Practical tasks: 0. Sample collection on the crime scene 1.DNA extraction 2.DNA amplification (PCR) 3.DNA staining (gel electrophoresis) 4.Analysis of samples

„People lie but evidence doesn’t lie” Types of evidences: -indirect (e.g. photo) -direct (e.g. hair) -„cold evidence” (hair, textile) -„hot evidence” (DNA)

Human Identity Testing or DNA Fingerprinting Forensic cases -- matching suspect with evidence Paternity testing -- identifying father Mass disasters -- putting pieces back together Historical investigations Missing persons investigations Military DNA “dog tag” Convicted felon DNA databases Involves generation of DNA profiles usually with the same core STR (short tandem repeat) markers

Forensic Sciences 1.Forensic Psychiatry and Mental Illness 2.Forensic Pathology 3.Forensic Engineering 4.Forensic Toxicology 5.Forensic Criminalistics 6.Forensic Entomology 7.Forensic Odontology 8.Forensic Epidemiology etc.

Basis of DNA Profiling The genome of each individual is unique (with the exception of identical twins!) and is inherited from parents Probe subsets of genetic variation in order to differentiate between individuals (statistical probabilities of a random match are used) DNA typing must be performed efficiently and reproducibly (information must hold up in court) Current standard DNA tests do not look at genes – little/no information about race, predisposal to disease, or phenotypical information (eye color, height, hair color) is obtained, but 3rd generation sequencing techniques do it in the future?

First cases for DNA identification

Brief History of Forensic DNA Methods 1900s: Landsteiner: Discovered the ABO blood groups: revolution in forensic methodology. Power of discrimination: s: RFLP+DNA detected via Southern blotting. Power of discrimination: in the range of for a six probe analysis. Alec Jeffreys developed first “DNA Profiling” for disease markers Mid-1980s: The Colin Pitchfork Case in the UK: the first DNA evidance used by court. Two young women raped and murdered in Narborough, England. 5,000 local men are asked to provide blood/saliva samples.1st exoneration and conviction on forensic DNA evidence by Jeffreys Problems with RFLP testing requires a relatively large amount of HMW DNA (~50ng = thousands of cells). Not ideal for forensic evidence, in which small, degraded samples are common Sir Alec Jeffreys

1984: developing of PCR by Karry Mullis Works with lower quantity (1-2ng), lower quality samples, than RFLP But power of discrimination goes from not good enough for databasing 1986: PCR on STR: Non-coding, 4-7 nucleotide sequences which vary greatly from person to person in the number of repeating units! Requires <1ng of DNA to type STR loci, power of discrimination ranges from World population is 10 9 so bring on the database! 1987 FBI with NIH began collaborative research to establish DNA identification techniques: The Combined DNA Index System (CoDIS): A database of DNA profiles from violent felons and crime scene samples. Database currently contains about 9M data from crime scenes. 1990s: DNA analysis was considered an “infallible” prosecution tool. “In rape cases, when the semen has been matched with the defendant’s and the chance that it came from another person is 33 billion to 1, you don’t need a jury.” Robert Brower, defense attorney. Brief History of Forensic DNA Methods

The O.J. Simpson case On June 12, 1994, O.J.’s ex girlfriend Nicole Brown and her new friend Ronald Goldman were found dead outside Brown's condominium The trial of the century convened: “Dollars v DNA” or California v OJ Simpson. 1995: OJ Simpson verdict: 'Not guilty'

Speed of Analysis (Technology) Power of Discrimination (Genetics) Low High SlowFast Comparison of Markers Used in Forensic Biology RFLP Multi-Locus Probes ABO blood groups Multiplex PCR of STRs mtDNA PCR Figure 1.1, J.M. Butler (2005) Forensic DNA Typing, 2 nd Edition © 2005 Elsevier Academic Press

