The comet assay detects strand breaks and not all damage is in the form of strand breaks. Some other types of damage can be converted to strand breaks with the use of lesion-specific endonucleases. Some types of damage, such as base or phosphate alkylations, will decompose to form strand breaks in a process dependent on pH and time of alkaline treatment. These are called ‘alkali-labile sites’ or ALS. (Intermediates in base excision repair, the base-less sugars left by glycosylase action, are also ALS.)
The most commonly used concentration of NaOH in the comet assay, 0.3 M, gives a pH of around 13, which is enough to convert many types of ALS into strand breaks. 0.03 M NaOH (pH around 12.1) is much less efficient. Thus it can be said, as an approximation, that 0.03 M NaOH will detect mainly strand breaks, while 0.3 M will also detect ALS.
Not all base damage is alkali-labile, and not all ALS are equally alkali-labile.
Regarding optimal pH and incubation time, we do not know the whole story, and research remains to be done. It is likely that different types of damage will have different time and pH optima.
"FAQ by Andrew Collins, Gunnar Brunborg and Jonas Nygren, 2006, NewGeneris FP7-project"