Forenisc
identification
John J. O’Leary
MD, PhD, MSc, MA, FRCPath,
FFPathRCPI, FTCD.
Trinity College Dublin
Topics
 Friction ridge identification
 Forensic dentistry
 Facial recognition and re-construction
systems
 DNA fingerprinting
Forensic identification
 People can be identified by their fingerprints. We know this
due to the philosophy of Friction Ridge Identification which
states:
"Friction ridge identification is established through the
agreement of friction ridge formations, in sequence, having
sufficient uniqueness to individualize". Friction ridge
identification is also governed by four premises or
statements of fact:
Friction ridges
1. Friction ridges develop on the fetus in their
definitive form prior to birth.
2. Friction ridges are persistent throughout life
except for permanent scarring, disease or
decomposition after death.
3. Friction ridge paths and the details in small areas
of friction ridges are unique and never repeated.
4. Overall friction ridge patterns vary within limits
which allow for classification.
Fingerprints
Arch
Loop
Whorl
Arch – tented arch
Fingerprints
Forensic dentistry
 Forensic dentistry or forensic odontology is the
proper handling, examination and evaluation of
dental evidence. The evidence that may be
derived from teeth, is the age (in children) and
identification of the person to whom the teeth
belong. This is done using dental records or antemortem (prior to death) photographs.
Facial recognition system
 A facial recognition system is a computer
application for automatically identifying or
verifying a person from a digital image or a
video frame from a video source. One of the
ways to do this is by comparing selected
facial features from the image and a facial
database.
Facial recognition and reconstruction
DNA fingerprinting
DNA forensics
 DNA
 Chromosomes
 Nucleotides
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Adenosine (A)
Guanine (G)
Cytosine (C)
Thymidine (T)
The structure of DNA
The structure of DNA
The structure of DNA
Every chromosome
has a unique
signature
The sequence of DNA
DNA-RNA-Protein
DNA
Protein
RNA
What is DNA?
 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 (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 Review:
 DNA is a double-stranded molecule.
 The DNA strands are 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.
Chromosome facts
 Number of chromosome = 46
– 22 autosomes and 2 sex chromosomes
 One chromosome of each pair donated from
parents sperm or egg
 Sex chromosomes
– XY male: XX female
 Largest chromosome: chr 1-263 million base pairs
(bp)
 Smallest chromosome: chr Y - 59 million base
pairs (bp)
Gene facts
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Human genome = 3.4 billion base pairs
Number of human genes: approx 100,000
Genes vary in length: average 3,000 bp
Only 5% of human genome is coding and contains
genes
 Genes divided into exons and introns
 Much of the function of the genome unknown
 0.1% difference in DNA between individuals
Gene facts: repetitive genome units
 Minisatellites are molecular marker loci consisting of
tandem repeat units of a 10-50 base motif, flanked by
conserved endonuclease restriction sites
– DNA fingerprinting
– VNTR (Variable Number of Tandem Repeats)
 Microsatellites are simple sequence tandem repeats
(SSTRs). The repeat units are generally di-, tri- tetra- or
pentanucleotides. For example, a common repeat motif in
birds is ACn, where the two nucleotides A and C are
repeated in bead-like fashion a variable number of times (n
could range from 8 to 50)
– Simple sequence repeats (SSR)
– Simple sequence length polymorphisms (SSLP)
Other important gene regions
 Single nucleotide polymorphisms or SNPs
(pronounced "snips") are DNA sequence
variations that occur when a single nucleotide
(A,T,C,or G) in the genome sequence is altered.
For example a SNP might change the DNA
sequence AAGGCTAA to ATGGCTAA.
 For a variation to be considered a SNP, it must
occur in at least 1% of the population.
 SNPs, which make up about 90% of all human
genetic variation, occur every 100 to 300 bases
along the 3-billion-base human genome
Use of DNA forensics
 Identification purposes
 Identify crime suspects
 Exonerate persons wrongly accused of
crime
 Identify crime and catastrophe victims
 Establish paternity and other family
relationships
Factors Leading to DNA Degradation
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Time
Temperature
Humidity
Light
Exposure to chemicals
DNA as Physical Evidence
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Perspective
Recognition of Evidence
Collection of Physical Evidence
Preservation of Physical Evidence
Preparation of the Physical Evidence
Evaluation and Quantification of the
Evidence
Individualization:
 Evidence that exhibit traits that are are so
unique that when considered alone or in
combination with other traits can reduce the
evidence source from a class to one
individual.
 Evidence that can indicate that two samples
share a common unique source or origin.
Association:
 “Description of the relationship between two
objects items, or people.”
 Concept used in a crime scene analysis for
reconstruction.
 “Involves the evaluation of evidence to infer
a common source.”
 Does not prove a crime.
