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4/5/11
1
Forensic DNA profiling: mitochondrial DNA and STR analyses
Daniele Podini [email protected]
Outline • History of Forensic DNA Analysis • STRs • mtDNA
– Structure and funcCon – mtDNA for human idenCficaCon – AnalyCcal process
• ExtracCon • AmplificaCon • Sequencing • Results and InterpretaCon
• QuesCons
Where did it all start?
• Mendel showed that the inheritance of certain traits follows particular laws which were later named after him.
• The significance of Mendel's work was not recognized until the turn of the 20th century.
• The independent rediscovery of these laws formed the foundation of the modern science of genetics.
James Watson & Francis Crick
Using x-ray diffusion data they proposed the double helix or spiral staircase structure of the DNA molecule in 1953
Base-Pairing • In double-stranded DNA the
bases are paired up – As with Ts and – Gs with Cs
• The bases are held together by hydrogen bonds – These bonds are weak
(∼1/10 the strength of a C-C bond)
The Human Genome
Nuclear DNA
3 billion bp
High Power Of DiscriminaCon
Mitochondrial DNA
16.5 Kbp
High Copy Number
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Early 1980s: Restriction Fragment Length Polymorphism (RFLP)
• Genetic variation in the distance between restriction enzyme sites
• Template DNA digested by enzymes, electrophoresed, detected via Southern blotting
• Power of discrimination in the range of 106-108 for a six probe analysis
Sir Alec Jeffreys
Mechanisms
for RFLPs
Mid-1980s: The Colin Pitchfork Case
Two young women raped and murdered in Narborough, England
5,000 local men are asked to provide blood/saliva samples
1st exoneraCon and convicCon on forensic DNA evidence
THEN THERE WAS PCR
Polymerase Chain Reaction PCR
• Polymerase Chain Reaction = molecular Xeroxing
• “Amplify” the desired DNA fragment(s)
• Increased sensitivity • 1988 FBI starts DNA section
Dr. Kary Mullis Eccentric Genius
1985
h\p://www.youtube.com/watch?v=L51UvB5za7c
h\p://www.karymullis.com/pcr.shtml
Repeat unit 2-‐7 b.p. in length Repeated 5-‐40 Cmes
Length appropriate for PCR ~ 400 nt
Highly polymorphic
Spread throughout the genome
Short Tandem Repeats1991
10 repeats
GATA
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STRBase
h\p://www.cstl.nist.gov/biotech/strbase/
Types of STR Repeat Units
• DinucleoCde • TrinucleoCde • TetranucleoCde • PentanucleoCde • HexanucleoCde
(CA)(CA)(CA)(CA)
(GCC)(GCC)(GCC) (AATG)(AATG)(AATG) (AGAAA)(AGAAA)
(AGTACA)(AGTACA)
Requires size based DNA separa1on to resolve different alleles from one another
Short tandem repeat (STR) = microsatellite = simple sequence repeat (SSR)
Short Tandem Repeat (STR) Markers
TCCCAAGCTCTTCCTCTTCCCTAGATCAATACAGACAGAAGACAGGTGGATAGATAGATAGATAGATAGATAGATAGATAGATAGATAGATAGATATCATTGAAAGACAAAACAGAGATGGATGATAGATACATGCTTACAGATGCACAC
= 12 GATA repeats (“12” is all that is reported)
Target region (short tandem repeat)
7 repeats
8 repeats
9 repeats
10 repeats
11 repeats
12 repeats
13 repeats
The number of consecu>ve repeat units can vary between people
An accordion-‐like DNA sequence that occurs between genes
5
D5S818
10 repeats
8 repeats
Short Tandem Repeats
GATA
LOCUS
ALLELE
Profile 8/10
Mitochondrial DNA
The Mitochondrion Cytoplasmic organelle
Double membrane
Outer membrane – porin proteins for the transportaCon of materials.
Inner membrane – highly folded (increased surface area) and highly impermeable.
