Abstract
Vaginal swabs taken in rape cases usually contain epithelial cells from the victim and sperm from the assailant and forensic DNA analysis requires separation of sperm from these cell mixtures. PH-20, which is a glycosylphosphatidylinositol-anchored hyaluronidase located on the head of sperm, has important functions in fertilization. Here we describe a newly developed method for sperm isolation using anti-PH-20 antibody-coupled immunomagnetic beads (anti-PH-20 IMBs). Optical microscopy and scanning electron microscopy showed the IMBs recognized the head of sperm specifically and exhibited a great capacity to capture sperm cells. However, we found it necessary to incubate the IMB–sperm complex with DNase I before sperm lysis in order to remove any female DNA completely. We compared the sensitivity of anti-PH-20 IMBs in sperm and epithelial cell discrimination to those coated with a different anti-sperm antibody (anti-SP-10, anti-ADAM2 or anti-JLP). Only the anti-PH-20 IMBs succeeded in isolating sperm from cell mixtures at a sperm/epithelial cell ratio of 103:105. Further, our method exhibited greater power and better stability for sperm isolation compared to the traditional differential lysis strategy. Taken together, the anti-PH-20 IMB method described here could be effective for the isolation of sperm needed to obtain a single-sourced DNA profile as an aid to identifying the perpetrator in sexual assault cases.
Highlights
Vaginal swabs taken in rape cases usually contain a mixture of epithelial cells from the victim and sperm from the assailant
Earlier transmission electron microscopy and immunogold labeling experiments showed PH-20 was located on the plasma membrane over the entire head of human sperm, but not on the midpiece or tail sections [13, 14], which make it a perfect target for isolating sperm with nuclear DNA from cell mixtures
To establish such a method for biomagnetic sperm isolation, an anti-PH-20 antibody was immobilized on commercial magnetic beads with covalent amide bonds and incubated with a simulated cell mixture containing equal concentrations (105/mL) of sperm and female epithelial cells
Summary
Vaginal swabs taken in rape cases usually contain a mixture of epithelial cells from the victim and sperm from the assailant. As stated by Butler, “by physically separating the perpetrator's sperm cells from the victim's epithelial cells, the perpetrator's DNA can be enriched and isolated from even a vast preponderance of victim's cells” [8]. This method is time-consuming, requires expensive instruments and skilled workers, which make it unsuitable for use as a routine method. The IMBs prepared by Li et al with an antibody against the motile sperm domain-containing protein 3 (MOSPD3) required the concentration of sperm to be as high as 105 cells/ mL for obtaining a full DNA profile of the male donor [12]. The new anti-PH20 IMB method provides an alternative solution to the traditional differential lysis approach for genotyping sexual assault cell mixtures
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
