Use of laser microdissection greatly improves the recovery of DNA from sperm on microscope slides

Traditionally, sperms are isolated from vaginal cell mixtures by preferential extraction methods. Although these methods work well when there is a reasonable amount of DNA present, they are problematic when there are limited amounts (ca. 250 pg). In particular, the analysis of sperm from microscope slides has proven difficult. Here, we describe the use of laser capture microdissection (LM) for the isolation of spermatozoa from microscope slides containing sperms and vaginal cells. Such slides are frequently an important source of evidential material during the forensic investigation of rape and other sexual assaults. Low copy number (LCN) PCR was used to compare profiles of sperm DNA prepared using LM and preferential lysis. LM was found to outperform preferential lysis in 15 out of 16 samples. The application of LM to the processing of actual casework slides, and in particular the potential use of LM for the analysis of old cases, is discussed. Finally, 77 post-coital slides were processed in order to accurately assess the robustness of the technique. There was a significant association between the quality of the male profile recovered and time since intercourse that was independent of the number of sperms analysed, suggesting that the DNA was degraded even though the spermhead was intact.     

Results of a collaborative study of the EDNAP group regarding the reproducibility and robustness of the Y-chromosome STRs DYS19, DYS389 I and II, DYS390 and DYS393 in a PCR pentaplex format

A collaborative exercise was carried out by the European DNA Profiling Group (EDNAP) in the frame work of the STADNAP program, i.e. standardization of DNA profiling in Europe, in order to evaluate the performance of a Y-chromosome STR pentaplex, which includes the loci DYS19, DYS389 I and II, DYS390 and DYS393 and to determine whether uniformity of results could be achieved among different European laboratories.Laboratories were asked to analyze the five Y-STRs using singleplex and multiplex conditions in three bloodstains and one mixed stain (95% female and 5% male).All the laboratories reported the same results even for the mixed stain included in the exercise. This demonstrates the reproducibility and robustness of Y-chromosome STR typing even with multiplex formats and proves the usefulness of Y-STR systems for analyzing mixed stains with a male component.A total of 930 male samples from 10 different populations from Europe were also analysed for all the loci included in the pentaplex. Eight of these ten populations also included haplotype data.As for single gene analysis, haplotype diversity was higher in Germany and Italy and lower in Western European countries and Finland.Pairwise haplotype analysis shows the Finnish departure from the rest of the populations and a relatively homogeneity in the other European populations with F(ST) estimates lower than 0.05.UPGMA analysis shows an association of Western European population (Ireland, UK, Portugal and Galicia) on the one hand and central European populations on the other.