forenisc identification john j. o’leary md, phd, msc, ma, frcpath, ffpathrcpi, ftcd
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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. - PowerPoint PPT PresentationTRANSCRIPT
Forenisc identification
John J. O’LearyMD, PhD, MSc, MA, FRCPath,
FFPathRCPI, FTCD.
Trinity College Dublin
TopicsTopics
Friction ridge identificationFriction ridge identification Forensic dentistryForensic dentistry Facial recognition and re-construction Facial recognition and re-construction
systemssystems DNA fingerprintingDNA fingerprinting
Forensic identificationForensic identification People can be identified by their fingerprints. We know this People can be identified by their fingerprints. We know this
due to the philosophy of Friction Ridge Identification which due to the philosophy of Friction Ridge Identification which states: states:
"Friction ridge identification is established through the "Friction ridge identification is established through the agreement of friction ridge formations, in sequence, having agreement of friction ridge formations, in sequence, having sufficient uniqueness to individualize". Friction ridge sufficient uniqueness to individualize". Friction ridge identification is also governed by four premises or identification is also governed by four premises or statements of fact:statements of fact:
Friction ridgesFriction ridges
1. Friction ridges develop on the fetus in their 1. Friction ridges develop on the fetus in their definitive form prior to birth. definitive form prior to birth.
2. Friction ridges are persistent throughout life 2. Friction ridges are persistent throughout life except for permanent scarring, disease or except for permanent scarring, disease or decomposition after death. decomposition after death.
3. Friction ridge paths and the details in small areas 3. Friction ridge paths and the details in small areas of friction ridges are unique and never repeated. of friction ridges are unique and never repeated.
4. Overall friction ridge patterns vary within limits 4. Overall friction ridge patterns vary within limits which allow for classification. which allow for classification.
FingerprintsFingerprints
Arch Loop Whorl Arch – tented arch
FingerprintsFingerprints
Forensic dentistryForensic dentistry
Forensic dentistryForensic dentistry or or forensic odontologyforensic odontology is the is the proper handling, examination and evaluation of proper handling, examination and evaluation of dental evidence. The evidence that may be dental evidence. The evidence that may be derived from teeth, is the age (in children) and derived from teeth, is the age (in children) and identification of the person to whom the teeth identification of the person to whom the teeth belong. This is done using dental records or ante-belong. This is done using dental records or ante-mortem (prior to death) photographs. mortem (prior to death) photographs.
Facial recognition systemFacial recognition system
A A facial recognition systemfacial recognition system is a computer is a computer application for automatically identifying or application for automatically identifying or verifying a person from a digital image or a verifying a person from a digital image or a video frame from a video source. One of the video frame from a video source. One of the ways to do this is by comparing selected ways to do this is by comparing selected facial features from the image and a facial facial features from the image and a facial database. database.
Facial recognition and reconstructionFacial recognition and reconstruction
DNA fingerprintingDNA fingerprinting
DNA forensicsDNA forensics
DNADNA ChromosomesChromosomes NucleotidesNucleotides
– Adenosine (A)Adenosine (A)– Guanine (G)Guanine (G)– Cytosine (C)Cytosine (C)– Thymidine (T)Thymidine (T)
The structure of DNAThe structure of DNA
The structure of DNAThe structure of DNA
The structure of DNAThe structure of DNA
Every chromosome Every chromosome has a unique has a unique
signaturesignature
The sequence of DNAThe sequence of DNA
RNA
DNA
Protein
DNA-RNA-ProteinDNA-RNA-Protein
What is DNA?What is DNA?