Forensic DNA testing systems today: STR: DNA regions with short repeat units (usually 2-6 bp in length) are called Short Tandem Repeats (STR). STRs are found surrounding the chromosomal centromere (the structural center of the chromosomes) STR is the standard DNA testing system for human identification. Beginning in 1996, the FBI Laboratory launched a nationwide forensic science effort to establish core STR loci for inclusion within the national database known as CODIS (Combined DNA Index System). Y-STR: STR found on the male specific Y-Chromosome. It is inherited through the male lineage. Y-STR can be used for sexual assault and other cases where identifying the males contributing to the sample is critical to the case. Mitochondrial DNA: Found in non-nucleic cells such as hair shaft with little or no root tissues. mtDNA is inherited through the female line, but can be found in both females and males. mtDNA is used to test difficult samples such as hair, bone and teeth, from which degraded DNA or non-nucleic DNA is found. It is also used for historically important cases like the Romanovs and the unknown soldier from the Vietnam war. Mini-STR: This testing system is an alternative approach developed for testing small fragments of DNA, and is especially useful for degraded biological evidence. Difficult samples, such as those recovered from mass disasters like the World Trade Center, can be successfully analyzed with mini-STR. SNPs: single nt polymorphism

Minisatellite Marker (D1S80) GAGGACCACCAGGAAG Repeat region Flanking regions 16 bp repeat unit STR Marker (TH01) TCAT Repeat region Flanking regions 4 bp repeat unit Figure 5.1, J.M. Butler (2005) Forensic DNA Typing, 2 nd Edition © 2005 Elsevier Academic Press

2 repeats 3 repeats AGACTAGACATT AGATTAGGCATT (AATG)(AATG)(AATG) (AATG)(AATG) (A) Length polymorphism: VNTR or STR – Technique: (VNTR-PCR) (B) Sequence polymorphism: SNP – Technique: (AS-PCR) Figure 2.5, J.M. Butler (2005) Forensic DNA Typing, 2 nd Edition © 2005 Elsevier Academic Press Polymorphism on the homologe chromosome

„Genghis Khan” argument: Lower power of discrimination - paternal relatives all share the same Y-STR haplotype (10% of Central Asian males share the same Y- STR haplotype, thought to belong to Genghis Khan) Y-STRs Problem: ~99% of classical violent crimes are committed by men DNA Mixtures of male suspect and female victim can pose an analytical challenge, especially when the female contribution is much greater than the male = preferential amplification Test for markers found only on the Y-chromosome. Only male DNA is amplified

Heavy (H) strand Light (L) strand Figure 10.1, J.M. Butler (2005) Forensic DNA Typing, 2 nd Edition © 2005 Elsevier Academic Press Hypervariable Region2 Hypervariable Region1 Mitochondria Pros Single-cell sensitivity because each cell contains ~1000 mitochondria Especially useful for shed hairs, burnt remains Can be used to establish kinship directly because entire complement of mtDNA is maternally inherited Pros Single-cell sensitivity because each cell contains ~1000 mitochondria Especially useful for shed hairs, burnt remains Can be used to establish kinship directly because entire complement of mtDNA is maternally inherited Cons Heteroplasmy - more than one mtDNA type manifesting in different tissues in the same individual Lower power of discrimination - maternal relatives all share the same mtDNA Cons Heteroplasmy - more than one mtDNA type manifesting in different tissues in the same individual Lower power of discrimination - maternal relatives all share the same mtDNA Mitochondrial DNA (mtDNA)

Single Nucleotide Polymorphisms (SNPs) Point mutations (base substitutions) found in 1% or more of the population 5 million identified in human genome Detected on micro-array plates with fluorescent tags (all or nothing response) ~50 SNPs provides same power of discrimination as 13 STR loci Certain SNPs used as predictors of ancestry/ethnicity by a private sector lab (DNA Witness)

Sources of our Biological Evidence Blood Semen Saliva Urine Hair Teeth Bone Tissue Blood stain Only a very small amount of blood (3ul) is needed to obtain a DNA profile

