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Chapter 3

Chapter 3. Sample Collection and Preparation (DNA Extraction and Quantification). ©2002 Academic Press. Material. Reference. Blood and blood stains. Budowle 1995. Semen and semen stains. Budowle 1995. Bones. Gill 1994. Teeth. Alvarez 1996. Hair with root. Higuchi 1988. Hair shaft.

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Chapter 3

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

  2. Material Reference Blood and blood stains Budowle 1995 Semen and semen stains Budowle 1995 Bones Gill 1994 Teeth Alvarez 1996 Hair with root Higuchi 1988 Hair shaft Wilson 1995 Saliva (with nucleated cells) Sweet 1997 Urine Benecke 1996 Feces Hopwood 1996 Debris from fingernails Wiegand 1993 Muscle tissue Hochmeister 1998a Cigarette butts Hochmeister 1991 Postage stamps Hopkins 1994 Envelope sealing flaps Word 1997 Dandruff Herber 1998 Fingerprints Van Oorschot 1997 Personal Items: Razor blade, chewing gum, wrist watch, ear wax, toothbrush Tahir 1996 Sources of Biological Evidence ©2002 Academic Press

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

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

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

  6. 20 ng 0.63 ng 10 ng 1.25 ng 2.5 ng 5 ng 5 ng 2.5 ng 10 ng 1.25 ng 20 ng 0.63 ng Slot Blot Quantitation Unknown Samples Calibration standards Calibration standards ~2.5 ng ©2002 Academic Press

  7. 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

  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. TPOX TH01 CSF D7 D16 D3 D5 D13 Penta D D8 D18 FGA VWA D21 AMEL Penta E Blood Stain PCR Amplification with Fluorescent STR Kits and Separation with Capillary Electrophoresis DNA Quantitation using Slot Blot Genotyping by Comparison to Allelic Ladder Overview of Steps Involved in DNA Typing ©2002 AP, Elsevier

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