Forenisc identification John J. O’Leary MD, PhD, MSc, MA, FRCPath, FFPathRCPI, FTCD.

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

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


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

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

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

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

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.

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)

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

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

Forenisc identification John J. O’Leary MD, PhD, MSc, MA, FRCPath, FFPathRCPI, FTCD.

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