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.
2.
3.
4.
5.
6.
7.
8.
Forensic Psychiatry and Mental Illness
Forensic Pathology
Forensic Engineering
Forensic Toxicology
Forensic Criminalistics
Forensic Entomology
Forensic Odontology
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
Sir Alec
Jeffreys
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
Brief History of Forensic DNA Methods
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.
The O.J. Simpson case
The trial of the century convened: “Dollars v DNA” or California v
OJ Simpson.
1995: OJ Simpson verdict: 'Not guilty'
Comparison of Markers Used in Forensic
Biology
High
RFLP
Multi-Locus Probes
Multiplex PCR
of STRs
Power of
Discrimination
(Genetics)
mtDNA PCR
ABO
blood groups
Low
Slow
Speed of Analysis
(Technology)
Figure 1.1, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press
Fast
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)
Flanking regions
Repeat region
GAGGACCACCAGGAAG
16 bp repeat unit
STR Marker (TH01)
Flanking regions
Repeat region
TCAT
4 bp repeat unit
Figure 5.1, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press
Polymorphism on the homologe chromosome
(A) Length polymorphism: VNTR or STR – Technique: (VNTR-PCR)
---------(AATG)(AATG)(AATG)------------------(AATG)(AATG)----------
3 repeats
2 repeats
(B) Sequence polymorphism: SNP – Technique: (AS-PCR)
--------AGACTAGACATT--------------AGATTAGGCATT------Figure 2.5, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press
Y-STRs
Test for markers found only on the Y-chromosome. Only male DNA is amplified
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•
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
„Genghis Khan” argument:
Lower power of discrimination - paternal
relatives all share the same Y-STR haplotype
(10% of Central Asian males share the same YSTR haplotype, thought to belong to Genghis
Khan)
16024
16365
Hypervariable Region1
1
73
340
HV2
HV1
Hypervariable Region2
Control region (D-loop)
16024
OH
T
Heavy (H)
strand
576
F 12S
rRNA
cyt b
Mitochondrial
1/16,569 DNA
P
22 tRNAs
2 rRNAs
V
16S
(mtDNA)
rRNA
13 genes
Pros
• Single-cell sensitivity because
E each cell contains ~1000 mitochondria
L1
• Especially useful for ND6
shed hairs, burnt remains
ND5
ND1 is maternally
• Can be used to establish kinshipMitochondria
directly because entire complement of mtDNA
inherited
L2
I
S2
Q
H
M
OL
Light (L)
A
ND4
ND2
Cons
N
strand
Heteroplasmy - more than one mtDNA type manifesting in different
C tissues in the same individual
Y mtDNA W
Lower power of discrimination - maternal relatives all share the same
ND4L
“16,569” bp
R
ND3
9-bp
deletion
S1
COI
G
COIII
ATP6
ATP8
COII
K
Figure 10.1, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press
D
Single Nucleotide Polymorphisms
(SNPs)
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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
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•
•
•
•
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•
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
DNA-extraction protocols
ORGANIC
CHELEX
SDS, DTT, EDTA and
Blood
stain
proteinase K
Filter Paper
Blood
stain
Apply blood to
paper and allow
stain to dry
Water
INCUBATE (56 oC)
Centrifuge
PUNCH
INCUBATE (ambient)
Phenol,
chloroform,
isoamyl alcohol
Centrifuge
REMOVE supernatant
VORTEX
5%
Chelex
Centrifuge
TRANSFER aqueous (upper) phase to new
tube
TE buffer
WASH Multiple Times with
extraction buffer
REMOVE supernatant
INCUBATE (56 oC)
INCUBATE (100 oC)
PCR
Reagents
Centrifuge
CONCENTRATE sample
(Centricon/Microcon-100 or ethanol
precipitation)
Centrifuge
QUANTITATE DNA
QUANTITATE DNA
PERFORM PCR
PERFORM PCR
Figure 3.1, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press
(NO DNA QUANTITATION TYPICALLY
PERFORMED WITH UNIFORM SAMPLES)
PERFORM PCR
Remove a
portion of
the mixed
stain
Differential DNA extraction of sperms from victim’s epithelial cells
SDS, EDTA and
proteinase K
(cell lysis buffer)
Incubate
at 37 oC
Centrifuge
Perpetrator’s sperm
mixed with victim’s
epithelial cells
sperm
pellet
SDS, EDTA and
proteinase K + DTT
“Male Fraction”
REMOVE
supernatant
DTT lyses
sperm
heads
DTT: Dithiothreitol: breaks down bisulfide bonds of sperm head
Figure 3.2, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press
sperm
pellet
“Female Fraction”
What is the technical basis of STR product differentiation?
Laser
Capillary filled with
polymer solution
Detectio
n
window
Capillary Array Electrophoresis
(cathode)
5-20 kV
+
(anode)
Outlet
Buffer
Inlet
Buffer
Data
Acquisition
Sample tray
Sample tray moves automatically beneath
the cathode end of the capillary to deliver
each sample in succession
Capillaries
Primers are labeled
Electrodes for Injection
CODIS
(Combined DNA Index System, developed by FBI and NIH)
Chromosome numb.
Sex specific marker
FBI’s core STR Loci: 13
The new expanded STR loci used by FBI, InterPol, etc.
D8S1179
D21S11
(12 alleles)
(24 alleles)
D3S1358
(8 alleles)
D19S433
(15 alleles)
D7S820
CSF1PO
(10 alleles)
(10 alleles)
TH01
D13S317
D16S539
(10 alleles)
(8 alleles)
(9 alleles)
VWA
TPOX
(14 alleles)
(8 alleles)
AMEL
D5S818
FGA low
(2 alleles)
(10 alleles)
(19 alleles)
D2S1338
100 bp
139bp
160 bp
250 bp*
Green panel
(14 alleles)
Yellow panel
D18S51
(23 alleles)
Red panel
FGA high
(9 alleles)
150 bp
200 bp
Blue panel
300 bp
LIZ-labeled GS500 DNA sizing standard
Figure 5.6, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press
340 bp
Orange panel
350 bp
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.