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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

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Fundamentals of Forensic DNA Typing

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

  2. 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.

  3. 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

  4. 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

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

  6. Centrifuge Remove a portion of the mixed stain Differential Extraction SDS, EDTA and proteinase K (cell lysis buffer) Incubate at 37 oC 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 “Male Fraction” “Female Fraction” sperm pellet

  7. 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…

  8. 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?

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