Differential extraction (also known as differential lysis) refers to the process by which the DNA from two different types of cells can be extracted without mixing their contents. The most common application of this method is the extraction of DNA from vaginal epithelial cells and sperm cells from sexual assault cases in order to determine the DNA profiles of the victim and the perpetrator. Its success is based on the fact that sperm cells pack their DNA using protamines (rather than histones) which are held together by disulfide bonds. The protamines sequester DNA from spermatozoa, making it more resilient to DNA extraction than DNA from epithelial cells.
After determining that sperm cells are present (typically through staining and light microscopy) in a vaginal/rectal sample, the subject's epithelial cells are lysed by a standard DNA extraction method, like a phenol/chloroform extraction and their DNA extracted through normal means. The epithelial DNA in solution is removed and saved, while the sperm cell's DNA precipitates with the attached protamines. Differential extraction uses a chemical called dithiothreitol (DTT) to disrupt the sulfur bonds in the protamines in order to release its DNA. Once the DNA is detached from the protamines, it is prone to standard DNA extraction methods. This creates two different DNA fractions from one sample, that of the victim and that of the perpetrator.
However, the described method is difficult to carry out because it is both very labor-intensive and time-consuming, leading to a build-up of untested rape kits. An estimated 500.000 rape kits alone in the US.[1] Greenspoon et al.[2] reported improvements in sample processing efficiency and throughput using robotic automation. However, associated costs for implementation in conjunction with low-throughput quantities of samples may be impractical for forensic laboratories and cannot be justified.[3]
Recently a multistep nucleic acid extraction procedure was introduced to provide DNA lysates of highest quality.[4] A self-sealing membrane allows a stepwise release and separation of DNA from mixed specimens. Implemented in a spin-column system, it is ideally suitable for DNA extraction procedures involving differential extraction of forensic samples such as epithelium, saliva or blood vs. sperms. Simple and reliable extraction protocols for both, stained samples as well as gynecological swabs, respectively, overcome the often claimed difficulties in differential extraction (e.g. losing a sperm pellet through several washing steps).
Furthermore, an early qualified decision whether the process of a differential extraction is worth the time and efforts is possible due to gradual buffer separation. As an immunological pre-test for semen in a sample of sexual assault can be carried out using the identical sample.
^Horsman KM, Barker SLR, Ferrance JP, Forrest KA, Koen KA, Landers JP. Separation of Sperm and Epithelial Cells in a Microfabricated Device: Potential Application to Forensic Analysis of Sexual Assault Evidence. Anal. Chem. 77, 742 749 (2005).
^Greenspoon SA, Ban JD, Sykes K, Ballard EJ, Edler SS, Baisden M, Covington BL. Application of the BioMek 2000 Laboratory Automation Workstation and the DNA IQ System to the extraction of forensic casework samples. J. Forensic Sci. 49, 29-39 (2004).
^Voorhees JC, Ferrance JP, Landers JP. Enhanced elution of sperm from cotton swabs via enzymatic digestion for rape kit analysis. J. Foren. Sci. 51, 574-579 (2006).
^Nature Methods “Differential lysis in a single-tube extraction process for accurate forensic profiling”, 8 May 2012
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