Final Figures

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Final Figures
Forensic DNA Typing, 3rd Edition Volume I

• Fundamentals of Forensic DNA Typing

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Law Enforcement
Scientific Analysis
Legal Proceedings
Police Agencies (local, state, federal)
Forensic Laboratory
Court System
Legal framework and precedent
Validated scientific tests
Laws and police training
Other Forensic Disciplines
Judge
Investigators/ Detectives
CSI
Prosecution
DNA Unit DNA Analysts
Defense
Scientific report(s) completed
Conviction or exoneration
Trial
Evidence submitted
Evidence returned
References submitted
Research (introduces new methods)
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Crime committed
Suspect developed
Biological material transferred
May match another (K)
May match another (K)
Reference (Known) sample K
Evidence (Question) sample Q
Database Search
Steps Involved
Collection
Q U A L I T Y A S S U R A N C E
Exclusion (no match)
Sample Storage
Q ? K
Extraction
Q
K
DNA Profile Comparison
Quantitation
Amplification
Q K
STR Markers
Inclusion (match)
Separation/ Detection
Report (with statistical weight)
Data Interpretation
Court
Plea
Profile put on database
Profile put on database
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The Human Genome
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(A) Sequence polymorphism
--------AGACTAGACATT------- --------AGATTAGGCATT-
------
(B) Length polymorphism
---------(AATG)(AATG)(AATG)----------
3 repeats
---------(AATG)(AATG)----------
2 repeats
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Maternal Contribution
Sex chromosome
Egg (haploid)
mtDNA
Paternal Contribution
Sex chromosome
Sperm (haploid)
or
Nuclear DNA
Sex chromosomes
Mitochondrial DNA
Autosomes
Zygote (diploid)
Male Childs Full Genome
mtDNA
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Resulting genotype combinations and frequencies
Mother gametes (egg)
A
a
q
p


