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short presentation about dna profiling
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Forensic DNA Typingor
Did you kill (rape…) that person?
How DNA can “definitively” say.
Adapted from:
National Institutes of Science & Technology
http://www.cstl.nist.gov/div831/strbase/intro.htm
Brief History of Forensic DNA Typing
• 1980 - Ray White describes first polymorphic RFLP marker (Restriction Fragment Length Polymorphism [alleles]).
Different RFLP for different people• 1985 - Alec Jeffreys discovers multilocus VNTR (variable number of
tandem repeats) probes.• 1985 - first paper on PCR• 1988 - FBI starts DNA casework• 1991 - first STR paper (renaming of VNTR– could be larger repeats, STR 4-6 bp’s.
now using mostly 4 bases )
• 1995 - FSS (Forensic Science Service-UK) starts UK DNA database• 1998 - FBI launches CODIS (Combined DNA Information Service)
Now FBI use 13 loci: PCR identifies it: in the quadrillions – except for identical. Except for police mistakes, it’s done deal.
RFLP’s: Sickle Cell hemoglobin
Case 1: Screening for the sickle-cell gene Sickle cell disease is a genetic disorder in which both genes in the patient encode the amino acid valine (Val) in the sixth position of the beta chain (betaS) of the hemoglobin molecule. "Normal" beta chains (betaA) have glutamic acid at this position. The only difference between the two genes is the substitution of a T for an A in the middle position of codon 6. This •converts a GAG codon (for Glu) to a GTG codon for Val and •abolishes a sequence (CTGAGG, which spans codons 5, 6, and 7) recognized
and cut by one of the restriction enzymes.
Brief History of Forensic DNA Typing
• 1980 - Ray White describes first polymorphic RFLP (Restriction Fragment Length
Polymorphism) marker—detect to transferring to membrane. Probe w southern blot (radiological). Diff. RFLP for dif. People. Single rflp
• 1985 - Alec Jeffreys discovers multilocus VNTR (variable number of tandem repeats) probes (stat. very impressive identical 4-6 bp that are spec. 7 and 9 repeat, one from mom and dad, on chrom. 1- nowadays use pcr- but flanking sequence that is unique to chromo1)). Jeffreys almost ident. Typing. Now use PCR.
• 1985 - first paper on PCR (Kerry Mullis)• 1988 - FBI starts DNA casework • 1991 - first STR paper ( renaming of VNTR– could be larger
repeats, STR 4-6 bp’s. now using mostly 4 bases )• 1995 - FSS (Forensic Science Service-UK) starts UK DNA
database• 1998 - FBI launches CODIS (Combined DNA Information
Service) database. Now FBI use 13 loci: PCR identifies it: in the quadrilians – except for identical.
DNA Use in Forensic Cases
• Most are rape cases (>2 out of 3)
• Looking for match between evidence and suspect
• Must compare victim’s DNA profile
•Mixtures must be resolved
•DNA is often degraded (stored wet- have mold, nuclease)
•Inhibitors to PCR are often present
Challenges
Human Identity Testing
• Forensic cases -- matching suspect with evidence
• Paternity testing -- identifying father
• Historical investigations• Missing persons investigations• Mass disasters -- putting pieces back together
• Military DNA “dog tag”• Convicted felon DNA databases
Sample Obtained from Crime Scene or
Paternity Investigation Biology
DNAExtraction
DNAExtraction
DNAQuantizatio
n
DNAQuantizatio
n
PCR Amplificationof Multiple STR
markers
PCR Amplificationof Multiple STR
markers
TechnologySeparation and Detection of
PCR Products(STR Alleles)
Sample Genotype
Determination
GeneticsComparison of Sample
Genotype to Other Sample Results
Comparison of Sample Genotype to Other
Sample Results
If match occurs, comparison of DNA profile to population databases
If match occurs, comparison of DNA profile to population databases
Generation of Case Report with Probability
of Random Match
Generation of Case Report with Probability
of Random Match
Steps in DNA Sample Processing
Sources of Biological Evidence
• Blood• Semen• Saliva• Urine• Hair• Teeth (useful in fires).
• Bone (there are cells. Decalcify it.
100,000 year old- has DNA. Has Dinosaur!)
• TissueAll felony arrests- cheek swab.