Inner Matrix – several copies of mtDNA
FuncCon of the mitochondrion: ProducCon of ATP Apoptosis – programmed cell death ElongaCon of fa\y acids OxidaCon of epinephrine (adrenaline) DegradaCon of tryptophan Heme synthesis Heat producCon
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mtDNA Genome 16,569 bp
h\p://www.mitomap.org/
Coding Region 13 polypepCdes 2 rRNAs 22 tRNAs
All necessary for OXPHOS
Highly compact (few intergenic spaces)
LocaCon and Copy Number of mtDNA
• Found within the mitochondria in the cellular cytoplasm.
• On average 4-‐5 copies of mtDNA molecules per mitochondria (range of 1-‐15 mtDNA copies).
• Number of mitochondria vary by cell type (e.g., muscles have more…).
• Generally, hundreds of mitochondria per cell.
Control region (D-‐loop)
1/16,569
cyt b
ND5 ND6
ND4
ND4L
ND3
COIII ATP6
ATP8 COII
12S rRNA
16S rRNA
ND1
ND2
COI
OH
9-‐bp dele1on
OL
F
V
L1
I Q M
W
A N
C Y
S1
D K
G
R
H S2
L2
E
P
T
HV1 HV2
16024 16365 73 340
16024 576
“16,569” bp
1
22 tRNAs
2 rRNAs
13 genes
Figure 10.1, J.M. Butler (2005) Forensic DNA Typing, 2nd EdiCon © 2005 Elsevier Academic Press
Forensic Focus Control Region (16024-‐576) Original Reference Sequence
• Human mtDNA was first sequenced in 1981 in Frederick Sanger’s lab located in Cambridge, England.
• Authors for this paper (Nature 1981, 290:457-‐465) were listed in alphabeCcal order so Stan Anderson was the first author.
• This sequence has come to be referred to as the “Anderson” sequence (GenBank accession: M63933).
• This first sequence is someCmes called the Cambridge Reference Sequence (CRS).
• This revised Cambridge Reference Sequence (rCRS) is now the accepted standard for comparison.
Maternal Inheritance of mtDNA
• FerClizing sperm contributes only nuclear DNA.
• Cellular components including the mitochondria in the cytoplasm come from the mother’s ovum.
• Any sperm mitochondria that may enter a ferClized egg are selecCvely destroyed due to a ubiquiCn tag added during spermatogenesis.
• Barring mutaCon, a mother passes her mtDNA type on to her children.
Summary – mtDNA CharacterisCcs
• High copy number of mtDNA.
• Circular molecule
• Maternal inheritance of mtDNA.
• Lack of recombinaCon.
• High mutaCon rate compared to single copy nucDNA.
4/5/11
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Methods for Measuring mtDNA VariaCon
• Low-‐resoluCon RFLP (1980s)
• High-‐resoluCon RFLP (1990s)
• Sequence analysis of HV1 and HV2 within control region (1991-‐present)
• Sequence analysis of complete mtDNA genome (2000-‐present)
Role of mtDNA Compared to Autosomal STRs
• Autosomal STRs provide a higher power of discriminaQon and are the preferred method whenever possible
• Due to high copy number, mitochondrial DNA (mtDNA) may be the only source of surviving DNA in highly degraded specimens or low quanCty samples such as hair shass
• A mtDNA result is be\er than no result at all…
Candidates for mtDNA TesCng
• Shed hairs lacking root bulb or a\ached Cssue
• Fragments of hair shass.
• Aged bones or teeth that have been subjected to long periods of exposure.
• Crime scene stains or swabs that were unsuccessful for nuclear DNA tesCng.
• Tissues (muscle, organ, skin) that were unsuccessful for nuclear DNA tesCng.
Terry Melton – InternaConal Symposium on the ApplicaCon of DNA Technologies in AnalyCcal Sciences
Mitochondrial DNA as a Means of IdenCficaCon
Why go to mtDNA?
• Disadvantages – mtDNA is not a posiCve form of idenCficaCon
(You have many maternal relaCves!!)
– Easily contaminated with modern DNA
ContaminaCon
• Modern DNA can easily be introduced and overwhelm target DNA from the sample. – Due to the sensiCvity of the reacCon
• Appropriate controls must be implemented to assure that the mtDNA sequence being reported is authenCc.
• Laboratories need to be designed to lessen the chances of contaminaCon.
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Why go to mtDNA?
• Disadvantages – mtDNA is not a posiCve form of idenCficaCon
(You have many maternal relaCves!!)
