103114 1 Forensic DNA Analysis Part 1 The science.docx

10/31/14
1
Forensic DNA Analysis
Part 1: The science behind DNA
profiling
Part 2: The case of the Romanovs
Part 3: Analyzing the certainty of DNA
profile comparisons
Part 4: Uncertainties in DNA profiling
and privacy issues
Part 1: The science behind
DNA Profiling
*Frumkin et al., Forensic Science International: Genetics, 2010,
4(2) pp. 95-103
*
*
*
‡‡Bright and Petricevic, Forensic Science International,
2004, 145, pp.7-12.
‡ ‡
‡ ‡

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DNA is one of many forms of
physical evidence
• Fingerprints
• Blood
• Guns
• Knives
• Bullets
• Wrecked cars
• Human bones
• Bruises
• Clothing
• Computers
• Papers
Image: Simon Howden /
FreeDigitalPhotos.net
Collection of DNA evidence
• Trace amounts of DNA can be
recovered from
– Hair follicles
– Scrapings of dead skin (e.g
from under someone’s
fingernails)
– Saliva

– Semen
– Blood
– Bone fragments buried for
hundreds of years (and
including teeth)
• Collection of DNA evidence (from a
crime scene for example) must be
done carefully to avoid:
– Contamination
– Degradation
– The mixing up of samples
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DNA is small
• A DNA molecule is ~20 nm (1
nm = 1.0 x 10-9 meters) wide
• Found in the nucleus of a cell
• Human cells are ~5-30 µm (1
µm = 1.0 x 10-6 m) wide, too
small to be visible to the naked
eye
• There is about 6 pg DNA in
typical human cell (1 pg = 1.0 x
10-12 g)

Illustration of a DNA double helix
Illustration of an
animal cell
~20 nm
~30 µm
Why is DNA good evidence?
• Every person’s DNA sequence is
unique
• The “sequence” of the DNA refers
to how the DNA basepairs are
arranged, like letters in a word or
words in a sentence
• Homozygotic (ie “Identical”) twins
have DNA sequences that are the
most similar
• Blood relatives have DNA
sequences that are more similar
than unrelated individuals
• Unrelated individuals have DNA
that is VERY different from each
other, making DNA more helpful in
crime scene investigation than less
“individual-specific” evidence such
as hair color or clothing fibers
Cartoon drawing of
DNA double helix

DNA basepairs
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4
How Mendelian Inheritance Influences
DNA sequence
• You have 2 copies of every
chromosome
• You inherit one copy (and its
sequence) from each of your
parents
• Which of each of your
parents’ two copies you
inherit is random
• So for a given chromosome,
if Mom is “chocolate/
chocolate” and Dad is
“vanilla/strawberry” then
you can be either
“chocolate/vanilla” or
“chocolate/strawberry”
Mom Dad
Ch1 Ch1

Ch1 Ch1
You
(possibility #1)
You
(possibility #2)
Or
How Mendelian Inheritance Influences DNA sequence
• The chromosomes segregate
(separate) independently with respect
to each other during cell division
• This “independent segregation”
phenomenon greatly increases the
number of possible combinations
• The many possible combinations is
the main reason why siblings look
different than each other (unless they
are identical twins) even though they
have the same parents.
Mom Dad
You
(possibility #1)
You
(possibility #2)
Ch1 Ch2 Ch1 Ch2

Ch1 Ch2 Ch1 Ch2
You
(possibility #3)
You
(possibility #4)
Ch1 Ch2 Ch1 Ch2
…etc
… etc
Or Or Or
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Methods of Analyzing
Differences in DNA Sequences
• An older method is called
“RFLP” analysis (Restriction
Fragment Length
Polymorphism)
• RFLP analysis makes use of
pieces of DNA that are
different sizes between
individuals and uses the
differences to distinguish

people
• A later modification of this was
called “VNTR” analysis
(Variable Number Tandem
Repeat) and which was in use
around the time of the OJ
Simpson trial in 1995
National Institutes of Health website
The red bands represent DNA molecules; they appear
at different vertical positions on the grey “Southern
blot” because they are differently sized.
Large
Medium
Small
Methods of Analyzing Differences in DNA Sequences
• These days, analysis is usually
done by “STR” analysis (Short
Tandem Repeat)
• This method also uses
differences in DNA size, but the
differences are smaller (a few
DNA base pairs instead of
thousands)
• STR analysis requires a much
smaller sample size (~1 ng) and it

