Dr. Kwok Cheong CHUNG Department of Biology The Chinese University of Hong Kong

1. 6th International Junior Science Olympiad (IJSO) Forensic Science Dr. Kwok Cheong CHUNG Department of Biology The Chinese University of Hong Kong

2. Contents • Forensic Science Fields • Forensic Science Techniques • DNA Forensics • Blood-typing and DNA Analysis • Restriction Fragment Length Polymorphisms (RFLP) • Polymerase Chain Reaction (PCR) • Mitochondrial DNA • Short Tandem Repeats (STR) • Automated DNA Sequencing

3. Notes to Teachers • Learning objectives • To let students know the various fields in forensic science (0.5 hr) • To let students know the techniques used in forensic sciences (1 hr) • To let students know the techniques in DNA Forensics: (2 hrs) • Blood-typing and DNA Analysis • Restriction Fragment Length Polymorphisms (RFLP) • Polymerase Chain Reaction (PCR) • Mitochondrial DNA • Short Tandem Repeats (STR) • Automated DNA Sequencing • Time allocation: 3.5 hrs

4. Learning Outcomes After studying this topic students will be able to: • know the various fields in forensic science • know the techniques used in forensic sciences • know the techniques in DNA Forensics: • Blood-typing and DNA Analysis • Restriction Fragment Length Polymorphisms (RFLP) • Polymerase Chain Reaction (PCR) • Mitochondrial DNA • Short Tandem Repeats (STR) • Automated DNA Sequencing

6. Forensic Science Techniques - Biology • Cell theory • Serology – blood composition & blood types • Dental forensics (forensic ondotology) - bite marks & human identification • Human body systems • Circulatory system • Health and illness • Misc. body fluids, tissues & hairs • Autopsy • Finger prints (dactylography), lip prints • Animals and habitats – Insects (forensic entomology) • Genetics and DNA • DNA fingerprinting analysis • DNA sequencing

7. Forensic Science Techniques - Chemistry • Elements, compounds, and mixtures • Toxicology • Drugs and poisons • Polymers • Analysis of ink (chromatography) • Drug analysis • Chemical reactions • Arson/Explosive analysis • Thermochemistry • Arson 縱火 • Identification of unknown metals

8. Forensic Science Techniques- Physics • Forces and motion • Accident investigation (velocity, acceleration, vectors) • Behavior of fluids • Blood-spatter analysis • Light and image formation: forensic photography • Electrical circuits - arson investigation • Thermodynamics • Arson • Metal identification • Temperature and heat • Structural forensics • Projectile motion: ballistics

9. Misc. Forensic Science Techniques • Prints • Shoeprints • Toolmarks and glove prints • Soil analysis • Hard drive imaging • Creating a duplicate of hard drive contents allowing analysis of data that has been deleted

10. Challenges of Forensic Data Mapping • Recognition of usefulness of crime scene material • Compatibility between case data, databases and mapping applications • Communication between crime analysts and forensic scientists • Legal Issues, information access and sharing

11. DNA Forensics • DNA is the chemical substance which makes up our chromosomes and controls all inheritable traits (eye, hair and skin color) • DNA is different for every individual except identical twins • DNA is found in all cells with a nucleus (muscle cells, white blood cells, soft tissue cells, bone cells, hair root cells and spermatozoa) • Half of a individual’s DNA/chromosomes come from the father & the other half from the mother • DNA is a double-stranded molecule made of four different building blocks • An individual’s DNA remains the same throughout life • In specific regions on a DNA strand each person has a unique sequence of DNA or genetic code

12. Nuclear DNA 3.2 billion bp Sex-chromosomes Autosomes Two Types of DNA Used • Nucleic DNA • In nucleus of cells • Individual specific • Mitochondrial DNA (mtDNA) • Found in mitochondria • From maternal side • Not as specific – shows maternal side only

13. 3’ 5’ Coding strand Complimentary strand 5’ 3’ DNA is made of nucleotides (A, C, G, & T) that are anti-parallel Replication/Transcription Direction

14. DNA Molecular Structure & Make-up C

15. Some Genetics Terms • Recombinant DNA - DNA molecules which are formed as a result of incorporating DNA from two or more sources into a single molecule • Restriction enzymes - chemicals that cut DNA into fragments that can later be incorporated into another DNA strand; ~150 different kinds • Polymer – long-chained molecule (e.g. DNA) • Polymerase - enzyme that is used to assemble new strands of DNA to the original/parent strand

16. Some Genetics Terms • A probe is a specific single strand DNA or RNA fragment which can bind with the sample DNA or RNA for detection ATCCGATCG-------- • Source of probe - synthesized, cloning genomic DNA or cDNA, as well as RNA • A probe must be labeled before hybridization • radioactive : αorγ32P • nonradioactive: biotin, digoxigenin, fluorescent dye

17. Blood-typing and DNA Analysis • 1901: Human blood groups identified by Karl Landsteiner • Major problem of ABO blood typing: blood protein markers are not found in semen • 1909: Chromosomes discovered to carry hereditary information • 1980: David Botstein and others used RFLP to construct a human gene map • 1984: Kary Mullis invented PCR methods, DNA fingerprinting was developed by Jeffries • 1987: First time DNA evidence was used to convict a person in the US (The Pitchfork Case)

18. Blood-typing and DNA Analysis • 1987: 1987 FBI with NIH began collaborative research to establish DNA identification techniques • 1988: FBI set up their own laboratory • Established detailed laboratory protocols • Performed validation studies

19. DNA Identification - Uses • Investigations of criminal cases involving victims • Assault 攻擊 • Kidnapping • Robbery • Rape • Murder • Catastrophe victims • Paternity / family relationships • Identify endangered and protected species • Detect bacteria/organisms that may pollute the air, water, food, and soil • Match organ donors with recipients • Determine pedigree for seed / livestock breeds • Authenticate consumables such as caviar and wine

