practical tasks: 0. sample collection on the crime scene 1.dna extraction 2.dna amplification (pcr)...
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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?
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
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.
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