ORGANIC Filter Paper CHELEX Blood stain PUNCH WASH Multiple Times with extraction buffer PERFORM PCR PCR Reagents SDS, DTT, EDTA and proteinase K INCUBATE (56 o C) Phenol, chloroform, isoamyl alcohol QUANTITATE DNA Apply blood to paper and allow stain to dry Blood stain VORTEX (NO DNA QUANTITATION TYPICALLY PERFORMED WITH UNIFORM SAMPLES) Water INCUBATE (ambient) 5% Chelex INCUBATE (100 o C) REMOVE supernatant INCUBATE (56 o C) QUANTITATE DNA PERFORM PCR Centrifuge REMOVE supernatant TRANSFER aqueous (upper) phase to new tube CONCENTRATE sample (Centricon/Microcon-100 or ethanol precipitation) Centrifuge TE buffer Figure 3.1, J.M. Butler (2005) Forensic DNA Typing, 2 nd Edition © 2005 Elsevier Academic Press DNA-extraction protocols

Perpetrator’s sperm mixed with victim’s epithelial cells Centrifuge REMOVE supernatant SDS, EDTA and proteinase K (cell lysis buffer) Remove a portion of the mixed stain SDS, EDTA and proteinase K + DTT Incubate at 37 o C sperm pellet DTT lyses sperm heads “Male Fraction” “Female Fraction” sperm pellet Figure 3.2, J.M. Butler (2005) Forensic DNA Typing, 2 nd Edition © 2005 Elsevier Academic Press Differential DNA extraction of sperms from victim’s epithelial cells DTT: Dithiothreitol: breaks down bisulfide bonds of sperm head

Laser Inlet Buffer Capillary filled with polymer solution 5-20 kV -+ Outlet Buffer Sample tray Detectio n window (cathode) (anode) Data Acquisition Sample tray moves automatically beneath the cathode end of the capillary to deliver each sample in succession Capillaries Electrodes for Injection Capillary Array Electrophoresis What is the technical basis of STR product differentiation? Primers are labeled

CODIS (Combined DNA Index System, developed by FBI and NIH) Sex specific marker Chromosome numb.

The new expanded STR loci used by FBI, InterPol, etc. FBI’s core STR Loci: 13

D3S1358 (8 alleles) VWA (14 alleles) D16S539 (9 alleles) D2S1338 (14 alleles) Blue panel Green panel Yellow panel Orange panel D21S11 (24 alleles) D8S1179 (12 alleles) D18S51 (23 alleles) TH01 (10 alleles) FGA low (19 alleles) FGA high (9 alleles) 250 bp* 139bp 200 bp 160 bp 300 bp 340 bp 350 bp 150 bp LIZ-labeled GS500 DNA sizing standard 100 bp Red panel D19S433 (15 alleles) D5S818 (10 alleles) TPOX (8 alleles) D13S317 (8 alleles) D7S820 (10 alleles) CSF1PO (10 alleles) AMEL (2 alleles) Figure 5.6, J.M. Butler (2005) Forensic DNA Typing, 2 nd Edition © 2005 Elsevier Academic Press

How do we interprete the CODIS file STR data? GeneScan view Genotyper view Allele call (repeat number) determined by comparison of peak size (bp) to allelic ladder allele peak sizes run under the same electrophoretic conditions Peak height in relative fluorescence units (RFUs)

Forensic science of future or today? A Genome-Wide Association Study Identified Five Loci (PRDM16, PAX3, TP63, C5orf50, and COL17A1) Influencing Facial Morphology in Europeans. e.g.:PAX3 influencing the position of the nasion Citation: Liu F, van der Lijn F, Schurmann C, Zhu G, Chakravarty MM, et al. (2012) A Genome-Wide Association Study Identifies Five Loci Influencing Facial Morphology in Europeans. PLoS Genet 8(9): e