DIRECT VISUALIZATION OF ASCORBIC ACID INDUCING DOUBLE�STRANDED BREAKS IN SINGLE NUCLEAR DNA USING SINGEL�CELL PULSED FIELD GEL ELECTROPHORESIS

Satoru Kaneko; Joji Yoshida; Kiyoshi Takamatsu

Volume : V, Issue : XI, November - 2015

DIRECT VISUALIZATION OF ASCORBIC ACID INDUCING DOUBLE�STRANDED BREAKS IN SINGLE NUCLEAR DNA USING SINGEL�CELL PULSED FIELD GEL ELECTROPHORESIS

Satoru Kaneko, Joji Yoshida, Kiyoshi Takamatsu

Abstract :

 To demonstrate the significance of instability analyses for single nuclear DNA, the action of hydroxyl radical generated by sodium ascorbate (SA)/transition metals was directly visualized using single–cell pulsedfield gel electrophoresis (SCPFGE). Human sperm were embedded in a thin film of agarose and digested with trypsin. SCPFGE pulled out several dozen long chain DNA fibers from the origin. After memane extraction, DNA was still packed tightly with nuclear proteins. However, following the administration of SA, the DNA was cleaved into fious fragments and shortened to granular fragments in a dose–dependent manner. The lowest observed adverse effect level was visualized that large fious fragments were separated beyond the anterior end of the long chain fiber bundles. Swelling of DNA clusters after trypsin digestion facilitated the permeation of low–molecular weight agents and promoted the reaction; however, the presence of Cu or Ni were necessary as co–factors. Since the retention of hydroxyl radicals is very short, they must react at the proximal DNA. The reaction was suppressed by EDTA as well as edaravone, a hydroxyl radical scavenger. If some transition metals are bound to nuclear proteins, the above difference may be explicable that they diffused during trypsin digestion together with degraded peptides. SCPFGE is a sensitive analytical approach to assess not only naturally–occurring but also chemically induced DNA fragmentation in a single nucleus.