DNA Analysis
DNA
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DNA = Deoxyribonucleic Acid
Located in CHROMOSOMES in the nucleus
of cells
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What is a chromosome?
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Where do we get them from?
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Tightly packed genetic information
One from each parent!
Genes – portions of DNA that code for traits
and functions
35,000 genes in human body
What is that stuff?
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DNA is a POLYMER made up of
nucleotides
What is a Polymer?
 Where have we seen these before?
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Nucleotide (The DNA monomer)
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Nucleotides are made up of three parts
Sugar (deoxyribose)
 Phosphate
 Base (A,T,G,C)
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There are approximately 100 million
nucleotides in the average DNA molecule!
Nucleotide
A, G, C or T
Forms sugar
Phosphate
Backbone
What makes DNA
Different from
RNA?
Bases (A, G, T, C)
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Purines
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Pyrimidines
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Thymine (T)
Cytosine (C)
Bases bind with SPECIFICITY!!
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Adenine (A)
Guanine (G)
A-T
G-C
Each base contains nitrogen thus they are
sometimes referred to as NITROGENOUS bases
A View of base pairing
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A:T
A View of Base Pairing
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G:C
What is important about base
pairs?
Can predict sequence of one strand based
on the sequence of the other.
 Responsible for Replication and
Transcription
 Repair of damaged DNA
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Order in the Court!
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The specific sequence of nucleotides of all
human beings is 99.9% the SAME!!
It is that 0.1% difference that makes each
person unique
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What is the exception to this rule?
What is so important about this sequence?
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It is a code for amino acids  proteins
What might happen if the bases in the
complementary strand were not ordered
correctly?
Match the sequence!
A
TCGACTAACCGAC
T A G C T G A T T G G C T G
Forensic Use of DNA
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Uses of DNA fingerprinting
ID suspects whose DNA may match evidence
at crime scene
 Clear persons wrongfully accused
 ID crime and catastrophe victims
 Establish paternity and other familial relations
 Match organ donors with recipients in
transplant programs
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RFLP
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Restriction Fragment Length
Polymorphism
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Characterize fragments and calculate the
statistical probability that two people could
have the same fragment sequence
Recombinant DNA Technology
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Recombinant DNA relies on the ability of certain
chemicals, known as restriction enzymes, to cut DNA
into fragments that can later be incorporated into another
DNA strand.
Restriction enzymes can be thought of as highly
specialized scissors that cut a DNA molecule when it
recognizes a specific sequence of bases.
Once a portion of the DNA strand has been cut out with
the aid of a restriction enzyme, the next step in the
recombinant DNA process is to insert the isolated DNA
segment into a foreign DNA strand, usually that of a
bacterium.
As the bacteria multiply rapidly, copies of the altered
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DNA are passed on to all descendants.
RFLP
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Length differences associated with relatively long
repeating DNA strands are called restriction
fragment length polymorphisms (RFLP) and form
the basis for one of the first DNA typing
procedures.
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.
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A Positive RFLP Test
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Once the DNA molecules have been cut up
by a restriction enzyme, the resulting
fragments are sorted out by electrophoresis.
The smaller DNA fragments will move at a
faster rate on the gel plate than the larger
ones.
The fragments are then transferred to a nylon
membrane in a process called Southern
blotting.
To visualize the RFLPs, the nylon sheet is
treated with radioactive probes containing a
base sequence complementary to the RFLPs
being identified (a process called
hybridization).
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A Positive RFLP Test
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Next, the nylon sheet is placed against X-ray
film and exposed for several days.
When the film is processed, bands appear
where radioactive probes stuck to fragments
on the nylon sheet.
A typical DNA fragment pattern will show two
bands (one RFLP from each chromosome).
When comparing the DNA fragment patterns
of two or more specimens, one merely looks
for a match between the band sets.
A high degree of discrimination can be
achieved by using a number of different
probes and combining their frequencies.
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Which Suspect, A
or B, cannot be
excluded from the
class of potential
perpetrators of
this assault?
When forensic scientists examine
DNA in the lab, each sample
appears as a unique sequence of
dark bars. Patterns of bars are
compared to find a match. In the
hypothetical example shown here,
it looks like suspect #2 left some
DNA at the crime scene.
In DNA fingerprinting,
scientists collect samples of
DNA from different sources —
for example, from a hair left
behind at the crime scene and
from the blood of victims and
suspects. They then narrow in
on the stretches of repetitive
DNA scattered throughout these
samples. The profile of
repetitive regions in a particular
sample represents its DNA
fingerprint, which ends up
looking a bit like a barcode.
Each bar in the barcode
represents one particular stretch
of repetitive DNA. Since these
repetitive regions are common
in the genome and highly
variable from individual to
individual, no two people
(except identical twins) will
have exactly the same set of
repetitive regions and, hence,
the same DNA fingerprint.
What to do
when
there’s not
much
there…
PCR Testing
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Polymerase chain reaction is the outgrowth of
knowledge gained from an understanding of how
DNA strands naturally replicate within a cell.
For the forensic scientist, PCR offers a distinct
advantage in that it can amplify minute quantities
of DNA many millions of times.
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1. Denature - DNA is
heated to separate
strands
1. Anneal - primers
(short strands of DNA
used to target specific
regions of DNA for
replication) are added
which HYBRIDIZE
with the strands
2. Extend - DNA
polymerase and free
nucleotides are added
to rebuild each of the
separated strands
3. Rinse and Repeat! 
(25-30x)
PCR and RFLP
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PCR technology cannot be applied to
RFLP DNA typing.
