A silanized mica substrate suitable for high-resolution fiber FISH analysis by scanning near-field optical/atomic force microscopy.
We applied a novel silanized mica substrate with an extremely flat surface constructed according to Sasou et al. (Langmuir 19, 9845-9849 (2003)) to high-resolution detection of a specific gene on a DNA fiber by scanning near-field optical/atomic force microscopy (SNOM/AFM). The interaction between the substrate and fluorescence-dye conjugated peptide nucleic acid (PNA) probes, which causes fluorescence noise signal, was minimal. By using the substrate, we successfully obtained a fluorescence in situ hybridization signal from the ea47 gene on a λphage DNA labeled with an Alexa 532-conjugated 15-base PNA probe. As the results, no fluorescence noises were observed, indicating that the surface adsorbed almost none of the PNA probe. The combination of the substrate and SNOM/AFM is an effective tool for visualizing DNA sequences at nanometer-scale resolution.
Nanobiotechnology Laboratory, National Food Research Institute, National Agriculture and Food Research Organization, Tsukuba, Ibaraki, Japan. ssugi@affrc.go.jp
Shigeru
S
Sugiyama
Megumi
M
Fukuta
Tamaki
T
Hirose
Toshio
T
Ohtani
Tomoyuki
T
Yoshino
D000538:Aluminum Silicates / Q000737:chemistry*; D010582:Bacteriophage lambda / Q000235:genetics; D004247:DNA / Q000032:analysis; D005456:Fluorescent Dyes / Q000737:chemistry; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D008855:Microscopy, Electron, Scanning; D020135:Peptide Nucleic Acids / Q000737:chemistry; D012821:Silanes / Q000737:chemistry*
D000538:Aluminum Silicates; D005456:Fluorescent Dyes; D020135:Peptide Nucleic Acids; D012821:Silanes; D004247:DNA; C011934:mica