MtDNA typing of single-sperm cells isolated by micromanipulation

Some sexual assault crimes constitute a problem for the legal institutions confronted with the DNA analysis of such cases. Often, sperm cells are found in the victim's vaginal tract during medical examination but their successful genotyping is compromised by the huge excess of the victim's epithelial cells as well as by the degradation of genomic DNA present in sperm cells as a consequence of female immune response. Mitochondrial DNA present in the mid-piece of sperm cells might be useful in some specific cases in order to differentiate the donors of a semen sample. The high number of copies per cell and its circular nature that may confer some protection from the action of exonucleases make it more suitable for cases where few cells are available and/or the DNA is degraded. We have developed a novel strategy for typing mtDNA from single-sperm cells. Specific amplification of male mitochondrial DNA is ensured by use of sequence specific primers designed on the basis of mitochondrial single nucleotide polymorphisms existent throughout the control region. The strategy was applied to single-sperm cells isolated by micromanipulation from slides smeared with vaginal swabs taken immediately after sexual intercourse of voluntary couples. After sequencing the PCR products, it was possible to obtain a match between the DNA sequence from the buccal swab and the DNA sequence of the single sperm-cell, for each voluntary man. With this new strategy, the problem of contamination with DNA from the victim observed when using universal primers was completely overtaken. This method will probably allow the resolution of multiple-rapist crimes, where the collected sperm cells can be separately typed.