1. DNA profiling is the use of molecular genetics methods to determine the exact genotype of a DNA sample to distinguish one human being from another Used in forensics to investigate crime scenes, missing persons, mass disasters, paternity, evolutionary links, … Crime scenes often contain biological evidence (blood, semen, hair, saliva, bones, skin) from which DNA can be extracted Humans have ~3 billion bp and >99.5% do not vary, however a small percentage (<0.5%) does differ Varying regions are called “polymorphic” due to nt differences between one individual and another and polymorphisms are in regions of our chromosome (called loci) that do not have any known function - this DNA is satellite DNA (specifically short tandem repeats/microsatellite) Crime Scene Investigation DNA Profiling / DNA Fingerprinting

2. Ways to detect unique sequences within mammalian DNA 1. Restriction digestion of chromosomal DNA works for some organisms BUT not in mammals: EX: humans have 3 billion base pairs with 1 million restriction fragments formed from a single restriction enzyme digest, nt differences between people will result in different restriction digestion products - but TOO difficult to isolate a single band on a gel from this large number of fragments 2. To characterize a specific gene use blot hybridization Crime Scene Investigation DNA Profiling / DNA Fingerprinting

3. Crime Scene Investigation DNA Profiling / DNA Fingerprinting

4. RFLPs Genome of each of us is unique (exception: identical twins) Variation in sequences between individuals is most pronounced in DNA that does not code for proteins Hypervariable regions called “polymorphic sites” Polymorphic sites - variation due to small insertions, deletions, or point mutations in restriction sites SO.. Restriction fragment with a polymorphic site may differ from one person to the next resulting in: RESTRICTION FRAGMENT LENGTH POLYMORPHISMS (RFLPs) Pattern of bands on a blot hybridization sometimes called a “DNA fingerprint” because can use it to identify an individual Crime Scene Investigation DNA Profiling / DNA Fingerprinting

5. History of Forensics Early use of science for criminal investigations - use of photographs to document crime scenes Past ~100 years - use of fingerprinting First genetic evidence collected for investigations - blood group typing (A, B, AB, and O) Test is very fast and straight-forward. BUT 40% of population is type O, so not useful if several suspects are type O. 1980s - first use of DNA-based forensic test called restriction fragment length polymorphism analysis (RFLP); discovered by Alec Jeffries, focus on VNTRs (variable number of tandem repeats) Crime Scene Investigation DNA Profiling / DNA Fingerprinting

6. Crime Scene Investigation DNA Profiling / DNA Fingerprinting

7. Restriction fragment length polymorphism analysis (RFLP) Crime Scene Investigation DNA Profiling / DNA Fingerprinting

8. Restriction fragment length polymorphism analysis (RFLP) Crime Scene Investigation DNA Profiling / DNA Fingerprinting

9. Restriction fragment length polymorphism analysis (RFLP) Crime Scene Investigation DNA Profiling / DNA Fingerprinting

10. Restriction fragment length polymorphism analysis (RFLP) Crime Scene Investigation DNA Profiling / DNA Fingerprinting

11. Restriction fragment length polymorphism analysis (RFLP) Crime Scene Investigation DNA Profiling / DNA Fingerprinting

12. Restriction fragment length polymorphism analysis (RFLP) Crime Scene Investigation DNA Profiling / DNA Fingerprinting

13. Restriction fragment length polymorphism analysis (RFLP) Although technique is powerful and has great discriminating potential, it is laborious, cannot be easily automated and is time-consuming Depends on DNA sequences being several hundred base pairs in length, so DNA must be of reasonable quality (not degraded) and it requires a large amount of DNA (not always able to get enough DNA from the crime scene) SO turn to using STRs - short tandem repeat DNA (smaller repeat lengths than VNTRs) Easier to analyze than RFLPs because: 1. due to small size they are easily replicated by PCR 2. Because they can be amplified by PCR, STR tests can be automated, with several tests being run at the same time 3. Even degraded DNA can give OK results 4. Even a single piece of hair (or a drop of blood, cheek cell, or piece of bone) can give enough genetic material for successful analysis Crime Scene Investigation DNA Profiling / DNA Fingerprinting

14. Advances in PCR have made a huge impact MODERN FORENSIC DNA PROFILING MAKES IT POSSIBLE TO DISTINGUISH ANY TWO PEOPLE ON THE PLANET (EXCEPT IDENTICAL TWINS), LIVING OR DEAD Crime Scene Investigation DNA Profiling / DNA Fingerprinting Polymerase chain reaction (PCR) Denature dsDNA Anneal primers to now ssDNA Polymerization with thermostable DNA pol (DNA synthesis - get 2 copies from 1) One cycle can be repeated over & over After cycle 30, > 1 billion identical molecules (2 30 = 1.07 x 10 9 )

15. Crime Scene Investigation DNA Profiling / DNA Fingerprinting

16. Crime Scene Investigation DNA Profiling / DNA Fingerprinting DNA profiling performed at many loci to improve the power of discrimination CODIS - COmbined DNA Index System - is a federally maintained database of DNA obtained from crime scenes and convicted violent offenders; started by FBI in 1998 CODIS examines 13 loci, or markers, that are uniformly distributed across the human genome Also includes AMEL (determines gender) The 13 loci reveal no medical or health info about a person; called “anonymous” markers

17. Crime Scene Investigation DNA Profiling / DNA Fingerprinting Why 13 loci?

18. Crime Scene Investigation DNA Profiling / DNA Fingerprinting This week’s Lab Scene: the Highway Motel, Room #13 The motel manager hears loud voices, a woman screams, and a shot rings out. The manager runs to the window in time to see the receding lights of a car leaving in a hurry. The door to room #13 is open. The manager runs to the open door to see a man lying face down in a pool of blood. He calls 911. The police arrive, and begin to examine the crime scene. An apparent homicide, but with no obvious clues as to who committed to crime. Or..? A forensic specialist is called in to examine the crime scene and evidence is collected. In addition, four suspects samples are collected. You will perform PCR analysis on a single locus, the BXP007 locus - start this TODAY Run PCR products on a 3% agarose gel, visualize products, compare to the allele ladder and to the crime scene sample, determine who committed the crime! In addition to running your PCR products on the agarose gel you will also run your restriction digests from the genetic engineering lab.