DNA is the chemical substance which makes up DNA is the chemical substance which makes up our chromosomes and controls all inheritable our chromosomes and controls all inheritable traits (eye, hair and skin color)traits (eye, hair and skin color)
DNA is different for every individual except DNA is different for every individual except identical twinsidentical twins
DNA is found in all cells with a nucleus (white DNA is found in all cells with a nucleus (white blood cells, soft tissue cells, bone cells, hair blood cells, soft tissue cells, bone cells, hair root cells and spermatozoa)root cells and spermatozoa)
Half of a individual’s DNA/chromosomes come Half of a individual’s DNA/chromosomes come from the father & the other half from the mother.from the father & the other half from the mother.
DNA Review:DNA Review:
DNA is a double-stranded molecule.DNA is a double-stranded molecule. The DNA strands are made of four different The DNA strands are made of four different
building blocks.building blocks. An individual’s DNA remains the same An individual’s DNA remains the same
throughout life.throughout life. In specific regions on a DNA strand each In specific regions on a DNA strand each
person has a unique sequence of DNA or person has a unique sequence of DNA or genetic code.genetic code.
Chromosome factsChromosome facts Number of chromosome = 46Number of chromosome = 46
– 22 autosomes and 2 sex chromosomes22 autosomes and 2 sex chromosomes One chromosome of each pair donated from One chromosome of each pair donated from
parents sperm or eggparents sperm or egg Sex chromosomes Sex chromosomes
– XY male: XX femaleXY male: XX female Largest chromosome: chr 1-263 million base pairs Largest chromosome: chr 1-263 million base pairs
(bp)(bp) Smallest chromosome: chr Y - 59 million base Smallest chromosome: chr Y - 59 million base
pairs (bp)pairs (bp)
Gene factsGene facts
Human genome = 3.4 billion base pairsHuman genome = 3.4 billion base pairs Number of human genes: approx 100,000Number of human genes: approx 100,000 Genes vary in length: average 3,000 bpGenes vary in length: average 3,000 bp Only 5% of human genome is coding and contains Only 5% of human genome is coding and contains
genesgenes Genes divided into exons and intronsGenes divided into exons and introns Much of the function of the genome unknownMuch of the function of the genome unknown 0.1% difference in DNA between individuals0.1% difference in DNA between individuals
MinisatellitesMinisatellites are molecular marker loci consisting of are molecular marker loci consisting of tandem repeat units of a 10-50 base motif, flanked by tandem repeat units of a 10-50 base motif, flanked by conserved endonuclease restriction sitesconserved endonuclease restriction sites– DNA fingerprinting DNA fingerprinting – VNTR (Variable Number of Tandem Repeats) VNTR (Variable Number of Tandem Repeats)
MicrosatellitesMicrosatellites are simple sequence tandem repeats are simple sequence tandem repeats (SSTRs). The repeat units are generally di-, tri- tetra- or (SSTRs). The repeat units are generally di-, tri- tetra- or pentanucleotides. For example, a common repeat motif in pentanucleotides. For example, a common repeat motif in birds is ACn, where the two nucleotides A and C are birds is ACn, where the two nucleotides A and C are repeated in bead-like fashion a variable number of times (repeated in bead-like fashion a variable number of times (nn could range from 8 to 50)could range from 8 to 50)– Simple sequence repeats (SSR)Simple sequence repeats (SSR)– Simple sequence length polymorphisms (SSLP) Simple sequence length polymorphisms (SSLP)
Gene facts: repetitive genome unitsGene facts: repetitive genome units
Other important gene regionsOther important gene regions Single nucleotide polymorphisms or Single nucleotide polymorphisms or SNPs SNPs
(pronounced "snips") are DNA sequence (pronounced "snips") are DNA sequence variations that occur when a single nucleotide variations that occur when a single nucleotide (A,T,C,or G) in the genome sequence is altered. (A,T,C,or G) in the genome sequence is altered. For example a SNP might change the DNA For example a SNP might change the DNA sequence Asequence AAAGGCTAA to AGGCTAA to ATTGGCTAA. GGCTAA.
For a variation to be considered a SNP, it must For a variation to be considered a SNP, it must occur in at least 1% of the population. occur in at least 1% of the population.
