Forenisc identification

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• Forenisc identification
• John J. OLeary
• MD, PhD, MSc, MA, FRCPath, FFPathRCPI, FTCD.
• Trinity College Dublin

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Topics

• Friction ridge identification
• Forensic dentistry
• Facial recognition and re-construction systems
• DNA fingerprinting

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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

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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.

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Fingerprints
Arch
Loop
Whorl
Arch tented arch
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Fingerprints
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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
  ante-mortem (prior to death) photographs.

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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.

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Facial recognition and reconstruction
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DNA fingerprinting
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DNA forensics

• DNA
• Chromosomes
• Nucleotides
• Adenosine (A)
• Guanine (G)
• Cytosine (C)
• Thymidine (T)

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The structure of DNA
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The structure of DNA
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The structure of DNA
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Every chromosome has a unique signature
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The sequence of DNA
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DNA-RNA-Protein
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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 individuals DNA/chromosomes come from
  the father the other half from the mother.

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DNA Review

• DNA is a double-stranded molecule.
• The DNA strands are made of four different
  building blocks.
• An individuals DNA remains the same throughout
  life.
• In specific regions on a DNA strand each person
  has a unique sequence of DNA or genetic code.

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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)

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Gene facts

• 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

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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)

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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

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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

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Factors Leading to DNA Degradation

• Time
• Temperature
• Humidity
• Light
• Exposure to chemicals

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DNA as Physical Evidence

• Perspective
• Recognition of Evidence
• Collection of Physical Evidence
• Preservation of Physical Evidence
• Preparation of the Physical Evidence
• Evaluation and Quantification of the Evidence

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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.

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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.

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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.

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Sources of DNA for Testing

• Blood
• Semen
• Tissue
• Bone (Marrow)
• Hair Root
• Saliva
• Urine
• Tooth (Pulp)

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How is DNA typing done

• Strict anti-contamination procedures
• Standard operating procedure for every forensic
  DNA test
• Dedicated laboratory facilities
• Contact DNA tracing

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DNA technologies used in forensic investigations

• RFLP
• PCR-RFLP
• STR analysis
• Mitochondrial DNA (mtDNA)
• Y-chromosome analysis
• SNP genotyping

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DNA technologies used in forensic investigations

• RFLP
• PCR-RFLP
• VNTRs
• HLA-DQ
• STR analysis
• Mitochondrial DNA (mtDNA)
• Y-chromosome analysis
• SNP genotyping

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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.

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Basis of RFLP analysis
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Basis of RFLP analysis
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A Schematic Representation of RFLP and Southern
Blot of a Single-locus VNTR
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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).
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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)---GAATTC- Jill 1 -GAATTC---(8.2
kb)---GCATGCATGCATGCATGCAT---(4.2 kb)---GAATTC-
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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)-GAATTC-Jill
2 -GAATTC--(1.8 kb)-GAATTC--(1.2
kb)--GCATGCATGCATGCATGCAT--(1.3 kb)-GAATTC-
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RFLP analysis
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Use of optimum number of loci for RFLP analysis
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DNA technologies used in forensic investigations

• RFLP
• PCR-RFLP
• VNTRs
• HLA-DQ
• STR analysis
• Mitochondrial DNA (mtDNA)
• Y-chromosome analysis
• SNP genotyping

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PCR -RFLP
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PCR -RFLP
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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

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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.

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A Typical DNA Profile
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DNA technologies used in forensic investigations

• RFLP
• PCR-RFLP
• VNTRs
• HLA-DQ
• STR analysis
• Mitochondrial DNA (mtDNA)
• Y-chromosome analysis
• SNP genotyping

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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

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DNA technologies used in forensic investigations

• RFLP
• PCR-RFLP
• VNTRs
• HLA-DQ
• STR analysis
• Mitochondrial DNA (mtDNA)
• Y-chromosome analysis
• SNP genotyping

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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)

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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

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Example of STR Multiplex
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The odds that 2 individuals will have the same 13
loci DNA profile is one in one billion
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Validation of STR Techniques

• 1991Fluorescent STR markers first described
• 1993First STR kit available
• 1996First multiplex STR kits available
• 199713 core STR loci defined Y-chromosome STR
  described
• 1999Multiplex STR kits validated
• 2000FBI and other labs stop running RFLP and
  convert to multiplex STRs.

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DNA technologies used in forensic investigations

• RFLP
• PCR-RFLP
• VNTRs
• HLA-DQ
• STR analysis
• Mitochondrial DNA (mtDNA)
• Y-chromosome analysis
• SNP genotyping

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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 mothers egg
• Fathers sperm contributes only nuclear DNA
• Important tool in missing person investigations

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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)

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DNA technologies used in forensic investigations

• RFLP
• PCR-RFLP
• VNTRs
• HLA-DQ
• STR analysis
• Mitochondrial DNA (mtDNA)
• Y-chromosome analysis
• SNP genotyping

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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

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DNA technologies used in forensic investigations

• RFLP
• PCR-RFLP
• VNTRs
• HLA-DQ
• STR analysis
• Mitochondrial DNA (mtDNA)
• Y-chromosome analysis
• SNP genotyping

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SNP genotyping
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Polymorphism analysis chips
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Polymorphism analysis chips
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Polymorphism analysis SNP chromosomal coverage
chr 20
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Polymorphism analysis chips
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Comparison of DNA Typing Methods and Power of
Discrimination
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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

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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

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The future for Forensics

• RNA based Genomics
• Proteomics

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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

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RNA degradationand cellular death
Signal intensities of 28 S and 18 S rRNA are
reduced, baseline is increased with degradation.
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Using RNA
Sample number RNA Concentration (ng/?l) A260280
11124 674 2.0
RNA concentration 700 rrna ratio 28s/18s 2
Agarose gel and UV spectroscopy results and
Agilent Bioanalyser
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RNA degradation assays
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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).
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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 peri-mortem
  period

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Protein identification workflow
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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

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MALDI-TOF mass spectrometry - Matrix Assisted
Laser Desorption/Ionization-Time of Flight Mass 
Spectrometry
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Types of protein arrays
Antibody-Pair Protein Arrays Single
Antibody/Labeled Sample Protein Arrays
Cellular Lysate Protein Arrays Peptide Arrays
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