19. Crime Scene Investigation DNA Profiling / DNA Fingerprinting Set up PCR in previous lab period: Items needed: Ice bath containing one yellow tube labeled MMP (blue liquid inside), 5 other tubes labeled CS (purple tube), A (green tube), B (blue tube), C (orange tube), and D (pink tube). 5 PCR tubes (these are smaller than we usually use, 0.5 microliter size) 5 normal size microfuge tubes used as adaptors (1.5 mL size) Foam float Marker P20 pipettor PCR block

20. Crime Scene Investigation DNA Profiling / DNA Fingerprinting Label PCR tubes (0.5 microliter size) “CS”, “A”, “B”, “C”, and “D” Also put your group name on the tube Put 0.5 microliter tubes inside 1.5 mL tubes and put 1.5 mL tubes into foam float and set on ice, keep all samples on ice throughout the set up of these samples Set up the tubes as follows: PCR tube labelDNA templateMaster mix + primers* CS20 microliters crime scene DNA (purple tube) 20 microliters MMP (yellow tube) A20 microliters suspect A DNA (green tube)20 microliters MMP (yellow tube) B20 microliters suspect B DNA (blue tube) 20 microliters MMP (yellow tube) C20 microliters suspect C DNA (orange tube) 20 microliters MMP (yellow tube) D20 microliters suspect D DNA (pink tube) 20 microliters MMP (yellow tube) Add all the DNA templates to their respective tubes first - change tips for each DNA! Next add MMP to each tube, mix by flicking the tube and/or by gently pipetting the the solution up and down - use a fresh tip each time!! I will start the PCR cycles up in my research lab *MASTER MIX + PRIMERS contains Taq DNA polymerase buffer, MgCl 2, dNTPs, Taq DNA polymerase, DNA primers specific for the BXP007 locus

21. Crime Scene Investigation DNA Profiling / DNA Fingerprinting PCR cycle: STEPFUNCTIONTEMPTIME# of CYCLES Initial denaturationDenature94˚C2 min1 cycle Thermal cyclingDenature94˚C30 sec Anneal 52˚C 30 sec Extend 72˚C 1 min Final extensionExtend72˚C10 min1 cycle HoldHold4˚Chours } 35 cycles

22. Crime Scene Investigation DNA Profiling / DNA Fingerprinting On Wednesday - electrophorese PCR products Materials needed: 3% agarose gel with ethidium bromide 5 PCR samples from before break 1x TAE running buffer Orange G loading dye “LD” (orange liquid) Allele ladder “allele ladder” (also orange liquid!) P20 pipettor Gel box Power supply

23. Crime Scene Investigation DNA Profiling / DNA Fingerprinting Electrophorese PCR products 1. Add 10 microliters of Orange G loading dye “LD” to each PCR reaction tube (5 of these); mix well 2. Set up gel apparatus, rotate gel from pouring position to loading position, add 1x TAE to gel box, carefully pull out comb 3. Load 20 microliters of each sample into wells on the gel Lane 1Allele ladder20 uL Lane 2crime scene20 uL Lane 3suspect A20 uL Lane 4suspect B20 uL Lane 5suspect C20 uL Lane 6suspect D20 uL 4. Run gel at 120 volts for ~45-60 minutes (do not let orange dye front migrate off the gel) 5. Visualize results on UV light box

24. Crime Scene Investigation DNA Profiling / DNA Fingerprinting Study questions: 1. Why do you need to perform PCR on DNA evidence from a crime scene? 2. Did your samples all generate PCR products? If not, give reasons to explain why. 3. Does the crime scene DNA sample have a genotype that matches any of the suspects? If so, which one matches? 4. If you had a pool of 13 suspects and only one suspect had a genotype that matched the BXP007 locus found at the crime scene would you be satisfied that you had identified the perpetrator? Why or hy not? Explain.

26. USEFUL WEBSITES Folding of RNA molecules http://www.bioinfo.rpi.edu/applications/mfold/old/rna/form1.cgi Molecular Biology tools http://www.molbiol.net/ http://www.molbiol.net/biolinks/alphabar/PCR%2520and%2520Primer%2520Design.shtml http://ntdb.chem.cuhk.edu.hk/tools.htm Oligonucleotide calculator http://members.aol.com/_ht_a/lucatoldo/myhomepage/JaMBW/3/1/9/index.html http://www-medlib.med.utah.edu/masspec/mongo.htm http://www.schepartzlab.yale.edu/ Protein information http://www.basic.nwu.edu/ Chemistry calculators http://users.pandora.be/educypedia/education/calculators.htm Good websites of “tools” http://www.csb.yale.edu/people/steitz/Toolkit/toolkit.htm http://szewczak.com/ Plasmid information MacPlasmap