Dr. Kwok Cheong CHUNG Department of Biology

The Chinese University of Hong Kong

Forensic Science

6 th International Junior Science Olympiad (IJSO)

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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

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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

– Accident investigation (velocity, acceleration, vectors)

• Behavior of fluids – Blood­spatter analysis

• Light and image formation: forensic photography • Electrical circuits ­ arson investigation • Thermodynamics

– 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”

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Forensic mtDNA Analysis

• Steps to obtain a mtDNA sequence from a sample:

1. Primary Visual Analysis 2. Sample Preparation 3. DNA extraction 4. Polymerase Chain Reaction (PCR)

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/

• 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/TechRF LP.shtml 39