- Course Introduction
- 01: Evidence & DNA
- 02: Forensic Biology
- 03: DNA Extraction & Quantitation
- Quantitation
President's DNA Initiative
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Advancing Justice Through DNA Technology
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Advancing Justice Through DNA Technology
DNA Analyst Training
Differential
Home > DNA Extraction & Quantitation > Chelex® 100 Extraction > Extraction Process >Differential
Generally, sexual assault evidence is a mixture of epithelial cells and semen. The QAS Standard 9.1.3 (FO) requires the laboratory to have a procedure for the differential extraction of stains that contain semen. Many 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.
Click here to read more about organic extraction in Subject 03, Module 02.
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 F1 fraction, the epithelial cell fraction, or the female 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 F2, sperm fraction, or male fraction. DTT prevents oxidation of thiol (SH) groups and reduces disulfides to dithiols.
