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DNA Structure
https://www.youtube.com/watch?v=qy8dk5iS1f0
What do these items have to do
with one another?
Deoxyribonucleic Acid (DNA)
Forensic Science
Introduction to DNA
• Like fingerprints, DNA is unique to each
individual  individual evidence!
• DNA can definitively link a suspect to a victim or
crime scene.
• Chromosomes are threadlike structures composed
of DNA
• The primary unit of heredity is called a gene
Video
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Structure of DNA
double
helix
• DNA (deoxyribonucleic acid) is polymer of
repeating units called nucleotides
– A nucleotide consists of
• Sugar-phosphate backbone
– Deoxyribose sugar
– Phosphate
• Nitrogenous base
– adenine, guanine, cytosine, thymine
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The Bases
• Four types of bases used
in DNA: adenine (A),
guanine (G), cytosine
(C), and thymine (T).
• Complementary base
pairing occurs between A
and T, and the base C
with G in double
stranded DNA.
The Bases
• DNA’s structure is a
double-stranded helix as
discovered by Rosalind
Franklin, Crick and
Watson.
• Chargaff discovered that
A always pairs with T
and C always pairs with
G.
Structure of DNA
• The long strands of DNA are coiled upon
themselves into chromosomes
– When paired, chromosomes resemble the letter X
• Humans have 23 pairs of chromosomes,
including 2 sex chromosomes
– Females are XX
– Males are XY
DNA at Work
• DNA codes for proteins
• Proteins are formed by amino acids (monomers)
in a long chain.
• The sequence of amino acids determines the
shape and function of the protein.
DNA at Work
• A codon (three nucleotides)
codes for a particular
amino acid.
 G-A-G codes for the
amino acid glutamine,
while C-G-T codes for
alanine.
Intracellular
DNA
Replication
(Inside the cell)
• DNA duplicates itself in
the nucleus prior to cell
division.
• DNA replication begins
with the unwinding of
the DNA strands of the
double helix.
• Many enzymes are
involved in unwinding
the DNA (helicases), and
assembling the new DNA
strands (polymerases).
Extracellular DNA Replication
(Outside the cell)
Why would a forensic scientist want to replicate
DNA?
This can be valuable when the amount of
evidence is minimal. Millions of copies of
DNA can be made from a single speck of
blood.
Extracellular Replication Polymerase Chain Reaction (PCR)
• A technique for making many copies of a
specific piece of DNA to be analyzed forensically
– Can amplify very minute quantities of DNA millions
of times!
• This method works by cycling
through different temperatures
• A device called a thermocycler controls the
temperatures, allowing for fast and accurate
copying of DNA
PCR
•
The outcome is a doubling of the number of
DNA strands.
• Heating, cooling, and strand rebuilding is
repeated typically 25 to 30 times, yielding more
than one million copies of the original DNA
molecule.
• Each cycle takes less than two minutes from
start to finish
Video - https://www.youtube.com/watch?v=0HCWmD7Mv8U
PCR
• PCR uses:
primer
DNA polymerase
(taken from bacteria)
nitrogen bases
(Adenine, Thymine,
Cytosine and
Guanine) to copy a
segment of DNA.
o
o
o
• Very little DNA is
needed (0.2 μl)
• All the ingredients
are placed into the
thermal-cycler.
• Thermal-cycler heats
the DNA sequence to
unravel the strand.
• The A, T, C and G
will pair with the
sample in the correct
order, the copy will
be released when the
cycle cools.
Steps of PCR
1. Extract DNA
2. Denature
3. Anneal.
4. Extend.
Polymerase Chain Reaction (PCR)
The steps of PCR
• Denaturation:
– Extracted and purified DNA is heated to “unzip”
(separate) the double helix
– This is done at high temperature, about 94°C
Polymerase Chain Reaction (PCR)
The steps of PCR
• Annealing:
– Short template pieces called “primers” bind with
the separated strands for new DNA to build upon.