– Easily contaminated with modern DNA
– Time-‐consuming and costly
Nuclear DNA Analysis
• 24-‐36 hours • ~$100 per sample • Use commercially available kits for processing
Sample CollecCon Laboratory
Profile generaCon
MtDNA Analysis
• 1-‐6 weeks post-‐submission to the laboratory
• ~$1,000 per sample • Custom designed primers
Sample CollecCon Laboratory
Profile generaCon
Why go to mtDNA?
• Advantages – Maternally inherited
• The pool of potenCal references is greatly increased.
Limited references available for nDNA Maternal inheritance
4/5/11
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Why go to mtDNA?
• Advantages – Maternally inherited
• The pool of potenCal references is greatly increased. – Numerous copies of the mitochondrial DNA genome in each cell.
Why go to mtDNA?
• Advantages – Maternally inherited
• The pool of potenCal references is greatly increased. – Numerous copies of the mitochondrial DNA genome in each cell.
– Small genome size and mulCple copies increase chances of recovering DNA from degraded samples.
Extract mtDNA from evidence (Q) sample
PCR Amplify HV1 and HV2 Regions
Sequence HV1 and HV2 Amplicons
(both strands)
Confirm sequence with forward and reverse strands
Note differences from Anderson (reference) sequence
Compare with database to determine haplotype frequency
Compare Q and K sequences
QuesQon Sample
Reference Sample
Extract mtDNA from reference (K) sample
PCR Amplify HV1 and HV2 Regions
Sequence HV1 and HV2 Amplicons
(both strands)
Confirm sequence with forward and reverse strands
Note differences from Anderson (reference) sequence
Performed separately and preferably
aCer evidence is completed
Process for EvaluaCon of mtDNA Samples
Figure 10.4, J.M. Butler (2005) Forensic DNA Typing, 2nd EdiCon © 2005 Elsevier Academic Press
Pre-‐ and post-‐PCR SeparaCon
Pre-‐PCR
ExtracCons & AmplificaCon set-‐ups
Post-‐PCR
Thermalcyclers & Sequencing
AmplificaCon ReacCons
Amplified Product
Pre-‐ and post-‐PCR SeparaCon
Pre-‐PCR
ExtracCons & AmplificaCon set-‐ups
Post-‐PCR
Thermalcyclers & Sequencing
AmplificaCon ReacCons
Amplified Product
Degraded Skeletal Remains
What to choose and how to generate a full mtDNA profile.
4/5/11
8
Degraded Skeletal Remains
• Sample SelecCon • ExtracCon Methods
• AmplificaCon Strategies
• Sequencing Strategies
Degraded Specimens
• In general terms all skeletal remains are degraded.
• Some are more degraded then others due to environmental stressors.
• Prudent sample selecCon will increase the rate of success.
Environment
• Recovery sites vary – Extreme condiCons
• Salt-‐water marshes • Glaciers
– High/Low temperatures
– Repeated freezing and thawing
– High/Low pH – High water levels – Salt or brackish water
Environment
• Remains may be – On the surface – Buried in soil or other substrates – Highly fragmented – Subjected to burning or high heat – Exposed to fuel or other chemicals
– Disturbed or moved by humans or animals
– Animal destrucCon (feeding)
Sample SelecCon
• Unknown skeletal remains – Remains are examined and samples selected by anthropologists or medical examiners
SelecCng samples for analysis
• What are the best skeletal elements to use for analysis?
4/5/11
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Bone Structure
• Bones with dense corCcal structure tend to have a greater success rate. – Compact bone may inherently afford greater protecCon for it’s deeper layers.
– Trabecular bone and elements composed of thin corCcal bone have a greater surface area
• Cranial fragments vary in success – Formed of a layer of trabecular bone sandwiched between two layers of corCcal bone
– Temporal and occipital tend to have denser corCcal bone
Bones SubmiUed for Analysis
DenCCon • Dental remains provide a parCcular challenge – The enamel gives a greater protecCon to the denCn from which the DNA is extracted.
– Anecdotally shown to provide copious quanCCes of DNA from even medieval era remains.
– Require a lot of handling.
