Autoradiographic evidence of differential loss of BUdR-substituted DNA after UV exposure in FPG harlequin staining

Autoradiographic analysis of CHO cells labelled with ³H]TdR or ³H]BUdR shows that only ³H]BUdR label is removed from metaphase chromosomes after FPG staining. ³H]BUdR is differentially removed from the bifilary labelled chromatid (BB) compared with the unifilary labelled chromatid (TB). UV treatment alone removes the label and produces harlequin staining and if the UV step is omitted from the FPG staining technique no loss of label or harlequin staining occurs. The heat treatment step (60 °C in 2 × SSC) removes further label, reducing the ratio of grains BB/TB to 0.8:1.0 and improving the differential staining. Over-treatment with heat produces paler staining chromatids without altering this ratio. The differential loss of BUdR-substituted DNA through UV photolysis and extraction in solution appears to be the cause of the differential harlequin staining of chromatids in this technique.