Fundamentals of Forensic DNA Typing

Slides prepared by John M. Butler

June 2009

Chapter 5DNA Extraction

Chapter 5 – DNA ExtractionChapter 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

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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

ORGANIC FTA PaperCHELEX

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 dryBlood stain

VORTEX

(NO DNA QUANTITATION TYPICALLY PERFORMED WITH

UNIFORM SAMPLES)

Water

INCUBATE (ambient)

5% Chelex

INCUBATE (100 oC)

REMOVE supernatant

INCUBATE (56 oC)

QUANTITATE DNA

PERFORM PCR

PERFORM PCR

Centrifuge

Centrifuge

Centrifuge

Centrifuge

REMOVE supernatantTRANSFER aqueous (upper) phase to new tube

CONCENTRATE sample (Centricon/Microcon-100 or ethanol

precipitation)

Centrifuge

TE buffer

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Perpetrator’s sperm mixed with victim’s

epithelial cells

Centrifuge

REMOVE supernatant

SDS, EDTA and proteinase K

(cell lysis buffer)

Remove a portion of the mixed stain

SDS, EDTA and proteinase K + DTT

Incubate at 37 oC

sperm pellet

DTT lyses sperm heads

“Male Fraction” “Female Fraction”sperm pellet

Differential ExtractionJo

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Evidence (female fraction)

Evidence (male fraction)

Suspect

Victim

Differential extraction used to separate sperm (male fraction) from vaginal epithelial cells (female fraction)

male

female

male

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?