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2004, Cilt 2, Sayı 3, Sayfa(lar) 097-103
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The Comparison of µ-FADU and COMET Methods in DNA Damage Analysis
Abdulkerim Bedir1, Birşen Bilgici2, Zafer Yurdakul2, Bilge Ş. Gürsel2, Muhlise Alvur1
1Ondokuz Mayıs Üniversitesi Tıp Fakültesi Biyokimya Anabilim Dalı, Samsun
2Ondokuz Mayıs Üniversitesi Tıp Fakültesi Radyasyon Onkolojisi Anabilim Dalı, Samsun

µ-FADU (Fluorometric Analysis of DNA Unwinding) and COMET are methods developed for the rapid detection of DNA damage and repair capacity. Both are based on time dependent alkaline DNA unwinding under appropriate denaturing conditions. COMET and µ-FADU are fluorometric and spectroflurometric assays, respectively. Tail length and tail moment are assessed by the COMET method, while the decrease of fluorescent intensity are determined by FADU. There are lots of different methods for detecting DNA damage. In this study, we selected and compared µ-FADU and COMET methods for DNA damage. The lymphocytes were isolated from whole blood collected with EDTA tubes (2000 cell/µL). Total (T), background (B) and standart P tubes irradiated by Cobalt 60 (P1→ 1 Gy, P2→ 2 Gy, P3→ 3 Gy, P4 → 4 Gy, P5 → 5 Gy) were prepared for µ-FADU and COMET assays. While µ-FADU method was performed to all tubes in LightCycler (Roche), COMET was applied only to standart P tubes irradiated by different dosages and assessed by fluorescence microscope (Nikon E800). The resulting DNA damage was assessed quantitatively bath by COMET and µ FADU methods. In µ-FADU method, the fluorescence of T and B tubes were the highest and lowest, respectively. The fluorescence of standart P tubes were in the decreasing order as follows: P1 > P2 > P3 > P4 > P5. As for COMET method, tail length and tail moment correlated with the -radiation dosage. Both methods were compared statistically by correlation and linear regression analysis. A significant relation was detected between methods (r = 0.9525, p = 0.004). In this study, we showed that DNA damage can be detected in Light Cycler with the µ-FADU method using low volume (20 µL) as compared with the traditional FADU method. In addition to this, we compared µ-FADU and COMET methods and found that they were in good harmony with each other.

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