AA
aA
A
qp
p2
p
Father gametes (sperm)
a
Aa
aa
q
pq
q2
Punnett square
Freq (A) p
(p q)2 p2 2pq q2
p q 1
Freq (a) q
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Frequency of A allele (p)
1.0
0.8
0.6
0.4
0.2
0.0
AA
aa
p2
0.8
q2
Aa
0.6
2pq
Frequency of genotype in population
0.4
0.2
1.0
0.0
0.2
0.4
0.6
0.8
Frequency of a allele (q)
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(a)
(c)
(b)
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Mothers Blood Type
A B AB O
A A or O A,B,AB, or O A,B, or AB A or O
B A,B,AB, or O B or O A,B, or AB B or O
AB A,B, or AB A,B, or AB A,B, or AB A or B
O A or O B or O A or B O
Childs Blood Type
Fathers Blood Type
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HaeIII
HinfI
PstI
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TMB (colorless)
Colored precipitate
HRP
Strepavidin
PCR product (denatured)
Biotin
GTCCAGTCG
3
hybridization
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(A)
Control dot
Subtype allele specific dots
Nominal allele specific dots
(B)
1.2/3
AB
AB
BB
AB
BC
S dot
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Differential Extraction
Remove a portion of the mixed stain
SDS, EDTA and proteinase K (cell lysis buffer)
Incubate at 37 oC
Perpetrators sperm mixed with victims
epithelial cells
sperm pellet
REMOVE supernatant
SDS, EDTA and proteinase K DTT
DTT lyses sperm heads
Male Fraction
Female Fraction
sperm pellet
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(a)
Too much DNA amplified
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TaqMan probe
Polymerization and Strand Displacement
Probe Cleavage (release of reporter dye)
Fluorescence occurs when reporter dye and
quencher dye are no longer in close proximity
Completion of Polymerization
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Nc No (1 E)c
If efficiency is close to 100 (E 1), then the
product copy number (Nc) doubles the target copy
number (No) with each cycle (c).
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(a)
(b)
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(a) Simultaneous amplification of three locations
on a DNA template
(b) Resolution of PCR products with size-based
separation method
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(a)
PCR product size generated
Fluorescent dye
DNA template containing STR marker
Forward PCR primer
Reverse PCR primer
GATA
GATA
GATA
GATA
Flanking regions
STR repeat region
(b)
4 repeat units
5 repeat units
6 repeat units
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1 2 3 4 5 6 5-TTTCCC
TCAT TCAT TCAT TCAT TCAT TCAT TCACCATGGA-3 3-AAA
GGG AGTA AGTA AGTA AGTA AGTA AGTA AGTGGTACCT-5
6 5 4 3
2 1
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PCR product size (bp)
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Fluorescent dye at 5end
For each linker unit added, there is an apparent
migration shift of 2.5 bp
Primer sequence
3-end
5-end
Non-nucleotide linkers (mobility modifiers)
PCR amplification generates a labeled PCR product
containing the mobility modifiers
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Size overlap
(a) COfiler kit allele relative size
ranges
6
15
CSF1PO
JOE-labeled (green)
279.65 bp
317.67 bp
6
15
D7S820
NED-labeled (yellow)
256.01 bp
292.62 bp
(b) Identifiler kit allele relative size
ranges
10 non-nucleotide linkers 25 bp shift
6
15
6
15
D7S820
CSF1PO
6FAM-labeled (blue)
6FAM-labeled (blue)
255.15 bp
291.58 bp
304.69 bp
341.84 bp
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30 bp shift in size
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Female X, X
11 Mixture 3X 1Y
Male X, Y
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(a)
(b)
Fluorescent dNTPs are incorporated into both
strands of PCR product
(c)
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JOE (green)
FAM (blue)
TAMRA (yellow)
ROX (red)
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(a)
Scan number
Region shown below
Relative Fluorescence Units
(b)
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Capillaries
Electrodes for Injection
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?1 S25-L25 244.34 - 244.46 -0.12 bp ?2
SOL - L28 257.51-256.64 0.87 bp c ?1
-?2 -0.12-0.87 0.99 bp
28.1
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Decide on Number of Samples and Ethnic/Racial
Grouping
Usually gt100 per group
Often anonymous samples from a blood bank
Gather Samples
Analyze Samples at Desired Genetic Loci
Summarize DNA types
Determine Allele Frequencies for Each Locus
See Table 11.1
Perform Statistical Tests on Data
Hardy-Weinberg equilibrium for allele independence
Linkage equilibrium for locus independence
Ethnic/ Racial Group 1
Ethnic/ Racial Group 2
Examination of genetic distance between
populations
Use Database(s) to Estimate an Observed DNA
Profile Frequency
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National Level
State Level
Local Level
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150 bp smaller
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Lineage Markers
Y-Chromosome (passed on complete, but only by
sons)
Mitochondrial (passed on complete, but only by
daughters)
Autosomal (passed on in part, from all
ancestors)
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Female-Male Mixture Performance with Autosomal
vs. Y-Chromosome DNA Markers
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?
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Control region (D-loop)
16024
16365
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340
438
574
1
HV1
HV2
HV3
576
16024
cyt b
1/16,569
mtGenome 16,569 bp
Coding region
Light (L) strand
Heavy (H) strand
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Compare Q and K sequences
Compare with database to determine haplotype
frequency
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(a) mtDNA Sequences Aligned with rCRS (positions
16071-16140)
(b) Reporting Format with Differences from rCRS
Sample Q 16093C 16129A
Sample K 16093C 16129A
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Louise of Hesse-Cassel
16169T/C
16169T/C
Georgij Romanov
Tsar Nicholas II
Tsarina Alexandra
Prince Philip Duke of Edinburgh
Mitotype 16111T 16357C 263G 315.1C
Mitotype 16126C 16169T 16294T 16296T 73G 263G 315.
1C
Xenia Cheremeteff-Sfiri
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(a) Mendelian Inheritance
(b) Example
11,12
8,14
12,14
8,11
12,14
14
14
11
14
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Obligate paternal allele
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(a)
Parentage (Paternity) Testing
Random man
Alleged father
Mother (known parent)
?

• Rules of Inheritance
• Child has two alleles for each autosomal marker
  (one from mother and one from biological father)
• Child will have mothers mitochondrial DNA
  haplotype (barring mutation)
• Child, if a son, will have fathers Y-chromosome
  haplotype (barring mutation)

child
(b)
Reverse Parentage Testing (Missing Persons
Investigation)
Alleged mother
Alleged father
?
Missing child
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Solution Additional Markers (Y-chromosome, more
STRs) and Multiple Reference Samples
Core Competency
Standard STR Typing (DNA Profile)
Familial Searching Attempts (fishing for brothers
or other relatives)
Direct Matching (or Parentage)
Sufficient DNA quantity (ng)
Touch DNA Attempts (poor quality, mixtures,
low-level stochastic effects)
Solution Replicate Testing
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(a) Hypothesis Testing Decisions
Truth about the population
Decision based on sample examined
H0 True
H0 False
Correct decision Type II error
Type I error Correct decision
Accept H0
Reject H0 (Accept H1)
(b) Example
Defendant
Saint
Sinner
Courtroom Verdict
Correct decision Wrongfully acquitted
Wrongfully accused Correct decision
Not Guilty
Guilty