will work on old or degraded
samples making it more useful in
crime scene investigation
• Analyzing more sites in one
comparison increases the
certainty of the analysis.
• The FBI uses 13 STR sites (ie the
13 “CODIS loci”).
http://www.cstl.nist.gov/biotech/strbase/fbicore.htm
The 13 loci used by the FBI.
“CODIS” is the “Combined DNA
Index System”…the specific group
loci (shown above) usually used by
law enforcement.
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Separate
strands
(denature)
Add primers
(anneal)
Make copies
(extend
primers)

Repeat Cycle,
Copying DNA
Exponentially
Starting DNA
Template
5’
5’
3’
3’
5’
5’
5’ 3’ 3’
3’ 3’ 5’
Forward primer
Reverse primer
Figure 4.2, J.M. Butler (2005) Forensic DNA Typing, 2nd
Edition © 2005 Elsevier Academic Press
STR Analysis
• Portions of
the trace DNA
sample are
copied many

times using a
laboratory
technique
called the
“Polymerase
Chain
Reaction” (P
CR)
STR Analysis
*Frumkin et al., Forensic Science International: Genetics, 2010,
4(2) pp. 95-103
*
6 8
** From Figure 6.10, J.M. Butler (2005)
Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic
Press
6
8
• Following PCR, the resulting DNA
samples are abundant enough to
have their sequence read by a
DNA analyzer
• The readings are peaks on an
“electropherogram” as shown to
the right
• The peaks represent DNAs of

different sizes that were
separated
• Peak sizes are calibrated against
a set of “size markers” (i.e. DNA
molecules of known size)
• DNA from different people will
exhibit differences in the numbers
of copies of the tandem repeat
regions.
• The differing numbers of copies
create size differences in the
peaks
• Even DNA from the two copies of
the chromosomes in one person
will give different peaks if the
donor individual is heterozygous.
One chromosome
(vanilla)
The other copy of the
same chromosome
(strawberry)
**
**
The DNA repeat region

stutter peak
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Allele
• Any of the possible forms in which a gene for a
specific trait can occur. In almost all animal cells, two
alleles for each gene are inherited, one from each
parent. Paired alleles (one on each of two paired
chromosomes) that are the same are called
homozygous, and those that are different are called
heterozygous. In heterozygous pairings, one allele is
usually dominant, and the other recessive. Complex
traits such as height and longevity are usually caused
by the interactions of numerous pairs of alleles, while
simple traits such as eye color may be caused by just
one pair.
An Example of STR
Analysis: Amelogenin DNA
is Used to Determine Sex
• Amelogenin is a protein required
for the formation of tooth enamel
• Both men and women have
amelogenin
• DNA coding for amelogenin is
located on the sex chromosomes

• A woman’s sex chromosomes
are “XX”
• A man’s sex chromosomes are
“XY”
Electron micrograph of an “amelogenin gel matrix”
Fincham, A.G., Moradian-Oldak, J. and Simmer, J.P., Journal of
Structural Biology, 1999, 126, pp. 270-299.
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Sequencing of Amelogenin DNA
• DNA corresponding to amelogenin
is slightly longer on the Y-
chromosome than on the X
• STR analysis of DNA from a male
will show two separate signals
(one signal each deriving from the
X and Y chromosomes,
respectively)
• DNA from a woman shows only
the shorter X signal since both X
chromosomes report the same
result X ≈ 103 DNA base pairs
Y ≈ 109 DNA base pairs

What results look
like for a male:
Frumkin et al., Forensic Science International: Genetics, 2010,
4(2) pp. 95-103
Another example of how differences in
DNA sizes can be used in DNA profiling:
Paternity testing
• This one is the older
“VNTR” analysis
• For every VNTR locus that
can be analyzed in the
laboratory, you will bear one
of the two signals each of
your parents do
• You will not bear any VNTRs
not borne by at least one of
your parents
Morling, N. and Hansen, H., Int. J. Leg. Med., 1993, 105, pp.
189-196.
La
dd
er
La
dd
er

