Transcript DNA Spring08

DNA
The Indispensable Forensic Tool
1
Introduction
• Genetic information is carried in the form of
DNA in all cellular organisms.
• DNA is the genetic code that imparts our
individuality.
2
Introduction
• Increasing knowledge of the human genome
and improved detection technologies and the
development of the polymerase chain
reaction (PCR) technique have increased the
sensitivity, speed and discrimination
potential of DNA profiling.
3
Introduction
• For identification of an individual, we can
examine specific regions of DNA as they vary
from person to person, in order to create a
DNA profile or fingerprint of the individual in
question.
• There is an extremely low probability that
another person has the same DNA profile for
the given set of regions.
4
Introduction
• DNA as a tool for forensic identification has broad
applications, including:
– identification of potential suspects or subjects
involved in a crime or who may have
contributed to a crime scene stain
– exoneration of wrongly accused persons
– identifying family relationships or establishing
paternity
– identifying victims of crime, war, catastrophe or
other death investigations
5
Objectives
• Understand the principles of Forensic DNA profiling
• Have an understanding of the range of profiling
methods
• Know the method of sample processing and sample
limitations
• Understand the significance of DNA profiling
results and the limitations of the data
• Know the methods used in DNA extraction and
Quantization
• Know the components of a Genetic analyzer
• Be familiar with DNA databases available and their
applications
• Understand the applications of Mitochondrial DNA
6
Classical Genetics
1 Children resemble their parents.
2 Genes come in pairs.
3 Genes don't blend.
4 Some genes are dominant.
5 Genetic inheritance follows rules.
6 Genes are real things.
7 All cells arise from pre-existing cells.
8 Sex cells have one set of chromosomes; body
cells have two.
7
Classical Genetics
9 Specialized chromosomes determine
gender.
10 Chromosomes carry genes.
11Genes get shuffled when chromosomes
exchange pieces.
12 Evolution begins with the inheritance of
gene variations.
13 Mendelian laws apply to human beings.
14 Mendelian genetics cannot fully explain
human health and behavior.
8
Individual Characteristics
15 DNA and proteins are key molecules of the
cell nucleus.
16 One gene makes one protein.
17 A gene is made of DNA.
18 Bacteria and viruses have DNA too.
19 The DNA molecule is shaped like a twisted
ladder.
20 A half DNA ladder is a template for copying
the whole.
9
Molecules of Genetics
21 RNA is an intermediary between DNA and
protein.
22 DNA words are three letters long.
23 A gene is a discrete sequence of DNA
nucleotides.
24 The RNA message is sometimes edited.
25 Some viruses store genetic information in
RNA.
10
Molecules of Genetics
26 RNA was the first genetic molecule.
27 Mutations are changes in genetic
information.
28 Some types of mutations are
automatically repaired.
11
Command & Control of Genetics
29 DNA is packaged in a chromosome.
30 Higher cells incorporate an ancient
chromosome.
31 Some DNA does not encode protein.
32 Some DNA can jump.
33 Genes can be turned on and off.
12
Command & Control of Genetics
34 Genes can be moved between species.
35 DNA responds to signals from outside
the cell.
36 Different genes are active in different
kinds of cells.
37 Master genes control basic body plans.
13
Command & Control of Genetics
38 Development balances cell growth and
death.
39 A genome is an entire set of genes.
40 Living things share common genes.
41 DNA is only the beginning for
understanding the human genome.
14
Principles of Heredity
DNA
15
Principles of Heredity
• Genes are the basic unit of heredity.
• Each gene by itself or with others controls the
development of a specific characteristic in the
new individual.
• Genes are located on chromosomes.
• Human cells contain 46 chromosomes which are
mated in 23 pairs. The only case in which this is
not so is human reproductive cells which have
23 chromosomes. During the fertilization of the
egg, the reproductive cells combine to form the
zygote which will then have 46 again.
16
Principles of Heredity
• The egg cell always contains the X chromosome,
but the sperm cell may have either the X or a Y
chromosome. The combining of an XX will
develop into a female, and XY will develop into a
male.