Cleaning the Sample
• The exterior of the bone fragment needs to be cleaned of any possible contaminants: – Dirt – Plant material – Extraneous DNA – Dried Tissue
Cleaning
• An easy way to clean the surface is using a sanding bit in a Dremel tool.
How far to clean?
• Everything on the surface needs to come off, along with the spongy bone.
• But, you’ll hit a point where there is no solid bone les.
4/5/11
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Other Cleaning Methods
• Bleaching – Bones can be subjected to a bleach sonicaCon to remove external contaminants.
– A fresh water sonicaCon should follow to get rid of the bleach or DNA can be lost.
• “DNA Off” or other DNA removal products
Too much?
• Aggressive cleaning can remove or otherwise damage available DNA.
ExtracCon Methods
• Numerous extracCon methods available. • Involve different methods of –
– pulverizing the samples
– removing the DNA from the samples
• Different starCng quanCCes of bone can also be used.
PulverizaCon Methods • Freezer Mill
– Uses liquid nitrogen and a magnet to pulverize the bone into a very fine powder.
– Disadvantage: • Requires storage and handling of liquid nitrogen. • Grinders and sample vials are reused – potenCal contaminaCon.
PulverizaCon Method
• Waring Blender Cup – Also grinds bone to a relaCvely fine powder – Disadvantage: Cups are reused, so there is a possibility of contaminaCon.
“Freeing” the DNA
• AFDIL now uses a complete demineralizaCon protocol – Demineralizes the bone matrix.
• Other chemical/physical treatments are commercially available to more easily acquire the DNA. – Silica gel – Charge Switch™ – DNA IQ™
4/5/11
11
ExtracCon of Skeletal Remains
• The powdered bone is extracted with – Proteinase K and extracCon buffer (Demin)
– Overnight at 56°C • DNA is removed from the extracCon buffer with – a series of washes with Phenol/ Chloroform/ Isoamyl alcohol
– PurificaCon of product with filters.
PCR -‐ Primer Design Origin 16024 576
HV1 F15971/R16410
HV2 F15/R389
Primer Design Origin 16024 576
HV1 F15971/R16410
HV2 F15/R389
PS1 F15989/R16251
PS2 F16190/R16410
PS3 F15/R285
PS4 F155/R389
PS5 F16381/R16569
Primer Design Origin 16024 576
HV1 F15971/R16410
HV2 F15/R389
PS1 F15989/R16251
PS2 F16190/R16410
PS3 F15/R285
PS4 F155/R389
mps1a mps2a
mps1b mps2b
mps3a mps4a
mps3b mps4b
mps5a
mVR1 mVR2
PS5 F16381/R16569
Science of DNA Sequencing
dideoxyNTP (ddNTP)
• Fred Sanger – developed the dideoxy method of sequencing in the 1970s… sCll used today.
AGCTGCATGCAATT-‐-‐-‐-‐-‐ TCGAC
-‐OH
dNTP G
-‐OH PPP
ddNTP G
-‐H PPP
* ddNTP
T
-‐H PPP
*
Template
Primer
A blend of dNTPs and ddNTPs
4/5/11
12
AGCTGCATGCAATT-‐-‐-‐-‐-‐ TCGAC
-‐OH
dNTP G
-‐OH PPP
ddNTP G
-‐H PPP
* ddNTP
T
-‐H PPP
*
IncorporaCon
AGCTGCATGCAATT-‐-‐-‐-‐-‐ TCGACG
-‐H
dNTP G
-‐OH PPP
ddNTP T
-‐H PPP
*
TerminaCon of the reacCon!
Fragment = TCGACG
AGCTGCATGCAATT-‐-‐-‐-‐-‐ TCGAC
-‐OH
dNTP G
-‐OH PPP
ddNTP G
-‐H PPP
* ddNTP
T
-‐H PPP
*
IncorporaCon
AGCTGCATGCAATT-‐-‐-‐-‐-‐ TCGACG
-‐OH
ddNTP G
-‐H PPP
* ddNTP
T
-‐H PPP
*
IncorporaCon
AGCTGCATGCAATT-‐-‐-‐-‐-‐ TCGACGT
-‐H
ddNTP G
-‐H PPP
*
TerminaCon of the reacCon!
Fragments = TCGACG TCGACGT
1 bp difference!
