Practical tasks:

0. Sample collection on the crime scene1. 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

Involves generation of DNA profiles usually with the same core STR (short tandem repeat) markers

Forensic Sciences1. Forensic Psychiatry and Mental Illness 2. Forensic Pathology3. Forensic Engineering4. Forensic Toxicology5. Forensic Criminalistics6. Forensic Entomology7. Forensic Odontology8. 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: 10-3

1980s: RFLP+DNA detected via Southern blotting. Power of discrimination: in the range of 106-108 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 102-106...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 13-15 STR loci, power of discrimination ranges from 1014-1023. World population is 109 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

Slow Fast

Comparison of Markers Used in Forensic Biology

Comparison of Markers Used in Forensic Biology

RFLPMulti-Locus Probes

ABO blood groups

Multiplex PCRof STRs

mtDNA PCR

Figure 1.1, J.M. Butler (2005) Forensic DNA Typing, 2nd 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

http://www.forensicdnacenter.com

Minisatellite Marker (D1S80)

GAGGACCACCAGGAAGGAGGACCACCAGGAAG

Repeat region

Flanking regions

16 bp repeat unit

STR Marker (TH01)

TCATTCAT

Repeat region

Flanking regions

4 bp repeat unit

Figure 5.1, J.M. Butler (2005) Forensic DNA Typing, 2nd 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, 2nd 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

Control region (D-loop)

1/16,569

cyt b

ND5ND6

ND4

ND4L

ND3

COIIIATP6

ATP8 COII

12S rRNA

16S rRNA

ND1

ND2

COI

OH

9-bp deletion

OL

F

V

L1

IQ

M

W

AN

CY

S1

DK

G

R

HS2

L2

E

P

T

HV1 HV2

16024 16365 73 340

16024 576

“16,569” bp

1

22 tRNAs

2 rRNAs

13 genesHeavy (H) strand

Light (L) strand

Figure 10.1, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

Hypervariable Region2Hypervariable 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

ConsHeteroplasmy - more than one mtDNA type manifesting in different tissues in the same individualLower 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

CHELEXBlood stain

PUNCH

WASH Multiple Times with extraction buffer

PERFORM PCR

PCR Reagents

SDS, DTT, EDTA and proteinase K

INCUBATE (56 oC)

Phenol,chloroform,

isoamyl alcohol

QUANTITATE DNA

Apply blood to paper and allow

stain to dryBlood stain

VORTEX

(NO DNA QUANTITATION TYPICALLY PERFORMED WITH UNIFORM SAMPLES)

Water

INCUBATE (ambient)

5% Chelex

INCUBATE (100 oC)

REMOVE supernatant

INCUBATE (56 oC)

QUANTITATE DNA

PERFORM PCRPERFORM PCR

Centrifuge

Centrifuge

Centrifuge

Centrifuge

REMOVE supernatantTRANSFER 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, 2nd 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 oC

sperm pellet

DTT lyses sperm heads

“Male Fraction” “Female Fraction”sperm pellet

Figure 3.2, J.M. Butler (2005) Forensic DNA Typing, 2nd 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

InletBuffer

Capillary filled with polymer solution

5-20 kV- +

OutletBuffer

Sample tray

Detection 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 bp160 bp 300 bp 340 bp 350 bp150 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, 2nd 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): e1002932.