M
ot
he
r
M
ot
he
r
Ch
ild
Ch
ild
Ex
cl
ud
ed
m
an
Ex
cl
ud

ed
m
an
No
n-
ex
cl
ud
ed
m
an
No
n-
ex
cl
ud
ed
m
an
Case 1 Case 2
..the

so-called
“obligate
paternal
allele”
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Summary of Part 1 of DNA
Profiling
• DNA evidence is just one type of evidence
• DNA can be valuable evidence because:
– each person’s DNA is unique
– testing requires only a small DNA sample
– the sample can be old and somewhat degraded
• STR analysis is currently the most commonly used
method of DNA testing and relies on small variations in
lengths of DNA that vary between individuals
• Individuals that are blood relatives have DNA that is more
similar than unrelated people and our detailed knowledge
of this enables DNA tests with different purposes (i.e.
crime scene investigations vs paternity testing)
Testing for blood at a crime
scene
• CSI’s normally try to detect,
photograph and sample
blood stains directly

• When blood is too hard to
detect directly, trace blood
can be detected by spraying
with luminol and an oxidizer
(e.g. peroxide…. H2O2)
• Drawback is that the luminol/
oxidizer mix will also react
with other things that aren’t
blood (ferricyanide,
horseradish, plant roots and
some bleaches, for example)
• Another drawback is that
contaminating the blood
sample with luminol spray
can limit or prevent other
tests being run on the blood
Photo by"
David Muelheims,"
Germany"
I.A.B.P.A. News March, 2007
500 mL of horse blood poured
on the ground and monitored for
16 months in an experiment to
determine how long “post-
deposition” a bloodstain
exposed to the elements would
be detectable with luminol
Chemical structure of luminol

C
C
C
C
C
C
C
N
N
C
NH2 O
O
H2O2 O2 H2O
Fe from hemoglobin
in blood as catalyst
Reaction between
oxygen and luminol
gives off blue light
H
H
H
H

H
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Testing for blood at a crime scene
• The luminol is deprotonated by the base (OH-)
in mixture and becomes the dianion
• When located in near physical proximity (ie on
a blood stain), a catalyst such as the iron in
hemoglobin from blood, disproportionates
peroxide into molecular oxygen and water
• Anything that can catalyze the formation of
oxygen from peroxide will be a source of
background (eg. Horseradish peroxidase in
horseradish, as is commonly used in
chemiluminescence detection of Western
blots).
• The O2 evolved by the disproportionation of
hydrogen peroxide reacts with the dianionic
form of the luminol to give a diphthalate in a
triplet excited state
• The triplet excited state undergoes an
intersystem crossing to the singlet excited
state which then gives off a blue photon when
returning to the ground state

• This mechanism which involves intersystem
crossing technically makes the luminol glow a
“phosphorescence” event because
intersystem crossing is a “forbidden
transition”. That it is a phosphorescence
event is one of the reasons why the glow lasts
a long time and is visible for 30 sec or so in
the context of crime scene investigation.
H2O2 O2 H2O
Fe from hemoglobin
in blood as catalyst
Wikipedia.com
Assignment
• Read Article:
“Establishing the
identity of Anna
Anderson
Manahan”
Forensic DNA Part 4
1
Part 4: Uncertainties in DNA Profiling
and Privacy Issues
• Limitations of using DNA evidence:
– What DON’T DNA testing results

prove?
– How far can data be trusted?
– How and when should forensic teams
entertain alternative explanations for
their data?
• Privacy:
− Protection of 4th amendment rights
(search and seizure) in acquisition of
DNA samples
− Possible negative or unintended
consequences associated with
archiving DNA and maintaining large
DNA sample databases
Uncertainties in DNA Profiling
• Case 1: Allele dropout
due to sample
degradation (ambiguous
data)
• Case 2: The Phantom of
Heilbronn (inadvertent
contamination)
http://en.wikipedia.org/wiki/
Phantom_of_Heilbronn
• Case 3: Artificial DNA
(nefarious contamination)

