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The presented case report describes the exclusion of the suspect of a sexual offence by means of methods of molecular genetics. Pretests for prostate-specific antigen performed at the beginning of the investigation and cytological sperm tests were negative. Nevertheless, by combining the methods of differential lysis and DNA quantification a small number of spermatozoa could be demonstrated in the trace evidence. Subsequently, the profile of the woman's boyfriend, with whom she had had unprotected vaginal intercourse two days before the incident, could be detected in the fraction of the hard lysis.