Chapter 3

Sample Collection and Preparation (DNA Extraction and Quantification)

2002 Academic Press

Sources of Biological Evidence

Material Blood and blood stains Reference Budowle 1995

Semen and semen stains Bones Teeth Hair with root Hair shaft Saliva (with nucleated cells) Urine Feces Debris from fingernails Muscle tissue Cigarette butts Postage stamps Envelope sealing flaps Dandruff Fingerprints Personal Items: Razor blade,
chewing gum, wrist watch, ear wax, toothbrush

Budowle 1995 Gill 1994 Alvarez 1996 Higuchi 1988 Wilson 1995 Sweet 1997 Benecke 1996 Hopwood 1996 Wiegand 1993 Hochmeister 1998a Hochmeister 1991 Hopkins 1994 Word 1997 Herber 1998 Van Oorschot 1997 Tahir 1996
2002 Academic Press

Organic Extraction Procedure

Blood stain
SDS, DTT, EDTA and

INCUBATE (56 oC) Centrifuge

proteinase K

Phenol, chloroform, isoamyl alcohol

VORTEX Centrifuge

TRANSFER aqueous (upper) phase to new tube TE buffer

CONCENTRATE sample
(Centricon/Microcon-100 or ethanol precipitation)

Centrifuge

QUANTITATE DNA

PERFORM PCR

2002 Academic Press

Chelex Extraction
Blood stain Water INCUBATE
(ambient)

Centrifuge REMOVE supernatant

5% Chelex

INCUBATE (56 oC) INCUBATE (100 oC) Centrifuge QUANTITATE DNA PERFORM PCR

2002 Academic Press

FTA Paper
Apply blood to paper and allow stain to dry WASH Multiple Times with extraction buffer PUNCH REMOVE supernatant

PCR Reagents

(NO DNA QUANTITATION REQUIRED)

PERFORM PCR
2002 Academic Press

Slot Blot Quantitation

Unknown Samples Calibration standards 20 ng
~2.5 ng

Calibration standards 0.63 ng

10 ng
5 ng 2.5 ng 1.25 ng 0.63 ng

1.25 ng
2.5 ng 5 ng 10 ng 20 ng

2002 Academic Press

Calculation of DNA Quantities in Genomic DNA

Important values for calculations: 1 bp = 618 g/mol A: 313 g/mol; T: 304 g/mol; A-T base pairs = 617 g/mol G: 329 g/mol; C: 289 g/mol; G-C base pairs = 618 g/mol 1 genome copy = ~3 x 109 bp = 23 chromosomes (one member of each pair) 1 mole = 6.02 x 1023 molecules Standard DNA typing protocols with PCR amplification of STR markers typically ask for 1 ng of DNA template. How many actual copies of each STR locus exist in 1 ng?

1 genome copy = (~3 x 109 bp) x (618 g/mol/bp) = 1.85 x 1012 g/mol = (1.85 x 1012 g/mol) x (1 mole/6.02 x 1023 molecules) = 3.08 x 10-12 g = 3.08 picograms (pg)

Since a diploid human cell contains two copies of each chromosome, then each diploid human cell contains ~6 pg genomic DNA
2002 Academic Press

1 ng genomic DNA (1000 pg) = ~333 copies of each locus (2 per 167 diploid genomes)

Sample Sources
Blood Blood stains Semen and semen stains Skin or other tissue Bone, teeth, hair Feces, urine, saliva

FTA Paper for sample storage and DNA extraction

2002 Academic Press

Steps to Sample Processing

DNA Extraction
organic phenol/chloroform Chelex FTA paper

DNA Quantitation (slot blot) PCR Amplification Separation/Detection Genotype Determination

2002 Academic Press

Overview of Steps Involved in DNA Typing

TH01 D7 D13 D21 D8 TPOX CSF D16 D18 FGA

D3 AMEL

D5 VWA

Penta D Penta E

Blood Stain

PCR Amplification with Fluorescent STR Kits and Separation with Capillary Electrophoresis

DNA Quantitation using Slot Blot

2002 AP, Elsevier

Genotyping by Comparison to Allelic Ladder