Sanger Sequencing 3’-TAAATGATTCC-5’
ATT
ATTTACTAA
ATTTACT ATTTAC
ATTT ATTTA
AT
ATTTACTA
ATTTACTAAG ATTTACTAAGG
A DNA template 5’ 3’
Primer anneals
Extension produces a series of ddNTP terminated products each one base different in length
Each ddNTP is labeled with a different color fluorescent dye
Sequence is read by no1ng peak color in electropherogram (possessing single base resolu1on)
Figure 10.5, J.M. Butler (2005) Forensic DNA Typing, 2nd EdiCon © 2005 Elsevier Academic Press
4/5/11
13
InterpreQng and ReporQng mtDNA Results
Data Review and EdiCng
Trimming of data (primer sequences too)
Data Review and EdiCng
rCRS
Differences from rCRS are noted by the sosware
ReporCng Differences from rCRS
rCRS
489 T-‐C 493 A-‐G
Point MutaCons are listed as differences
from the rCRS
DeleCons
• DeleCons – report the posiCon and bases deleted…
rCRS
523 A-‐del 524 C-‐del
InterpretaConal Issues -‐ Heteroplasmy
• Heteroplasmy – the presence of more than one mtDNA type in an individual (Melton 2004).
• Once thought to be rare, heteroplasmy exists (at some level) in all Cssues (Melton 2004).
• Especially important in hair analysis (semi-‐clonal).
4/5/11
14
InterpretaConal Issues -‐ Heteroplasmy
• Two types: Length (most common) and Point Heteroplasmy.
AAACCCCCCCCCTCCCCCCGCTTC
AAACCCCCCC : : :TCCCCCGCTTC
Sequence 1
rCRS
303 310 315
Sequence 2 AAACCCCCCCCCCTCCCCCCGCTTC
Sequence 1 has 9 Cs before 310T Sequence 2 has 10 Cs before 310T
“Out of phase!”
HV2 Length Heteroplasmy
AAACCCCCCCCCTCCCCCCGCTTC Sequence 1 Sequence 2 AAACCCCCCCCCCTCCCCCCGCTTC
“Out of phase!”
Dr. CinCa Friedman, São Paulo, Brasil
Double coverage is important to determine sequences surrounding HV1, HV2, HV3 C-‐stretches.
Point Heteroplasmy
16093 (C/T)
16086 16101
Figure 10.9, J.M. Butler (2005) Forensic DNA Typing, 2nd EdiCon © 2005 Elsevier Academic Press
“Hotspot” for heteroplasmy “Y” Pyrimidine
OriginaCon of Heteroplasmy
Chinnery et al. (2000) Trends in GeneCcs
Ovum – 100K mitochondria
Very li\le mito growth unCl implantaCon
Females – produce ~7 million ova during fetal development only a few hundred become mature oocytes
Heteroplasmic VariaCon
Sekiguchi et al. (2003) 16291
Buccal Swabs
Heteroplasmy DetecCon
• DetecCon of heteroplasmy – sequencing can detect only to ~10% level.
• Other methods (e.g. Denaturing Gradient Gel Electrophoresis) are much more sensiCve.
4/5/11
15
Famous Case Involving Heteroplasmy
IdenQficaQon of the Romanov Remains (the Last Russian Czar)
Tsarina Alexandra
Tsar Nicholas II
Xenia Cheremeteff-Sfiri
Prince Philip Duke of Edinburgh
Georgij Romanov
Mitotype 16111T 16357C 263G 315.1C
Mitotype 16126C 16169T 16294T 16296T 73G 263G 315.1C
16169T/C
16169T/C Louise of
Hesse-Cassel
SOURCES: Gill et al. (1994) Nature Gene1cs, 6, 130‑135.; Ivanov et al. (1996) Nature Gene1cs, 12, 417‑420; Stone, R. (2004) Science, 303, 753.