• Just like our chromosomes, the genes they carry
also come in pairs. When the chromosomes pair
up, so do the genes that they carry (eye color with
eye color, and hair color with hair color)
• Alternative forms of genes that influence a given
characteristic and are aligned with one another on
a chromosome pair are known as alleles.
17
Principles of Heredity
• An example of these allele genes is human blood
types which are categorized with the A-B-O system.
• For example, a gene pair consisting of similar genes
is called homozygous (AA,BB)
• A pair that consists of two different genes is said to
be heterozygous (AO)
• When there is a heterozygous pair, the dominant
gene is the one that will show. The A and B genes
are said to be dominant and the O is recessive.
18
Principles of Heredity
• A pair of allele genes together make up what
is called the genotype (which is the genetic
make up) Phenotypes are someone’s
physical characteristics that we would be able
to see.
• Genotypes can be determined by studying
someone’s family history.
• If the genotypes of both parents are known,
the genotypes of their possible offspring can
be somewhat predicted.
19
Principles of Heredity
•
A simple way of predicting
genotypes is the Punnett
square.
•
Although genotyping is useful
for studying the transmission
of genes it isn’t directly
related to criminal
investigations.
•
It is more useful during
paternity testing which would
be disputed in civil, not
criminal court.
20
Packaging of Bio Evidence
• Before the collection of biological evidence
begins, it is important that it be photographed
and recorded on sketches.
• Wearing disposable latex gloves while handling
the evidence is required.
• Clothing from victim and suspect with blood
evidence must be collected.
• The packaging of biological evidence in plastic or
airtight containers must be avoided because the
accumulation of residual moisture could
contribute to the growth of DNA-destroying
bacteria and fungi.
21
Packaging of Bio Evidence
• Each stained article should be packaged separately
in a paper bag or in a well-ventilated box.
• Dried blood is best removed from a surface by
using a sterile cotton swab lightly moistened with
distilled water that is air dried before being placed
in a swab box, then a paper or manila envelope.
• All biological evidence should be refrigerated or
stored in a cool location until delivery to the
laboratory.
• Standard/reference DNA specimens must also be
collected, such as blood or the buccal swab
22
(swabbing the mouth and cheek).
DNA in a Nutshell
• DNA is constructed as a very large molecule made by
linking a series of repeating units called nucleotides.
• A nucleotide is composed of a sugar, a phosphorouscontaining group, and a nitrogen-containing molecule
called a base.
• Four types of bases are associated with the DNA
structure: adenine (A), guanine (G), cytosine (C), and
thymine (T).
• As a result, adenine pairs with thymine and guanine
pairs with cytosine.
• This concept is known as base pairing.
23
DNA in a Nutshell
• Each group of three nucleotides in a DNA
sequence codes for a particular amino acid.
– Example: G-A-G codes for the amino acid
glutamine, while C-G-T codes for alanine.
– If a nucleotide is “changed”, for example a T is
substituted for A and G-A-G becomes G-T-G, the
“wrong” amino acid is placed in the protein
– This is the basis for many diseases and health
issues.
24
How nucleotides can be linked to form a DNA strand.
S designates the sugar component, which is joined with
phosphate groups (P) to form the backbone of DNA. Projecting
from the backbone are four bases: A, adenine; G, guanine; T,
25
thymine; and C, cytosine.
DNA in a Nutshell
• DNA duplicates itself prior to cell division.
• DNA replication begins with the unwinding of the
DNA strands of the double helix.
• The ability to multiply small bits of DNA now means
that sample size is no longer a limitation in
characterizing DNA recovered at a crime scene.
• All humans have the same type of repeats in DNA,
but there is tremendous variation in the number of
repeats each of us have.
26
A representation of a
DNA double helix.
Notice how bases G and
C pair with each other,
as do bases A and T.
This is the only
arrangement in which
two DNA strands can
align with each other in
a double-helix
configuration.
27
(a) A string of amino acids composes one of the protein chains of
hemoglobin. (b) Substitution of just one amino acid for another in
the protein chain results in sickle-cell hemoglobin.
