Current U.S. National guidelines allow for the collection of sexual assault evidence up to 5 days after the incidence occurs. In these cases, the ability to obtain an autosomal STR (aSTR) profile of the male donor in these cases diminishes as the time interval increases. This inability to recover an aSTR profile from the semen donor is not due to a complete lack of sperm cells, as studies have frequently shown that sperm persists in the vaginal canal or cervix up to 10 days post coitus. Thus, the inability to recover an aSTR profile of the sperm donor is likely due to a low quantity of sperm cells and/or degradation of sperm cells, which pose significant problems to existing DNA extraction and typing methods. A typical DNA workflow for this type of evidence in a forensic casework laboratory includes the use of a differential extraction to separate sperm cells from non-sperm cells. These often harsh extraction methods can cause degraded and fragile sperm cells to be prematurely lysed into the non-sperm cell fraction. The significant amounts of vaginal epithelial cells in the sample can overwhelm the minute number of sperm cells present in this fraction, resulting in a complete masking of the male profile. For most sexual assault samples collected more than 48-72 hours after an incident, Y-STR analysis might be used instead of aSTR analysis, as it allows for an increased time frame of DNA recovery by detecting only the male donor Y-haplotype, circumventing the need for a differential extraction and avoiding potential competition during amplification. However, Y-STR loci are part of the non-recombining region of the Y-chromosome, and thus do not have the same discrimination power of aSTR loci. Therefore, enhanced methods for the recovery of sperm cells that allow researchers to bypass the limitations of a typical DNA workflow and obtain an aSTR profile need to be developed. This study aimed to do so by utilizing enhanced, non-standard methods to collect individual sperm cells via direct physical recovery from semen containing samples. Optimized direct lysis and micro-volume aSTR amplification were also used in order to obtain DNA profiles of the sperm donor. The quality of the obtained DNA profiles was evaluated using metrics such as allele recovery, stutter occurrence and percentage, and drop-in allele levels. Using these developed methods, the ability to analyze single sperm cells was demonstrated and the minimum number of sperm cells required in order to obtain probative and reliable DNA profiles was determined.


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Graduation Date





Ballantyne, John


Master of Science (M.S.)


College of Medicine


Burnett School of Biomedical Sciences

Degree Program





CFE0008579; DP0024255





Release Date


Length of Campus-only Access

1 year

Access Status

Masters Thesis (Open Access)