2. Forensic DNA Fingerprinting: Using Restriction Enzymes

3. Stan Hitomi Coordinator – Math & Science Principal – Alamo School San Ramon Valley Unified School District Danville, CA Kirk Brown Lead Instructor, Edward Teller Education Center Science Chair, Tracy High School and Delta College, Tracy, CA Bio-Rad Curriculum and Training Specialists: Sherri Andrews, Ph.D. sherri_andrews@bio-rad.com Damon Tighe. damon_tighe@bio-rad.com Leigh Brown, M.A. leigh_brown@bio-rad.com Forensic DNA Fingerprinting Kit Instructors

4. Why Teach DNA Fingerprinting? Real-world connections Tangible results Link to careers and industry Easy integration to current AP Lab 6, and new AP Big Idea 3 – Genetics and Information Transfer Easy to turn into an inquiry-based activity Standards-based

5. Forensic DNA Fingerprinting Kit Advantages Use of real restriction enzymes and electrophoresis of real DNA fragments Lab can completed in two 45 minute sessions Sufficient materials for 8 student workstations

6. S cience T echnology E ngineering M ath I nquiry

7. DNA structure DNA restriction analysis (RFLP) Agarose gel electrophoresis Molecular weight determination Simulation of DNA Fingerprinting Plasmid mapping Enzyme kinetics Enzyme Structure and function The Forensic DNA Fingerprinting Kit Can Help You Teach:

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

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

10. DNA Fingerprinting Procedure Overview

11. Laboratory Quick Guide

12. DNA Fingerprinting Procedures Day One

13. DNA Fingerprinting Procedures Day Two

14. DNA Fingerprinting Procedures Day Three

15. Deoxyribonucleic Acid (DNA)

16. DNA Schematic

17. Engaging your students Phosphate Sugar Base

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

19. 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) Palindrome Restriction site Fragment 1 Fragment 2

20. 5 vs 3 Prime Overhang Generates 5 prime overhang Enzyme cuts

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

22. 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

23. 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

24. Restriction Fragment Length Polymorphism RFLP Allele 1 Allele 2 GAATTC GTTAAC GAATTC GTTAAC CTGCAG GAGCTC CGGCAG GCGCTC PstIEcoRI 123 3 Fragment 1+2 Different Base Pairs No restriction site + MA-1A-2 Electrophoresis of restriction fragments M: Marker A-1: Allele 1 Fragments A-2: Allele 2 Fragments

25. Student Inquiry Questions to consider: –How important is each step in the lab protocol? –What part of the protocol can I manipulate to see a change in the results? –Possible variables: enzyme concentration substrate concentration incubation temp or time enzyme or DNA UV exposure methylated plasmid agarose concentration buffer concentration running time. –How do I insure the changes I make is what actually affects the outcome (importance of controls). –Write the protocol. After approval – do it!

26. Student Inquiry More Advanced Questions: What can I learn about these plasmids? Can I use these plasmids to successfully transform bacteria? Can I ligate these plasmids together and successfully transform bacteria? Can I do a restriction digest on pGLO plasmid? Can I determine the plasmid map using different enzymes?

27. Student Inquiry Teacher Considerations What materials and equipment do I have on hand, and what will I need to order? –Extra agarose, DNA, different / more restriction enzymes? –Water bath (different temps) –Other supplies depending on student questions (mini prep, thermal cyclers, etc) –Consider buying extras in bulk or as refills – many have 1 year + shelf life. What additional prep work will I need? –Order supplies –Pour gels How much time do I want to allow? –Limited time? Have students read lab and come up with inquiry questions and protocol before they start. Collaborative approach. –Will you need multiple lab periods? –Will everyone need the same amount of time?

28. Plasmid Map and Restriction Sites BamHI: EcoRI: HindIII: EcoRI+Hind III: 1 linear fragment; 7367bp 2 fragments; 863bp / 6504bp 3 fragments; 721bp/2027bp/3469bp 5 fragments; 721bp/863bp/947bp/1659bp/2027bp BamHI 7367bp EcoRI 863bp 6504bp Hind III 721bp 2027bp 3469bp EcoRI+ HindIII 2027bp 1659bp 947bp 863bp 721bp

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

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

31. Classroom obstacle course

32. 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

33. 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

34. Bio-Rad’s Electrophoresis Equipment Electrophoresis Cells Power Supplies Precast Agarose Gels PowerPac ™ MiniPowerPac ™ Basic PowerPac ™ HCPowerPac ™ Universal Mini-Sub ® Cell GTWide Mini-Sub Cell GT

35. Webinars Enzyme Kinetics — A Biofuels Case Study Real-Time PCR — What You Need To Know and Why You Should Teach It! Proteins — Where DNA Takes on Form and Function From plants to sequence: a six week college biology lab course From singleplex to multiplex: making the most out of your realtime experiments explorer.bio-rad.com/Webinars