D.N.A. Box 10.2, J.M. Butler (2005) Forensic DNA Typing, 2nd EdiCon © 2005 Elsevier Academic Press
AFDIL – ConfirmaCon of FSS
TSAR
GEORGIJ
Dr. Thomas Parsons
Extract mtDNA from evidence (Q) sample
PCR Amplify HV1 and HV2 Regions
Sequence HV1 and HV2 Amplicons
(both strands)
Confirm sequence with forward and reverse strands
Note differences from Anderson (reference) sequence
Compare with database to determine haplotype frequency
Compare Q and K sequences
QuesQon Sample
Reference Sample
Extract mtDNA from reference (K) sample
PCR Amplify HV1 and HV2 Regions
Sequence HV1 and HV2 Amplicons
(both strands)
Confirm sequence with forward and reverse strands
Note differences from Anderson (reference) sequence
Performed separately and preferably
aCer evidence is completed
Process for EvaluaCon of mtDNA Samples
Figure 10.4, J.M. Butler (2005) Forensic DNA Typing, 2nd EdiCon © 2005 Elsevier Academic Press
InterpretaCon of mtDNA Results
• Once the sequence has been generated (Q and K), and the differences from the rCRS are noted, what next?
SWGDAM Guidelines for Mitochondrial DNA (mtDNA) NucleoCde Sequence InterpretaCon
(1) Exclusion
(2) Inconclusive
(3) Cannot Exclude (Failure to Exclude)
InterpretaCon of mtDNA Results
• Exclusion – if there are two or more nucleoCde differences between the quesConed and known samples, the samples can be excluded as originaCng from the same person or maternal lineage.
Q TATTGCACAG K TATTGTACGG
Exclusion
Sample Q Sample K
6 T-‐C 9 G-‐A 263 A-‐G 315.1 C
263 A-‐G 315.1 C
4/5/11
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InterpretaCon of mtDNA Results
• Inconclusive – if there is one nucleoCde difference between the quesConed and the known samples, the result will be inconclusive.
Q TATTGCACGG K TATTGTACGG
Sample Q Sample K
6 T-‐C 263 A-‐G 315.1 C
263 A-‐G 315.1 C
Inconclusive
InterpretaCon of mtDNA Results
• Cannot Exclude – if the sequences from quesConed and known samples under comparison have a common base at each posiCon or a common length variant in the HV2 C-‐stretch, the samples cannot be excluded as originaCng from the same person or the same maternal lineage.
Q TATTGTACGG K TATTGTACGG
Sample Q Sample K
152 T-‐C 263 A-‐G 315.1 C
152 T-‐C 263 A-‐G 315.1 C
Cannot Exclude
ReporCng StaCsCcs
• When “cannot exclude” is the interpretaCon, then a staCsCcal esCmate is needed in order to weigh the significance of the observed match
• CounCng method is most common approach used and involves counCng the number of Cmes that a parCcular mtDNA haplotype (sequence) is seen in a database
• The larger the number of unrelated individuals in the database, the be\er the staCsCcs will be for a random match frequency esCmate.
Control region (D-‐loop)
1/16,569
cyt b
ND5 ND6
ND4
ND4L
ND3
COIII ATP6
ATP8 COII
12S rRNA
16S rRNA
ND1
ND2
COI
OH
9-‐bp dele1on
OL
F
V
L1
I Q M
W
A N
C Y
S1
D K
G
R
H S2
L2
E
P
T
HV1 HV2
16024 16365 73 340
16024 576
“16,569” bp
1
22 tRNAs
2 rRNAs
13 genes
Figure 10.1, J.M. Butler (2005) Forensic DNA Typing, 2nd EdiCon © 2005 Elsevier Academic Press
Forensic Focus
Uses of Haplogroup Typing
www.payvand.com
h\p://www.rcfp.org/
Anthropological Studies
Forensic Identification
Mass Graves/ Commingled Remains
h\p://www.dccam.org
KEY:
EU = European
AS = Asian
AF = African
NA = Native American L3
M
10398G, 10400T
13263G
5178A
7600A 4833G
C
D
E G
AS/NA
AS/NA
AS AS
N
8272-8280 del
A10398, C10400
C7028
1719A 663G
12406A
X
H
F
A B
EU
EU/AS/NA
AS
AS/NA AS/NA
10398G, 1719A
I
EU
G3594 10398G, 7028T
3594T, 10398G, 7028T
L1/L2
AF
AF/EU/AS
4/5/11
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Acknowledgments
Many thanks to • Organizers • Mike Coble NIST (Former chief research secCon AFDIL)
QUESTIONS? [email protected]