SNPs, which make up about 90% of all human SNPs, which make up about 90% of all human genetic variation, occur every 100 to 300 bases genetic variation, occur every 100 to 300 bases along the 3-billion-base human genome along the 3-billion-base human genome
Use of DNA forensicsUse of DNA forensics
Identification purposesIdentification purposes Identify crime suspectsIdentify crime suspects Exonerate persons wrongly accused of Exonerate persons wrongly accused of
crimecrime Identify crime and catastrophe victimsIdentify crime and catastrophe victims Establish paternity and other family Establish paternity and other family
relationshipsrelationships
Factors Leading to DNA DegradationFactors Leading to DNA Degradation
TimeTime TemperatureTemperature HumidityHumidity LightLight Exposure to chemicalsExposure to chemicals
DNA as Physical EvidenceDNA as Physical Evidence
PerspectivePerspective Recognition of EvidenceRecognition of Evidence Collection of Physical EvidenceCollection of Physical Evidence Preservation of Physical EvidencePreservation of Physical Evidence Preparation of the Physical EvidencePreparation of the Physical Evidence Evaluation and Quantification of the Evaluation and Quantification of the
EvidenceEvidence
Individualization:Individualization:
Evidence that exhibit traits that are are so Evidence that exhibit traits that are are so unique that when considered alone or in unique that when considered alone or in combination with other traits can reduce the combination with other traits can reduce the evidence source from a class to one evidence source from a class to one individual.individual.
Evidence that can indicate that two samples Evidence that can indicate that two samples share a common unique source or origin.share a common unique source or origin.
Association:Association:
““Description of the relationship between two Description of the relationship between two objects items, or people.”objects items, or people.”
Concept used in a crime scene analysis for Concept used in a crime scene analysis for reconstruction.reconstruction.
““Involves the evaluation of evidence to infer Involves the evaluation of evidence to infer a common source.”a common source.”
Does not prove a crime.Does not prove a crime.
Traits that Indicate IndividualityTraits that Indicate Individuality
Fingerprints - are a result of several genes Fingerprints - are a result of several genes and other non-genetic events. Has been and other non-genetic events. Has been accepted as unique for each individual accepted as unique for each individual (even identical twins) (even identical twins)
DNA - early results suggested individuality DNA - early results suggested individuality except in identical twins; but in reality more except in identical twins; but in reality more like a partial print.like a partial print.
Sources of DNA for TestingSources of DNA for Testing
BloodBlood SemenSemen TissueTissue Bone (Marrow)Bone (Marrow) Hair RootHair Root SalivaSaliva UrineUrine Tooth (Pulp)Tooth (Pulp)
Strict anti-contamination proceduresStrict anti-contamination procedures Standard operating procedure for every Standard operating procedure for every
forensic DNA testforensic DNA test Dedicated laboratory facilitiesDedicated laboratory facilities Contact DNA tracingContact DNA tracing
How is DNA typing doneHow is DNA typing done
DNA technologies used in forensic DNA technologies used in forensic investigationsinvestigations
RFLPRFLP PCR-RFLPPCR-RFLP STR analysisSTR analysis Mitochondrial DNA (mtDNA)Mitochondrial DNA (mtDNA) Y-chromosome analysisY-chromosome analysis SNP genotypingSNP genotyping
DNA technologies used in forensic DNA technologies used in forensic investigationsinvestigations
RFLPRFLP PCR-RFLPPCR-RFLP VNTRsVNTRs HLA-DQHLA-DQ STR analysisSTR analysis Mitochondrial DNA (mtDNA)Mitochondrial DNA (mtDNA) Y-chromosome analysisY-chromosome analysis SNP genotypingSNP genotyping
Restriction Enzymes (biological Restriction Enzymes (biological catalysts) cut DNA whenever they catalysts) cut DNA whenever they encounter a specific DNA sequence.encounter a specific DNA sequence.
