, and 55 °C for lanes 10–13) with different amplification times (0.5, 1, 1.5, and 2 h), and in the absence of DNA polymerase (lane 1). d The schematic diagram depicting the preparation for lssDNAc and hybridization workflow.