28
Replication of DNA. The strands of the original DNA molecule
are separated, and two new strands are assembled.
29
DNA in a Nutshell
• Typically, a core sequence consists of 15
to 35 bases in length and repeats itself
up to a thousand times.
• The key to understanding DNA typing lies
in the knowledge that numerous
possibilities exist for the number of times
a particular sequence of base letters can
repeat itself on a DNA strand.
30
DNA Profiling
By extracting DNA and digesting
it with a restriction enzyme that
cut the DNA outside but not
within the repeat block, the size
of each block of block of
repeats could be determined by
electrophoresis, and the term
restriction fragment length
polymorphism (RFLP) was
applied.
31
DNA Profiling
The first application of RFLP analysis was
in the study of genetic diseases.
Many RFLP sequences were mapped to a
specific location on a chromosome.
The gene associated with a genetic
disease, i.e., Huntington’s disease, could
be located by studying the transmission
of many RFLP sequences through the
generations of a family.
32
The DNA RFLP typing
process.
33
DNA Profiling
Forensic serology was utilizing protein
and enzyme genetics for the purpose of
identification.
The source of a body fluid or tissue could
be narrowed down by determining the
ABO type or by studying variants in a few
enzymes
These were separated by electrophoresis
and visualized using an enzymatic
activity-based color reaction.
34
DNA Profiling
Most of the markers of forensic interest were
located on blood cells and were therefore
not useful in characterizing saliva, semen or
vaginal secretions.
The ABO types were present in these body
fluids, but only in “secretors” who make up
about 80% of the population.
The fragile nature of proteins also leads to loss
of enzymatic activity and detectability after
short periods of time in adverse
environmental conditions.
35
DNA Profiling
1985, Sir Alex Jeffreys was credited with
the first practical forensic application of
human DNA analysis.
Jeffries used the RFLP method to identify
the semen donor in a serial rapehomicide case in Britain. This first use of
forensic DNA profiling is described in
Joseph Wambaugh's The Blooding.
36
DNA Profiling
In 1986, two companies in the US began
offering commercial DNA testing. Lifecodes
and Cellmark developed RFLP methods using
2 different restriction enzymes. Due to the
use of different restriction enzymes, data
generated by the two companies could not be
inter-compared.
The FBI went online in 1988 with a method
using the restriction enzyme Hae III which has
become fairly standardized among state and
local forensic laboratories
37
Restriction Fragment Length
Polymorphism
The RFLP method identifies variation in the number
of repeats in tandem repeat tracts.
After size fragmentation by gel electrophoresis, DNA
is transferred to a nylon membrane by the
Southern blot method.
38
Restriction Fragment Length
Polymorphism
Southern Blot
39
Clean Technique
•Similar to what Microbiology uses.
•Prevents cross-contamination
•Surface areas where samples are
processed should be cleaned with a fresh
10% bleach solution or appropriate
disinfectant cleaner.
•All instruments used to process forensic
samples (e.g., forceps, scissors, scalpel &
razor blades, bone cutting equipment,
pipettes' and metal probes) must be
cleaned.
40
Clean Technique
Samples should always be placed on clean surfaces
and in sterile tubes.
Waste that may contain amplified DNA should be
disposed of with great care. Contamination of
extraction areas with amplified product is the most
difficult kind of contamination to eradicate after it
has occurred.
Finally the foot traffic in the lab where analysis is
being conducted should be limited
41
Sampling
Questioned samples should be processed
separately from reference standards, by
completing the analysis of questioned samples
at a different time in a different location than
reference standards.
Eliminates the possibility of crosscontamination or sample switching
42
Sample Size & Sampling Size
•The preservation & conservation of evidence is
important to forensic DNA quality assurance.
•The most effective challenge to a DNA test is reanalysis by a second independent laboratory.
•Before testing the DNA analyst must decide
whether there is sufficient sample for one or more
tests. (When the quantity of sample is sufficient, a
portion of the sample is set aside and preserved
for possible future re-analysis).
43
Degraded DNA
If samples are thought to
contain partially degraded
DNA, more sample may be
needed to obtain useful
results. Environmental
conditions before, during
and after the deposition of
a stain and even in
collection, packaging and
storage greatly affect its
quality.
