1. Fundamentals of Forensic DNA Typing
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

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. 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.

6. 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”

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?