– This occurs at ~65°C
Polymerase Chain Reaction (PCR)
The steps of PCR
• Extend/Elongation:
– DNA Polymerase adds free nucleotides from the
surrounding solution onto the template primers
– In this way, new strands are built out of the original
2 separated stands
– This happens at 72°C
new DNA
strands
Polymerase Chain Reaction (PCR)
• Each step only requires a few minutes
• The thermocycler machine cycles through
these temperatures for several hours
• Each cycle doubles the number of copied
DNA strands
PCR is specific to your
“region of interest.” Different
primers will selectively
amplify different genes
DNA Typing
• DNA typing (a.k.a. DNA Fingerprinting) was
developed by British geneticist Sir Alec Jeffreys
in 1984.
• This technique converts DNA into readable bands
on a gel
With these bands, we
can compare suspect
and crime scene DNA,
or child and possible
father, etc.
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DNA Typing
DNA typing is a method in which DNA is converted
into a series of bands that distinguishes each
individual.
Only one-tenth of a single percent of DNA (about
three million bases) differ from one person to the
next. Scientists use these regions to generate a
DNA profile of an individual.
Uses of DNA Profiling
To identify potential suspects
To identify crime and casualty victims
To establish paternity
To exonerate individuals
To match organ donors
jyokum 2013
DNA Typing
• Portions of the DNA molecule contain
sequences of bases that are repeated numerous
times, known as tandem repeats.
• Tandem repeats – region of a chromosome that
contains multiple copies of a DNA sequence that
repeats
• To a forensic scientist, these tandem repeats
distinguish one individual from another through
DNA-typing.
DNA Typing
• Tandem repeats are found throughout our
genome between the coding areas of DNA.
• What is important to understand is that all
humans have the same repeat regions, but there
is tremendous variation in the number of
repeats and each person has a unique number
in each region of their genome.
DNA Typing
“Fingerprinting” 1985—Alec Jeffreys
Forensic scientists aimed efforts at
characterizing the following repeat segments:
• RFLP – restriction fragment length
polymorphism
• STR – short tandem repeats
RFLP—Restriction Fragment
Length Polymorphisms
Restriction enzymes are used to cut DNA into smaller
fragments that can then be separated and characterized
for identification.
1) Extract—separate DNA from the cell
2) Cut—use of restriction enzymes to make shorter
base strands
3) Sort—by size using electrophoresis (DNA separation technique)
4) Analyze—the specific alleles for identification
Video - https://www.youtube.com/watch?v=jMLIaxxY6-8
RFLP Analysis
Electrophoresis
• A technique used to separate DNA fragments
Electrophoresis
• In the lab, DNA molecules are cut by
restriction enzymes into fragments of
various sizes. Restriction enzymes cut at
specific sequences throughout the DNA.
• The resulting fragments are forced to move
along a gel-coated plate under the influence
of an electrical potential
Electrophoresis
• DNA samples are injected into a gel
• An electrical current is moved through a gel
• DNA molecules moves because it is negatively
charged
• DNA molecules are sorted by size.
• The smaller, lighter molecules will move the
farthest on the gel.
jyokum 2013
Electrophoresis
https://www.youtube.
com/watch?v=lgmq_
HsuZIU
Electrophoresis
• After the fragments have
“migrated” across the gel,
the gel can be stained to
show the “bands” or
fragments easily
• Then comparisons can be
made
– Example: crime scene
sample to suspect
Short Tandem Repeats (STR)
•
STR is another method of DNA typing.
•
STRs are locations (loci) on the chromosome that
contain short sequences of two to five bases that
repeat themselves in the DNA molecule.
•
The advantages of this method over RFLP are:
1. provides greater discrimination
2. requires less time
3. a smaller sample size,
4. DNA is less susceptible to degradation.
Short Tandem Repeats
(STRs)
• A common method of DNA typing
• STR’s are less susceptible to degradation and can be
recovered from bodies or stains that have been
subject to extreme decomposition
• With the technology of PCR one can extract and
amplify a combination of different STR’s. More on
this later…
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STRs
• In forensic laboratories, there are thirteen
STR loci that are typically used to create a
genetic fingerprint of an individual.
