A look at an automated method to overcome the limitations of standard differential extraction using phenol: chloroform or organic extraction.

BACKGROUND
Currently, evidence from sexual assault cases comprises a large portion of samples in the DNA-typing backlog in the United States. A 2003 report commissioned by the U.S. National Institute of Justice indicated that more than 430,000 unsolved sexual assault cases with forensic DNA evidence remained unprocessed at that time. In addition, the National Crime Victimization Survey for 2006, released by the U.S. Department of Justice, indicates that there were approximately270,000 sexual assaults with victims 12 and older throughout 2006. Emphasis continues to be placed on finding the means to work through these samples, with federal funding being made available for both sample outsourcing and automated processing to help meet the demand. As laboratories work to reduce their case backlogs, they continue to receive additional cases and must address the need for quick and efficient processing of samples from sexual assault cases.

Sexual assault samples are among the most difficult and time-consuming to analyze because frequently, they are mixtures of body fluids from multiple individuals. These samples may be comprised of semen and epithelial cells in addition to blood. The challenge with these samples is to separate the sperm and non-sperm fractions efficiently. Separation is necessary to obtain a clean male DNA profile from the semen donor, reducing the need for time-consuming mixture analysis. A number of methods have been developed to achieve this goal.

ORGANIC DIFFERENTIAL EXTRACTION
This method is by far the most common method used in crime laboratories today. The standard differential extraction method begins with an initial incubation in digestion buffer containing proteinase K to selectively lyse epithe-lial cells. Sperm cells are then separated from the released epithelial DNA by centrifugation, removal of the super-natant containing epithelial DNA, and extensive washing of the sperm pellet. The washed sperm are then lysed, releasing the DNA. Finally, DNA present in both fractions is purified using organic extraction. This manual extraction method is time-consuming, and the steps are difficult to automate. A small set of eight samples requires eight hours of processing time, including approximately three hours of hands-on time.

Although organic extraction is the most common method used by crime laboratories, this differential extraction method is very technique-dependent and the quality of results can vary between analysts. The sperm pellet washing steps can be inefficient at removing soluble DNA from the cell pellet, leading to incomplete separation of sperm and non-sperm fractions, particularly in samples that contain large numbers of the victim’s cells relative to sperm cells. In addition, the time-consuming nature of the process precludes this method as a viable solution in an efficiency-minded laboratory.