Use of an automated sequencer to determine the sequence specificity of DNA damage

An automated sequencer was used to determine the sequence specificity of DNA damage caused by hedamycin in the plasmid pUC19 using a linear amplification/Taq DNA polymerase method. Previously, manual DNA sequencers have been in widespread use to investigate the sequence specificity of a DNA damaging agent. Manual DNA sequencers are restricted in the length of DNA sequence that can be read at base pair resolution for densitometry. An automated sequencer can greatly expand on the length of analysable DNA sequence. An additional important capability of the automated sequencer, is the ability to quantitate the intensity of damage at each base pair site. Thus we have used the automated sequencer to elucidate the sequence specificity of DNA damage for 300 bp. We have carried out an extended analysis of the sequence specificity of hedamycin DNA damage and found that the sequence 5'-cGt-3', tGt and cGg are preferentially damaged. The sequence specificity of cisplatin was also investigated.