Traits that Indicate Individuality
 Fingerprints - are a result of several genes
and other non-genetic events. Has been
accepted as unique for each individual
(even identical twins)
 DNA - early results suggested individuality
except in identical twins; but in reality more
like a partial print.
Sources of DNA for Testing
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Blood
Semen
Tissue
Bone (Marrow)
Hair Root
Saliva
Urine
Tooth (Pulp)
How is DNA typing done
 Strict anti-contamination procedures
 Standard operating procedure for every
forensic DNA test
 Dedicated laboratory facilities
 Contact DNA tracing
DNA technologies used in forensic
investigations
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RFLP
PCR-RFLP
STR analysis
Mitochondrial DNA (mtDNA)
Y-chromosome analysis
SNP genotyping
DNA technologies used in forensic
investigations
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RFLP
PCR-RFLP
VNTRs
HLA-DQ
STR analysis
Mitochondrial DNA (mtDNA)
Y-chromosome analysis
SNP genotyping
Basis of RFLP analysis
 Restriction Enzymes (biological
catalysts) cut DNA whenever they
encounter a specific DNA sequence.
 Gel electrophoresis separates the
fragments of DNA according to their
length.
Basis of RFLP analysis
Basis of RFLP analysis
A Schematic Representation of RFLP and
Southern Blot of a Single-locus VNTR
In the segment of DNA shown below, you can see the elements of an RFLP:
a target sequence flanked by a pair of restriction sites.
When this segment of DNA is cut by EcoR I, three restriction fragments are
produced, but only one contains the target sequence which can be bound by the
complementary probe sequence (purple).
Let's look at two people and the segments of DNA they carry that contain
this RFLP (for clarity, we will only see one of the two stands of DNA).
Since Jack and Jill are both diploid organisms, they have two copies of
this RFLP. When we examine one copy from Jack and one copy from Jill,
we see that they are identical:
Jack 1: -GAATTC---(8.2 kb)---GCATGCATGCATGCATGCAT---(4.2 kb)--GAATTCJill 1: -GAATTC---(8.2 kb)---GCATGCATGCATGCATGCAT---(4.2 kb)--GAATTC-
When we examine their second copies of this RFLP, we see that they are
not identical. Jack 2 lacks an EcoR I restriction site that Jill has 1.2 kb
upstream of the target sequence (difference in italics).
Jack 2: -GAATTC--(1.8 kb)-CCCTTT--(1.2 kb)--GCATGCATGCATGCATGCAT--(1.3 kb)GAATTCJill 2: -GAATTC--(1.8 kb)-GAATTC--(1.2 kb)--GCATGCATGCATGCATGCAT--(1.3 kb)GAATTC-
RFLP analysis
Use of optimum number of loci
for RFLP analysis
DNA technologies used in forensic
investigations
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RFLP
PCR-RFLP
VNTRs
HLA-DQ
STR analysis
Mitochondrial DNA (mtDNA)
Y-chromosome analysis
SNP genotyping
PCR -RFLP
Polymerase Chain Reaction (PCR)
PCR -RFLP
In 1984, Alec Jeffreys developed
“DNA Fingerprinting”
 Was searching for disease markers
 Applied the technique to personal
identification
 Demonstrated that the DNA could be
retrieved from old dried blood stains
 Applied the technique to high-profile
forensic tests
RFLP Methods: commentary
 Have a high power of discrimination:
20-80 different alleles may be possible at
one location; analyzed in combination can
be used to determine an individualized
type.
 RFLP procedures are labor intensive:
multi-locus probes are difficult to
automate: single-locus probes can be
used in serial fashion.
 Require ample supply of high grade DNA.
A Typical DNA Profile
Molecpath
DNA technologies used in forensic
investigations
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RFLP
PCR-RFLP
VNTRs
HLA-DQ
STR analysis
Mitochondrial DNA (mtDNA)
Y-chromosome analysis
SNP genotyping
VNTRs
(variable number of tandem repeats)
 Minisatellites are molecular marker loci
consisting of tandem repeat units of a 10-50
base motif, flanked by conserved
endonuclease restriction sites
– VNTR (Variable Number of Tandem Repeats)
 Popular from 1985-1995
 Required relatively large amounts of DNA
DNA technologies used in forensic
investigations
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RFLP
PCR-RFLP
VNTRs
HLA-DQ
STR analysis
Mitochondrial DNA (mtDNA)
Y-chromosome analysis
SNP genotyping
STRs
 Short Tandem Repeats: Repeating units of
an identical DNA sequence, length is often
between 2 – 5 bp in length. The repeat
units are arranged in direct succession of
each other, and the number of repeat units
varies between individuals (subgroup of
VNTRs)
Multiplex STRs
 High power of Discrimination
 Rapid Analysis
 Analysis can be automated and 3 or more
locations can be analyzed at a time.
