USE OF PULSED-FIELD GEL-ELECTROPHORESIS TO INVESTIGATE FACTORS INFLUENCING THE MEASUREMENT OF DNA DOUBLE-STRAND BREAKS IN HUMAN BRAIN-TUMORSPECIMENS

We used pulsed-field gel electrophoresis combined with densitometry to investigate variables influencing the measurement of DNA double-stran d breaks (dsb) in human brain tumour cells and frozen tissue. Our stud ies showed that this system worked best when we ran the gel at 60 V fo r 22-25 h at 18 degrees C with a pulse time of 15s in 1xTris-acetic ac id-EDTA buffer. Because the densitometric analysis worked well only wh en DNA concentrations were within a certain range, we developed a quic k assay using a DNA-specific dye (TO-PRO-1) to determine concentration s before making agarose plugs. When we used our optimal procedure to m easure dsb in six frozen human brain tumour specimens, we found that r adiation-resistant tumours had significantly more initial dsb than did radiation-sensitive tumours. The number of residual dsb could not be ascertained because the process of freezing the specimens destroyed ds b repair ability.