Authors
Graciela Spivak, Jia Guo, and Philip Hanawalt
Summary
We have developed a method to synthesize strand-specific, fluorescent DNA probes that bind defined genomic sequences. PCR with biotin-labelled forward primers and natural reverse primers permits purification of the individual strands; incorporation of aminoallyl-dUTP results in exposed amino groups to which Alexa fluorophores are bound. The probes are utilized in a specialized Comet-FISH approach, combining single-cell electrophoresis with fluorescence in situ hybridization to detect low, physiologically relevant levels of DNA strand breaks at the single molecule level. The probes bind to the termini of DNA segments of interest. Adjacent probe signals indicate intact DNA strands; separation of the signals implies strand breaks. Global genomic repair can be measured simultaneously in the same cells. We have used our method to document transcription-coupled repair of UV-induced cyclobutane pyrimidine dimers and potassium bromate-induced 8-oxoGuanine in the ATM gene in human fibroblasts. This ultrasensitive assay can be completed within one week.Further details
The protocol was published on Protocol Exchange on 11 September 2013. To see the entire protocol, click on the source link.Advertisement
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