The alkaline version of single cell gel electrophoresis (comet) assay is widely 
used for evaluating DNA damage at the individual cell level. The standard 
alkaline method of the comet assay involves deproteinization of cells embedded 
in agarose gel using a high salt-detergent lysis buffer, followed by 
denaturation of DNA and electrophoresis using a strong alkali at pH>13 [N.P. 
Singh, M.T. McCoy, R.R. Tice, E.L. Schneider, A simple technique for 
quantitation of low levels of DNA damage in individual cells, Exp. Cell. Res. 
175 (1988) 184-191]. However, a recent report showed that a strong alkali 
treatment results in simultaneous deproteinization of cells and denaturation of 
genomic DNA [P. Sestili, C. Martinelli, V. Stocchi, The fast halo assay: an 
improved method to quantify genomic DNA strand breakage at the single 
cell-level, Mutat. Res. 607 (2006) 205-214]. This study was carried out to test 
whether the strong alkali deproteinization of cells could replace the high 
salt-detergent lysis step used in the standard method of the alkaline comet 
assay. Peripheral blood lymphocytes from 3 healthy individuals were irradiated 
with gamma rays at doses varying between 0 and 10 Gy. Following irradiation, the 
comet assay was performed according to the standard alkaline method (pH>13) and 
a modified method. In the modified method, agarose embedded cells were treated 
with a strong alkali (0.3M NaOH, 0.02 M Trizma and 1mM EDTA, pH>13) for 20 min 
to allow deproteinization of cells and denaturation of DNA. This was followed by 
electrophoresis using the same alkali solution to obtain comets. DNA damage 
expressed in terms of comet tail length, percentage of DNA in comet tail and 
tail moment obtained by the standard alkaline method and the modified method 
were compared. In both methods, DNA damage showed a good correlation with the 
dose of gamma ray. The results indicate a satisfactory sensitivity of the 
modified method in detecting radiation-induced DNA damage in human peripheral 
blood lymphocytes.