Forensic DNA Part 4
2
Case 1: Allele dropout due to
sample degradation (ambiguous data)
• Often one or both
alleles at a locus
cannot be
sequenced due to
sample degradation
• How do crime labs
handle ambiguous
data?
6 8
Heterozygous alleles
are well balanced
*
8
Allele 6 amplicon
has “dropped out”
Figure 6.10, J.M. Butler (2005) Forensic DNA Typing, 2nd
Edition © 2005 Elsevier Academic Press
• Crime scenes are often messy and DNA evidence is often
old or has been exposed to harsh chemical environments,
fire, or years of weather and sunlight by the time collected

*If this region of DNA is degraded in just one copy of the
chromosome,
the corresponding allele will not amplify and will not be
observed in the
results.
6
8
6
8
Case 2: “The Phantom of
Heilbronn” (inadvertent contamination)
• Mitochondrial DNA testing in more than
a dozen crimes (including several
murders) in eastern Europe between
1993-2008 suggested a single
perpetrator
• The German government spent $18
million dollars investigating leads
• In 2009, the DNA source was traced to a
woman in a cotton swab packing plant
who had handled the swabs used to
collect all the associated crime scene
evidence
• An inability on the part of the
investigators to entertain other possible
scenarios to explain their DNA results

caused an multi-year “goose chase”
wasting valuable resources that might
have solved other crimes
Krimsky and Simoncelli, Genetic Justice: DNA Data Banks,
Criminal Investigations and Civil Liberties. Columbia
University Press, 2011.
Heilbronn
Forensic DNA Part 4
3
Case 3: Artificial DNA and the creation
of a fabricated human DNA profile
• A paper published in 2009 demonstrated
that it is possible to prepare an artificial
DNA sample in test tube
• They made artificial DNA samples from:
– a real human DNA sample
– by piecing together DNA from a library of
known alleles
• The authors also offer a solution which is
to test DNA methylation which
distinguishes between DNA from a human
and artificial DNA.
• In principle a crime scene could be
contaminated with artificial DNA and the

lack of authenticity would go undetected
using existing forensic technology
4(2) pp. 95-103
Case 3: Artificial DNA
4(2) pp. 95-103
Fake “touch” sample
Fake saliva sample
Fake blood sample
Donor of blood for
Fake blood sample
(doesn’t match fake DNA
found in sample)
Donor of saliva for
fake saliva sample
(doesn’t match fake
DNA found in sample)
Forensic DNA Part 4
4
Privacy Issues: protection of 4th amendment
rights against “search and seizure”

The right of the people to be secure in
their persons, houses, papers, and
effects, against unreasonable searches
and seizures, shall not be violated, and
no warrants shall issue, but upon
probable cause, supported by oath or
affirmation, and particularly describing
the place to be searched, and the
persons or things to be seized.
Privacy Issues: protection of 4th amendment
rights against “search and seizure”
• Surreptitious sampling of DNA occurs when
law enforcement gathers DNA samples that
have been “shed” (skin, saliva, hair
follicles) without first obtaining a warrant
• In the U.S., there is no legal protection for
“abandoned” DNA samples
– e.g. DNA from saliva found on the
mouth of a beverage bottle or the butt of
a cigarette
• Several legal cases have addressed this
issue, most of the ones involving DNA
evidence have sided with law enforcement
but other arenas of “privacy invasion” have
sided with the citizen….
– State of Washington v. Athan
– Kyllo v. United States
• What do you think?

Forensic DNA Part 4
5
Privacy Issues: possible unintended
consequences
• Early government DNA databases initially consisted of
murderers and rapists
• In 2006, President George W. Bush signed into law the
“DNA Fingerprint Act of 2005” which removed an earlier
provision prohibiting the upload to CODIS of arrestees
who had NOT been charged or convicted of crimes
• Since 2009, in California, a sample of DNA is taken from
you if you are arrested for any felony (even if never
charged or convicted)
• Currently in the UK people arrested or detained for any
crimes (…but not necessarily charged or convicted), have
DNA samples taken and the UK database now contains
more than 7.5% of the population
• “Familial DNA searches” can be conducted in which DNA
from crime scenes is compared against that of known
felons…which sometimes eventually fingers family
members of known criminals…which subjects those from
larger families to greater genetic surveillance and, in
essence, increases the sizes of all the databases to
include innocent family members of known criminals

