Gopal Krishna R Dhondalay PhD

​Summary

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Within the last decade, the Single Cell Gel Electrophoresis (SCGE) or Comet Assay has been used with increasing popularity to investigate the level of DNA damage in terms of strand breaks and alkali labile sites in biomonitoring, genotoxicity testing, molecular epidemiology, ecogenotoxicology, fundamental research in DNA damage and repair as well as in biodosimetry, attracting adherents by its simplicity, sensitivity, speed, versatility and economy. In this biodosimetric study using comet assay, to evaluate the genetic damage caused on Human Peripheral Blood Lymphocytes exposed to chronic low therapeutic levels of in vitro gamma 60Co (Control, 0.1 Gy, 0.25 Gy, 0.5 Gy, 1.0 Gy, 2.0 Gy, 3.0 Gy and 4.0 Gy) ionizing radiation and to develop standard dose-response curve, the cells were embedded in agarose on microscopic slides and further processed for lysis (pH 10) using detergent and high salt to from nucleoids containing supercoiled loops of DNA linked to the nuclear matrix and alkaline electrophoresis (pH 13.1) resulted in structures resembling comets, observed by fluorescent microscopy. The likely basis for this is that the loops containing a break lose their supercoiling and become free to extend toward anode during electrophoresis. DNA damage was assessed in terms of head length and tail length using conventional image-analysis software, Adobe Photoshop 7.0, Adobe Systems. Total of 50 comets under each dose were analyzed which showed considerable decrease in head length and increase in tail length as the dose increased, and a linear regression curve for head length (R2=0.8479), tail length (R2=0.9615) and tail/head ratio (R2=0.9257) was obtained. In general, the comet assay is considered as suitable and a rapid test for DNA-damaging potentials in biodosimetric studies.