Cellular extraction and initial end-joining reaction conditions were optimized with restriction enzyme cleaved DNA to maximize ligation activity. Several parameters affecting ligation were examined including extract protein concentration, substrate concentration, ATP utilization, reaction time, temperature, and effect of ionic strength. Similar reactions were performed with the bleomycin-linearized substrate. In all cases, end-joined molecules ranging from dimers to higher molecular weight forms were produced and observed directly in agarose gels stained with Vistra Green and imaged with a FluorImager 595. This method permits detection of less than or equal to 0.25 ng double-stranded DNA per band directly in post-electrophoretically stained agarose gels. Therefore, the optimized end joining reactions required only 100 ng or less of substrate DNA, and up to 50% conversion of substrate to product was achieved.

The DSB end structure was shown to directly affect repair of the strand break. Bleomycin-induced DSBs were repaired at a 6-fold lower rate than blunt-ended DNA, and initiation of the reaction lagged behind that of the blunt-end rejoining reaction. Recent experiments have shown repair of DSBs produced by g-rays to be 15-fold less efficient than for DSBs produced by restriction enzyme. While repair of the high-LET-like DSB produced by 125I was near the lower limit of detection. Thus, as the cytotoxicity of the DNA damaging agent increases, the DSB created by the agent is less efficiently repaired.

Repair efficiency is also dependent upon the repair capacity of the cellular extract employed as a source of repair enzymes. These repair activities are known to vary from tissue to tissue, and person to person.

Therefore, by using patient samples as a source of enzyme activities, our method might be employed clinically as a predictive assay for patient sensitivity to DNA damaging agents. Knowledge of a patient's sensitivity to DNA damaging agents may permit more effective choices to be made when selecting treatment options in cases of cancer, and other diseases where DNA damaging agents are commonly used.