Fundamentals of Forensic DNA Typing Chapter 5 DNA Extraction Fundamentals of Forensic DNA Typing Slides prepared by John M. Butler June 2009

Chapter 5 – DNA Extraction Chapter Summary DNA extraction involves separating the nucleic acids in a cell away from proteins and other cellular materials. Different methodologies widely used by forensic DNA scientists include organic, Chelex, or solid-phase extraction. Post-extraction filtration is sometimes used to concentrate low amounts of recovered DNA sample. A differential extraction that exploits chemical differences in sperm cell coatings can be used with sexual assault evidence to separate sperm from epithelial cells. Laser-capture microdissection technologies now enable physical separation of cells through selective recovery of individual sperm or other cells. It is important with any DNA extraction technique to remove as many substances as possible that could interfere with downstream testing and cause the extracted DNA molecules to break down over time.

DNA Extraction DNA is extracted from proteins that protect it in the nucleus of a cell Chemicals are added to digest the protecting proteins and produce “naked” DNA molecules The final solution looks like a tube of water http://projects.nfstc.org/gallery/main.php?g2_itemId=675

Primary DNA Extraction Methods Organic Chelex Solid-phase FTA (paper bind/wash/retention for direct PCR) Qiagen (silica bind/wash/release with vacuum filtration or centrifugation) DNA IQ and PrepFiler (silica bind/wash/release with magnetic bead capture) Differential extraction – separation of non-sperm and sperm fractions based on absence or presence of DTT to break open the sperm cell coating

TRANSFER aqueous (upper) phase to new tube ORGANIC FTA Paper CHELEX Blood stain PUNCH WASH Multiple Times with extraction buffer PERFORM PCR PCR Reagents SDS, DTT, EDTA and proteinase K INCUBATE (56 oC) Phenol, chloroform, isoamyl alcohol QUANTITATE DNA Apply blood to paper and allow stain to dry VORTEX (NO DNA QUANTITATION TYPICALLY PERFORMED WITH UNIFORM SAMPLES) Water INCUBATE (ambient) 5% Chelex INCUBATE (100 oC) REMOVE supernatant Centrifuge TRANSFER aqueous (upper) phase to new tube CONCENTRATE sample (Centricon/Microcon-100 or ethanol precipitation) TE buffer John M. Butler (2009) Fundamentals of Forensic DNA Typing, Figure 5.1 Figure 5.1 Schematic of commonly used DNA extraction processes.

Differential Extraction Remove a portion of the mixed stain SDS, EDTA and proteinase K (cell lysis buffer) Incubate at 37 oC Centrifuge Perpetrator’s sperm mixed with victim’s epithelial cells sperm pellet John M. Butler (2009) Fundamentals of Forensic DNA Typing, Figure 5.2 REMOVE supernatant SDS, EDTA and proteinase K + DTT DTT lyses sperm heads Figure 5.2 Schematic of differential extraction process used to separate male sperm cells from female epithelial cells. “Male Fraction” sperm pellet “Female Fraction”

Differential extraction used to separate sperm (male fraction) from vaginal epithelial cells (female fraction) female Evidence (female fraction) Evidence (male fraction) male Suspect male Victim female The four samples typically associated with a forensic DNA case…

Chapter 5 – Points for Discussion Would there be advantages to direct sample testing without DNA extraction? Why are PCR inhibitors problematic? What is the purpose of DTT in an extraction procedure? Describe some situations where differential extraction will be help separate mixture components in a sexual assault case. How are hair and bone DNA extraction more challenging than blood or saliva?