2. Stan Hitomi Director, Edward Teller Education Center UC Davis / Lawrence Livermore National Laboratory, Livermore, CA Kirk Brown Lead Instructor, Edward Teller Education Center Science Chair, Tracy High School and Delta College, Tracy, CA Sherri Andrews, Ph.D. North Carolina School of the Arts Winston-Salem, NC Forensic DNA Fingerprinting Kit Instructors

3. Why Teach DNA Fingerprinting? Standards-based Real-world connections Tangible results Link to careers and industry Laboratory extensions

4. Forensic DNA Fingerprinting Workshop Time Line Restriction digest of DNA samples Introduction to DNA Fingerprinting Electrophoresis on Agarose gels Analysis and interpretation of results

5. DNA Fingerprinting Real World Applications Crime scene Human relatedness Paternity Animal relatedness Anthropology studies Disease-causing organisms Food identification Human remains Monitoring transplants

6. DNA structure DNA restriction analysis Agarose gel electrophoresis Molecular weight determination Simulation of DNA Fingerprinting The Forensic DNA Fingerprinting Kit Can Help You Teach:

7. DNA Fingerprinting Procedures Overview

8. DNA Fingerprinting Procedures Day One

9. DNA Fingerprinting Procedures Day Two

10. DNA Fingerprinting Procedures Day Three

11. Chromosomes are Comprised of DNA and Proteins

12. Karyotyping Indentifies Genetic Anomalies

13. DNA is Tightly Packaged into Chromosomes Which Reside in the Nucleus

14. Model of DNA DNA is Comprised of Four Base Pairs

15. Deoxyribonucleic Acid (DNA)

16. DNA Schematic O CH 2 O P O O O Base CH 2 O P O O O Base OH Sugar O 5’ phosphate 3’ hydroxyl

17. Genes are Composed of Exons and Introns

18. Introns are Spliced Out of RNA During Transcription

19. DNA Restriction Enzymes Evolved by bacteria to protect against viral DNA infection Endonucleases = cleave within DNA strands Over 3,000 known enzymes

20. Enzyme Site Recognition Each enzyme digests (cuts) DNA at a specific sequence = restriction site Enzymes recognize 4- or 6- base pair, palindromic sequences (eg GAATTC) Palindrone Restriction site Fragment 1 Fragment 2

21. 5 vs 3 Prime Overhang Generates 5 prime overhang Enzyme cuts

22. Common Restriction Enzymes EcoRI – Eschericha coli – 5 prime overhang Pstl – Providencia stuartii – 3 prime overhang

23. The DNA Digestion Reaction Restriction Buffer provides optimal conditions NaCI provides the correct ionic strength Tris-HCI provides the proper pH Mg 2+ is an enzyme co-factor

24. DNA Digestion Temperature Why incubate at 37°C? Body temperature is optimal for these and most other enzymes What happens if the temperature istoo hot or cool? Too hot = enzyme may be denatured (killed) Too cool = enzyme activity lowered, requiring longer digestion time

25. Agarose Electrophoresis Loading Electrical current carries negatively- charged DNA through gel towards positive (red) electrode Power Supply Buffer Dyes Agarose gel

26. Agarose Electrophoresis Running Agarose gel sieves DNA fragments according to size –Small fragments move farther than large fragments Power Supply Gel running

27. Bio-Rad’s Electrophoresis Equipment Electrophoresis Cells Power Supplies Precast Agarose Gels PowerPac ™ JuniorPowerPac ™ Basic PowerPac ™ HCPowerPac ™ UniversalPowerPac ™ 3000 Mini-Sub ® Cell GTWide Mini-Sub Cell GT

28. Analysis of Stained Gel Determine restriction fragment sizes Create standard curve using DNA marker Measure distance traveled by restriction fragments Determine size of DNA fragments Identify the related samples

29. Molecular Weight Determination Size (bp)Distance (mm) 23,00011.0 9,40013.0 6,50015.0 4,40018.0 2,30023.0 2,00024.0 Fingerprinting Standard Curve: Semi-log

30. DNA Fingerprinting Lab Extensions Independent studies Plasmid DNA isolation (mini-preps) Plasmid mapping using restriction enzymes Southern blot analysis Introductory labs to electrophoresis: Kool-Aid/FastBlast pH indicator in buffer