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

2. Topics  Friction ridge identification  Forensic dentistry  Facial recognition and re-construction systems  DNA fingerprinting

3. 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:

4. 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.

5. Fingerprints ArchLoopWhorlArch – tented arch

6. Fingerprints

7. 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.

8. 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.

9. Facial recognition and reconstruction

10. DNA fingerprinting

14. DNA forensics  DNA  Chromosomes  Nucleotides –Adenosine (A) –Guanine (G) –Cytosine (C) –Thymidine (T)

15. The structure of DNA

18. Every chromosome has a unique signature

19. The sequence of DNA

20. RNA DNA Protein DNA-RNA-Protein

21. 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.

22. 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.

24. 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)

25. 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

26.  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

27. 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

28. 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

29. Factors Leading to DNA Degradation  Time  Temperature  Humidity  Light  Exposure to chemicals

30. 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

31. 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.

32. 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.

33. 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.

34. Sources of DNA for Testing  Blood  Semen  Tissue  Bone (Marrow)  Hair Root  Saliva  Urine  Tooth (Pulp)

37.  Strict anti-contamination procedures  Standard operating procedure for every forensic DNA test  Dedicated laboratory facilities  Contact DNA tracing How is DNA typing done

38. DNA technologies used in forensic investigations  RFLP  PCR-RFLP  STR analysis  Mitochondrial DNA (mtDNA)  Y-chromosome analysis  SNP genotyping

39. DNA technologies used in forensic investigations  RFLP  PCR-RFLP  VNTRs  HLA-DQ  STR analysis  Mitochondrial DNA (mtDNA)  Y-chromosome analysis  SNP genotyping

40.  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

43. A Schematic Representation of RFLP and Southern Blot of a Single-locus VNTR

44. 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).

45. 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:

46. 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-

47. RFLP analysis

48. Use of optimum number of loci for RFLP analysis

49. DNA technologies used in forensic investigations  RFLP  PCR-RFLP  VNTRs  HLA-DQ  STR analysis  Mitochondrial DNA (mtDNA)  Y-chromosome analysis  SNP genotyping

50. Polymerase Chain Reaction (PCR) PCR -RFLP

52. 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

53. 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.

54. A Typical DNA Profile

55. Molecpath

56. DNA technologies used in forensic investigations  RFLP  PCR-RFLP  VNTRs  HLA-DQ  STR analysis  Mitochondrial DNA (mtDNA)  Y-chromosome analysis  SNP genotyping

57. 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

58. DNA technologies used in forensic investigations  RFLP  PCR-RFLP  VNTRs  HLA-DQ  STR analysis  Mitochondrial DNA (mtDNA)  Y-chromosome analysis  SNP genotyping

59. 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)

60. 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

61. Example of STR Multiplex

62. The odds that 2 individuals will have the same 13 loci DNA profile is one in one billion

63. 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.

64. DNA technologies used in forensic investigations  RFLP  PCR-RFLP  VNTRs  HLA-DQ  STR analysis  Mitochondrial DNA (mtDNA)  Y-chromosome analysis  SNP genotyping

65.  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)

66.  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)

67. DNA technologies used in forensic investigations  RFLP  PCR-RFLP  VNTRs  HLA-DQ  STR analysis  Mitochondrial DNA (mtDNA)  Y-chromosome analysis  SNP genotyping

68. 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

69. DNA technologies used in forensic investigations  RFLP  PCR-RFLP  VNTRs  HLA-DQ  STR analysis  Mitochondrial DNA (mtDNA)  Y-chromosome analysis  SNP genotyping

70. SNP genotyping

72. Polymorphism analysis chips

74. Polymorphism analysis: SNP chromosomal coverage chr 20

75. Polymorphism analysis chips

76. Comparison of DNA Typing Methods and Power of Discrimination

77. 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

78. 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

82. The future for Forensics  RNA based Genomics  Proteomics

83. 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

84. 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

85. 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

86. RNA degradation assays

87. 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).

88. 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

89. Protein identification workflow

90. 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

91. MALDI-TOF mass spectrometry - Matrix Assisted Laser Desorption/Ionization- Time of Flight Mass Spectrometry

92. Antibody-Pair Protein Arrays Single Antibody/Labeled Sample Protein Arrays Types of protein arrays Cellular Lysate Protein Arrays Peptide Arrays