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Forenisc identification John J. O’Leary 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: "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 ArchLoopWhorlArch – 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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Every chromosome has a unique signature
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The sequence of DNA
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RNA DNA Protein 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 individual’s 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 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.
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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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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) Gene facts: repetitive genome units
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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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Strict anti-contamination procedures Standard operating procedure for every forensic DNA test Dedicated laboratory facilities Contact DNA tracing How is DNA typing done
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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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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
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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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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:
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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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Polymerase Chain Reaction (PCR) 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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Molecpath
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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 1991—Fluorescent STR markers first described 1993—First STR kit available 1996—First multiplex STR kits available 1997—13 core STR loci defined; Y- chromosome STR described 1999—Multiplex STR kits validated 2000—FBI 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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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)
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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: 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 degradation: and cellular death Signal intensities of 28 S and 18 S rRNA are reduced, baseline is increased with degradation. Intact RNASlightly degradedSeverely degraded
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Using RNA Agarose gel and UV spectroscopy results and Agilent Bioanalyser Sample number RNA Concentrat ion (ng/ l) A260:280 111246742.0 RNA concentration = 700 rrna ratio [28s/18s] = 2
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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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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
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MALDI-TOF mass spectrometry - Matrix Assisted Laser Desorption/Ionization- Time of Flight Mass Spectrometry
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Antibody-Pair Protein Arrays Single Antibody/Labeled Sample Protein Arrays Types of protein arrays Cellular Lysate Protein Arrays Peptide Arrays
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