20. RFLP • Restriction Fragment Length Polymorphisms • Fragment lengths of repeating bases result from using restriction enzymes • 1st method used in forensic science

21. RFLP Process • Need large amount of DNA • DNA is treated with restriction enzyme • Cut DNA is then separated using electrophoresis • DNA bands transferred to Nylon Membrane (Southern blotting) • Radioactive DNA probe is added to membrane (hybridization) • X-ray film placed next to membrane for a couple of days • X-ray DNA film fragments then measured samples along with control • RFLP strands used are typically thousands of bases long

22. RFLP Process

23. PCR DNA Typing Technique • Polymerase Chain Reaction • Now being used more than RFLP • Requires only small amount of DNA • Produces large amount of DNA • Can be used to aid other techniques • Uses electrophoresis • Best on strands no longer than a couple of hundred bases long

24. PCR Process • Heat DNA template to ~94°C • DNA becomes denatured • Annealing • Add primers (short strands of DNA) to separated strands • Primers combine or hybridize by lowering temp • Extension • DNA polymerase (directs rebuilding of DNA strand) • Mixture of free nucleotides • dNTPs • pH buffer, salt, Mg2+ • Heat to ~55-72 °C • Repeat process 25-30 times • > 1 billion copies to be made (32 cycles)

25. Simple Overview of PCR Amplification

26. Schematic Representation of PCR • Rate of PCR 2n

27. Short Tandem Repeats (STR) • Use DNA sections with repeat bases (2-7) • Uses capillary electrophoresis • Visualized as peaks on a graph • Advantages • Better discrimination than RFLP • Faster result time • Low mutation rates • Only ~1 nanogram needed

28. How STRs Appear as a Result of Analysis

29. Databases of Forensic Data • National Forensic Databases (US) • Combined DNA Index System (CODIS) • Automated Fingerprint Identification System (AFIS) • PDQ (paint) • National DNA Index System (NDIS) • National Integrated Ballistics Information Network (NIBIN) • National Law Enforcement Telecommunications Systems (NLETS) • National Crime Information Center (NCIC ) • Financial Crimes Enforcement Network (FinCEN)

30. CODIS • Combined DNA Index System in USA • National DNA I.D. system • All profiles stored in CODIS are generated using STR analysis • Has three levels • Local • State • Federal

31. FBI uses 13 different DNA loci1:53,581,500,000,000,000,000 probability

32. Example of using STR Analysis in Forensics • 49 murders in Seattle area 1982-1984 • Bodies discarded in woods • Task force investigated for years • In 2001 DNA breakthrough led to further investigation including microscopic analysis of artifacts recovered with bodies • Green paint spheres found on artifacts • High end spray paint • Linked bodies to each other • Linked bodies to a common location / source - a single truck painting plant • PCR-based STR analysis was used to convict the killer

33. Mitochondrial DNA in Forensics • Mitochondria • Organelles which are responsible for cellular respiration (ATP production) • Have a double membrane, cristae (folds), a matrix, and their own DNA • Mitochondria of the sperm cell do not enter the egg at fertilization • Mitochondrial DNA (mtDNA) codes for proteins and enzymes used by the mitochondria • Nuclear DNA also codes for enzymes used in the mitochondria

34. Nuclear DNA vs. Mitochondrial DNA • Nuclear DNA • found in nucleus of the cell • double helix • bounded by a nuclear envelope • 2 sets of 23 chromosomes • DNA packed into chromatin • used with evidence such as saliva, semen, blood • maternal and paternal • can “discriminate between individuals of the same maternal lineage” • Mitochondrial DNA • found in mitochondria of the cell • circular • free of a nuclear envelope • each mitochondria may have several copies of the single mtDNA molecule • DNA is not packed into chromatin • used with evidence such as hair, bones, teeth, and body fluid • maternal only • cannot “discriminate between individuals of the same maternal lineage”

35. Forensic mtDNA Analysis • Steps to obtain a mtDNA sequence from a sample: • Primary Visual Analysis • Sample Preparation • DNA extraction • Polymerase Chain Reaction (PCR) Amplification • Postamplification Quantification of the DNA • Automated DNA Sequencing • Data Analysis

36. Postamplification Purification and Quantification • Purification is performed by using filtration devices that remove the excess reagents used in the PCR from the sample • Quantification is performed by using capillary electrophoresis (CE), which compares the amount of DNA in the PCR product to a known DNA standard to determine the concentration of the DNA in the PCR-amplified sample

37. Automated DNA Sequencing • Dideoxy Terminator (Sanger’s) Method: • similar to PCR amplification • terminator bases tagged with a fluorescent dye are added in addition to free nucleotides • terminator bases with the OH group replaced with H group in the sugar moiety • normal bases compete with the terminator bases for incorporation into the growing DNA strand, resulting in a collection of DNA products that differ in size by one base and have a fluorescent labeled base at the end position

38. Automated DNA Sequencing • Results of automated DNA sequence analysis using fluorescent dyes

39. References for Further Studies • Forensic science • http://en.wikipedia.org/wiki/Forensic_science • A Free And Comprehensive Guide To The World Of Forensic Science • http://www.all-about-forensic-science.com/ • Polymerase chain reaction • http://en.wikipedia.org/wiki/Polymerase_chain_reaction • Principle of the PCR • http://users.ugent.be/~avierstr/principles/pcr.html • PCR: The polymerase chain reaction • http://www.horizonpress.com/pcr/ • Restriction Fragment Length Polymorphism (RFLP) • http://www.ncbi.nlm.nih.gov/projects/genome/probe/doc/TechRFLP.shtml