To determine whether the same concentration of humic acid ca
To determine whether the same concentration of humic Micafungin causes changes in PCR efficiency, we performed another experiment in which the humic acid was added to the PCR mix after bisulfite conversion. All samples that were amplified in the presence of humic acid (thick lines) show a decrease in PCR efficiency, as indicated by a reduction in the slope of the amplification curve (Fig. 6A). Even though the presence of inhibitor in the amplification decreased PCR efficiency as seen by a reduction in the slope in Fig. 6A, it did not cause a shift in the melting curve (Fig. 6B). The TM of DNA extracted from semen samples (blue in Fig. 6B) is still lower when compared with the TM values of DNA extracted from blood (red in Fig. 6B) and saliva (green in Fig. 6B) regardless of the presence of humic acid (thick lines). Thompson and coworkers  speculated that changes in the melt curve can depend on the size and GC content of the amplicon. Our amplicon has a size of 91 bp and a GC content of 21% for complete methylated DNA strands (blood and saliva cells) and 14% for unmethylated DNA strands (semen). Pionzio and McCord  concluded that long amplicons with low GC content are more prone to display effects of inhibition. Our amplicon is considered to be short and with low GC content. Therefore, it demonstrates robustness for melt curve analysis even when the PCR efficiency is reduced by the presence of inhibitors.
Conclusion This HRM analysis method is able to amplify and distinguish DNA from semen even when only 1 ng of input DNA is bisulfite converted. Lower levels of input DNA can also provide useful results. When tested with DNA that is not bisulfite converted, our primers failed to amplify the DNA, which proves that incomplete conversions will not result in false results possibly leading to misidentification of a body fluid. The results also show that when inhibitors like humic acid are coextracted with the DNA, the cleanup step performed as part of the bisulfite conversion step is capable of eliminating detrimental effects given that no decrease in the amplification efficiency was observed. Furthermore, even when humic acid was added to the amplification step following bisulfite conversion, the HRM method provided reliable results, with TM values similar to those obtained in the absence of inhibitor. These results demonstrate that HRM analysis can be a promising technology to identify the tissue source of a given DNA sample.
Acknowledgments The authors thank the volunteers who participated in this study. The authors also thank Mark Guilliano of Qiagen for technical support. Deborah S. B. S. Silva was supported by FAPERGS/CAPES and CNPq (Conselho Nacional de Desemvolvimento Científico e Tecnológico–modalidade Doutorado Sanduíche). This study was supported by funding provided by award 2012-D1-BX-K018 from the National Institute of Justice, USA. Points of view in the document are those of the authors and do not necessarily represent the official view of the U.S. Department of Justice.
Introduction In rape and sexual assault cases it is routine to examine clothing of the victims in order to detect seminal stains for the assignment of the perpetrator. During the last years at the Institute of Legal Medicine in Ulm, we worked on sexual assault cases involving murder by corpse drowning in the bathtub or in public water bodies. Normally, those corpses are recovered only shortly after the crime and clothing of the victim is submitted for DNA examination. Because BFI is essential for the prosecution of rape and sexual assault cases, our experimental work aimed to analyze the possibility of co-analysis – BFI and STR profiling – of seminal fluid-stained cloths that have been immersed in water for different time periods. Therefore, two different types of fabric (cotton and synthetic fiber) were subjected to flowing water for up to 7days. Methods for BFI included conventional approaches like Phosphatesmo and RSID as well as molecular RNA-based detection of seminal fluid specific mRNA targets that have been introduced into the forensic community in the last years , , .