Gel electrophoresis separates the Gel electrophoresis separates the fragments of DNA according to their fragments of DNA according to their length.length.
Basis of RFLP analysisBasis of RFLP analysis
Basis of RFLP analysisBasis of RFLP analysis
Basis of RFLP analysisBasis of RFLP analysis
A Schematic Representation of RFLP and A Schematic Representation of RFLP and Southern Blot of a Single-locus VNTRSouthern 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).
Jack 1: -GAATTC---(8.2 kb)---GCATGCATGCATGCATGCAT---(4.2 kb)---GAATTC-
Jill 1: -GAATTC---(8.2 kb)---GCATGCATGCATGCATGCAT---(4.2 kb)---GAATTC-
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:
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-
RFLP analysisRFLP analysis
Use of optimum number of loci Use of optimum number of loci for RFLP analysisfor RFLP analysis
DNA technologies used in forensic DNA technologies used in forensic investigationsinvestigations
RFLPRFLP PCR-RFLPPCR-RFLP VNTRsVNTRs HLA-DQHLA-DQ STR analysisSTR analysis Mitochondrial DNA (mtDNA)Mitochondrial DNA (mtDNA) Y-chromosome analysisY-chromosome analysis SNP genotypingSNP genotyping
Polymerase Chain Reaction (PCR)
PCR -RFLPPCR -RFLP
PCR -RFLPPCR -RFLP
In 1984, In 1984, Alec JeffreysAlec Jeffreys developed developed “DNA Fingerprinting”“DNA Fingerprinting”
Was searching for disease markersWas searching for disease markers Applied the technique to personal Applied the technique to personal
identificationidentification Demonstrated that the DNA could be Demonstrated that the DNA could be
retrieved from old dried blood stainsretrieved from old dried blood stains Applied the technique to high-profile Applied the technique to high-profile
forensic testsforensic tests
RFLP Methods: commentaryRFLP Methods: commentary
Have a high power of discrimination: Have a high power of discrimination: 20-80 different alleles may be possible at 20-80 different alleles may be possible at one location; analyzed in combination can one location; analyzed in combination can be used to determine an individualized be used to determine an individualized type.type.
RFLP procedures are labor intensive: RFLP procedures are labor intensive: multi-locus probes are difficult to multi-locus probes are difficult to automate: single-locus probes can be automate: single-locus probes can be used in serial fashion.used in serial fashion.
Require ample supply of high grade DNA.Require ample supply of high grade DNA.
A Typical DNA ProfileA Typical DNA Profile
Molecpath
DNA technologies used in forensic DNA technologies used in forensic investigationsinvestigations
RFLPRFLP PCR-RFLPPCR-RFLP VNTRsVNTRs HLA-DQHLA-DQ STR analysisSTR analysis Mitochondrial DNA (mtDNA)Mitochondrial DNA (mtDNA) Y-chromosome analysisY-chromosome analysis SNP genotypingSNP genotyping
VNTRsVNTRs(variable number of tandem repeats)(variable number of tandem repeats) MinisatellitesMinisatellites are molecular marker loci are molecular marker loci
consisting of tandem repeat units of a 10-50 consisting of tandem repeat units of a 10-50 base motif, flanked by conserved base motif, flanked by conserved endonuclease restriction sitesendonuclease restriction sites– VNTR (Variable Number of Tandem Repeats)VNTR (Variable Number of Tandem Repeats)
Popular from 1985-1995Popular from 1985-1995 Required relatively large amounts of DNARequired relatively large amounts of DNA
DNA technologies used in forensic DNA technologies used in forensic investigationsinvestigations
RFLPRFLP PCR-RFLPPCR-RFLP VNTRsVNTRs HLA-DQHLA-DQ STR analysisSTR analysis Mitochondrial DNA (mtDNA)Mitochondrial DNA (mtDNA) Y-chromosome analysisY-chromosome analysis SNP genotypingSNP genotyping
STRsSTRs