Privacy Issues: possible unintended
consequences
“The power to assemble a permanent
national DNA database of all
offenders who have committed any of
the crimes listed [in 18 U.S.C.] has
catastrophic potential. If placed in the
hands of an administration that
chooses to ‘exalt order at the cost of
liberty’…the database could be used
to repress dissent or, quite literally, to
eliminate political opposition.”
-Judge Stephen R. Reinhardt in
United States v. Kincade (2004).
What are some possible negative consequences of acquiring
and archiving DNA samples of the population-at-large?
DNA Profiling Part 3
1
Part 3: Analyzing the certainty of
DNA profile comparisons

At the end of the DNA data analysis in which two samples are
being
compared to decide if they match, the analyst concludes one of
the
following:
- “cannot exclude”
- “can exclude”
- “inconclusive/uninterpretable”
If the answer is “cannot exclude” (I.e. there was a DNA match),
a
statistical interpretation is also provided which may be stated
similarly
to the following:
“There is a 1 in 300 million chance that suspect and crime scene
DNA derive from different persons by chance”
This number refers to a calculation of the “random match
probability”
Alleles in DNA profiling
• Allele: A specific sequence
of DNA observed in nature.
In the top example these
would be chocolate, vanilla
and strawberry.
• In DNA profiling, each allele
is assigned a number (as in

bottom example).
• The allele’s number
normally refers to how many
copies of the repeated
stretch of DNA has been
observed in the STR.
• Allele range: The entire
cohort of different alleles
observed at a specific
chromosomal locus.
• The allele range in this
example would be 8-14.
Mom Dad Alleles
Allele
range
The “CSF1PO locus”
Chr1 copy1 Chr1 copy2
2
Allele frequencies
• Allele frequency: The proportion of all
copies of a locus that is made up of a
particular sequence variation.

– Alleles are not all observed in nature in the
same proportions
– Cataloging which ones are actually
observed and how often helps determine
how rare they are
– The rarer the allele, the less likely it will be
observed by chance in two unrelated
people
• If Mom and Dad are the only two people
in the whole world….the allele
frequencies for the chromosome shown
would be the following:
– chocolate = 0.5
– vanilla = 0.25
– strawberry = 0.25
Mom Dad
Allele
frequencies
Chr1 copy1 Chr1 copy2
Allele frequencies:
• Sum to “1” or “100%” at a given locus
(depending on how table shows info)
• Are a mathematical expression of the
likelihood of observing that particular
allele in a randomly chosen DNA
sample

• Using the example shown on the right,
a frequency of “25.369” means that
25.369% of the time, a scientist testing
DNA will observe 10 copies of the
tandem repeat of DNA associated with
the CSF1PO locus (if all DNA donors
are Caucasian)
• Tables of allele frequencies are used
which are broken down according to
broad ethnic populations (eg.
Caucasian, African American, Hispanic,
Asian) Total 99.999
Allele
frequencies
Locus
Budowle et al J Forensic Sci, 1999, 44(6), pp. 1277-86.
3
How do they narrow it down to
“1 chance in 300 million”?
• Multiple loci are analyzed
• The overall probability (of two
samples being from different
people by chance if they give the

same result…) is known as the
“random match probability”.
• The random match probability
assumes complete randomness of
all the alleles in the human
population and is computed using
the “basic product rule”:
…so what does THAT mean?
P = 2
n A1A2B1B2C1C2....N1N2
The Basic Product Rule
• Overall probability is represented by
“P”
• Each locus being analyzed is
represented by the letters A, B, C etc.,
• The letters A, B, C etc each appear
twice because there are two copies of
each chromosome
• The two copies of each chromosome
can either bear the same allele or
different alleles and are represented by
subscripts “1” and “2” ie. A1A2
• The 2n represents a necessary doubling
required per locus… because in the
laboratory it cannot be distinguished
which allele came from the maternal
chromosome and which from paternal
and they count as separate possibilities

when tabulating all the possible
combinations
P = 2
P = 2
P = 2
P = 2
Mom Dad
…was it …or
Mom Dad
4
Example “basic product rule” calculation
• Imagine analyzing two
DNA samples (e.g
suspect DNA and crime
scene DNA) for the 3
loci shown
• For the three loci, you
obtain the following

results for both
samples
– DS1358 = Alleles 16 &
17*
– VWA = Alleles 17 & 18
– FGA = Alleles 21 & 22
• HOW SURE are you
that the DNA match is
not by chance?
Crime
Scene
Suspect
*
*Two alleles are typically observed per locus because ~80% of
people are heterozygous at these loci I.e. “vanilla, strawberry”.
Allele frequency tables from the literature (same ones FBI uses)
Example of
“basic product
rule calculation”
• Multiply the observed allele
frequencies (expressed as
decimal) together according
to the equation

