The goal of this proposal was to examine the mechanisms for PCR inhibition and degradation and their effects on forensic DNA typing. The effects of these problems are well known; poor amplification and allele dropout. However, there are very few studies in the forensic literature that explore the issue of how inhibitors produce poor PCR results and even less is known about the mechanisms for degradation commonly present in typical forensic samples. A better understanding of these inhibition mechanisms could lead to the development of more sensitive, more robust analytical protocols.
In this proposal we performed controlled studies to clarify the mechanisms of environmental and chemical degradation and PCR inhibition on single source samples and mixtures. To do this we utilized real time PCR and HPLC/EC to evalutate the mechanisms of DNA degradation, oxidative damage and PCR inhibition on the recovery of STR profiles. Both degraded and pristine DNA were examined. In particular we performed the following experiments: 1) An analysis of the effects of various inhibitors on PCR amplification using real time PCR with high resolution DNA melt curves. 2) an analysis of the effect of natural and enzymatic degradation on PCR profiles. 3) An analysis of the effect of chemical oxidation on DNA profiles and 4) a correlation between PCR inhibition and DNA amplification.
Our overall conclusions are that 1) Environmental damage to DNA in tissue samples occurs rapidly to the point that DNA becomes nearly unrecoverable. The template in such samples breaks down to very small pieces in as little as 3 weeks.
2) The effects of oxidative damage on such samples was minimal. We utilized HPLC with electrochemical detection to monitor base damage to heavily degraded tissue samples. No oxidation of DNA bases was found for environmentally degraded DNA, although it was present in saliva samples.
3) . The combination of real time PCR and DNA melt curves is an effective tool for the detection of PCR inhibition and permits classification of various inhibitors based on their behavior. Our experiments on the effect of DNA template sequence, DNA template length and inhibitor concentration reveal that PCR inhibitors may affect STR results in several different fashions. Real time PCR results reveal that PCR inhibitors can affect Taq polymerase reactions reducing the total amount of DNA produced and/or can bind DNA, resulting in a loss of available template.
4) The effects of DNA binding also appear to be sequence and/or length specific. PCR inhibitors that mainly affect taq tend to inhibit DNA by affecting the largest alleles first, while inhibitors that bind DNA may affect smaller alleles as well as larger ones.
5) It has been widely reported that MiniSTRs improve resistance to PCR inihibition. Based on our results, a caveat should be that such improvements may depend on the type of inhibition. Sequence specific inhibition may still cause problems even with reduced sized amplicons
