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DNA Extraction and Quantitation for Forensic Analysts


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Chelex® 100 Extraction Process - Differential
National Institute of Justice (NIJ) (see reuse policy).

Generally, sexual assault evidence is a mixture of epithelial cells and semen. One of the FBI's Quality Assurance Standards requires the laboratory to have a procedure for the differential extraction of stains that contain semen. Some laboratories have adopted a Chelex differential extraction procedure for these sample types. 09

Proteinase K, mentioned in the non-differential procedure, lyses epithelial and white blood cells but not sperm cells under the extraction conditions. It is particularly useful in organic extraction procedures as it maintains activity in the presence of denaturing agents such as sodium dodecyl sulfate (SDS), a component of extraction or digest buffers. Similarly, Proteinase K is not inactivated by metal chelating agents.

Read more about Organic Extraction in this course.

After extracting the sample in digest buffer and Proteinase K, the sample is centrifuged. The supernatant contains DNA from the lysed epithelial and white blood cells. The supernatant containing the cell lysate is subjected to extraction using 5% Chelex® 100. This fraction is treated as a single sample and referred to as the epithelial cell fraction, or the non-sperm fraction, depending upon laboratory procedures.

Sperm DNA is wound around proteins called protamines, which are analogous to histones. Protamines contain a high concentration of cysteine residues and disulfide bonds, making the sperm head and its DNA more resistant to the effects of Proteinase K. The pellet from the original extraction contains the sperm components from the sample and is subjected to a second incubation in buffer containing Proteinase K and dithiothreitol (DTT). This fraction is treated as a second sample referred to as the sperm fraction. DTT prevents oxidation of thiol (SH) groups and reduces disulfides to dithiols.

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