• Convert expression to a
probability
..that the Suspect and Crime Scene samples derived from
different people by chance
(I.e. that there was a “random match”).
P = 2n D3S1358 Allele 16 and 17 frequencies⎡ ⎣ ⎤ ⎦
VWA Allele 17 and 18 frequencies⎡ ⎣ ⎤ ⎦
21 and 22 frequencies⎡ ⎣ ⎤ ⎦
P = 23 0.23153( )× 0.21182( )⎡ ⎣ ⎤ ⎦
.22194( )⎡ ⎣ ⎤ ⎦ ⎡ ⎣ ⎤ ⎦
= 7.49 × 10
−4 = 0.000749
0.000749 =
7.49
10,000
=
1
1335
≈ 1 in 1335 chance
P = 2
n A1A2B1B2C1C2

5
More loci analyzed = greater
certainty that DNA samples do
not match by chance
• In previous example with 3 loci we narrowed the
probability to: ~ 1 in 1335
• A few things to point out:
– Every additional locus tested decreases the
probability of a random match.
– The results of the calculation are VERY
DEPENDENT on the actual alleles observed in the
analysis.
– Allele frequencies vary with ethnic background.
– The random match probability is not an “index
of guilt” and does not even address the likelihood
that the defendant is the source of the DNA. Its
sole purpose is to help a jury address the
hypothesis that a DNA match between crime
scene and suspect was observed BY CHANCE!
0.000749 =

7.49
10,000
=
1
1335
≈ 1 in 1335 chance
Group work on basic product rule
calculation
• Break into groups of ~4 people
• Work through the following calculation: Imagine analyzing 3
sets of 2-DNA samples
(suspect and crime scene DNA) using 4 loci, and the allele
frequencies below:
1. 7,8; 13,14; 14,15; 25,26
2. 10,11;16,17; 18,19; 21,23
3. 11,12; 18,19; 19,20; 18,22
• Spend ~15 min. discussing within your group the following:
– Why analyzing more loci decreases the probability of a
random match.
– Why the calculation is only as good as the table(s).
– What you think the forensic expert probably does in cases in
which the ethnic
background of the possible perpetrator isn’t known.
– Are there negative consequences to incorrectly calculating
the number? What
– Why does a “match” between crime scene and suspect DNA

not necessarily
mean the suspect is guilty?
6
Group work on basic product rule
calculation
• Break into groups of ~4 people
• Work through the calculation provided on the
worksheet
• Spend ~15 minutes discussing within your
group the following:
– Why analyzing more loci decreases the
probability of a random match.
– Why the calculation is only as good as the
table(s).
– What you think the forensic expert
probably does in cases in which the
ethnic background of the possible
perpetrator isn’t known.
– Are there negative consequences to
incorrectly calculating the number? If so,
what might some of them be?

– Why does a “match” between crime
scene and suspect DNA not necessarily
mean the suspect is guilty?
1
Part 2 of DNA Profiling: the
case of the Romanovs
• The last Tsar of Russia,
Nicholas II had a wife,
Alexandra, four daughters,
Olga, Tatiana, Maria and
Anastasia and a son, Alexei
• After a lengthy period of civil
unrest, and amidst the
devastation of WWI, Nicholas II
abdicated his throne in March,
1917
• First the provisional (Karensky)
government was in power for a
few months but in October
1917 the Bolsheviks took over
during the Bolshevik Revolution
Exile and Murder
• The ex-Tsar and his family
were eventually imprisoned in

the “Ipatiev House” in
Ekaterinburg, a small town in
the Ural mountains
• On July 17, 1918 the family
was taken to a basement room
of the house and murdered
• The locations of the bodies
were a Russian state secret
for about 70 years
• There were persistent rumors
throughout the 20th century
that two of the children, Alexei
and Anastasia, had possibly
survived.
Ipatiev House
Room
where
murders
took
place
romanov-memorial.com
awesomestories.com
2