• Although controversial, the profile is kept in
DNA databases. In the United States, the
genetic fingerprints are kept in various
Combined DNA Index Systems (CODIS)
databases ranging from the smaller, local
levels to the national level.
• The CODIS loci analyzed by STR analysis.
Notice they are spread over 14 chromosomes,
and that two are on the X and Y chromosomes.
CODIS
• The Combined DNA Index
System is maintained by the
Federal Bureau of Investigation.
As of June 2012, CODIS
maintained over 9.7 million
offender profiles, 1.1 million
arrestee profiles and 436,000
forensic profiles. Profiles
maintained in CODIS are
compiled from both suspects and
evidence, and therefore are used
to help solve criminal cases. Also
as of June 2012, CODIS has
produced over 182,200 "hits,"
assisting in more than 174,600
investigations.
• Profiles of missing persons are
also maintained in CODIS. The
true power of STR analysis is in
its statistical power of
discrimination. Because the 13
loci are independently assorted,
the laws of probabilities can be
applied. This means that if
someone has the genotype of
ABC at three independent loci,
then the probability of having
that specific genotype is the
probability of having type A
times the probability of having
type B times the probability of
having type C. This has resulted
in the ability to generate match
probabilities of 1 in a quintillion.
Combined DNA Information System
(CODIS)
• CODIS maintains a
database of DNA profiles
from
–convicted offenders
–unsolved crime scene
evidence
–profiles of missing
persons
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Possible Sources of DNA
•
•
•
•
•
•
•
•
•
•
•
Skin
Sweat
Blood
Mucus
Saliva
Tissue
Semen
Urine
Hair (root)
Ear Wax
Vaginal or rectal cells
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Collecting and Packaging
Biological Evidence
• Photograph evidence first
• Wear gloves at all times
• Package each stained article separately
in paper or a well-ventilated box (to
avoid bacterial or fungal growth)
• Remove dried blood using a sterile
swab moistened with distilled water
• Store biological evidence in the
refrigerator or a cool location until it is
delivered to the lab
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(end)
(stop)
Isolating DNA
• The first step to DNA sequencing is isolating
DNA
• Perform DNA Extraction Lab
DNA Sequencing: PCR
Polymerase Chain Reaction
(PCR)
• A technique for making many copies of a
specific piece of DNA to be analyzed forensically
– Can amplify very minute quantities of DNA millions
of times!
• This method works by cycling
through different temperatures
• A device called a thermocycler controls the
temperatures, allowing for fast and accurate
copying of DNA
(stop)
PCR Simulation
Extracellular DNA Replication
(Outside the cell)
• Polymerase chain
reaction (PCR) is a
technique for replicating
a small DNA sample
found at a crime scene.
• The ability to multiply
small bits of DNA means
that a single sample is no
longer a limitation in
analyzing crime scene
DNA.
Steps of PCR
1. Denature. The first step requires a high temperature to
denature and separate the double stranded DNA. This is
done by heating the sample to 92-94oC.
2. Anneal. The second step requires lowering the
temperature to allow annealing (binding) of the primers
to the single stranded DNA. The optimal annealing
temperature is 45-55oC.
3. Extend. The third step requires DNA synthesis by DNA
polymerase. The optimal temperature is about 75-80oC.
The rate of primer extension by polymerase is about 50100 nucleotides/sec.
Resources
• Saferstein, Richard. Forensic Science: An Introduction. New
Jersey: Pearson Prentice Hall, 2008
• Saferstein, Richard. Forensic Science: An Introduction. 2nd
ed. New Jersey: Pearson Prentice Hall, 2011
• Saferstein, Richard. Criminalistics: An Introduction to
Forensic Science. 8th ed. Upper Saddle River, NJ; Pearson
Prentice Hall, 2004
• http://law2.umkc.edu/faculty/projects/ftrials/clinton/lewinskyd
ress.html
• http://www.trutv.com/library/crime/notorious_murders/famous
/simpson/index_1.html
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