Time course and inhibitors of Hae III digestion in the forensic laboratory

Forensic restriction fragment length polymorphism (RFLP) profiling has become standardized in the United States and Canada to the extent that all federal, state and local forensic laboratories employ the restriction enzyme Hae III. Forensic DNA samples are sometimes resistant to digestion with Hae III. This problem is typically ascribed to sample characteristics and is dealt with through prolonged incubation with Hae III, repeated incubations and/or re-extraction. We determined and extensively tested the maximum useful duration of Hae III incubation for several types of samples. We also examined the effect of common DNA extraction reagents and laboratory procedures on subsequent restrictability of DNA. No benefit whatsoever was found to continuing digestion reactions past 1 hour. Certain laboratory reagents were found to inhibit Hae III activity at extremely low concentrations whereas others were found to have little effect on the success of the restriction digestion. Significantly, no contaminating reagent or other laboratory procedure was found to cause loss of Hae III specificity, whether enzyme activity was compromised or not. The results of this study allow the streamlining of the RFLP procedure and provide a basis for understanding how laboratory procedures may adversely affect Hae III activity and DNA digestion.