Protamine mRNA as molecular marker for spermatozoa in semen stains

Cytological detection of spermatozoa with subsequent DNA analysis is the most important biological evidence in sexual crimes when suitable samples are available. Immunological and enzymatic detection of semen-specific proteins may be helpful but cannot replace specific identification of spermatozoa. We have recently shown that detection of cell-specific gene expression can be used to identify menstrual blood. In this paper we demonstrate that the basic nucleoproteins protamine 1 and 2 are suitable markers for spermatozoa because they are exclusively expressed in the haploid genome and that protamine mRNA can be detected in semen stains by the highly sensitive reverse transcriptase-polymerase chain reaction (RT-PCR). With semi-nested PCR, 10-100 spermatozoa are theoretically sufficient to provide positive amplification results, with hot-start PCR at least 100-1,000 cells are required corresponding to an average semen volume of 0.01-0.1 microl. This new method thus allows specific identification of spermatozoa with molecular biology tools and may broaden the spectrum of investigations in the forensic laboratory.