Short Tandem Repeats: Repeating units of Short Tandem Repeats: Repeating units of an identical DNA sequence, length is often an identical DNA sequence, length is often between 2 – 5 bp in length. The repeat between 2 – 5 bp in length. The repeat units are arranged in direct succession of units are arranged in direct succession of each other, and the number of repeat units each other, and the number of repeat units varies between individuals (subgroup of varies between individuals (subgroup of VNTRs)VNTRs)
Multiplex STRsMultiplex STRs
High power of DiscriminationHigh power of Discrimination Rapid AnalysisRapid Analysis Analysis can be automated and 3 or more Analysis can be automated and 3 or more
locations can be analyzed at a time.locations can be analyzed at a time. FBI (USA) uses 13 specific STR regions for FBI (USA) uses 13 specific STR regions for
CODISCODIS 6 of the 13 loci are used by the British 6 of the 13 loci are used by the British
Forensic Science ServiceForensic Science Service
Example of STR Multiplex Example of STR Multiplex
The odds that 2 individuals will have the same 13 loci DNA profileis one in one billion
Validation of STR TechniquesValidation of STR Techniques 1991—Fluorescent STR markers first 1991—Fluorescent STR markers first
describeddescribed 1993—First STR kit available1993—First STR kit available 1996—First multiplex STR kits available1996—First multiplex STR kits available 1997—13 core STR loci defined; Y-1997—13 core STR loci defined; Y-
chromosome STR describedchromosome STR described 1999—Multiplex STR kits validated1999—Multiplex STR kits validated 2000—FBI and other labs stop running RFLP 2000—FBI and other labs stop running RFLP
and convert to multiplex STRs.and convert to multiplex STRs.
DNA technologies used in forensic DNA technologies used in forensic investigationsinvestigations
RFLPRFLP PCR-RFLPPCR-RFLP VNTRsVNTRs HLA-DQHLA-DQ STR analysisSTR analysis Mitochondrial DNA (mtDNA)Mitochondrial DNA (mtDNA) Y-chromosome analysisY-chromosome analysis SNP genotypingSNP genotyping
Can be used on samples not suitable for RFLP or Can be used on samples not suitable for RFLP or STR analysisSTR analysis
mtDNA is present in mitochondriamtDNA is present in mitochondria All mothers have the same mtDNA as their All mothers have the same mtDNA as their
daughtersdaughters The mitochondria of each new embryo comes from The mitochondria of each new embryo comes from
the mother’s eggthe mother’s egg Father’s sperm contributes only nuclear DNAFather’s sperm contributes only nuclear DNA Important tool in missing person investigationsImportant tool in missing person investigations
Mitochondrial DNA (mtDNA)Mitochondrial DNA (mtDNA)
Mitochondrial DNA (mtDNA)Mitochondrial DNA (mtDNA)
Lowest power of discriminationLowest power of discrimination Longest sample processing timeLongest sample processing time Can be very helpful in forensic cases Can be very helpful in forensic cases
involving severely degraded DNA involving severely degraded DNA samplessamples
Sometimes mitochondria are Sometimes mitochondria are heteroplasmic (more than one kind of heteroplasmic (more than one kind of mitochondria in a person or in a cell)mitochondria in a person or in a cell)
DNA technologies used in forensic DNA technologies used in forensic investigationsinvestigations
RFLPRFLP PCR-RFLPPCR-RFLP VNTRsVNTRs HLA-DQHLA-DQ STR analysisSTR analysis Mitochondrial DNA (mtDNA)Mitochondrial DNA (mtDNA) Y-chromosome analysisY-chromosome analysis SNP genotypingSNP genotyping
Y chromosome analysisY chromosome analysis
The Y chromosome is passed directly from The Y chromosome is passed directly from the father to the sonthe father to the son
Analysis of genetic markers on the Y Analysis of genetic markers on the Y chromosome is useful for tracing chromosome is useful for tracing relationships between males and for relationships between males and for analysing biological material from multiple analysing biological material from multiple male contributorsmale contributors
