The typical differential extraction procedure utilized by the forensic science community to extract male deoxyribonucleic acid (DNA) from the sperm cells of the perpetrator separately from female DNA from the epithelial cells of the victim is both time-consuming and labor-intensive. This has contributed greatly to the backlog of unanalyzed sexual assault evidence collection kits (SAECK) seen in many laboratories today and has encouraged research in new methods that are more efficient and more effective in achieving better sperm DNA recovery.
The Cotton Lab has developed a Temperature-Controlled Differential Extraction (TCDE) procedure geared towards attaining better sperm recovery and better distribution of male DNA in the sperm fraction (SF) to generate a single source or distinguishable male profile. The TCDE protocol is a direct-lysis procedure that utilizes highly temperature-controlled enzymes, or enzymes that are active at or near their optimal temperatures. This procedure has been previously shown to decrease extraction time significantly and to extract samples that are suitable for downstream analysis.
This research specifically attempted to modify the TCDE procedure in the hopes of obtaining higher sperm DNA recovery and eliminating previous concerns of too much sperm being retained by the cotton swab material. It also compared a slightly modified TCDE procedure where the material fraction (MF) and SF are kept as separate fractions (the Separate Method) and a method that results in a recombined MF and SF (Recombined Method) to see if there was a greater distribution of the total male DNA eluted into the SF. Preliminary experimentation with swabs prepared with semen was performed to help make effective modifications. Then, vaginal swabs from eight different female donors were prepared with semen to mimic forensic casework samples and extracted using the Separate and Recombined Methods for comparison of the two extraction methods.
Despite unusual epithelial cell lysis results for some samples, the quantitation of the fractions by quantitative polymerase chain reaction (qPCR) showed that for approximately half of the samples extracted using the Separate Method, a majority of total male DNA was eluted into the SF. For these samples, a single source or distinguishable male profile can be generated. However, it was also demonstrated that even with good separation, a very small proportion of the female DNA in the SF still overwhelms the male DNA that is present in much smaller amounts, particularly for the Recombined Method where there are only two fractions.
Though further experimentation is necessary, these modifications proved effective in achieving high sperm recovery in the SF and generating a distinguishable male profile when extracting samples using the Separate Method. This research has confirmed that the TCDE procedure can be faster and less labor intensive while still producing clean DNA profiles in downstream analysis, and thus has the potential to be implemented in forensic laboratories after some of the concerns are addressed.
| Identifer | oai:union.ndltd.org:bu.edu/oai:open.bu.edu:2144/46317 |
| Date | 09 June 2023 |
| Creators | Ruigrok, Erin Kasey |
| Contributors | Cotton, Robin W. |
| Source Sets | Boston University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
| Rights | Attribution-NoDerivatives 4.0 International, http://creativecommons.org/licenses/by-nd/4.0/ |
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