A novel co-amplification system for simultaneous amplification of 23 Y-STR and identification of spermatozoa

In alleged sexual assault cases, identification of the presence of spermatozoa at the crime scene, or on items of eventual significance, or associated with the body of the victim, is integral to the forensic investigation to support or refute the proposition that sexual act has occurred. A 3-plex MSRE-PCR (methylation-sensitive restriction enzyme-PCR) system has been developed previously to identify spermatozoa based on the presence or absence of DNA methylation. This assay showed that 0.1 ng of DNA from a semen extract was sufficient to identify the presence of spermatozoa even when there was excessively more DNA isolated from vaginal fluid than DNA from a semen extract (80 ng/0.1 ng) or a mix of the menstrual blood/semen DNA (5 ng/0.1 ng). In this study, we combine spermatozoa detection with co-amplification of 23 Y-STR loci. We perform standard validation steps to present a novel test that saves time and uses the same sample for both DNA typing and spermatozoa detection in the same reaction. The combined assay can identify Y-STR and spermatozoa simultaneously using just 0.1 ng semen DNA, even in the presence of 5 ng of DNA from a female (male/female:1/50). No other body fluid tested, such as saliva, gave a result for the presence of spermatozoa. A total of 9 non-probative forensic samples from 7 sexual assault cases were tested by this co-amplification system. In all cases, the same sperm-positive data were obtained, concordant with our previous study analyzed by only 3-plex MSRE-PCR, and the Y-STR results were also consistent with that analyzed by only PowerPlex® Y23 kit. The co-amplification will be beneficial for the limited samples in many criminal cases.