DNA technologies used in forensic DNA technologies used in forensic investigationsinvestigations
RFLPRFLP PCR-RFLPPCR-RFLP VNTRsVNTRs HLA-DQHLA-DQ STR analysisSTR analysis Mitochondrial DNA (mtDNA)Mitochondrial DNA (mtDNA) Y-chromosome analysisY-chromosome analysis SNP genotypingSNP genotyping
SNP genotypingSNP genotyping
Polymorphism analysis Polymorphism analysis chipschips
Polymorphism analysis chipsPolymorphism analysis chips
Polymorphism analysis: Polymorphism analysis: SNP chromosomal coverageSNP chromosomal coverage
chr 20
Polymorphism analysis chipsPolymorphism analysis chips
Comparison of DNA Comparison of DNA TypingTyping Methods Methods and Power of Discriminationand Power of Discrimination
Statistical and population issuesStatistical and population issues
The sib ruleThe sib rule– Upper limit of match probabilityUpper limit of match probability
Individualisation (uniqueness)Individualisation (uniqueness)– frequency of a profile is considerably less than frequency of a profile is considerably less than
the reciprocal of the population size: profile is the reciprocal of the population size: profile is uniqueunique
Identification on a databaseIdentification on a database
Forensic DNA data basesForensic DNA data bases
Primary concern: privacyPrimary concern: privacy DNA provides information in relation toDNA provides information in relation to
– Genetic predisposition to diseaseGenetic predisposition to disease– Predisposition to behaviourPredisposition to behaviour– ParentageParentage
Questions in relation to DNA storage and useQuestions in relation to DNA storage and use STR DNA described as ‘junk DNA’: but could be STR DNA described as ‘junk DNA’: but could be
used for genetic susceptibility in the futureused for genetic susceptibility in the future
The future for ForensicsThe future for Forensics
RNA based GenomicsRNA based Genomics ProteomicsProteomics
RNA based approaches in RNA based approaches in Forensic MedicineForensic Medicine
RNA to establish time of deathRNA to establish time of death RNA (cDNA) chip analyses: to look at gene RNA (cDNA) chip analyses: to look at gene
pathway dysfunction in death and in causes pathway dysfunction in death and in causes of deathof death
Allelic expression analyses to forensically Allelic expression analyses to forensically identify personsidentify persons
RNA degradation:RNA degradation:and cellular deathand cellular death
Signal intensities of 28 S and 18 S rRNA are reduced, baseline is increased with degradation.
Intact RNA Slightly degraded Severely degraded
Using RNAUsing RNA
Agarose gel and UV spectroscopy results and
Agilent Bioanalyser
Sample Sample numbernumber
RNA RNA ConcentratConcentration (ng/ion (ng/l)l)
A260:280A260:280
1112411124 674674 2.02.0
RNA concentration = 700rrna ratio [28s/18s] = 2
RNA degradation assaysRNA degradation assays
Expression ArraysExpression 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 Proteomic approaches in Forensic MedicineForensic Medicine
Proteome signature profiling in death and in Proteome signature profiling in death and in the examination of the cause of deaththe examination of the cause of death
Proteome disease profilingProteome disease profiling Use of organ and disease specific protein Use of organ and disease specific protein
arraysarrays Examining enzyme activity in the peri-Examining enzyme activity in the peri-
mortem periodmortem period
Protein identification workflowProtein identification workflow
• 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
Traditional protein identification
MALDI-TOF mass spectrometry - Matrix Assisted Laser Desorption/Ionization-
Time of Flight Mass Spectrometry
Antibody-Pair Protein Arrays
Single Antibody/Labeled Sample Protein Arrays
Types of protein arrays
Cellular Lysate Protein Arrays
Peptide Arrays