Differential Method

Differential Method

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Differential extraction methods are used to separate spermatozoa from other cell types. Spermatozoa are more difficult to lyse than other cells and conditions can be set so that all cells except spermatozoa are lysed. The supernatant containing the DNA from these cells is removed from the sperm cells, which can then be lysed separately.

The differential extraction steps are:

Optional wash step

Some laboratories have incorporated an optional wash step at the beginning of the procedure to remove cellular debris and contaminants. The sample is gently washed in a buffer and detergent and the supernatant is removed (wash fraction). This can be done under refrigerated conditions or at room temperature.

Non-sperm cell lysis

An extraction buffer containing a buffer, detergent, and Proteinase K is added to the sample and incubated. This step lyses all cells except spermatozoa. The supernatant containing the DNA from the lysed cells (fraction 1) is removed after pelleting the spermatozoa. The sperm pellet is often washed numerous times with a buffer to remove excess DNA from this lysis step. If this wash is not done, it is not unusual to see a low level of fraction 1 DNA in fraction 2. If any of the sperm cells are weak or otherwise compromised, these may lyse in the first step, leaving a low level of fraction 2 DNA in fraction 1.

Sperm cell lysis

The pelleted sperm cells are lysed under more stringent conditions, using a buffer, detergent, DTT, and a higher concentration of Proteinase K (fraction 2), and are subsequently incubated.
Both fractions (including the wash fraction, if appropriate) are extracted separately with the phenol/chloroform/isoamyl alcohol combination and purified.08

The success of differential extraction depends on the sperm head resisting the processes that readily lyse epithelial and white blood cells. Separating the sources of DNA from different contributors to a stain, namely a male donor and female victim, lessens the difficulty associated with mixture interpretation during data analysis, source attribution, and/or statistical calculations. The value of differential extraction is demonstrated by the requirement in the Quality Assurance Standards (QAS) for inclusion in the laboratory’s recorded procedures (see Standard 9.1.3). Details of the method are provided in the laboratory manual.

A commercial kit for differential extraction is available from Promega. The Differex™ System, includes a proprietary separation solution and the use of a spin basket. The procedure begins with a Proteinase K digestion to lyse non-sperm cells. The sample and buffer are placed into a spin basket within the tube containing the proprietary solution (which is not miscible with the aqueous buffer). Through centrifugation, the sperm cells are isolated as a pellet, while the non-sperm DNA remain in solution. The difference between this system and routine differential extraction is that the separation solution acts as a selective membrane (without the risk of clogging). After the removal of the solution containing non-sperm DNA, the sperm DNA is isolated using the DNA IQ™ System, described later in this subject.

Click here to go to the Differex™ System website.

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