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The RFLP strands are too long, often
numbering in the thousands of bases.
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PCR is best used with DNA strands that
are no longer than a couple of hundred
bases.
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PCR Advantages
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One advantage in moving to shorter DNA
strands is that they would be expected to be
more stable and less subject to degradation
brought about by adverse environmental
conditions.
The long RFLP strands tend to readily break
apart under the adverse conditions not
uncommon at crime scenes.
PCR also offers the advantage in that it can
amplify minute quantities of DNA, thus
overcoming the limited sample size problem
often associated with crime scene evidence.
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DNA Typing
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Portions of the DNA molecule contain sequences of
bases that are repeated numerous times, known as
tandem repeats.
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To a forensic scientist, these tandem repeats offer a
means of distinguishing one individual from another
through DNA typing.
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Tandem repeats seem to act as filler or spacers
between the coding regions of DNA.
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What is important to understand is that all humans
have the same type of repeats, but there is
tremendous variation in the number of repeats each
of us have.
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Short Tandem Repeats (STRs)
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The latest method of DNA typing, short tandem
repeat (STR) analysis, has emerged as the
most successful and widely used DNA
profiling procedure.
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STRs are locations on the chromosome that
contain short sequences that repeat themselves
within the DNA molecule.
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They serve as useful markers for identification
because they are found in great abundance
throughout the human genome.
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STR Advantages
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STRs normally consist of repeating
sequences of 3 to 7 bases in length, and the
entire strand of an STR is also very short,
less than 450 bases in length.
This means that STRs are much less
susceptible to degradation and may often be
recovered from bodies or stains that have
been subjected to extreme decomposition.
Also, because of their shortness, STRs are
ideal candidates for multiplication by PCR,
thus overcoming the previously mentioned
limited-sample-size problem often associated
with crime-scene evidence.
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The Power of STR
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What makes STRs so attractive to forensic
scientists is that hundreds of different types of
STRs are found in human genes.
The more STRs one can characterize, the
smaller will be the percentage of the population
from which a particular combination of STRs can
emanate.
This gives rise to the concept of multiplexing.
Using the technology of PCR, one can
simultaneously extract and amplify a
combination of different STRs.
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Number of repeats is
transferred just like any other
DNA material
Standardizing STR Testing
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Currently, U.S. crime laboratories have
standardized on 13 STRs for entry into a
national database (CODIS).
A high degree of discrimination and even
individualization can be attained by analyzing a
combination of STRs (multiplexing) and
determining the product of their frequencies.
With STR, as little as 125 picograms of DNA is
required for analysis.
This is 100 times less than that normally
required for RFLP analysis.
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CODIS
CODIS STRs and Probability
STR
African
American
American
Caucasian
D351358
0.097
0.080
VWA
0.074
0.068
1/X = 0.097
FGA
0.036
0.041
SOLVE FOR X
TH01
0.114
0.080
TPOX
0.091
0.207
X = 1/0.097 = 10.3
CFS1PO
0.079
0.128
D5S818
0.121
0.166
This means the DNA matches one
in 10.3 people
D13S317
0.139
0.081
D7S820
0.087
0.067
D8S1179
0.080
0.069
D21S11
0.042
0.041
D18S51
0.032
0.032
D16S539
0.076
0.091
1/X = value
Think about class evidence…how
do we narrow down the pool of
possible suspects?
Y- STR
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Another tool available in the arsenal of the
DNA analyst is the ability to type STRs
located on the Y chrosome, which is male
specific.
More than 20 different Y-STR markers have
been identified.
Y-STRs will prove useful when multiple males
are involved in a sexual assault.
A Y-STR analysis will have only one band or
peak, rather than the conventional STR which
is derived from two chromosomes and has
two bands or peaks.
The Y-STR is therefore less complicated in
appearance and interpretation.
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Gould
Slabotnik
Function of CODIS
The four primary functions of the current CODIS software are:
 DNA profile entry and management: the function dealing with the
database DNA profiles.
 Searching: the function allowing a search of database DNA profiles.
 Match management: the function managing search results. For
example, it allows a laboratory to record and distinguish whether a
particular match is an offender hit or a forensic hit, and whether the
match is within or outside of the state.
 Statistical calculations: the function enabling laboratory personnel
to calculate profile statistics, based on the laboratory's or FBI's
population frequency data
Where is DNA found?
DNA is contained in blood, semen, skin
cells, tissue, organs, muscle, brain cells,
bone, teeth, hair, saliva, mucus,
perspiration, fingernails, urine, feces, etc.
 http://www.dna.gov/basics/
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Nuclear vs. Mitochondrial DNA
Oocyte Maturation
Nuclear DNA
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Nuclear DNA:
Found in nucleus of cells
 Contains 23 chromosome pairs
 Each parent contributes half
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Mitochondrial DNA
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Mitochondria are found
in all cells of the body
Provide 90% of energy
our bodies need to
function
Contain bits of DNA
passed down through
generations
Hundreds of thousands
of mitochondria per cell!!
What are the benefits?
What are the limitations?
Mitochondrial DNA
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Used in identifying remains
Generous amount present in bones
 Shows maternal lineage matches (will be
identical if related through the mother)
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