DNA in the Cell
Target Region for PCRTarget Region for PCR
chromosome
cell nucleus
Double stranded DNA molecule
Individual nucleotides
Make copies (extend primers)
Starting DNA Template
5’
5’
3’
3’
5’
5’
3’
3’
Add primers (anneal) 5’3’
3’5’
Forward primer
Reverse primer
DNA Amplification with the Polymerase Chain Reaction (PCR)
Separate strands
(denature)
5’
5’3’
3’
In 32 cycles at 100% efficiency, 1.07 billion copies of targeted DNA region are created
In 32 cycles at 100% efficiency, 1.07 billion copies of targeted DNA region are created
PCR (Polymerase Chain Reaction) Copies DNA Exponentially through Multiple Thermal Cycles
Original DNA target region
Heat
Cool
Heat
DNA Poly.dUTP
Oligo’s
1 copy2 copies
Cool 4 copiesHeat …
Short Tandem Repeats (STRs) (say chromo 3)
the repeat region is variable between samples while the flanking regions where PCR primers bind are constant
7 repeats
8 repeats
AATG
Homozygote = both alleles are the same length
Heterozygote = alleles differ and can be resolved from one another
Identical in all people
Identical in all people
170 bp170 bp195 bp195 bp
Different primer sets produce different PCR product sizes for the same STR allele
TCAT repeat unitTCAT repeat unit
Diff. PCR primers sets, can amplify the same region. Different companies sell different kits.
Choosing which STRs:Significant statistical variation – but not too many. Freq. that are measured in pop. : Loc 1 -10%. Loc 2 – 10%; locus 1+2 -1/100. Random
match with 13 primers 1/1013.
Variation Among STRs
Multiplex PCR• Over 10 Markers Can Be
Copied at Once
• Sensitivities to levels less than 1 ng of DNA
• Ability to Handle Mixtures and Degraded Samples
• Different Fluorescent Dyes Used to Distinguish STR Alleles with Overlapping Size Ranges
Most rxns: require 2 PCR (tubes) 7 or 8 primer pairs in one tube– need total of about 2 tubes for 13 different STRs.
$20-$25 per rxn in lab. $150 incl labor. Cost for forensic up to $1000.
An Example Forensic STR Multiplex Kit
D3 FGAvWA 5-FAM (blue)
D13D5 D7 NED (yellow)
A D8 D21 D18 JOE (green)
GS500-internal lane standard
ROX (red)
AmpFlSTR® Profiler Plus™Kit available from PE Biosystems (Foster City, CA)
9 STRs amplified along with sex-typing marker amelogenin in a single PCR reaction
100 bp 400 bp300 bp200 bpSize Separation
Col
or S
epar
atio
n
Available Kits for STR Analysis
• Kits make it easy for labs to just add DNA samples to a pre-made mix
• 13 CODIS core loci– Profiler Plus and COfiler (PE Applied Biosystems)– PowerPlex 1.1 and 2.1 (Promega Corporation)
• Increased power of discrimination– CTT (1994): 1 in 410– SGM Plus™ (1999): 1 in 3 trillion– PowerPlex ™ 16 (2000): 1 in 2 x 1017
ABI Prism 310 Genetic Analyzer
capillary
Syringe with polymer solution
Autosampler tray
Outlet buffer
Injection electrode
Inlet buffer
5 min from inj. to output.
Close-up of ABI Prism 310 Sample Loading Area
Autosampler Tray
Sample Vials
Electrode
Capillary
See Technology section for more information on CE
amelogenin
D19
D3
D8
TH01
VWA D21FGA
D16D18 D2
amelogeninD19
D3D8 TH01
VWA D21
FGA
D16D18 D2
Tw
o di
ffer
ent i
ndiv
idua
ls
DNA Size (base pairs)
Results obtained in less than 5 hours with a spot of blood the size of a pinhead
probability of a random match: ~1 in 3 trillion
Human Identity Testing with Multiplex STRs
Simultaneous Analysis of 10 STRs and Gender ID
AmpFlSTR® SGM Plus™ kit
D tells chromosome 21—happens to be down’s syndrome. 2 peaks cause heterozygotic
Amelogenin amel protein that happens to be on sex chromosome, tooth enamel– top: 2 peaks: x and y. (universal that two diff. people.) Bottom only 1 peak cause they have two X chromosomes.
STR Allele Frequencies
0
5
10
15
20
25
30
35
40
45
6 7 8 9 9.3 10
Caucasians (N=427)
Blacks (N=414)
Hispanics (N=414)
TH01 Marker
*Proc. Int. Sym. Hum. ID (Promega) 1997, p. 34
Number of repeats
Fre
qu
ency
FBI’s CODIS DNA Database
Combined DNA Index System --all 50 states can upload their convicted felony and seq. of unsolved cases…. In Florida to convicted felon.
• Used for linking serial crimes and unsolved cases with repeat offenders
• Launched October 1998• Links all 50 states• Requires >4 RFLP markers and/or 13 core STR markers• Current backlog of >600,000 samples
13 CODIS Core STR Loci with Chromosomal Positions
CSF1PO
D5S818
D21S11
TH01
TPOX
D13S317
D7S820
D16S539 D18S51
D8S1179
D3S1358
FGA
VWA
AMEL
AMEL
The End