Dr. Kwok Cheong CHUNG Department of Biology The Chinese University of Hong Kong Forensic Science 6 th International Junior Science Olympiad (IJSO) 1 Contents • Forensic Science Fields • Forensic Science Techniques • DNA Forensics – Bloodtyping and DNA Analysis – Restriction Fragment Length Polymorphisms (RFLP) – Polymerase Chain Reaction (PCR) – Mitochondrial DNA – Short Tandem Repeats (STR) – Automated DNA Sequencing 2 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) • Bloodtyping 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 3 Learning Outcomes • know the various fields in forensic science • know the techniques used in forensic sciences • know the techniques in DNA Forensics: – Bloodtyping and DNA Analysis – Restriction Fragment Length Polymorphisms (RFLP) – Polymerase Chain Reaction (PCR) – Mitochondrial DNA – Short Tandem Repeats (STR) – Automated DNA Sequencing After studying this topic students will be able to: 4 5 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 6
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– Bloodtyping and DNA Analysis – Restriction Fragment Length Polymorphisms (RFLP)
– Polymerase Chain Reaction (PCR) – Mitochondrial DNA – Short Tandem Repeats (STR) – Automated DNA Sequencing
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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) • Bloodtyping 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 3
Learning Outcomes
• know the various fields in forensic science • know the techniques used in forensic sciences • know the techniques in DNA Forensics:
– Bloodtyping and DNA Analysis – Restriction Fragment Length Polymorphisms (RFLP) – Polymerase Chain Reaction (PCR) – Mitochondrial DNA – Short Tandem Repeats (STR) – Automated DNA Sequencing
After studying this topic students will be able to:
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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
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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
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Forensic Science Techniques Physics • Forces and motion
– Arson – Metal identification – Temperature and heat – Structural forensics
• Projectile motion: ballistics 8
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
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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
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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 doublestranded 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 11
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
Autosomes Sex chromosomes
Nuclear DNA 3.2 billion bp
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DNA is made of nucleotides (A, C, G, & T) that are antiparallel Replication/Transcription Direction
3’
3’ 5’
5’ Coding strand
Complimentary strand
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DNA Molecular Structure & Makeup
C
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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 – longchained molecule (e.g. DNA)
• Polymerase enzyme that is used to assemble new strands of DNA to the original/parent strand
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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 : αorγ 32 P – nonradioactive : biotin, digoxigenin, fluorescent dye
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Bloodtyping 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)
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Bloodtyping 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
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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 19
RFLP • Restriction Fragment Length Polymorphisms – Fragment lengths of repeating bases result from using restriction enzymes
• 1st method used in forensic science
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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)
• Xray film placed next to membrane for a couple of days
• Xray DNA film fragments then measured samples along with control
• RFLP strands used are typically thousands of bases long
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RFLP Process
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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
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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, Mg 2+ – Heat to ~5572 ˚C
• Repeat process 2530 times – > 1 billion copies to be made (32 cycles) 24
Simple Overview of PCR Amplification
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Schematic Representation of PCR • Rate of PCR 2 n
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Short Tandem Repeats (STR) • Use DNA sections with repeat bases (27) • Uses capillary electrophoresis • Visualized as peaks on a graph • Advantages
– Better discrimination than RFLP – Faster result time – Low mutation rates – Only ~1 nanogram needed
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How STRs Appear as a Result of Analysis
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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)
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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
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FBI uses 13 different DNA loci 1:53,581,500,000,000,000,000 probability
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Example of using STR Analysis in Forensics
• 49 murders in Seattle area 19821984 • 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
– PCRbased STR analysis was used to convict the killer
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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
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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”
Amplification 5. Postamplification Quantification of
the DNA 6. Automated DNA Sequencing 7. Data Analysis
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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 PCRamplified sample
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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 37
Automated DNA Sequencing • Results of automated DNA
sequence analysis using fluorescent dyes
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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.allaboutforensicscience.com/