Adaptation of the TdT assay for semi‐quantitative flow cytometric detection of DNA strand breaks

Abstract

The enzyme Terminal Deoxynucleotidyl Transferase (TdT) is a DNA polymerase which can be used to label DNA strand breaks by the incorporation of a labelled nucleotide followed by a fluorescent detection step. The amount of label incorporated can then be assessed by flow cytometry. The mechanism of action of TdT, however, will allow the addition of varying numbers of nucleotides to the free 3' termini produced by DNA strand breaks. The substitution of Digoxigenin (DIG) labelled dideoxy-nucleotides for labelled deoxy-nucleotides in the TdT assay will limit the addition of label to a DNA break to a single nucleotide, thus ensuring a direct relationship between an increase in DNA strand breaks and an increase in fluorescence. We have used this adaptation of the TdT Assay to evaluate DNA damage incurred in lymphocytes, from patients with Chronic Lymphocytic Leukaemia (CLL), on exposure to UV irradiation and apoptosis-inducing drugs, fludarabine and 2-Chloro-2'-deoxyadenosine (2-CdA). This technique may give a good indication of the susceptibility of CLL patients to apoptosis inducing drugs, and hence an indication of the likely response to these therapies.