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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
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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
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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)
• 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
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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
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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
– 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
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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 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 11
has a unique sequence of DNA or genetic code
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
Nuclear DNA
3.2 billion bp
Sex-
Autosomes chromosomes
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DNA is made of nucleotides (A, C,
G, & T) that are anti-parallel
Replication/Transcription Direction
3’
5’
5’
3’
Coding strand
Complimentary
strand
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DNA Molecular Structure & Make-up
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 – long-chained 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 : αorγ32P
– nonradioactive : biotin, digoxigenin, fluorescent dye
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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)
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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
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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
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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
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•
•
•
•
•
•
•
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
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RFLP Process
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PCR DNA Typing Technique
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•
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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
–
–
–
–
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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)
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Simple Overview of PCR Amplification
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Schematic Representation of PCR
• Rate of PCR
2n
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Short Tandem Repeats (STR)
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•
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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
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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
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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
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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
• Mitochondrial DNA
– found in nucleus of the cell
– found in mitochondria of the cell
– double helix
– circular
– bounded by a nuclear
– free of a nuclear envelope
envelope
– each mitochondria may have
– 2 sets of 23 chromosomes
several copies of the single
mtDNA molecule
– DNA packed into chromatin
– DNA is not packed into
– used with evidence such as
chromatin
saliva, semen, blood
– used with evidence such as hair,
– maternal and paternal
bones, teeth, and body fluid
– can “discriminate between
– maternal only
individuals of the same
maternal lineage”
– 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.
2.
3.
4.
Primary Visual Analysis
Sample Preparation
DNA extraction
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 • Quantification is performed by
by using filtration
using capillary electrophoresis (CE),
devices that remove the
which compares the amount of
excess reagents used in
DNA in the PCR product to a
the PCR from the
known DNA standard to determine
sample
the concentration of the DNA in the
PCR-amplified 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
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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.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/TechRF
LP.shtml
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