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Chapter 3
Sample Collection and Preparation (DNA Extraction and Quantification)
©2002 Academic Press
Blood and blood stains Budowle 1995
Semen and semen stains Budowle 1995Bones Gill 1994Teeth Alvarez 1996Hair with root Higuchi 1988Hair shaft Wilson 1995Saliva (with nucleated cells) Sweet 1997Urine Benecke 1996Feces Hopwood 1996Debris from fingernails Wiegand 1993Muscle tissue Hochmeister 1998aCigarette butts Hochmeister 1991Postage stamps Hopkins 1994Envelope sealing flaps Word 1997Dandruff Herber 1998Fingerprints Van Oorschot 1997Personal Items: Razor blade, chewing gum, wrist watch, ear wax, toothbrush
Tahir 1996
Material Reference
Sources of Biological Evidence
©2002 Academic Press
Organic Extraction Procedure
Blood stain
SDS, DTT, EDTA and
proteinase KPhenol,
chloroform,isoamyl alcohol
QUANTITATE DNA
PERFORM PCR
INCUBATE (56 oC)
Centrifuge
VORTEX
Centrifuge
CONCENTRATE sample (Centricon/Microcon-100 or ethanol precipitation)
Centrifuge
TRANSFER aqueous (upper) phase to new tube
TE buffer
©2002 Academic Press
Chelex Extraction
Blood stain Water
INCUBATE (ambient)
5% Chelex
INCUBATE (100 oC)
REMOVE supernatant
INCUBATE (56 oC)
QUANTITATE DNA
PERFORM PCR
Centrifuge
Centrifuge
©2002 Academic Press
FTA Paper
WASH Multiple Times with extraction buffer
PCR Reagents
Apply blood to paper and allow
stain to dry
PUNCHREMOVE
supernatant
PERFORM PCR
(NO DNA QUANTITATION REQUIRED)
©2002 Academic Press
Slot Blot Quantitation
20 ng
10 ng
5 ng
2.5 ng
1.25 ng
0.63 ng20 ng
10 ng
5 ng
2.5 ng
1.25 ng
0.63 ng
Calibration standards
Calibration standards
Unknown Samples
~2.5 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 1 ng genomic DNA (1000 pg) = ~333 copies of each locus (2 per 167 diploid genomes)
©2002 Academic Press
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
DNA Quantitation using Slot Blot
AMEL
D3
TH01 TPOX
Penta D
Penta EFGAD21 D18
CSF
D16D7
D13D5VWA D8
PCR Amplification with Fluorescent STR Kits and Separation with Capillary Electrophoresis
Blood Stain
Overview of Steps Involved in DNA Typing
Genotyping by Comparison to Allelic Ladder©2002 AP, Elsevier