Did one of the
daughters survive?
• Anna Anderson (Manahan) appeared
mysteriously in 1921 and claimed to be the
youngest daughter of the Tsar, Grand
Duchess Anastasia
• She looked a lot like Anastasia, was about
the same age, and spoke fluent Russian
and German
• She also knew details of Anastasia’s life
that were not widely known outside the
Romanov family circle
• Some believed her to be Anastasia even to
her death in 1984 including Tatiana Botkin
the daughter of the Romanov family doctor.
Gravesites found near Ekaterinburg were
determined to be those of the Tsar and
his family The main grave found in 1991
contained remains of Tsar, Tsarina, 3
daughters and 4 servants
Another grave found in 2007 (a few hundred meters away)
contained remains of the son and one of the daughters
http://www.history.com/topics/russian-revolution/videos/
finding-the-romanovs
romanov-memorial.com awesomestories.com
Dr. Alexander Avdonin:

discoverer of 1st grave
3
Forensic work included analysis of bone fragments,
dental records, blood samples and DNA
romanov-memorial.com
*Rogaev, et al., PNAS, 2009, 106(13), pp. 5258-5263.
*
*
awesomestories.com
Mitochondrial DNA sequencing helped
determine the identities of the skeletons
found in the second grave
• Mitochondrial DNA is
passed only from
mother to child
• There must be a chain
of “unbroken maternal
lineage” established to
use mtDNA to link two
people genetically
• But a son’s mtDNA

can be compared to his
mother, grandmother,
great grandmother, etc.
• The red or black filled
circles and squares
represent familial
relationship groups that
can be confirmed by
mitochondrial DNA
sequencing
4
• DNA data show that
skeletons from both
graves match each
other as well as
maternally-linked
descendants from
Queen Victoria
Genetic Marker Database Alexei
Maria Tsarina Descendants
Across a row the sequences
are identical.
• Y-STR DNA analysis allows comparison of
paternally-linked family members
• Y-STR analysis showed that the skeleton

believed to be the son, Alexei, in one grave
matched the skeleton believed to be the Tsar
found in the other grave as well as DNA from a
bloodstained shirt.
Tsar Alexei
Paternal
DNA Testing
*
*They still had a shirt worn by Nicholas II during an
assassination attempt in Japan in 1891
that they knew had his blood on it! The stain on the shirt was
117 years old!!!
The “allele” is the same
for all samples at this
locus, consistent with
them deriving from
paternally related
individuals.
Many more alleles
coming up the same
strengthens the
conclusion the two
individuals were related.

5
Okay, so what about the identity of
Anna Anderson?
• Anna Anderson (Manahan)
appeared mysteriously in 1921
and claimed to be the youngest
daughter of the Tsar, Grand
Duchess Anastasia
• Tissue and hair samples from the
deceased Anna Anderson
Manahan were compared to living
relatives of the Romanovs and
DNA from the Tsar and Tsarina’s
skeletons found near Ekaterinberg
• The samples were also compared
to a living relative of Franzisca
Schanzkowska, a polish
ammunitions factory worker who
disappeared about the same time
as Anderson appeared in Berlin
DNA analysis of tissue samples from Anna
Anderson as compared to Tsar and Tsarina
• Are results from any one of the six loci
consistent with her BEING the Grand
Duchess Anastasia?
• Are results from any of the loci consistent

with her being a child of EITHER the Tsar
OR the Tsarina? Which ones? Why?
• Are results from any of the loci
inconsistent with her being the child of
either the Tsar or Tsarina? Which? Why?
Locus 1 2 3 4 5 6
6
DNA analysis of tissue samples from Anna Anderson
as compared to a relative of the Tsarina and a relative
of Franzisca Schanzkowska,
a WWI munitions worker from Poland
• Does this data establish her as directly related to
Carl Maucher (great nephew of Schanzkowska)?
• Does this data rule out that she is related to the
Tsarina?
• Does it matter that the “blood lines” are all
maternal?

103114 1 Forensic DNA Analysis Part 1 The science.docx

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