44
Degraded DNA
•If a drop of blood falls on a cloth and dries quickly, the DNA
will be well-preserved.
•If the drop of blood falls on cloth, which remains damp for
any appreciable period of time, microorganisms may degrade
the DNA in white blood cells (remember red blood cells don’t
contain DNA).
•The surface upon which the body fluid is deposited is
important. The DNA in human body fluid cells will be degraded
rapidly on items (rich in microorganisms) i.e.,:
foliage
soil
carpeting
any warm and moist surface
45
Degraded DNA
•Most agencies have evidence submission manuals
that include handling; the FBI Handbook can be
found at the following website:
http://www.fbi.gov/hq/lab/handbook/intro.htm
46
Relevance of the Evidence
The relevance of any evidence to
a particular case depends on the
close interactions between the
DNA analyst, the crime scene
investigator, the case
investigator and the prosecutor.
47
Relevance of the Evidence
•The crime scene investigator has a brief window
of opportunity to collect evidence that may be
relevant to the crime being investigated. The
investigator will collect a broad range of
evidence samples.
•The crime scene investigator or responding
officer must document unique information
relating to the state of the scene and evidence
collected.
48
Relevance of the Evidence
After preliminary investigation, the case
investigator may be able to provide information
about the offense, such as any prior relationship
of the victims and suspects, statements to
police, medical reports, and how various
evidentiary items relate to the crime.
49
Relevance of the Evidence
The prosecutor brings knowledge of defense theories,
pertinent case questions, legal standards of proof and an
overview of information about the case gleaned from many
different sources.
The DNA analyst contributes knowledge of the capabilities
and limitations of various DNA testing methods. The analyst
knows what items are most likely to produce profiles and
what interpretations might be made regarding those profiles.
The analyst can use DNA to determine the source of a
biological sample, but not when or under what circumstances
it was deposited.
50
Relevance of the Evidence
The most beneficial reason for allowing the DNA
analyst access to certain aspects of the case is in
enhancing his/her ability to select and test
evidence in an order consistent with its likelihood to
yield results and its probative quality.
Because stains are not always easy to see, crime
scenarios may help lead the analyst to the best
area for testing and save time for the analyst,
contributor and courts.
51
Relevance of the Evidence
Not uncommon to find blood from multiple
individuals on the clothing of violent
perpetrators.
Lacking evidence to connect the suspect to
the injured victim, the DNA analyst might
perform an exhaustive analysis of blood on the
victim’s clothing or other items from the scene,
as the suspect may have been injured during
the event.
52
Relevance of the Evidence
It is not uncommon for a perpetrator to
injure him/herself during a knife attack,
particularly when one hand is used to
control the victim while the other holds the
knife. Most physical struggles will result in
the transfer of some trace DNA evidence
from subject to victim.
53
Choosing the Technique
Currently there are three main varieties of
PCR-based forensic DNA typing.
The major technique used in most crime
labs is the STR multiplex which relies on
nuclear DNA and is applicable to all
nucleated specimen types capable of
yielding at least one nanogram of DNA.
 Samples might involve saliva, blood,
semen, vaginal secretions, body tissues,
spongy bone, and growth phase hairs with
roots or sheaths.
54
Choosing the Technique
A common evidence type not amenable to nuclear
STR testing is hair shafts. A hair root may contain a
tissue tag or nucleated cells that will give STR results,
but not the shaft.
The only DNA test capable of providing results on this
material is mitochondrial testing. (FBI started using in
1996) mtDNA testing is also the method of choice for
badly degraded samples such as old skeletal remains.
mtDNA is inherited through the maternal line. Any
maternally related individual or the children of a female
individual could serve as reference standards.
55
Choosing the Technique
In a mixture of male and female DNA where the female
component is expected to greatly exceed the male
component, Y-chromosome STRs are a technique of
choice. Based on Y-chromosome STR loci, the test can
indicate the presence of a male to corroborate a story or
yield a profile of a specified male.
