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Learn about the various fields and techniques in forensic science, including DNA forensics and analysis. Explore topics such as blood-typing, DNA sequencing, and forensic photography. This course will provide a comprehensive overview of forensic science.
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6th International Junior Science Olympiad (IJSO) Forensic Science Dr. Kwok Cheong CHUNG Department of Biology The Chinese University of Hong Kong
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
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
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
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
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
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
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
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
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
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
3’ 5’ Coding strand Complimentary strand 5’ 3’ DNA is made of nucleotides (A, C, G, & T) that are anti-parallel Replication/Transcription Direction
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
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
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)
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
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
RFLP • Restriction Fragment Length Polymorphisms • Fragment lengths of repeating bases result from using restriction enzymes • 1st method used in forensic science
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
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
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)
Schematic Representation of PCR • Rate of PCR 2n
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
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)
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
FBI uses 13 different DNA loci1:53,581,500,000,000,000,000 probability
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
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
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”
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
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
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
Automated DNA Sequencing • Results of automated DNA sequence analysis using fluorescent dyes
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