2. Forensic DNA Fingerprinting: Using Restriction Enzymes

4. Forensic DNA Fingerprinting Kit Advantages Standards Based Aligns with AP Biology Lab 6 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

5. DNA structure DNA restriction analysis (RFLP) Agarose gel electrophoresis Molecular weight determination Simulation of DNA Fingerprinting Plasmid mapping The Forensic DNA Fingerprinting Kit Can Help You Teach:

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

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

8. DNA Fingerprinting Procedure Overview

9. Laboratory Quick Guide

10. DNA Fingerprinting Procedures Day One

11. DNA Fingerprinting Procedures Day Two

12. DNA Fingerprinting Procedures Day Three

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 PO O O Base CH 2 O P O O O Base OH Sugar O Phosphate

17. What information is in our DNA? One copy of Homo sapiens DNA contains three billion base pairs located on 23 chromosomes. This amount of DNA could code for three million genes of average size (1000 bp). However, in reality, it only codes for approximately 50,000 genes. So, only a few percent of our DNA actually codes for protein! We don't know why this is so but it does provide us with some interesting observations on the sequence content. –DNA that codes for protein. These are the exons of genes ~ 2-3% –Non-coding single copy, non-repetitive~ 68% –Non-coding repetitive DNA ~ 30%. Of this: –2/3 Randomly repetitive (or 20% total DNA) –1/3 Tandemly repetitive (or 10% total DNA)

18. 1985 One type of this last group is called Variable Number Tandem Repeats or VNTR. This DNA is: Non-coding and 9-100bp in length –Repetitive –The repeating sequence is repeated next to itself (tandem) –The number of repeats is variable from person to person The VNTR DNA can be used in one method of obtaining a DNA fingerprint. This method is called Restriction Fragment Length Polymorphism and results in an RFLP Fingerprint

19. Other Repetitive Units ---1991 Many non- coding regions have no known function, and are sometimes referred to as "junk DNA." Microsatellites are one example of such "junk DNA.“ Microsatellites, or Simple Sequence Repeats (SSRs), are polymorphic loci present in DNA that consist of repeating units of 1-6 base pairs in length polymorphic DNAbase pairs Microsatellites occur in "non- coding" DNA, that is, DNA that is not transcribed into messenger RNA to code for proteins.

20. STR’s are similar to SSR’s however the number of repeating bases are less (usually 2-6 bp in length) The number of repeats of the SSR’s is highly variable between individuals.

23. 2-4

24. STR-PCR

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

26. 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

27. 5 vs 3 Prime Overhang Generates 5 prime overhang Enzyme cuts

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

30. 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

31. 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

32. 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

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

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

35. 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

36. 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

37. 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

38. Plasmid Map and Restriction Sites Laboratory Extensions 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

39. 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