This test is almost exclusively used on mixtures of male
and female DNA. Where semen does not contain
spermatozoa or the mixture consists of male saliva and
female secretions, nuclear STRs can discern an
interpretable mixture up to about a 1:10 ratio of male to
56
female DNA.
Comparison of Techniques
The US Department of Defense employs mtDNA typing through its Defense Prisoner of
War/Missing Personnel Office. http://www.dtic.mil/dpmo/family/dnatyping.htm
57
Organic Extraction
• Once the sample is chosen the
DNA must be extracted from
the sample before
electrophoresis can be
conducted.
58
Organic Extraction
Organic extraction is
appropriate for most
forensic specimens
including:
• Blood & blood stains
• Epithelial cells
• Saliva
• Vomit
•
•
•
•
Urine
Feces
Sweat
Semen (differential
extraction method)
• Hair
• Tissue
• Bone
59
Organic Extraction
• Organic extraction uses and steps:
– a digest buffer (SDS) to denature proteins
– serine (pro-K) to cleave the proteins into smaller
fragments. Pro-K digestion helps lyse the cells and
solubulizes components while denaturing the protein.
– alcohol, provides an alkaline environment and
precipitates the RNA and protein which separates it
from aqueous DNA, Phenol and chloroform.
– wash sample with buffer (TE)
– filter to remove leftover extraction reagents and
contaminants.
60
Organic Extraction
• The amount of sample used is based on the
sample type, concentration, substrate and
expected condition.
• Blood stains may be obtained from a cutting
of the fabric on which they were deposited
except when found on blue denim. The dye in
denim is a PCR inhibitor, so swabbing a
bloodstain on jeans is the best method of
recovery. Cotton swabs should be used for
DNA extraction.
61
Chelex Extraction
This method is appropriate for many forensic
specimens including:
• Blood & blood stains
• Epithelial cells
• Saliva
• Hair
• Semen (differential extraction method)
62
Chelex Extraction
• It is highly selective for heavy metals and
divalent cations versus monovalent cations. .
• The basic nature of Chelex resin keeps
extracted DNA in a single stranded state
available for concentration and/or
quantization.
63
Chelex Extraction
Blood
• Samples are first extracted in DI water
or saline to help free cells from the
substrate and to lyse any red blood
cells. The supernatant, which may
contain heme, can be discarded.
Washing steps help remove any
residual protein. Cells are then
incubated with Chelex and lysed
(broken apart) by boiling.
64
Chelex Extraction
• Epithelial Cells
• Chelex extraction is a rapid method for
extraction of DNA from epithelial cells found
on items such as cigarette butts, envelope
flaps, stamps, buccal swabs, beverage
containers and swabbings or cuttings from
other textiles or hard surfaces.
• Usually exposed to the environment
65
Chelex Extraction
• Hairs
• Hairs that just fall out are in telogen or resting
phase and do not possess nucleated root cells
or a tissue tag and will not provide sufficient
DNA for typing.
• Hairs in the growth or cessation stages
(anagen or catagen) may have nuclear DNA in
their roots.
• Hairs, forcibly removed, may or may not have
tissue tags.
66
Chelex Extraction
• It is very important to clean the hair
thoroughly to remove any foreign body fluids
or other materials. Retain the hair shaft as a
control.
• If nuclear DNA testing fails, the shaft may be
sent for mtDNA analysis. Approximately a 1cm
(2.54 cm=1 inch) length of hair, obtained form
the root end is necessary for analysis. Hairs
mounted on slides can also be recovered for
analysis.
67
Chelex Extraction
Sperm
• Mixtures containing
spermatozoa provide a unique
opportunity to separate the
DNA profiles of the contributors.
• After pelleting and washing the
sperm, a second extraction
employing DTT lyses the sperm
cells to release their DNA.
68
Organic Extraction
• Organic extraction of stains or
swabs either known or
suspected to contain sperm calls
for a slightly altered procedure.
The most commonly
encountered example would be
vaginal swabs from a positive
sexual assault case.