 FBI (USA) uses 13 specific STR regions for
CODIS
 6 of the 13 loci are used by the British
Forensic Science Service
Example of STR Multiplex
The odds that 2 individuals will have the same 13 loci DNA profile
is one in one billion
Validation of STR Techniques
 1991—Fluorescent STR markers first
described
 1993—First STR kit available
 1996—First multiplex STR kits available
 1997—13 core STR loci defined; Ychromosome STR described
 1999—Multiplex STR kits validated
 2000—FBI and other labs stop running
RFLP and convert to multiplex STRs.
DNA technologies used in forensic
investigations
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RFLP
PCR-RFLP
VNTRs
HLA-DQ
STR analysis
Mitochondrial DNA (mtDNA)
Y-chromosome analysis
SNP genotyping
Mitochondrial DNA (mtDNA)
 Can be used on samples not suitable for RFLP or
STR analysis
 mtDNA is present in mitochondria
 All mothers have the same mtDNA as their
daughters
 The mitochondria of each new embryo comes
from the mother’s egg
 Father’s sperm contributes only nuclear DNA
 Important tool in missing person investigations
Mitochondrial DNA (mtDNA)
 Lowest power of discrimination
 Longest sample processing time
 Can be very helpful in forensic cases
involving severely degraded DNA
samples
 Sometimes mitochondria are
heteroplasmic (more than one kind of
mitochondria in a person or in a cell)
DNA technologies used in forensic
investigations
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RFLP
PCR-RFLP
VNTRs
HLA-DQ
STR analysis
Mitochondrial DNA (mtDNA)
Y-chromosome analysis
SNP genotyping
Y chromosome analysis
 The Y chromosome is passed directly from
the father to the son
 Analysis of genetic markers on the Y
chromosome is useful for tracing
relationships between males and for
analysing biological material from multiple
male contributors
DNA technologies used in forensic
investigations
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RFLP
PCR-RFLP
VNTRs
HLA-DQ
STR analysis
Mitochondrial DNA (mtDNA)
Y-chromosome analysis
SNP genotyping
SNP genotyping
Polymorphism analysis
chips
Polymorphism analysis chips
Polymorphism analysis:
SNP chromosomal coverage
chr 20
Polymorphism analysis chips
Comparison of DNA Typing Methods
and Power of Discrimination
Statistical and population issues
 The sib rule
– Upper limit of match probability
 Individualisation (uniqueness)
– frequency of a profile is considerably less than
the reciprocal of the population size: profile is
unique
 Identification on a database
Forensic DNA data bases
 Primary concern: privacy
 DNA provides information in relation to
– Genetic predisposition to disease
– Predisposition to behaviour
– Parentage
 Questions in relation to DNA storage and use
 STR DNA described as ‘junk DNA’: but could be
used for genetic susceptibility in the future
The future for Forensics
 RNA based Genomics
 Proteomics
RNA based approaches in
Forensic Medicine
 RNA to establish time of death
 RNA (cDNA) chip analyses: to look at gene
pathway dysfunction in death and in causes
of death
 Allelic expression analyses to forensically
identify persons
RNA degradation:
and cellular death
Intact RNA
Slightly degraded
Severely degraded
Signal intensities of 28 S and 18 S rRNA are reduced,
baseline is increased with degradation.
Using RNA
Sample
number
RNA
Concentrat
ion (ng/l)
A260:280
11124
674
2.0
RNA concentration = 700
rrna ratio [28s/18s] = 2
Agarose gel and UV spectroscopy
results and Agilent Bioanalyser
RNA degradation assays
Expression Arrays
Colour representation of Applied
Biosystems 1700 grid formation
and layout:
▲=fluorescent signals (used for
gridding and quantitation),
□ = control,
+ = probe/target.
Magnified area of a 1700 array –
demonstrating chemiluminescent
quantitative ladder(arrow).
Proteomic approaches in
Forensic Medicine
 Proteome signature profiling in death and in
the examination of the cause of death
 Proteome disease profiling
 Use of organ and disease specific protein
arrays
 Examining enzyme activity in the perimortem period
Protein identification workflow
Traditional protein identification
• 2-dimensional polyacrylamide gel electrophoresis
(2D-PAGE)
• Peptide separation by high-performance liquid
chromatography (HPLC)
• Electrospray ionization (ESI)
• Matrix-assisted laser desorption and ionization (MALDI) by
mass spectrometry
MALDI-TOF mass spectrometry
- Matrix Assisted Laser Desorption/IonizationTime of Flight Mass Spectrometry
Types of protein arrays
Antibody-Pair Protein Arrays
Single Antibody/Labeled Sample Protein Arrays
Cellular Lysate Protein Arrays
Peptide Arrays