Chapter 5 DNA Extraction Fundamentals of Forensic DNA
- Slides: 8
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. Lasercapture 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.
http: //projects. nfstc. org/gallery/main. php? g 2_item. Id=675 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
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 Prep. Filer (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
ORGANIC SDS, DTT, EDTA and John M. Butler (2009) Fundamentals of Forensic DNA Typing, Figure 5. 1 Blood stain FTA Paper CHELEX proteinase K Blood stain Apply blood to paper and allow stain to dry Water INCUBATE (56 o. C) Centrifuge Phenol, chloroform, isoamyl alcohol PUNCH INCUBATE (ambient) Centrifuge REMOVE supernatant VORTEX Centrifuge 5% Chelex TRANSFER aqueous (upper) phase to new tube TE buffer CONCENTRATE sample (Centricon/Microcon-100 or ethanol precipitation) Centrifuge QUANTITATE DNA PERFORM PCR WASH Multiple Times with extraction buffer REMOVE supernatant INCUBATE (56 o. C) INCUBATE (100 o. C) PCR Reagents Centrifuge QUANTITATE DNA PERFORM PCR (NO DNA QUANTITATION TYPICALLY PERFORMED WITH UNIFORM SAMPLES) PERFORM PCR
John M. Butler (2009) Fundamentals of Forensic DNA Typing, Figure 5. 2 Remove a portion of the mixed stain Differential Extraction SDS, EDTA and proteinase K (cell lysis buffer) Incubate at 37 o. C Centrifuge Perpetrator’s sperm mixed with victim’s epithelial cells sperm pellet SDS, EDTA and proteinase K + DTT “Male Fraction” REMOVE supernatant DTT lyses sperm heads sperm pellet “Female Fraction”
Differential extraction used to separate sperm (male fraction) from vaginal epithelial cells (female fraction) female female Evidence (female fraction) Evidence (male fraction) Suspect Victim 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?