69
Chelex Differential Extraction
Chelex Differential Extraction
• As with organic differential extraction
methods, this procedure allows for differential
extraction of DNA from stains containing
sperm using Chelex resin.
• This sperm digest is then mixed with 5%
Chelex. After boiling with Chelex resin to
remove ions and inhibitors, the two fractions
of DNA are then ready to be concentrated and
or quantitated.
70
DNA Extraction Kit
• Most use either modified glass or magnetic
beads which in the presence of salts which
promote binding between DNA and the
specialized glass particles.
• Some kits are optimized for extraction from
specific substrates, such as blood or feces.
• Overall, these proprietary kits offer rapid
extraction, but may require additional washing
in order to isolate samples appropriate for
PCR.
71
Yield Gel Extraction
Yield Gel
• Quantization of DNA with ethidium bromide
visualization of DNA bands in an
electrophoretic submarine gel is used to
assess the total amount of total DNA present
(from any source) and the relative size
(condition) of the DNA fragments. This
technique helps to determine a ball-park
range of DNA quantities, preventing under- or
overloading of the slot blot
72
Slot Blot Extraction
Slot Blot for Human DNA Quantization
• Analysis to determine how much human DNA
is present. Aliquots of extracted DNA are
fixed to a positively charged nylon membrane
along with a serial dilution of known human
DNA standards. The limits of detection are
between 10 and 0.15 ng. Aliquots of DNA
samples having DNA quantities outside of this
range cannot be accurately measured.
73
Slot Blot Extraction
• Slot Blot Human DNA
Gel Plate
74
Fluorometric or Luminometric
Assessment
Fluorometric or Luminometric Assessment
• DNA intercalators in the form of dyes or
excitable enzymatic systems can be used to
label DNA for detection by
spectrophotometers, fluoremeters or
luminometers. Common research protocols.
Forensic DNA analysis tends not to use this
type of quantitation, as it is labor intensive
and not as reliable as other techniques.
75
Spectroscopic Methods
Spectroscopic Methods
• Once the DNA is isolated and extracted, the
amount of recovered DNA can be estimated
using spectroscopic methods.
• Nucleic acids have UV-Vis absorption
characteristics in the 240-280nm range due
to the chemical nature of the purine and
pyrimidine bases.
76
PCR DNA Quantitization
• Real Time PCR Method
(TaqMan®)for Amplifiable
Human DNA Quantization
The yield gel measures the amount and
condition of total DNA, and the slot blot
measures the amount of isolated higher primate
DNA, the RT PCR system measures the amount
of amplifiable human DNA
77
DNA Amplification
• Amplification of extracted and
quantitated DNA samples is a
process where DNA is copied
to generate sufficient sample
for analysis. Amplification
using PCR is a method of
chemically copying DNA.
• In forensic DNA analysis is
multiplex PCR short tandem
repeats.
78
DNA Amplification
• For animated descriptions of PCR see the
following websites:
• http://vector.cshl.org/Shockwave/pcran
whole.html
• http://www.people.virginia.edu/~rjh9u/
pcranim.html
• http://allserv.rug.ac.be/~avierstr/principl
es/pcr.html
79
The Genetic Amplifier
• The underlying premise by which
capillary electrophoresis operates
is that electrical species
(negatively charged DNA)
suspended in an electrolyte will
migrate according to an applied
electrical current. DNA fragments
should travel away from a
negatively charged electrode
(cathode) and toward a positively
charged electrode (anode).
CE is easily
automated and
reproducible, fast
and with high
separation
efficiency, with a
small sample size
requirement. 80
Databases
• One of goals of CODIS (the
FBI’s Combined DNA Index
System) is the creation of a
database of states’
offenders’ profiles. This
would aid in solving crimes
where there are no specific
suspects.
81
Databases
• The national DNA identification index was
authorized by the DNA Identification Act of
1994 and has been in operation since
October, 1998.
• Another important aspect of CODIS is its use
as a population statistics database, for
research and protocol development and for
quality control if personally identifiable
information is removed.
82
Databases
• CODIS is a computer software program
developed by the FBI that maintains
local, state, and national databases of
DNA profiles from convicted offenders,
unsolved crime scene evidence, and
profiles of missing persons.
