1. Chapter 3 Sample Collection and Preparation (DNA Extraction and Quantification) ©2002 Academic Press

2. Blood and blood stains Budowle 1995 Semen and semen stainsBudowle 1995 BonesGill 1994 TeethAlvarez 1996 Hair with rootHiguchi 1988 Hair shaftWilson 1995 Saliva (with nucleated cells)Sweet 1997 UrineBenecke 1996 FecesHopwood 1996 Debris from fingernailsWiegand 1993 Muscle tissueHochmeister 1998a Cigarette buttsHochmeister 1991 Postage stampsHopkins 1994 Envelope sealing flapsWord 1997 DandruffHerber 1998 FingerprintsVan Oorschot 1997 Personal Items: Razor blade, chewing gum, wrist watch, ear wax, toothbrush Tahir 1996 MaterialReference Sources of Biological Evidence ©2002 Academic Press

3. Organic Extraction Procedure Blood stain SDS, DTT, EDTA and proteinase K Phenol, chloroform, isoamyl alcohol QUANTITATE DNA PERFORM PCR INCUBATE (56 o C) Centrifuge VORTEX Centrifuge CONCENTRATE sample (Centricon/Microcon-100 or ethanol precipitation) Centrifuge TRANSFER aqueous (upper) phase to new tube TE buffer ©2002 Academic Press

4. Chelex Extraction Blood stain Water INCUBATE (ambient) 5% Chelex INCUBATE (100 o C) REMOVE supernatant INCUBATE (56 o C) QUANTITATE DNA PERFORM PCR Centrifuge ©2002 Academic Press

5. FTA Paper WASH Multiple Times with extraction buffer PERFORM PCR PCR Reagents Apply blood to paper and allow stain to dry PUNCH REMOVE supernatant PERFORM PCR (NO DNA QUANTITATION REQUIRED) ©2002 Academic Press

6. 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 Unknown Samples ~2.5 ng ©2002 Academic Press

7. Calculation of DNA Quantities in Genomic DNA Important values for calculations: 1 bp = 618 g/molA: 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 10 9 bp = 23 chromosomes (one member of each pair) 1 mole = 6.02 x 10 23 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 10 9 bp) x (618 g/mol/bp) = 1.85 x 10 12 g/mol = (1.85 x 10 12 g/mol) x (1 mole/6.02 x 10 23 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

8. 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

9. 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

10. DNA Quantitation using Slot Blot AMEL D3 TH01 TPOX Penta D Penta E FGA D21 D18 CSF D16 D7 D13 D5 VWA 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