83
Databases
• Likelihood ratios (LR) & match probabilities are
widely used statistical measurements used in
assessing or explaining the significance of DNA
evidence
• A powerful means of testing assumptions.
• Match probability – is the forensic sample & the
subject or suspect sample from the same person
& the match probability that they came from
different parties. MP is another presentation of
forensic calculations, calculated from the
frequencies of DNA markers in a database.
84
Databases
• As of April 2003, all states & the US Army & FBI
participated in National Data Index System (NDIS)
except for MS & RI. In the US, some states hold
voluntary samples, often provided for exclusion in
investigations, but the NDIS at the FBI consists only
of convicted offenders.
• CODIS is a coordinated system of local, state and
national databases, which enables crime labs to
exchange and compare profiles electronically.
• http://www.fbi.gov/hq/lab/codis/index1.htm
85
Significance
• The Evaluation of Forensic DNA Evidence, is
available online from the National Academies
Press at
http://www.nap.edu/catalog/5141.html.
Many labs employ software to aid in
appropriately crunching the numbers.
• PCR-based analyses has facilitated typing of minute
amounts of evidence, allowing for more matches to
be made between standards and crime scene
evidence and in cold hits.
86
Databases
• Occasionally or in the event a body is badly
decomposed and parental lineage is
unavailable, secondary standards known to
belong to the individual in question may be
used in casework, such as make-up applicators,
toothbrushes, jewelry, and other personal
items that might hold traces of the person’s
biological material.
87
mtDNA
• Mitochondrial DNA
(mtDNA) is a useful
tool in the event that
available samples are
degraded and
incapable of providing
nuclear DNA for
traditional forensic
DNA analysis.
mtDNA is a circular DNA molecule
found in the mitochondria of a cell.
While only one copy of nuclear
DNA exists in the cell nucleus , a
typical cell may have hundreds of
mitochondria.
88
mtDNA
• Because they are present in the female
gamete at conception, mtDNA is maternallylinked.
• 99.99% of mammalian mtDNA is inherited
from the mother.
• The other sect of mtDNA is incorporated as
the sperm carries approximately 100
mitochondria on its tail region as opposed to
the ~100,000 found in the oocyte (egg).
89
mtDNA
• The US Armed Forces Institute of Pathology has
outlined a DNA Outreach Program to insure
proper identification of fallen servicemen.
• Trial courts in some states have yet to
demonstrate admissibility of mtDNA evidence in
criminal cases, but with increasing technology
and general awareness, it is expected that
mtDNA analysis will rapidly join nDNA analysis
by PCR and RFLP or STR as an accepted tool in
forensic identification.
90
mtDNA
• Mitochondrial DNA typing does not
approach STR analysis in its discrimination
power and thus is best reserved for
samples, such as hair, for which STR
analysis may not be possible.
• Forensic analysis of mtDNA is more
rigorous, time consuming, and costly when
compared to nuclear DNA analysis.
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mtDNA
• Also, all individuals of the same maternal
lineage will be indistinguishable by mtDNA
analysis.
• Two regions of mtDNA have been found to be
highly variable and a procedure known as
sequencing is used to determine the order of
base pairs.
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mtDNA
 Tomb of the Unknown Soldier, Arlington, VA
President Reagan presided over the funeral, and presented the Medal of Honor to the
Vietnam Unknown. The President also acted as next of kin by accepting the interment flag at the
end of the ceremony.
The remains of the Vietnam Unknown were exhumed May 14, 1998. Based on mtDNA
testing, Department of Defense scientists identified the remains as those of AF 1Lt. Michael
Joseph Blassie, who was shot down near An Loc, VN, in 1972. The identification was announced
on June 30, 1998 and on July 10, his remains arrived home to his family in St. Louis, Missouri;
he was reinterred at Jefferson Barracks National Cemetery.
The crypt, which once held the remains of the Vietnam Unknown, has been replaced.
The original inscription of "Vietnam" and the dates of the conflict has been changed to
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"Honoring and Keeping Faith with America's Missing Servicemen.